SP17 binding proteins, including fully human anti-SP17 antibodies

Abstract

The present disclosure describes proteins that specifically bind to human sperm protein 17 (Sp17) with nanomolar affinity and high specificity. These proteins include recombinant human anti-Sp17 IgG, which are suitable for use as therapeutic antibodies to treat cancers that express Sp17 ectopic. Other Sp17 binding proteins, including antibody fragments, antibody conjugates, and fusion proteins, are also described.

Description

Cross-references to related applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 524,810, filed July 3, 2023, and U.S. Patent Application No. 18 / 408,414, filed January 9, 2024, the disclosures of which are incorporated herein by reference in their entirety. sequence list

[0002] This disclosure includes a sequence list named "Sequence_Listing_1200590019.xml", created on July 3, 2024, with a file size of 14,027 bytes. Technical Field

[0003] This invention relates to proteins that bind sperm protein 17 (SP17), these SP17-binding proteins being derived from anti-SP17Fab, including anti-SP17 monoclonal antibodies and SP17-binding conjugates and fusion proteins derived from the complementarity-determining region (CDR) of that Fab. The invention particularly relates to the use of SP17-binding proteins in the diagnosis and treatment of various health conditions, including cancer. Background Technology

[0004] The human SP17 gene, located on chromosome 11q24.2, encodes a highly conserved, antigenic 17.4 kDa protein, SP17, which is expressed in sperm. SP17 is involved in the acrosome reaction during fertilization. Immunohistochemical assays of tissue microarrays and reverse transcription-polymerase chain reaction (RT-PCR) of RNA panels from different tissues indicate that SP17 is expressed in normal testes and absent in other healthy tissues. SP17 has also been identified as a tumor antigen aberrantly expressed in multiple myeloma, lymphoma, ovarian cancer, and non-small cell lung cancer. Summary of the Invention

[0005] Various aspects of this disclosure relate to the discovery of a novel anti-SP17 Fab comprising a CDR having nanomolar affinity for SP17 and high specificity for SP17 relative to other human proteins. The CDR maintains its high affinity and fidelity in various antibody forms. Human antibodies containing these CDRs are undergoing preclinical development for use as cancer immunotherapeutic agents. These CDRs are also compatible with a full range of immunotherapeutic strategies, including antibody-drug conjugates, bispecific proteins (e.g., as T-cell connectors), and adoptive cell therapies (e.g., as CAR-T). These CDRs can also be cloned into antibodies for laboratory research and diagnostics. The following disclosure describes, for example, a chimeric IgG containing a mouse Fc region, which can be combined with an anti-mouse secondary antibody for various assays and diagnostics. Its applications in flow cytometry, immunohistochemistry, and Western blotting are described.

[0006] The foregoing background and summary sections are intended to provide a brief introduction to the subject matter and to outline some of the technical improvements and advantages it brings. The background and summary sections should not be construed as identifying essential features of the subject matter, nor should they be construed as limiting the interpretation of this specification or any patent claims based on this specification and ultimately granted. Brief description of the attached figures

[0007] The following figures may be used to further understand this specification. The figures are exemplary, and neither this specification nor any patent claims based on this specification and ultimately granted should be construed as being limited by the figures.

[0008] Figure 1 These are SDS-PAGE gel images of 2 micrograms of recombinant SP17 protein loaded with molecular weight standards (lane M) and under reducing conditions (lane 1) and non-reducing conditions (lane 2).

[0009] Figure 2 The image consists of two subplots. The left subplot is an SDS-PAGE gel stained with Coomassie Brilliant Blue, loaded with molecular weight standards (Mr lane), anti-SP17 antibody chAB2 under reducing conditions (lane 1), and anti-SP17 antibody chAB2 under non-reducing conditions (lane 2). The right subplot is a Western blot of the SDS-PAGE gel loaded with molecular weight standards (M lane) and anti-SP17 antibody chAB2 under reducing conditions (E lane), where chAB2 was detected by enhanced chemiluminescence (ECL) using anti-mouse secondary antibody.

[0010] Figure 3 It is a protein blot of an SDS-PAGE gel loaded with molecular weight standards (lane M) and SP17 protein (lanes 1, 2 and 3), in which SP17 protein was detected using anti-SP17 antibody chAB2.

[0011] Figure 4 The results consist of three subplots. The left subplot shows the flow cytometry results of ID8 cells incubated with anti-mouse fluorescein isothiocyanate (FITC) antibody but without primary antibody. The middle subplot shows the flow cytometry results of ID8 cells labeled with anti-SP17 antibody chAB2 and anti-mouse FITC antibody. The right subplot is a histogram showing that, compared to the ID8 cells incubated only with anti-mouse FITC antibody in the left subplot, the mean fluorescence intensity of ID8 cells labeled with chAB2 and anti-mouse FITC antibody in the middle subplot is increased.

[0012] Figure 5The chromatogram is a high-performance liquid chromatography (HPLC) chromatogram, indicating that the anti-SP17 antibody chAB2 formulation can be manufactured with a purity of 93%.

[0013] Figure 6 This is a surface plasmon resonance (SPR) plot of the anti-SP17 antibody chAB2 binding to recombinant 6His-SP17 protein. The lines from highest to lowest correspond to the fitted cycles of 6His-SP17 concentrations at 300 nmol, 100 nmol, 50 nmol, 25 nmol, 12.5 nmol, and 6.25 nmol. This data was used to calculate the binding rate constant (ka) of chAB2 to SP17 as 62,600 mol / s, the dissociation rate constant (kd) as 0.0001298 s / s, and the dissociation constant (KD) as 2.073 nmol.

[0014] Figure 7 These are SDS-PAGE gel images of anti-SP17 antibody SP17-AB2 loaded with molecular weight standards (lane M) and under reducing conditions (lane 1) and non-reducing conditions (lane 2).

[0015] Figure 8 It is a Western blot of protein on an SDS-PAGE gel loaded with molecular weight standard (lane M), SP17 protein (lane 1) and tumor cell lysate (lane 2), in which SP17 protein is detected using anti-SP17 antibody SP17-AB2.

[0016] Figure 9A , 9B 9C is a graph showing the immunotherapy concentration (x-axis, nanomolar) versus cytotoxicity against SK-OV-3 cells (y-axis, percentage of cytotoxicity), with the immunotherapy agents selected from the monomeric antibody SP17-AB2 (circle, ●), the monomeric immunoconjugate SP17-AB2-DXD (square, ■), the control human IgG4 antibody (positive triangle, ▲), and drutecan (inverted triangle, ▼).

[0017] Figure 10 This is a graph showing the relationship between the concentration of immunotherapy drugs (x-axis, nanomolar) and their cytotoxicity to ID8 cells (y-axis, percentage of cytotoxicity). The immunotherapy drugs compared in the graph include: the monomeric antibody SP17-AB2 (dot, ●), the immunoconjugate SP17-AB2-DXD (square, ■), hIgG4 (upper triangle, ▲), and deruxtecan (lower triangle, ▼). Detailed Implementation

[0018] Various aspects of this disclosure relate to antibodies exhibiting high affinity for human SP17 protein. These antibodies were developed using a Fab phage display biopanning strategy based on a library generated from peripheral blood of 120 healthy donors, as described in Example 1 below. This library contains approximately one trillion different combinations. The best-performing Fab in this library has the immunoglobulin heavy chain variable region (VH) with nucleotide sequence SEQ ID NO: 1 and the immunoglobulin light chain variable region (VL) with nucleotide sequence SEQ ID NO: 2, as shown in Table 1 below. The VH amino acid sequence is SEQ ID NO: 3, and the VL amino acid sequence is SEQ ID NO: 4. The VH and VL amino acid sequences are shown in Table 2 below, where CDRs are indicated in bold and underlined, and framed regions are indicated in plain text. CDR sequences are SEQ ID NO: 5-10 and are shown separately in Table 3 below. Those skilled in the art will recognize that, at least for some CDRs, the precise boundary between CDRs and frame regions is not clear, and therefore the CDRs described in Tables 2 and 3 and SEQ ID NO: 5-10 may contain one or more amino acids that may be more appropriately classified as frame regions rather than CDRs.

[0019] The nucleotide sequences (SEQ ID NO: 1 and 2) encoding the variable region of Fab, which exhibits optimal performance, were initially cloned into the mouse IgG2a heavy chain gene and the mouse κ light chain gene to express the chimeric monoclonal antibody (chAB2). The affinity of this chimeric antibody was approximately 2 nanomolar, as determined by surface plasmon resonance. Immunohistochemical analysis of this chimeric antibody in 33 normal human tissues showed a lack of detectable cross-reactivity in the variable region. Therefore, this chimeric antibody can be used for, for example, laboratory analyses, including immunohistochemistry, Western blotting, and flow cytometry (described below), as well as medical diagnostics, including enzyme-linked immunosorbent assay (ELISA).

[0020] Various aspects of this disclosure relate to a kit comprising a recombinant anti-SP17 antibody and a reporter molecule as described in any part of this disclosure, wherein the kit is configured for detecting SP17 in a biological sample. In some embodiments, the reporter molecule comprises (i) a conjugated dye or (ii) a conjugated fluorescent, phosphorescent, chemiluminescent, radioactive, or magnetic label. In some specific embodiments, the reporter molecule comprises a secondary antibody; the conjugated dye is an antibody-conjugated dye; and the conjugated fluorescent, phosphorescent, chemiluminescent, radioactive, or magnetic label is an antibody-conjugated fluorescent, phosphorescent, chemiluminescent, radioactive, or magnetic label. In some very specific embodiments, the kit is configured for detecting SP17 in a biological sample by immunohistochemistry, Western blotting, flow cytometry, magnetically activated cell sorting, ELISA, or chemiluminescent immunoassay.

