Tumor-specific bispecific immune cell engagers

Abstract

Disclosed herein are monospecific and bispecific antibodies that contain ALPPL2 / ALPP binding variable regions. The bispecific antibodies are composed of a monoclonal antibody that targets a tumor-specific cell surface antigen (ALPPL2 / ALPP) with exquisite tissue specificity and a monoclonal antibody that targets a cell surface molecule expressed by immune effector cells (e.g., CD3).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED PATENT APPLICATIONS This patent application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 247,014, filed September 22, 2021. U.S. Provisional Patent Application No. 63 / 247,014 is incorporated by reference for all purposes. [Background technology]

[0002] 2. Background of the Invention Identifying tumor-specific cell surface antigens has proven difficult, as the majority of tumor-associated antigens are also expressed in normal tissues. In our mesothelioma studies, we selected a phage antibody display library on the surface of live tumor cells and tumor tissues after counterselection against normal cells, and identified a panel of human antibodies that specifically bind to mesothelioma (An, F., et al. Mol Cancer Ther 7(3):569-78 (2008); Su, Y et al., Cancer Res., 2020 Aug 31). One of the antibodies, M25, binds only to tumors and not to any normal human tissues studied, except for placenta. We identified the tumor antigen that binds to M25 as human alkaline phosphatase placenta-like 2 (ALPPL2, also known as alkaline phosphatase germ cell (ALPG)), a member of the human alkaline phosphatase family (Su et al, 2020). Of the four members of this family, ALPPL2 and placental alkaline phosphate (ALPP) are virtually identical in amino acid sequence (98% homology) and have a very limited normal tissue expression pattern, being expressed only in placental trophoblasts. Both share a high degree of homology with intestinal alkaline phosphatase (ALPI) (87% homology) and some homology with the tissue-nonspecific liver / bone / kidney phosphatase ALPL (57% homology). M25 specifically binds to ALPPL2 and ALPP, but not to ALPI or ALPL (Su et al., 2020). The inventors performed immunohistochemistry (IHC) studies and showed that ALPPL2 is expressed in mesothelioma (Su et al., 2020) and several other tumors, such as seminoma, ovarian cancer, pancreatic cancer, gastric cancer, and colorectal cancer (WO2017095823A1; Hyrenius-Wittsten, A., et al., Science Translational Medicine 2021 Apr 28;13(591)), but not in other normal tissues except placental trophoblast, thus demonstrating exquisite tissue specificity. Thus, ALPPL2 is one of the rare cell surface antigens that can be classified as truly tumor-specific.To evaluate ALPPL2 as a potential therapeutic target, we constructed antibody-drug conjugates (ADCs) by conjugating a microtubule inhibitor to our anti-ALPPL2 human monoclonal antibody M25 and showed that the M25 ADC potently inhibited tumor cell proliferation in vitro and xenograft growth of mesothelioma cell lines in vivo (Su et al., 2020). Summary of the Invention

[0003] BRIEF SUMMARY OF THE INVETION In some aspects, the present disclosure provides an antibody comprising a variable region that specifically binds to ALPPL2 and ALPP. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region, and the complementarity determining region (CDR) 1, CDR2, and CDR3 of the heavy chain variable region are selected from the following set: SEQ ID NO:20, 21, and 22; SEQ ID NO:23, 24, and 25; SEQ ID NO:26, 27, and 28; SEQ ID NO:29, 30, and 31; SEQ ID NO:32, 33, and 34; SEQ ID NO:35, 36, and 37; SEQ ID NO:38, 39, and 40; SEQ ID NO:41, 42, and 43; SEQ ID NO:44, 45, and 46; SEQ ID NO: 47, 48, and 49; or SEQ ID NO:50, 51, and 52; and the CDR1, CDR2, and CDR3 of the light chain variable region are selected from the following set: SEQ ID NO:53, 54, and 55; SEQ ID NO:56, 57, and 58; SEQ ID NO:59, 60, and 61; SEQ ID NO:62, 63, and 64; SEQ ID NO:65, 66, and 67; SEQ ID NO:68, 69, and 70; SEQ ID NO:71, 72, and 73; or SEQ ID NO:74, 75, and 76 with the proviso that said antibody does not have the heavy chain variable region of M25FYIA and the light chain variable region of M25FYIA.