[0021] The human variable region was then cloned into the human IgG4 heavy chain gene and the human κ light chain gene to generate a human monoclonal antibody (SP17-AB2) suitable for use as a therapeutic antibody. The heavy chain constant region was mutated to include the Ser228Pro mutation to reduce non-specific interaction with the Fc receptor γ (FcγR). The intact heavy chain has the amino acid sequence shown in SEQ ID NO: 10, and the intact light chain has the amino acid sequence shown in SEQ ID NO: 11, as shown in Table 4 below.

[0022] Various aspects of this disclosure relate to recombinant anti-SP17 antibodies or antigen-binding fragments thereof that contain the aforementioned CDR, or have sequence homology (or sequence identity) with the VH and VL variable regions containing the aforementioned CDR. In all embodiments, the antibody or its antigen-binding fragment binds to human SP17.

[0023] The term "antibody" includes different classes of immunoglobulins (Ig) (e.g., IgA, IgG, IgM, IgD, and IgE) and subclasses (e.g., IgG2a and IgG4), and also includes fully human antibodies, chimeric antibodies, and their engineered variants. For example, this specification describes a chimeric human-mouse antibody (chAB2) and an engineered fully human antibody containing the Ser228Pro mutation (SP17-AB2).

[0024] The term "antigen-binding fragment" refers to the Fab fragment and a single-chain variable fragment (scFv). A scFv is a fusion protein consisting of two variable regions linked by a flexible linker, which retains antigen-binding properties equivalent to those of the Fab fragment. An example of a scFv is busizumab (also known as BEOVU®).

[0025] As used in this disclosure, the term "sequence homology" refers to the percentage of "positive matches" determined by the standard protein BLAST® over the full length of the sequence described in SEQ ID NO. The standard protein BLAST® is available at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp. BLAST® is commonly described in Altschul et al. (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein databasesearch programs", Nucleic Acids Res. 25:3389-3402, and Altschul et al. (2005), "Protein database searches using compositionally adjusted substitution matrices", FEBS J. 272:5101-5109. As used in this disclosure, the term "sequence consistency" refers to the percentage of exact matches over the full length of the sequence described in SEQ ID NO.

[0026] In all embodiments, the recombinant anti-SP17 antibody or its antigen-binding fragment comprises two variable domains. The two variable domains include a heavy chain variable region and a light chain variable region. In some specific embodiments, the heavy chain variable region has a percentage sequence homology (or sequence identity) with SEQ ID NO: 5, 6, and 7, and the light chain variable region has a percentage sequence homology (or sequence identity) with SEQ ID NO: 8, 9, and 10. In some very specific embodiments, the heavy chain variable region has a percentage sequence homology (or sequence identity) with SEQ ID NO: 3, and the light chain variable region has a percentage sequence homology (or sequence identity) with SEQ ID NO: 4.

[0027] In some embodiments, the antibody or its antigen-binding fragment comprises (1) a first variable domain comprising a VH CDR1 region, a VH CDR2 region, and a VH CDR3 region, wherein the VH CDR1 region comprises an amino acid sequence identical to at least 7, 8, 9, or 10 consecutive amino acids as described in SEQ ID NO: 5, the VH CDR2 region comprises an amino acid sequence identical to at least 7, 8, 9, or 10 consecutive amino acids as described in SEQ ID NO: 6, and the VH CDR3 region comprises an amino acid sequence identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 consecutive amino acids as described in SEQ ID NO: 7; and (2) a second variable domain comprising a VL CDR1 region, a VL CDR2 region, and a VL CDR3 region, wherein the VL CDR1 region comprises an amino acid sequence identical to at least 7, 8, 9, or 10 consecutive amino acids as described in SEQ ID NO: 5, the VH CDR2 region comprises an amino acid sequence identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive amino acids as described in SEQ ID NO: 7; and (2) a second variable domain comprising a VL CDR1 region, a VL CDR2 region, and a VL CDR3 region, wherein the VL CDR1 region comprises an amino acid sequence identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive amino acids as described in SEQ ID NO: 5. The VL CDR2 region contains an amino acid sequence identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive amino acids as described in SEQ ID NO: 8, and the VL CDR3 region contains an amino acid sequence identical to at least 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids as described in SEQ ID NO: 10.

[0028] In some embodiments, the recombinant anti-SP17 antibody comprises four variable domains, wherein the antibody comprises (1) a third variable domain comprising a VH CDR1 region, a VH CDR2 region, and a VH CDR3 region, wherein the VH CDR1 region comprises an amino acid sequence identical to at least 7, 8, 9, or 10 consecutive amino acids as described in SEQ ID NO: 5, the VH CDR2 region comprises an amino acid sequence identical to at least 7, 8, 9, or 10 consecutive amino acids as described in SEQ ID NO: 6, the VH CDR3 region comprises an amino acid sequence identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 consecutive amino acids as described in SEQ ID NO: 7; and (2) a fourth variable domain comprising a VL CDR1 region, a VLCDR2 region, and a VL CDR3 region, wherein the VL CDR1 region comprises an amino acid sequence identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive amino acids as described in SEQ ID NO: 7; The VL CDR2 region contains an amino acid sequence identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive amino acids as described in SEQ ID NO: 8, and the VL CDR3 region contains an amino acid sequence identical to at least 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids as described in SEQ ID NO: 10. Such antibodies include, for example, IgG antibodies.

[0029] In some embodiments, one or more of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 contain one or more conserved mutations. Conserved mutations are known in the art and include, for example, threonine to serine, isoleucine to valine or leucine, tyrosine to phenylalanine, aspartic acid to glutamic acid, asparagine to glutamine, arginine to lysine, and similar substitutions. In this disclosure, a conserved mutation is a "positive" match in the standard protein BLAST®, but not a homologous match.

[0030] Various aspects of this disclosure relate to a recombinant anti-SP17 antibody or an antigen-binding fragment thereof, comprising two variable domains, wherein: the antibody or antigen-binding fragment comprises a first variable domain containing an amino acid sequence having at least 90% sequence homology to SEQ ID NO: 3; the antibody or antigen-binding fragment comprises a second variable domain containing an amino acid sequence having at least 90% sequence homology to SEQ ID NO: 4; and the antibody or antigen-binding fragment binds to human SP17. In some specific embodiments, the first variable domain contains an amino acid sequence having at least 90% sequence homology to SEQ ID NO: 3; the second variable domain contains an amino acid sequence having at least 90% sequence homology to SEQ ID NO: 4; and the antibody or antigen-binding fragment binds to human SP17.

[0031] SEQ ID NO: 3 is a VH amino acid sequence comprising VH CDR1, VH CDR2, VH CDR3, and a frame region. In some embodiments, the VH amino acid sequence contains a mutation relative to SEQ ID NO: 3, such that the VH amino acid sequence has at least 90% but less than 100% sequence homology with SEQ ID NO: 3. In some specific embodiments, the VH amino acid sequence contains a mutation relative to SEQ ID NO: 3, such that the VH amino acid sequence has at least 90% but less than 100% sequence identity with SEQ ID NO: 3.

[0032] SEQ ID NO: 4 is a VL amino acid sequence comprising VL CDR1, VL CDR2, VL CDR3, and a frame region. In some embodiments, the VL amino acid sequence contains a mutation relative to SEQ ID NO: 4, such that the VL amino acid sequence has at least 90% but less than 100% sequence homology with SEQ ID NO: 4. In some specific embodiments, the VL amino acid sequence contains a mutation relative to SEQ ID NO: 4, such that the VL amino acid sequence has at least 90% but less than 100% sequence identity with SEQ ID NO: 4.

[0033] Mutations in the VH and / or VL amino acid sequences can be engineered, for example, to regulate the affinity of an antibody (or its antigen-binding fragment) for the SP17 antigen, to regulate expression or purification, to regulate half-life, or for other purposes.

[0034] In some embodiments, the first variable domain comprises an amino acid sequence having at least 95% sequence homology with SEQ ID NO: 3. In some specific embodiments, the first variable domain comprises an amino acid sequence having at least 97% sequence homology with SEQ ID NO: 3. In some more specific embodiments, the first variable domain comprises an amino acid sequence having at least 98% sequence homology with SEQ ID NO: 3. In some very specific embodiments, the first variable domain comprises an amino acid sequence having at least 99% sequence homology with SEQ ID NO: 3.

[0035] In some embodiments, the first variable domain comprises an amino acid sequence that is at least 95% sequence identical to SEQ ID NO: 3. In some specific embodiments, the first variable domain comprises an amino acid sequence that is at least 97% sequence identical to SEQ ID NO: 3. In some more specific embodiments, the first variable domain comprises an amino acid sequence that is at least 98% sequence identical to SEQ ID NO: 3. In some very specific embodiments, the first variable domain comprises an amino acid sequence that is at least 99% sequence identical to SEQ ID NO: 3.

[0036] In some embodiments, the second variable domain comprises an amino acid sequence having at least 95% sequence homology with SEQ ID NO: 4. In some specific embodiments, the second variable domain comprises an amino acid sequence having at least 97% sequence homology with SEQ ID NO: 4. In some more specific embodiments, the second variable domain comprises an amino acid sequence having at least 98% sequence homology with SEQ ID NO: 4. In some very specific embodiments, the second variable domain comprises an amino acid sequence having at least 99% sequence homology with SEQ ID NO: 4.

[0037] In some embodiments, the second variable domain comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 4. In some specific embodiments, the second variable domain comprises an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 4. In some more specific embodiments, the second variable domain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 4. In some very specific embodiments, the second variable domain comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 4.

[0038] In some embodiments, the recombinant anti-SP17 antibody comprises four variable domains, wherein the antibody comprises (1) a third variable domain comprising an amino acid sequence having at least 90% sequence homology (e.g., at least 95%, 97%, 98%, or 99% sequence homology) with SEQ ID NO: 3; and (2) a fourth variable domain comprising an amino acid sequence having at least 90% sequence homology (e.g., at least 95%, 97%, 98%, or 99% sequence homology) with SEQ ID NO: 4. In some specific embodiments, the third variable domain comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 95%, 97%, 98%, or 99% sequence identity) with SEQ ID NO: 3, and the fourth variable domain comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 95%, 97%, 98%, or 99% sequence identity) with SEQ ID NO: 4.

[0039] In some embodiments, the KD of the recombinant anti-SP17 antibody or its antigen-binding fragment with human SP17 is no greater than 25 nanomolars. In some specific embodiments, the KD of the recombinant anti-SP17 antibody or its antigen-binding fragment with human SP17 is no greater than 10 nanomolars. In some very specific embodiments, the KD of the recombinant anti-SP17 antibody or its antigen-binding fragment with human SP17 is no greater than 2.5 nanomolars.

[0040] In some implementations, KD is determined by surface plasmon resonance (e.g., using a Biacore™ instrument).

[0041] In some embodiments, the recombinant anti-SP17 antibody includes an IgG heavy chain constant region. In some specific embodiments, the recombinant anti-SP17 antibody includes an IgG2 heavy chain constant region. In some specific embodiments, the recombinant anti-SP17 antibody includes an IgG4 heavy chain constant region. In some very specific embodiments, the recombinant anti-SP17 antibody includes an IgG2a heavy chain constant region.

[0042] In some embodiments, the recombinant anti-SP17 antibody includes a κ or λ light chain constant region. In some specific embodiments, the recombinant anti-SP17 antibody includes a κ light chain constant region.

[0043] In some embodiments, the recombinant anti-SP17 antibody comprises a mouse heavy chain constant region and a mouse light chain constant region. In some specific embodiments, the recombinant anti-SP17 antibody comprises a mouse IgG heavy chain constant region and a mouse κ or λ light chain constant region. In some very specific embodiments, the recombinant anti-SP17 antibody comprises a mouse IgG2a heavy chain constant region and a mouse κ light chain constant region.

[0044] In some embodiments, the recombinant anti-SP17 antibody comprises a human heavy chain constant region and a human light chain constant region. In some specific embodiments, the recombinant anti-SP17 antibody comprises a human IgG heavy chain constant region and a human κ or λ light chain constant region. In some very specific embodiments, the recombinant anti-SP17 antibody comprises a human IgG4 heavy chain constant region and a human κ light chain constant region.

[0045] In some embodiments, the recombinant anti-SP17 antibody has a heavy chain with at least 90% sequence homology to SEQ ID NO: 11. In some specific embodiments, the recombinant anti-SP17 antibody has a heavy chain with at least 95% sequence homology to SEQ ID NO: 11. In some more specific embodiments, the recombinant anti-SP17 antibody has a heavy chain with at least 98% sequence homology to SEQ ID NO: 11. In some very specific embodiments, the recombinant anti-SP17 antibody has a heavy chain with at least 99% sequence homology to SEQ ID NO: 11.

[0046] In some embodiments, the recombinant anti-SP17 antibody has a heavy chain with at least 90% sequence identity to SEQ ID NO: 11. In some specific embodiments, the recombinant anti-SP17 antibody has a heavy chain with at least 95% sequence identity to SEQ ID NO: 11. In some more specific embodiments, the recombinant anti-SP17 antibody has a heavy chain with at least 98% sequence identity to SEQ ID NO: 11. In some very specific embodiments, the recombinant anti-SP17 antibody has a heavy chain with at least 99% sequence identity to SEQ ID NO: 11.

[0047] In some embodiments, the recombinant anti-SP17 antibody has a light chain with at least 90% sequence homology to SEQ ID NO: 12. In some specific embodiments, the recombinant anti-SP17 antibody has a light chain with at least 95% sequence homology to SEQ ID NO: 12. In some more specific embodiments, the recombinant anti-SP17 antibody has a light chain with at least 98% sequence homology to SEQ ID NO: 12. In some very specific embodiments, the recombinant anti-SP17 antibody has a light chain with at least 99% sequence homology to SEQ ID NO: 12.

[0048] In some embodiments, the recombinant anti-SP17 antibody has a light chain with at least 90% sequence identity to SEQ ID NO: 12. In some specific embodiments, the recombinant anti-SP17 antibody has a light chain with at least 95% sequence identity to SEQ ID NO: 12. In some more specific embodiments, the recombinant anti-SP17 antibody has a light chain with at least 98% sequence identity to SEQ ID NO: 12. In some very specific embodiments, the recombinant anti-SP17 antibody has a light chain with at least 99% sequence identity to SEQ ID NO: 12.

[0049] In some embodiments, the recombinant anti-SP17 antibody has (1) a heavy chain with at least 90% sequence homology to SEQ ID NO: 11, and (2) a light chain with at least 90% sequence homology to SEQ ID NO: 12. In some specific embodiments, the recombinant anti-SP17 antibody has (1) a heavy chain with at least 95% sequence homology to SEQ ID NO: 11, and (2) a light chain with at least 95% sequence homology to SEQ ID NO: 12. In some more specific embodiments, the recombinant anti-SP17 antibody has (1) a heavy chain with at least 98% sequence homology to SEQ ID NO: 11, and (2) a light chain with at least 98% sequence homology to SEQ ID NO: 12. In some very specific embodiments, the recombinant anti-SP17 antibody has (1) a heavy chain with at least 99% sequence homology to SEQ ID NO: 11, and (2) a light chain with at least 99% sequence homology to SEQ ID NO: 12.

[0050] In some embodiments, the recombinant anti-SP17 antibody has (1) a heavy chain with at least 90% sequence identity to SEQ ID NO: 11, and (2) a light chain with at least 90% sequence identity to SEQ ID NO: 12. In some specific embodiments, the recombinant anti-SP17 antibody has (1) a heavy chain with at least 95% sequence identity to SEQ ID NO: 11, and (2) a light chain with at least 95% sequence identity to SEQ ID NO: 12. In some more specific embodiments, the recombinant anti-SP17 antibody has (1) a heavy chain with at least 98% sequence identity to SEQ ID NO: 11, and (2) a light chain with at least 98% sequence identity to SEQ ID NO: 12. In some very specific embodiments, the recombinant anti-SP17 antibody has (1) a heavy chain with at least 99% sequence identity to SEQ ID NO: 11, and (2) a light chain with at least 99% sequence identity to SEQ ID NO: 12.

[0051] In some embodiments, the recombinant anti-SP17 antibody comprises two heavy chains and two light chains, wherein each of the two heavy chains has at least 90% sequence homology with SEQ ID NO: 11, and each of the two light chains has at least 90% sequence homology with SEQ ID NO: 12. In some specific embodiments, the recombinant anti-SP17 antibody comprises two heavy chains and two light chains, wherein each of the two heavy chains has at least 95% sequence homology with SEQ ID NO: 11, and each of the two light chains has at least 95% sequence homology with SEQ ID NO: 12. In some more specific embodiments, the recombinant anti-SP17 antibody comprises two heavy chains and two light chains, wherein each of the two heavy chains has at least 98% sequence homology with SEQ ID NO: 11, and each of the two light chains has at least 98% sequence homology with SEQ ID NO: 12. In some very specific embodiments, the recombinant anti-SP17 antibody comprises two heavy chains and two light chains, wherein each of the two heavy chains has at least 99% sequence homology with SEQ ID NO: 11, and each of the two light chains has at least 99% sequence homology with SEQ ID NO: 12. Such antibodies include, for example, IgG antibodies.

[0052] In some embodiments, the recombinant anti-SP17 antibody comprises two heavy chains and two light chains, wherein each of the two heavy chains has at least 90% sequence identity with SEQ ID NO: 11, and each of the two light chains has at least 90% sequence identity with SEQ ID NO: 12. In some specific embodiments, the recombinant anti-SP17 antibody comprises two heavy chains and two light chains, wherein each of the two heavy chains has at least 95% sequence identity with SEQ ID NO: 11, and each of the two light chains has at least 95% sequence identity with SEQ ID NO: 12. In some more specific embodiments, the recombinant anti-SP17 antibody comprises two heavy chains and two light chains, wherein each of the two heavy chains has at least 98% sequence identity with SEQ ID NO: 11, and each of the two light chains has at least 98% sequence identity with SEQ ID NO: 12. In some very specific embodiments, the recombinant anti-SP17 antibody comprises two heavy chains and two light chains, wherein each of the two heavy chains has at least 99% sequence identity with SEQ ID NO: 11, and each of the two light chains has at least 99% sequence identity with SEQ ID NO: 12. Such antibodies include, for example, IgG antibodies.

[0053] In some embodiments, the recombinant anti-SP17 antibody comprises at least four variable domains, wherein: the first and second variable domains are paired in the antibody such that the first and second variable domains bind to epitopes of human SP17, and the antibody comprises a third and a fourth variable domain that are paired in the antibody such that the third and fourth variable domains bind to different epitopes. Such antibodies include, for example, IgG-like bispecific antibodies, such as trivalent antibodies. The different epitopes may be, for example, CD3 epitopes, and the antibody can function as a therapeutic antibody by crosslinking SP17-expressing cancer cells with CD3-expressing T cells and Fc receptor-expressing leukocytes (such as monocytes, macrophages, natural killer cells, or dendritic cells), the leukocytes binding to the Fc region of the antibody. Examples of trivalent antibodies include caputoxumab (also known as REMOVAB®), which binds to the EpCAM cancer antigen, CD3, and the Fc receptor (see, for example, U.S. Patent No. 8,277,806, the entire contents of which are incorporated herein by reference). Those skilled in the art can design trivalent antibodies that bind to the SP17, CD3, and Fc receptors, for example, by replacing the variable region binding to EpCAM in caputazumab with the variable region shown in SEQ ID NOs: 3 and 4. Other trivalent antibodies binding SP17 can be similarly designed based on the known sequences of SEQ ID NOs: 3 and 4 and other existing trivalent antibodies. In some specific embodiments, the recombinant anti-SP17 antibody comprises an Fc region. In some specific embodiments, the distinct epitope is an extracellular epitope of CD3. In some very specific embodiments, the recombinant anti-SP17 antibody comprises an Fc region, and the distinct epitope is an extracellular epitope of CD3.