[0004] In some embodiments, the CDR1, CDR2, and CDR3 of the heavy chain variable region comprise SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25, respectively, and the CDR1, CDR2, and CDR3 of the light chain variable region comprise SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58, respectively; or the CDR1, CDR2, and CDR3 of the heavy chain variable region comprise SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28, respectively, and the CDR1, CDR2, and CDR3 of the light chain variable region comprise SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58, respectively; or the CDR1, CDR2, and CDR3 of the heavy chain variable region comprise SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46, respectively, and the CDR1, CDR2, and CDR3 of the light chain variable region comprise SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:73, respectively; or the CDR1, CDR2, and CDR3 of the heavy chain variable region comprise SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, respectively, and the CDR1, CDR2, and CDR3 of the light chain variable region comprise SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67, respectively; or the CDR1, CDR2, and CDR3 of the heavy chain variable region comprise SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43, respectively, and the CDR1, CDR2, and CDR3 of the light chain variable region comprise SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55, respectively; or The CDR1, CDR2, and CDR3 of the heavy chain variable region comprise SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37, respectively, and the CDR1, CDR2, and CDR3 of the light chain variable region comprise SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61, respectively.

[0005] In some embodiments, the heavy chain variable region is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11. In some embodiments, the light chain variable region is selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, and 19.

[0006] In some embodiments, the heavy chain variable region comprises SEQ ID NO:1 and the light chain variable region comprises SEQ ID NO:12; or the heavy chain variable region comprises SEQ ID NO:2 and the light chain variable region comprises SEQ ID NO:13; or the heavy chain variable region comprises SEQ ID NO:3 and the light chain variable region comprises SEQ ID NO:13; or the heavy chain variable region comprises SEQ ID NO:9 and the light chain variable region comprises SEQ ID NO:18; or the heavy chain variable region comprises SEQ ID NO:1 and the light chain variable region comprises SEQ ID NO:12; or the heavy chain variable region comprises SEQ ID NO:7 and the light chain variable region comprises SEQ ID NO:16; or the heavy chain variable region comprises SEQ ID NO:8 and the light chain variable region comprises SEQ ID NO:12; or The heavy chain variable region comprises SEQ ID NO:6, and the light chain variable region comprises SEQ ID NO:14.

[0007] In some embodiments, the antibody is an IgG, IgA, or IgE antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.

[0008] In some aspects, the antibody is a monospecific antibody. In some aspects, the antibody is linked to a cytotoxic agent. In some aspects, the cytotoxic agent is a radionucleotide.

[0009] In some embodiments, the antibody is a bispecific antibody comprising a second variable region that specifically binds to a second target protein, the bispecific antibody comprising a second heavy chain variable region and a second light chain variable region. In some embodiments, the second target protein is expressed on the surface of a human immune effector cell. In some embodiments, the second target protein is human CD3. In some embodiments, the CDR1, CDR2, and CDR3 of the second heavy chain variable region comprise SEQ ID NO:79, SEQ ID NO:80, and SEQ ID NO:81, and the CDR1, CDR2, and CDR3 of the second light chain variable region comprise SEQ ID NO:82, SEQ ID NO:83, and SEQ ID NO:84. In some embodiments, the second heavy chain variable region comprises SEQ ID NO:77, and the second light chain variable region comprises SEQ ID NO:78. In some embodiments, the bispecific antibody comprises SEQ ID NO:85 and SEQ ID NO:86. In some embodiments, the bispecific antibody comprises SEQ ID NO:87 and SEQ ID NO:88.

[0010] Pharmaceutical compositions comprising any of the antibodies described above or elsewhere herein are also provided.

[0011] Nucleic acids encoding any of the antibodies described above or elsewhere herein are also provided.

[0012] Vectors containing the nucleic acid sequences described above or elsewhere herein are also provided.

[0013] Also provided is a cell comprising a nucleic acid as described above or elsewhere herein, or a vector as described above or elsewhere herein, hi some aspects, the cell is a mammalian cell.

[0014] Also provided is a method for producing an antibody, comprising culturing a cell as described above or elsewhere herein under conditions that allow for the production of said antibody.