[0054] In this disclosure, the term "pairing" refers to the spatial proximity and orientation between VH and VL regions, which allows the VH and VL regions to simultaneously bind epitopes. For example, VH and VL regions can be paired in a Fab via a quaternary structure comprising one or more disulfide bonds and a non-covalent interaction between a heavy chain constant domain CH1 and a light chain constant domain CL. For example, VH and VL regions can also be paired as scFv fusion proteins, which typically include a flexible linker, such as polyglycine, to tether the VH and VL regions to a spatially proximate location and allow the VH and VL regions to simultaneously bind epitopes.

[0055] Various aspects of this disclosure relate to antibody conjugates comprising recombinant anti-SP17 antibodies as described in any part of this disclosure, wherein the recombinant anti-SP17 antibody is conjugated to a radioisotope or a pharmaceutically active agent.

[0056] In some embodiments, the recombinant anti-SP17 antibody is conjugated with a radioisotope. In some specific embodiments, the recombinant anti-SP17 antibody is conjugated with a radioisotope selected from actinium-225, astatine-211, bismuth-212, bismuth-213, copper-67, gallium-68, holmium-166, iodine-124, iodine-131, lutetium-177, samarium-153, technetium-99, terbium-149, and yttrium-90. Examples of antibodies conjugated with radioisotopes include tosimomab (also known as BEXXAR). ® ) and tivamozine (also known as ZEVALIN) ® Those skilled in the art can use known strategies to design antibodies conjugated to radioisotopes, such as strategies for conjugating iodine-131 in tosimomumab and yttrium-90 or indium-111 in timimomumab (see, for example, U.S. Patent Nos. 6,565,827 and 7,422,739, the entire contents of which are incorporated herein by reference).

[0057] In some embodiments, the recombinant anti-SP17 antibody is conjugated with a pharmaceutically active agent. In some specific embodiments, the recombinant anti-SP17 antibody is partially conjugated with a drug selected from calicheamicin, camptothecin, deruxtecan, doxorubicin, emtansine, exatecan, irinotecan, maleimidocaproyl monomethyl auristatin F, mertansine, monomethyl auristatin F, paclitaxel, PE38 (a truncated fragment of Pseudomonas exotoxin A), pyrrolobenzodiazepine, SN-38 (7-ethyl-10-hydroxycamptothecin), and vedotin. Examples of antibodies conjugated to pharmaceutically active agents include gemtuzumab ozomicin (also known as MYLOTARG). ® ) and trastuzumab metansine (also known as Kadcyla) ® Those skilled in the art can use known strategies to design antibodies conjugated to pharmaceutically active agents, such as strategies for conjugating calimoxycin to gemtuzumab and metansine to trastuzumab (see, for example, U.S. Patent Nos. 5,877,296 and 8,088,387, the entire contents of which are incorporated herein by reference).

[0058] Various aspects of this disclosure relate to pharmaceutical compositions comprising a recombinant anti-SP17 antibody as described in any part of this disclosure, its antigen-binding fragment or conjugate thereof, and a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers typically comprise water containing a dissolved solute, which buffers the pH and provides a metal cation and ionic strength to stabilize the antibody or other therapeutic agent of this disclosure. Such formulations are generally sterile, and the selection and preparation of such pharmaceutically acceptable carriers are well known. Solid forms comprising lyophilized therapeutic agents typically include, for example, metal cations, anions, and optionally polyols such as sugars (e.g., trehalose or glucose), which stabilize the therapeutic agent in the solid phase and during its recombination into an aqueous form. General guidance on selecting pharmaceutically acceptable carriers can be found, for example, in *REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY*, 22nd edition (edited by Allen Jr., Loyd V.), Pharmaceutical Press, 2012, and those skilled in the art will also refer to formulations of the therapeutic agents described in this disclosure and other existing therapeutic agents when selecting pharmaceutically acceptable carriers. Such guidance is usually available in scientific literature and existing product labels.

[0059] In some embodiments, the pharmaceutical composition is suitable for administration to a subject. In some specific embodiments, the pharmaceutical composition is suitable for administration to a human patient. In some very specific embodiments, the pharmaceutical composition is suitable for intravenous administration to a human patient.

[0060] In some embodiments, the pharmaceutical composition comprises an anti-SP17 antibody, and the antibody has a purity of at least 85% relative to the total protein in the pharmaceutical composition. In some specific embodiments, the pharmaceutical composition comprises an anti-SP17 antibody, and the antibody has a purity of at least 90% relative to the total protein in the pharmaceutical composition. In some very specific embodiments, the pharmaceutical composition comprises an anti-SP17 antibody, and the antibody has a purity of at least 93% relative to the total protein in the pharmaceutical composition.

[0061] In some implementations, purity is determined by chromatography. In some specific implementations, purity is determined by HPLC.

[0062] Various aspects of this disclosure relate to a kit comprising (1) a sealed container containing a pharmaceutical composition as described in any part of this disclosure, and (2) instructions for use of the pharmaceutical composition.

[0063] Various aspects of this disclosure relate to medical devices comprising pharmaceutical compositions as described in any part of this disclosure. In some embodiments, the medical device is a syringe, an intravenous cannula, or a drug-eluting implant.

[0064] Various aspects of this disclosure relate to methods for treating or preventing cancer in a subject, including identifying the subject as containing cells ectopically expressing SP17 and administering a pharmaceutical composition as described in any part of this disclosure. Identifying cells in the subject containing ectopically expressed SP17 includes, for example, identifying mRNA encoding SP17 by RT-PCR and identifying SP17 protein expression by flow cytometry and / or immunohistochemistry. Such methods can be advantageously used to determine ectopic expression of SP17 in cancer cells, for example, based on a pre-selection of one or more cancer cells to be analyzed, or based on the co-identification of ectopic Sp17 expression with a cancer phenotype, such as co-expression of Sp17 with another cancer antigen.

[0065] Various aspects of this disclosure relate to methods for modulating cells expressing SP17 in a human subject, comprising administering an immunotherapeutic agent to the human subject, wherein the immunotherapeutic agent is or comprises a recombinant anti-SP17 antibody or an antigen-binding fragment thereof as described in any part of this disclosure. In some embodiments, the method is a method for treating or preventing cancer in a human subject. In some specific embodiments, the method is a method for treating or preventing cancer in a human subject who has cancer and at least a portion of the cells expressing SP17 are cancer cells.

[0066] In some embodiments, the immunotherapy agent is administered in an effective amount that effectively induces cell death in at least a subset of cells expressing SP17, and the method modulates SP17-expressing cells by inducing cell death. In some specific embodiments, the immunotherapy agent comprises an Fc region, and the method induces cell death through the binding and activation of one or more leukocytes to SP17-expressing cells mediated by an Fc receptor. In some specific embodiments, the immunotherapy agent is conjugated to a radioactive isotope, and the method induces cell death by emitting radiation within or near SP17-expressing cells. In some specific embodiments, the immunotherapy agent is conjugated to a pharmaceutically active agent that is cytotoxic, and the method induces cell death by releasing the pharmaceutically active agent within or near SP17-expressing cells. In some very specific embodiments, the immunotherapy agent is conjugated to a pharmaceutically active agent via an unstable linker that is cytotoxic, and the method induces cell death by releasing the pharmaceutically active agent within or near SP17-expressing cells.

[0067] In some embodiments, the subject is a mammal. In some specific embodiments, the subject is a rodent, lagomorph, feline, canine, pig, sheep, goat, llama, cattle, horse, or primate. In some very specific embodiments, the subject is a human patient.

[0068] In some embodiments, the subject is male or female. In some specific embodiments, the subject is female. In some specific embodiments, the subject is male.

[0069] In some embodiments, the subject exhibits ectopic expression of SP17. In some specific embodiments, the subject has cancer, and the cancer cells ectopically express SP17.

[0070] In some implementations, the method includes identifying that the subject contains cells that ectopically express SP17.

[0071] In some embodiments, the method includes identifying the subject as having cells that ectopically express SP17, wherein the cells expressing SP17 ectopically comprise leukocytes, and the cancer is lymphoma, leukemia, or myeloma. In some specific embodiments, the method includes identifying the subject as having cells that ectopically express SP17, wherein the cells expressing SP17 ectopically comprise plasma cells, and the cancer is multiple myeloma. In some specific embodiments, the method includes identifying the subject as having cells that ectopically express SP17, wherein the cells expressing SP17 ectopically comprise lymphocytes, and the cancer is lymphoma. In some specific embodiments, the method includes identifying the subject as having cells that ectopically express SP17, wherein the cells expressing SP17 ectopically comprise ovarian cells, and the cancer is ovarian cancer. In some specific embodiments, the method includes identifying the subject as having cells that ectopically express SP17, wherein the cells expressing SP17 ectopically comprise lung epithelial cells, and the cancer is non-small cell lung cancer.

[0072] In some embodiments, the method includes identifying, prior to administration, that a tissue sample from the human subject contains RNA or SP17 protein encoding SP17.

[0073] In some implementations, the tissue sample is a blood sample.

[0074] In some embodiments, the tissue sample is a biopsy sample. In some specific embodiments, the tissue sample is a tumor biopsy sample. In some very specific embodiments, the tissue sample is a bone marrow biopsy sample. In some very specific embodiments, the tissue sample is an ovarian biopsy sample. In some very specific embodiments, the tissue sample is a lung biopsy sample.

[0075] In some embodiments, the method includes identifying that the tissue sample contains a cancer biomarker, wherein the cancer biomarker is neither RNA encoding SP17 nor SP17 protein.

[0076] In some embodiments, the method does not include identifying co-expression or co-localization of the cancer biomarker with RNA encoding SP17, and / or the method does not include identifying co-expression or co-localization of the cancer biomarker with SP17 protein. SP17 is unique because healthy women are known not to express SP17, and healthy men are known to express SP17 only in testicular tissue. Therefore, the detection of SP17 RNA or SP17 protein in biological samples other than testicular samples suggests the presence of a cancer-associated phenotype that can be treated with the immunotherapeutic agents of this disclosure, even in the absence of data suggesting SP17 expression on cells with a cancer-associated phenotype (e.g., by detecting SP17 expression on cells exhibiting cancer morphology, or by detecting co-expression or co-localization of SP17 with cancer biomarkers).

[0077] In some embodiments, identifying that the tissue sample contains RNA encoding SP17 includes RT-PCR. In some specific embodiments, identifying that the tissue sample contains RNA encoding SP17 includes RT-PCR, and the RT-PCR does not distinguish between cancer cells and healthy cells containing RNA encoding SP17.

[0078] In some embodiments, identifying the presence of the SP17 protein in the tissue sample includes ELISA. In some specific embodiments, identifying the presence of the SP17 protein in the tissue sample includes ELISA, and the ELISA does not distinguish between cancer cells containing RNA encoding SP17 and healthy cells.

[0079] In some embodiments, the cells ectopically expressing SP17 comprise leukocytes, and the cancer is lymphoma, leukemia, or myeloma. In some specific embodiments, the cells ectopically expressing SP17 comprise leukocytes, and the cancer is multiple myeloma or lymphoma. In some very specific embodiments, the cells ectopically expressing SP17 comprise plasma cells, and the cancer is multiple myeloma. In some very specific embodiments, the cells ectopically expressing SP17 comprise lymphocytes, and the cancer is lymphoma.

[0080] In some embodiments, the ectopic SP17-expressing cells comprise ovarian cells, and the cancer is ovarian cancer.

[0081] In some embodiments, the cells expressing SP17 ectopically comprise lung epithelial cells, and the cancer is non-small cell lung cancer.

[0082] In some embodiments, the administration is selected from intravenous, intramuscular, subcutaneous, intradermal, intraocular, parenteral, intraperitoneal, intrathecal, intralesional, intralesional, and intratumoral administration. In some specific embodiments, the administration is intravenous.

[0083] Various aspects of this disclosure relate to a recombinant nucleic acid that encodes a recombinant anti-SP17 antibody or an antigen-binding fragment thereof as described in any part of this disclosure.

[0084] Various aspects of this disclosure relate to a recombinant nucleic acid comprising a nucleotide sequence encoding a protein, said protein comprising an antigen-binding region, wherein (1) the antigen-binding region comprises a first variable domain and a second variable domain; (2) the first variable domain comprises a VH CDR1 region, a VH CDR2 region, and a VH CDR3 region, said VH CDR1 region comprising an amino acid sequence identical to at least 7, 8, 9, or 10 consecutive amino acids as shown in SEQ ID NO: 5, said VH CDR2 region comprising an amino acid sequence identical to at least 7, 8, 9, or 10 consecutive amino acids as shown in SEQ ID NO: 6, said VH CDR3 region comprising an amino acid sequence identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 consecutive amino acids as shown in SEQ ID NO: 7; and (3) the second variable domain comprises a VL CDR1 region, a VL CDR2 region, and a VLCDR3 region, said VL CDR1 region comprising an amino acid sequence identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive amino acids as shown in SEQ ID NO: 7. The VL CDR2 region contains an amino acid sequence identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive amino acids as shown in SEQ ID NO: 8, and the VL CDR3 region contains an amino acid sequence identical to at least 3, 4, 5, 6, or 7 consecutive amino acids as shown in SEQ ID NO: 9; and (4) the first variable domain and the second variable domain are paired in the antigen-binding region of the protein such that the antigen-binding region specifically binds to human SP17.

[0085] In some embodiments, the first variable domain comprises an amino acid sequence having at least 90% sequence homology with SEQ ID NO: 3. In some specific embodiments, the first variable domain comprises an amino acid sequence having at least 95% sequence homology with SEQ ID NO: 3. In some more specific embodiments, the first variable domain comprises an amino acid sequence having at least 98% sequence homology with SEQ ID NO: 3. In some very specific embodiments, the first variable domain comprises an amino acid sequence having at least 99% sequence homology with SEQ ID NO: 3.

[0086] In some embodiments, the first variable domain comprises an amino acid sequence that is at least 90% sequence identical to SEQ ID NO: 3. In some specific embodiments, the first variable domain comprises an amino acid sequence that is at least 95% sequence identical to SEQ ID NO: 3. In some more specific embodiments, the first variable domain comprises an amino acid sequence that is at least 99% sequence identical to SEQ ID NO: 3. In some very specific embodiments, the first variable domain comprises the same amino acid sequence as SEQ ID NO: 3.

[0087] In some embodiments, the second variable domain comprises an amino acid sequence having at least 90% sequence homology with SEQ ID NO: 4. In some specific embodiments, the second variable domain comprises an amino acid sequence having at least 95% sequence homology with SEQ ID NO: 4. In some more specific embodiments, the second variable domain comprises an amino acid sequence having at least 98% sequence homology with SEQ ID NO: 4. In some very specific embodiments, the second variable domain comprises an amino acid sequence having at least 99% sequence homology with SEQ ID NO: 4.

[0088] In some embodiments, the second variable domain comprises an amino acid sequence that is at least 90% sequence identical to SEQ ID NO: 4. In some specific embodiments, the second variable domain comprises an amino acid sequence that is at least 95% sequence identical to SEQ ID NO: 4. In some more specific embodiments, the second variable domain comprises an amino acid sequence that is at least 99% sequence identical to SEQ ID NO: 4. In some very specific embodiments, the second variable domain comprises the same amino acid sequence as SEQ ID NO: 4.

[0089] In some embodiments, the first variable domain comprises an amino acid sequence having at least 90% sequence homology with SEQ ID NO: 3, and the second variable domain comprises an amino acid sequence having at least 90% sequence homology with SEQ ID NO: 4. In some specific embodiments, the first variable domain comprises an amino acid sequence having at least 95% sequence homology with SEQ ID NO: 3, and the second variable domain comprises an amino acid sequence having at least 95% sequence homology with SEQ ID NO: 4. In some more specific embodiments, the first variable domain comprises an amino acid sequence having at least 98% sequence homology with SEQ ID NO: 3, and the second variable domain comprises an amino acid sequence having at least 99% sequence homology with SEQ ID NO: 4. In some very specific embodiments, the first variable domain comprises an amino acid sequence having at least 99% sequence homology with SEQ ID NO: 3, and the second variable domain comprises an amino acid sequence having at least 99% sequence homology with SEQ ID NO: 4.

[0090] In some embodiments, the first variable domain comprises an amino acid sequence that is at least 90% sequence identical to SEQ ID NO: 3, and the second variable domain comprises an amino acid sequence that is at least 90% sequence identical to SEQ ID NO: 4. In some specific embodiments, the first variable domain comprises an amino acid sequence that is at least 95% sequence identical to SEQ ID NO: 3, and the second variable domain comprises an amino acid sequence that is at least 95% sequence identical to SEQ ID NO: 4. In some more specific embodiments, the first variable domain comprises an amino acid sequence that is at least 99% sequence identical to SEQ ID NO: 3, and the second variable domain comprises an amino acid sequence that is at least 99% sequence identical to SEQ ID NO: 4. In some very specific embodiments, the first variable domain comprises the same amino acid sequence as SEQ ID NO: 3, and the second variable domain comprises the same amino acid sequence as SEQ ID NO: 4.

[0091] Various aspects of this disclosure relate to a recombinant nucleic acid comprising a nucleotide sequence encoding a protein, the protein comprising an antigen-binding region, wherein (1) the antigen-binding region comprises a first variable domain and a second variable domain; (2) the first variable domain comprises an amino acid sequence having at least 90% sequence homology with SEQ ID NO: 3 (e.g., at least 95%, 97%, 98%, or 99% sequence homology); (3) the second variable domain comprises an amino acid sequence having at least 90% sequence homology with SEQ ID NO: 4 (e.g., at least 95%, 97%, 98%, or 99% sequence homology); and (4) the antibody or its antigen-binding fragment binds to human SP17.

[0092] In some embodiments, the first variable domain comprises an amino acid sequence that is at least 90% sequence identical to SEQ ID NO: 3 (e.g., at least 95%, 97%, 98%, or 99% sequence identical), and the second variable domain comprises an amino acid sequence that is at least 90% sequence identical to SEQ ID NO: 4 (e.g., at least 95%, 97%, 98%, or 99% sequence identical).

[0093] In some embodiments, the protein is a recombinant anti-SP17 antibody as described anywhere in this disclosure.

[0094] In some embodiments, the protein is an antigen-binding fragment of a recombinant anti-SP17 antibody as described in any part of this disclosure.

[0095] In some embodiments, the protein is an IgG-like bispecific antibody or a portion thereof as described anywhere in this disclosure. In some specific embodiments, the protein is a trifunctional antibody or a portion thereof as described anywhere in this disclosure. For example, an IgG-like bispecific antibody (such as a trifunctional antibody) may be encoded by two different nucleic acids, for example: a first nucleic acid encoding a first heavy chain and a light chain that bind to a first epitope (e.g., SP17); a second nucleic acid encoding a second heavy chain and a light chain that bind to a second epitope (e.g., CD3); and the first heavy chain and light chain are expressed in a first cell line, and the second heavy chain and light chain are expressed in a second cell line, thereby avoiding mispairing between heavy chains and light chains that bind to different epitopes.

[0096] In some embodiments, the protein is a bispecific T-cell adaptor (BiTE). A BiTE is a fusion protein comprising a scFV that binds an antigen (e.g., SP17) and a domain that binds a cell surface protein expressed by T cells (e.g., CD3). Examples of BiTEs include blinatumomab (also known as BLINCYTO®) and tebentafusp (also known as KIMMTRAK®). Those skilled in the art can design BiTEs that bind SP17 and CD3, for example by replacing the variable region in blinatumomab that binds CD19 or the variable region in tebentafusp that binds glycoprotein 100 with the variable regions shown in SEQ ID NO: 3 and 4 (see, for example, U.S. Patent Nos. 10,517,960 and 11,597,766, the entire contents of which are incorporated herein by reference).

[0097] In some embodiments, the protein is a chimeric antigen receptor (CAR). A CAR is a fusion protein comprising (1) an N-terminal extracellular scFV that binds to an antigen (e.g., SP17), (2) a transmembrane α-helix, and (3) a C-terminal intracellular signaling domain, such as CD3-ζ and / or one or more intracellular signaling domains selected from CD27, CD28, CD134, and CD137. The intracellular signaling domain typically contains an immune receptor tyrosine activation motif (ITAM). Examples of CARs include tisagenlecleucel (also known as KYMRIAH®) and axicabtagene ciloleucel (also known as YESCARTA®). Those skilled in the art can design CARs that incorporate SP17, for example by replacing the variable region in salmon or acerencin that incorporates CD19 with the variable region shown in SEQ ID NO: 3 and 4 (see, for example, U.S. Patent Nos. 7,446,190, 7,741,465 and 9,499,629, the entire contents of which are incorporated herein by reference).

[0098] In some embodiments, the recombinant nucleic acid comprises a nucleotide sequence that is at least 70% identical to SEQ ID NO: 1. In some specific embodiments, the recombinant nucleic acid comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 1. In some more specific embodiments, the recombinant nucleic acid comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 1. In some very specific embodiments, the recombinant nucleic acid comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 1.

[0099] In some embodiments, the recombinant nucleic acid comprises a nucleotide sequence encoding an amino acid sequence having at least 90% sequence homology with SEQ ID NO: 11. In some specific embodiments, the recombinant nucleic acid comprises a nucleotide sequence encoding an amino acid sequence having at least 95% sequence homology with SEQ ID NO: 11. In some more specific embodiments, the recombinant nucleic acid comprises a nucleotide sequence encoding an amino acid sequence having at least 98% sequence homology with SEQ ID NO: 11. In some very specific embodiments, the recombinant nucleic acid comprises a nucleotide sequence encoding an amino acid sequence having at least 99% sequence homology with SEQ ID NO: 11.

[0100] In some embodiments, the recombinant nucleic acid comprises a nucleotide sequence encoding an amino acid sequence that has at least 90% sequence identity with SEQ ID NO: 11. In some specific embodiments, the recombinant nucleic acid comprises a nucleotide sequence encoding an amino acid sequence that has at least 95% sequence identity with SEQ ID NO: 11. In some more specific embodiments, the recombinant nucleic acid comprises a nucleotide sequence encoding an amino acid sequence that has at least 98% sequence identity with SEQ ID NO: 11. In some very specific embodiments, the recombinant nucleic acid comprises a nucleotide sequence encoding an amino acid sequence that has at least 99% sequence identity with SEQ ID NO: 11.

[0101] In some embodiments, the recombinant nucleic acid comprises a nucleotide sequence that is at least 70% identical to SEQ ID NO: 2. In some specific embodiments, the recombinant nucleic acid comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 2. In some more specific embodiments, the recombinant nucleic acid comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 2. In some very specific embodiments, the recombinant nucleic acid comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 2.

[0102] In some embodiments, the recombinant nucleic acid comprises a nucleotide sequence encoding an amino acid sequence having at least 90% sequence homology with SEQ ID NO: 12. In some specific embodiments, the recombinant nucleic acid comprises a nucleotide sequence encoding an amino acid sequence having at least 95% sequence homology with SEQ ID NO: 12. In some more specific embodiments, the recombinant nucleic acid comprises a nucleotide sequence encoding an amino acid sequence having at least 98% sequence homology with SEQ ID NO: 12. In some very specific embodiments, the recombinant nucleic acid comprises a nucleotide sequence encoding an amino acid sequence having at least 99% sequence homology with SEQ ID NO: 12.

[0103] In some embodiments, the recombinant nucleic acid comprises a nucleotide sequence encoding an amino acid sequence that has at least 90% sequence identity with SEQ ID NO: 12. In some specific embodiments, the recombinant nucleic acid comprises a nucleotide sequence encoding an amino acid sequence that has at least 95% sequence identity with SEQ ID NO: 12. In some more specific embodiments, the recombinant nucleic acid comprises a nucleotide sequence encoding an amino acid sequence that has at least 98% sequence identity with SEQ ID NO: 12. In some very specific embodiments, the recombinant nucleic acid comprises a nucleotide sequence encoding an amino acid sequence that has at least 99% sequence identity with SEQ ID NO: 12.

[0104] In some implementations, the recombinant nucleic acid includes a replication origin, wherein the recombinant nucleic acid is a plasmid.

[0105] Various aspects of this disclosure relate to a viral vector comprising a recombinant nucleic acid as described anywhere in this disclosure. In some embodiments, the vector is tropistic to human leukocytes. In some specific embodiments, the vector is tropistic to human T cells, natural killer cells, or monocytes. In some very specific embodiments, the viral vector is an adenovirus vector, an adeno-associated virus vector, a lentiviral vector, or a gamma-retroviral vector. The viral vector can be used, for example, to introduce a recombinant nucleic acid encoding a CAR into leukocytes to produce transgenic leukocytes expressing said CAR, for use in cancer immunotherapy.

[0106] Various aspects of this disclosure relate to a cell that contains recombinant nucleic acids as described in any part of this disclosure or viral vectors as described in any part of this disclosure.

[0107] In some embodiments, the cell is a prokaryote, such as *Escherichia coli*, and the cell is used to clone or amplify the recombinant nucleic acid. When the cell is a prokaryote, the recombinant nucleic acid typically includes an origin of replication for amplification and an antibiotic resistance gene to provide a selection advantage to cells containing the recombinant nucleic acid.

[0108] In some implementations, the cell is a bacterial cell.

[0109] In some embodiments, the cells are mammalian cells. In some specific embodiments, the cells are immortalized mammalian cell lines. In some very specific embodiments, the cells are CHO cells (Chinese hamster ovary cells), HEK cells (human embryonic kidney cells) (e.g., HEK293 cells), NSO cells (mouse myeloma cells), Sp2 / O cells (mouse myeloma cells), or PER.C6® cells (human retinal cells). Immortalized mammalian cell lines are commonly used to express proteins used as therapeutic agents, such as therapeutic antibodies (including, for example, the human anti-SP17 antibody described herein). Mammalian cells typically ensure the fidelity of tertiary and quaternary structures of variable regions and other regions of SP17-binding proteins, as well as the fidelity of post-translational modifications (e.g., glycosylation patterns). Mammalian cells can also be used, for example, to manufacture viral vectors as described herein. The recombinant nucleic acids can be transiently or stably present in the cells.

[0110] In some embodiments, the cell is a mammalian cell, and the mammalian cell expresses the protein.

[0111] In some embodiments, the cell is a mammalian cell that expresses a viral vector, and the recombinant nucleic acid contains a packaging signal for packaging the recombinant nucleic acid into the viral vector.

[0112] In some embodiments, the cell is a tissue culture cell. In some specific embodiments, the cell is a tissue culture cell, and the tissue culture cell expresses the protein. In some specific embodiments, the cell is a tissue culture cell; the tissue culture cell expresses a viral vector; and the recombinant nucleic acid contains a packaging signal for packaging the recombinant nucleic acid into the viral vector.

[0113] In some embodiments, the cells are peripheral blood mononuclear cells (PBMCs). In some specific embodiments, the cells are human PBMCs. In some specific embodiments, the cells are T cells, natural killer cells, monocytes, macrophages, or dendritic cells. In some very specific embodiments, the cells are human T cells, human natural killer cells, human monocytes, human macrophages, or human dendritic cells. PBMCs can be transfected with recombinant nucleic acids to express CARs, making PBMCs available for use in cancer immunotherapy, such as CAR-T cells, CAR-NK cells, CAR-monocytes, CAR-macrophages, or CAR-DCs.

[0114] In some embodiments, the cell is a leukocyte, and the protein encoded by the nucleotide sequence is a CAR. In some specific embodiments, the cell is a human leukocyte, and the protein encoded by the nucleotide sequence is a CAR. In some very specific embodiments, the cell is a human leukocyte, the protein encoded by the nucleotide sequence is a CAR, and the human leukocyte expresses the CAR.

[0115] In some embodiments, the cell is a PBMC, and the protein encoded by the nucleotide sequence is a CAR. In some specific embodiments, the cell is a human PBMC, and the protein encoded by the nucleotide sequence is a CAR. In some very specific embodiments, the cell is a human PBMC, the protein encoded by the nucleotide sequence is a CAR, and the human PBMC expresses the CAR.

[0116] In some embodiments, the cell is a T cell, natural killer cell, monocyte, macrophage, or dendritic cell, and the protein encoded by the nucleotide sequence is a CAR. In some specific embodiments, the cell is a human T cell, human natural killer cell, human monocyte, human macrophage, or human dendritic cell, and the protein encoded by the nucleotide sequence is a CAR. In some very specific embodiments, the cell is a human T cell, human natural killer cell, human monocyte, human macrophage, or human dendritic cell, the protein encoded by the nucleotide sequence is a CAR, and the cell expresses the CAR.

[0117] Various aspects of this disclosure relate to a Fab fragment binding to SP17, comprising a CDR having sequence homology with the amino acid sequences shown in SEQ ID NO: 3-6. The Fab may comprise, for example, amino acid sequences having at least 90%, 95%, 97%, 98%, or 99% sequence homology with the sequences shown in SEQ ID NO: 3-6, or amino acid sequences having at least 90%, 95%, 97%, 98%, or 99% sequence identity with the sequences shown in SEQ ID NO: 3-6. In some embodiments, the Fab fragment is encoded by a recombinant nucleic acid as described in any part of this disclosure.

[0118] Various aspects of this disclosure relate to an antibody comprising a Fab fragment as described anywhere in this disclosure. In some embodiments, the antibody is encoded by a recombinant nucleic acid as described anywhere in this disclosure.

[0119] Various aspects of this disclosure relate to a bispecific antibody comprising a CDR having sequence homology with the amino acid sequences shown in SEQ ID NO: 3-6. The bispecific antibody may comprise, for example, an amino acid sequence having at least 90%, 95%, 97%, 98%, or 99% sequence homology with the sequences shown in SEQ ID NO: 3-6, or an amino acid sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity with the sequences shown in SEQ ID NO: 3-6. In some embodiments, the bispecific antibody is encoded by a recombinant nucleic acid as described in any part of this disclosure.

[0120] Various aspects of this disclosure relate to a BiTE comprising a CDR having sequence homology with the amino acid sequences shown in SEQ ID NO: 3-6. The BiTE may comprise, for example, an amino acid sequence having at least 90%, 95%, 97%, 98%, or 99% sequence homology with the sequences shown in SEQ ID NO: 3-6, or an amino acid sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity with the sequences shown in SEQ ID NO: 3-6. In some embodiments, the BiTE is encoded by a recombinant nucleic acid as described in any part of this disclosure.

[0121] The various features of this disclosure have been generally and specifically described in the foregoing detailed description. The following examples provide specific instances of the preparation of the subject matter described herein. Through these examples, and in conjunction with the foregoing detailed description, those skilled in the art will immediately recognize variations of the methods described in the examples (e.g., by modifying BiTE, scFv, or chimeric antigen receptors instead of IgG). The following examples are illustrative only and should not limit this disclosure or any patent claims that are ultimately granted based on this disclosure. Any patent claims that are ultimately granted based on this disclosure should be defined by the features expressly recited in the context of the dependent claims and by the conventional principles of claim interpretation applicable to this disclosure. Example Example 1. Identification of human variable regions of anti-SP17 antibodies based on human diversity

[0122] A Fab phage display library was constructed from peripheral blood obtained from 120 healthy human donors. In short, the library was constructed by randomly combining nucleotide sequences encoding the variable regions of the immunoglobulin heavy chain and the immunoglobulin light chain. The library exhibits approximately one trillion possible combinations. Biopanning of the library identified 12 positive clones, and nucleotide sequencing of these clones identified two distinct clones. A single clone was identified by ELISA as binding to SP17. The nucleotide sequences of the human VH and VL regions of this antibody are listed in Table 1 below. The variable regions have the amino acid sequences shown in Table 2 and the CDRs shown in Table 3 below. Table 1. Nucleotide sequences of the anti-SP17 VH and VL regions identified in a Fab phage display library of approximately one trillion human Fab samples developed from peripheral blood samples of 120 human subjects via biopanning. Table 2. Amino acid sequences of the anti-SP17 VH and VL regions identified in a Fab phage display library of approximately one trillion human Fab samples developed from peripheral blood samples of 120 human subjects via biopanning. Table 3. Amino acid sequences of CDRs in the VH and VL regions identified in a Fab phage display library of approximately one trillion human Fab cells developed from peripheral blood samples of 120 human subjects via biopanning. Example 2. Engineering of mouse-human chimeric anti-SP17 IgG2a / KAPPA monoclonal antibodies

[0123] The nucleotide sequences encoding the VH and VL regions of Example 1 were cloned into the mouse IgG2a heavy chain gene and the mouse κ light chain gene, respectively, and expressed in CHO-S cells to generate a mouse-human chimeric antibody, named chAB2. Successful antibody generation was confirmed by SDS-PAGE and Western blotting using anti-mouse heavy chain and light chain antibodies and recombinant SP17 protein. Figure 2 and 3 The chAB2 antibody is reactive to the mouse SP17 protein expressed on the surface of the mouse ovarian cancer cell line ID8. Figure 4 HPLC analysis of the chAB2 antibody preparation showed a purity of 93%. Figure 5 The KD of chAB2 and SP17, determined using surface plasmon resonance with Biacore™ T200, was 2.073 nanomolar. Figure 6 ).

[0124] The specificity of the chAB2 antibody against SP17 was determined by immunohistochemistry using a normal tissue microarray composed of 33 normal tissues from 2-3 human donors for each tissue. Normal tissues included the brain, eye, adrenal gland, pituitary gland, thyroid gland, parathyroid gland, tonsils, thymus, spleen, heart, lung, larynx, esophagus, stomach, small intestine, colon, liver, pancreas, salivary glands, kidney, bone, skeletal muscle, skin, peripheral nerves, mesothelial cell cartilage, breast, ovary, endometrium, cervix, testis, and prostate. The chAB2 antibody bound to the testis but not to normal tissues. Example 3. Engineering of human anti-SP17 IgG4 / KAPPA monoclonal antibodies

[0125] The VH and VL nucleotide sequences from Example 1 were cloned into the human IgG4 heavy chain gene and the human κ light chain gene, respectively. A Ser228Pro mutation was introduced into the heavy chain to reduce non-specific Fc receptor γ binding. The resulting antibody was named SP17-AB2. Successful cloning was confirmed by sequence analysis. The amino acid sequences of the heavy and light chains are shown in Table 4, with the Ser228Pro mutation underlined in SEQ ID NO: 11. Successful expression of the SP17-AB2 antibody was confirmed by SDS-PAGE. Figure 7 The ability of the SP17-AB2 antibody to bind to the SP17 protein was confirmed by Western blotting. Figure 8 ). Table 4. Amino acid sequence of human anti-SP17 antibody SP17-AB2 Based on the above results, SP17-AB2 antibodies may be particularly suitable as therapeutic antibodies for treating cancers expressing SP17. Example 4. Engineering of anti-SP17 immunoconjugates with droncitacin

[0126] The SP17-AB2 antibody described in Example 3 was cross-linked with derutecan using the conjugation strategy described in U.S. Patent No. 10,729,782 (the entire contents of which are incorporated herein by reference) to generate the immunoconjugate SP17-AB2-DXD. Derutecan, a topoisomerase inhibitor, exhibits antitumor cytotoxicity based on its ixanotecan moiety, which is covalently cross-linked with SP17-AB2 via a linker and disulfide bond, as described in U.S. Patent No. 10,729,782. Successful preparation of the immunoconjugate was confirmed by reducing the immunoconjugate with excess dithiothreitol followed by mass spectrometry analysis. The results showed that the derutecan to SP17-AB2 ratio of SP17-AB2-DXD was approximately 7.93, indicating that, on average, a single SP17-AB2-DXD molecule contains approximately eight derutecan moieties. Notably, this drug-to-antibody ratio is significantly higher than that of most FDA-approved antibodies.

[0127] Prior to cytotoxicity assays, SK-OV-3 cells were seeded at 1000, 2000, or 3000 cells per well and cultured overnight. SK-OV-3 cells are SP17-expressing human ovarian cancer cells resistant to multiple chemotherapeutic agents, including cisplatin, doxorubicin, and diphtheria toxin. SK-OV-3 cells were treated with different concentrations of SP17-AB2 antibody, SP17-AB2-DXD immunoconjugate, control human IgG4 antibody (hIgG4), or drutecan (DXD) alone. Cells were cultured for 5 or 6 days, and then cytotoxicity was measured using a luminescence assay and an EnVision™ plate reader (PerkinElmer, Massachusetts, USA). The SP17-AB2-DXD immunoconjugate showed superior cytotoxicity compared to the naked SP17-AB2 antibody and the human IgG4 control. Figures 9A-9C (Table 5). Table 5. The IC50 and cytotoxicity of the immunoconjugate SP17-AB2-DXD against human ovarian cancer cells are superior to those of the naked antibody SP17-AB2.

[0128] Prior to the second cytotoxicity assay, ID8 cells were seeded at 250 or 300 cells per well and cultured overnight to establish approximately 90% confluence. Cells were then treated with different concentrations of SP17-AB2 antibody, SP17-AB2-DXD immunoconjugate, hIgG4, or derutecan alone. Cells were cultured for another 3 days before cytotoxicity was measured. The SP17-AB2-DXD immunoconjugate showed superior cytotoxicity compared to the naked SP17-AB2 antibody and human IgG4 control. Figure 10 (Table 6). Table 6. The immunoconjugate SP17-AB2-DXD showed superior cytotoxicity against mouse ovarian cancer cells compared to the naked antibody SP17-AB2.

[0129] No patent claim based on this disclosure and ultimately granted shall be construed as requiring any feature of the foregoing embodiments. No method described in the specification or claims shall be construed as requiring the steps to be performed in a particular order unless expressly stated otherwise. These methods shall be construed as supporting the performance of the steps in any order unless expressly stated otherwise.

[0130] Some features described in the context of different implementations may also be implemented in combination in a single implementation. Conversely, various features described in the context of a single implementation may also be implemented separately in multiple implementations, or in any suitable sub-combination. Furthermore, although some features may be described in a particular combination above and even initially claimed in that combination, one or more features in the combination of claims may be removed from that combination, and the combination of claims may instead refer to a sub-combination or a variant of that sub-combination.

[0131] The embodiment configurations described in this document do not represent all embodiments that can be implemented or fall within the scope of the claims. The term "embodiment" should be interpreted as meaning "used as an embodiment, example, or illustration," rather than preferred or preferred relative to other embodiments.

[0132] Articles such as "the," "a," and "an" can indicate singular or plural. When the word "or" is not preceded by "one of the following" (or other similar language that explicitly indicates that "or" is an exclusive use, e.g., explicitly indicating that only one of x or y is chosen), the word "or" should be interpreted as inclusive (e.g., "x or y" means one or both of x and y).

[0133] The term "and / or" should also be interpreted as inclusive (e.g., "x and / or y" means one or both of x and y). When "and / or" or "or" is used as a conjunction to connect groups containing three or more items, the group should be interpreted as including a single item, all items, or any combination or number of items.

[0134] The terms “have,” “contain,” and “include” should be interpreted as synonymous with the term “comprising” and are inclusive or open-ended so as not to exclude additional undescribed subject matter. The use of the foregoing four terms also discloses and supports narrower alternative implementations, in which these terms are replaced by “consisting of” or “substantially composed of,” the latter two being closed expressions used to exclude additional undescribed subject matter.

[0135] Unless otherwise stated, all numbers or expressions used in this specification (except for the claims), such as mathematical formulas or expressions representing concentration, ratio, quantity, etc., shall in all cases be understood to be modified by the term "about". At least, not intended to limit the application of the doctrine of equivalents to the claims, each numerical parameter modified by the term "about" in the specification or claims shall be interpreted in conjunction with the number of significant figures stated and using ordinary rounding methods. All disclosed scopes shall be understood to cover and support any subscopes stated in the claims and any and all individual numerical values ​​falling within that scope. For example, the scope "at least 90%" shall be interpreted to include support for at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, and at least 99.9%.

[0136] The terms recited in the claims shall be determined according to their ordinary and conventional meaning, which can be determined by referring to relevant entries in widely used general dictionaries, relevant technical references, meanings commonly understood by those skilled in the art, etc., and shall be understood to be the broadest meaning conferred by any of the foregoing sources or a combination thereof (e.g., two or more relevant references may be combined to provide the broadest meaning that such combination of references can give), but only subject to the following two exceptions: (a) when a term is used in a manner broader than its ordinary and conventional meaning, the term shall be given its ordinary and conventional meaning plus additional extended meaning, and (b) when a term is explicitly defined to have a different meaning by reciting the term and its definition together with the phrase "in this disclosure" or similar language, the term shall be limited to that definition. References to specific embodiments shall not invoke the foregoing exception (b) nor otherwise limit the scope of the terminology in the claims. Except where the foregoing exception (b) applies, nothing contained in this document shall be construed as a waiver, denial, or limitation of the scope of the claims.

[0137] The subject matter described in the claims is not equivalent to, nor should be construed as equivalent to, any implementation, feature, or combination of features described or illustrated in this document. The above conclusion holds even if only one implementation of that feature or combination of features is illustrated and described.

Claims

1. A recombinant anti-SP17 antibody, comprising: a heavy chain having at least 90% sequence homology to SEQ ID NO: 11; and a light chain having at least 90% sequence homology to SEQ ID NO: 12, wherein: the antibody binds to human sperm protein 17 (SP17).

2. A recombinant anti-SP17 antibody or antigen-binding fragment thereof, comprising two variable domains, wherein: the antibody or antigen-binding fragment thereof comprises a first variable domain comprising a VH CDR1 region comprising an amino acid sequence that is identical to at least 7 consecutive amino acids of the amino acid sequence set forth in SEQ ID NO: 5, a VH CDR2 region comprising an amino acid sequence that is identical to at least 7 consecutive amino acids of the amino acid sequence set forth in SEQ ID NO: 6, and a VH CDR3 region comprising an amino acid sequence that is identical to at least 7 consecutive amino acids of the amino acid sequence set forth in SEQ ID NO: 7; the antibody or antigen-binding fragment thereof comprises a second variable domain comprising a VL CDR1 region comprising an amino acid sequence that is identical to at least 7 consecutive amino acids of the amino acid sequence set forth in SEQ ID NO: 8, a VL CDR2 region comprising an amino acid sequence that is identical to at least 3 consecutive amino acids of the amino acid sequence set forth in SEQ ID NO: 9, and a VL CDR3 region comprising an amino acid sequence that is identical to at least 5 consecutive amino acids of the amino acid sequence set forth in SEQ ID NO: 10; and the antibody or antigen-binding fragment thereof binds to human sperm protein 17 (SP17).

3. The recombinant anti-SP17 antibody of claim 2, comprising four variable domains, wherein: the antibody comprises a third variable domain comprising a VH CDR1 region comprising an amino acid sequence that is identical to at least 7 consecutive amino acids of the amino acid sequence set forth in SEQ ID NO: 5, a VH CDR2 region comprising an amino acid sequence that is identical to at least 7 consecutive amino acids of the amino acid sequence set forth in SEQ ID NO: 6, and a VH CDR3 region comprising an amino acid sequence that is identical to at least 7 consecutive amino acids of the amino acid sequence set forth in SEQ ID NO: 7; and the antibody comprises a fourth variable domain comprising a VL CDR1 region comprising an amino acid sequence that is identical to at least 7 consecutive amino acids of the amino acid sequence set forth in SEQ ID NO: 8, a VL CDR2 region comprising an amino acid sequence that is identical to at least 3 consecutive amino acids of the amino acid sequence set forth in SEQ ID NO: 9, and a VL CDR3 region comprising an amino acid sequence that is identical to at least 5 consecutive amino acids of the amino acid sequence set forth in SEQ ID NO:

10. The antibody includes a fourth variable domain that includes a VL CDR1 region comprising an amino acid sequence that is identical to at least 7 consecutive amino acids of the amino acid sequence set forth in SEQ ID NO: 8, a VL CDR2 region comprising an amino acid sequence that is identical to at least 3 consecutive amino acids of the amino acid sequence set forth in SEQ ID NO: 9, and a VL CDR3 region comprising an amino acid sequence that is identical to at least 5 consecutive amino acids of the amino acid sequence set forth in SEQ ID NO:

10.

4. A recombinant anti-SP 17 antibody or antigen-binding fragment thereof comprising two variable domains, wherein: the antibody or antigen-binding fragment thereof includes a first variable domain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 3; the antibody or antigen-binding fragment thereof includes a second variable domain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 4; and the antibody or antigen-binding fragment thereof binds to human SP 17.

5. The recombinant anti-SP 17 antibody of claim 4, comprising four variable domains, wherein: the antibody includes a third variable domain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 3; and the antibody includes a fourth variable domain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:

4.

6. The recombinant anti-SP 17 antibody or antigen-binding fragment thereof of claim 2, having an off-rate (KD) of no more than 25 nanomolar for human SP 17.

7. The recombinant anti-SP 17 antibody or antigen-binding fragment thereof of claim 6, wherein the KD is determined by surface plasmon resonance.

8. The recombinant anti-SP 17 antibody of claim 2, comprising: a mouse IgG2a heavy chain constant region; and a mouse kappa light chain constant region.

9. The recombinant anti-SP 17 antibody of claim 2, comprising: a human IgG4 heavy chain constant region; and a human kappa light chain constant region.

10. The recombinant anti-SP 17 antibody of claim 9, wherein: the antibody has one heavy chain that is at least 90% identical in sequence to SEQ ID NO: 11; and the antibody has one light chain that is at least 90% identical in sequence to SEQ ID NO:

12.

11. The recombinant anti-SP 17 antibody of claim 9, comprising two heavy chains and two light chains, wherein: each of the two heavy chains is at least 90% identical in sequence to SEQ ID NO: 11; and each of the two light chains is at least 90% identical in sequence to SEQ ID NO:

12. each of the two light chains has at least 90% sequence identity to SEQ ID NO:

12.

12. The recombinant anti-SP 17 antibody of claim 2, comprising at least four variable domains, wherein: the first variable domain pairs with the second variable domain in the antibody such that the first variable domain and the second variable domain bind one epitope of human SP 17; and the antibody comprises a third variable domain and a fourth variable domain that pair in the antibody such that the third variable domain and the fourth variable domain bind a different epitope.

13. An antibody conjugate comprising the recombinant anti-SP 17 antibody of claim 2, wherein the recombinant anti-SP 17 antibody is conjugated to a radioisotope.

14. The antibody conjugate of claim 13, wherein the radioisotope is actinium-225, astatine-211, bismuth-212, bismuth-213, copper-67, gallium-68, holmium-166, iodine-124, iodine-131, lutetium-177, samarium-153, technetium-99, terbium-149, or yttrium-90.

15. An antibody conjugate comprising the recombinant anti-SP 17 antibody of claim 2, wherein the recombinant anti-SP 17 antibody is conjugated to a pharmaceutically active agent.

16. The antibody conjugate of claim 15, wherein the antibody is conjugated to a group selected from the group consisting of calicheamicin, camptothecin, deruxtecan, doxorubicin, emtansine, exatecan, irinotecan, maleimidocaproyl monomethyl auristatin F, mertansine, monomethyl auristatin F, paclitaxel, PE38 (Pseudomonas exotoxin A truncated fragment), pyrrolobenzodiazepine, SN-38 (7-ethyl-10-hydroxy camptothecin), and vedotin.

17. A pharmaceutical composition comprising: one of the following: (1) the recombinant anti-SP 17 antibody of claim 2, or an antigen binding fragment thereof; or (2) the antibody conjugate of claim 2; and, a pharmaceutically acceptable carrier.

18. A kit comprising: a sealed container comprising the pharmaceutical composition of claim 17; and instructions for use of the pharmaceutical composition.

19. A medical device comprising the pharmaceutical composition of claim 17, wherein the medical device is a syringe, an intravenous cannula, or a drug-loaded implant.

20. A kit comprising the recombinant anti-SP 17 antibody of claim 2 and a reporter group comprising one of: (1) a conjugated dye; or (2) a conjugated fluorescent, phosphorescent, chemiluminescent, radioactive, or magnetic label, wherein the kit is configured to detect SP 17 in a biological sample.

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