[0015] Also provided are methods of killing cancer cells, comprising contacting the cancer cells with an antibody as described above or elsewhere herein. In some embodiments, the antibody is a bispecific antibody comprising a second variable region that specifically binds to a second target protein, the second target protein being human CD3, the antibody comprising a second heavy chain variable region and a second light chain variable region, and binding of the antibody brings the cancer cells into close proximity with peripheral blood mononuclear cells (PBMCs) expressing CD3. In some embodiments, the PBMCs are T cells. In some embodiments, the cancer cells are mesothelioma cells, testicular cancer cells, endometrial cancer cells, pancreatic cancer cells, ovarian cancer cells, non-small cell lung cancer cells, gastric cancer cells, or colon cancer cells.

[0016] In some embodiments, the CDR1, CDR2, and CDR3 of the second heavy chain variable region comprise SEQ ID NO:79, SEQ ID NO:80, and SEQ ID NO:81, and the CDR1, CDR2, and CDR3 of the second light chain variable region comprise SEQ ID NO:82, SEQ ID NO:83, and SEQ ID NO:84. In some embodiments, the second heavy chain variable region comprises SEQ ID NO:77 and the second light chain variable region comprises SEQ ID NO:78. In some embodiments, the antibody comprises SEQ ID NO:85 and SEQ ID NO:86. In some embodiments, the antibody comprises SEQ ID NO:87 and SEQ ID NO:88.

[0017] In some aspects, the antibody is linked to a cytotoxic agent. In some aspects, the cytotoxic agent is a radionucleotide.

[0018] In some aspects, the cancer cells are in a human who has cancer cells, and the antibody is administered to the human, thereby killing the cancer cells.

[0019] Also provided is a chimeric antigen receptor (CAR)-expressing human cell, wherein the CAR comprises a heavy chain variable region and a light chain variable region as described above or elsewhere herein. In some embodiments, the human cell is a T cell, a natural killer cell, or a macrophage.

[0020] Also provided is a method for detecting tumor cells in a sample, the method comprising contacting the sample with an antibody as described above or elsewhere herein; and detecting specific binding of the antibody to the sample. [Brief description of the drawings]

[0021] [Figure 1] Biolayer Interferometry (BLI) measurements of the affinity of FYIA, FYIA_germ, FYIA_germ_6-6, and FYIA_germopt (aka SYLY) Fabs for human ALPPL2. The chip was loaded with Fabs followed by an association step with 5 nM human ALPPL2-Fc, followed by a dissociation step. The calculated affinity values ​​by curve fitting are 8.6 nM for FYIA, 13.0 nM for FYIA_germ, 0.88 nM for FYIA_germ_6-6, and 0.19 nM for FYIA_germopt. [Diagram 2]Binding of FYIA, FYIA_germ, and FYIA_germopt Fabs to live M28 cells. M28 cells were incubated with Fabs for 1 h at RT and binding was analyzed by flow cytometry. The calculated affinity values ​​by curve fitting are 30.55 nM for FYIA, 69.41 nM for FYIA_germ, and 0.97 nM for FYIA_germopt. [Diagram 3] Binding of FYIA, FYIA_germ, and FYIA_germopt Fabs to HEK293 cells stably transfected with human ALPI. HEK293-ALPI cells were incubated with Fabs for 1 h at RT and binding was analyzed by flow cytometry. No detectable binding was found. The ALPI-binding Fab, M25AD, was included in this study as a positive control. [Figure 4] Additional Fabs (FYIA_germ_SY and FYIA_germ_6-6) along with FYIA, FYIA_germ, and FYIA_germopt Fabs were also studied for binding to live M28 cells. M28 cells were incubated with the Fabs for 1 h at RT and binding was analyzed by flow cytometry. The calculated affinity values ​​by curve fitting are 1.27 nM for FYIA_germ_SY and 4.82 nM for FYIA_germ_6-6. [Diagram 5] Heat-induced aggregation assay. In its IgG1 form, FYIA has a higher aggregation temperature (Tagg) than daratumumab (73-74°C vs. 71-72°C). [Figure 6] Heat-induced aggregation assay. SYLY Fab showed improved thermal stability with a higher aggregation temperature (Tagg) than FYIA Fab (75-76°C vs. 73°C). [Figure 7]Therapeutic antibody profiler analysis of M25. The scores calculated based on the five developability guidelines derived from clinical-stage therapeutic values ​​are all within the favorable range. PPC: positively charged patch; PNC: negatively charged patch; SFvCSP: structural Fv charge symmetry parameter; PSH: surface hydrophobic patch. [Figure 8] FYIA Therapeutic Antibody Profiler analysis. Scores calculated based on five developability guidelines derived from clinical stage therapeutic values ​​are all within the favorable range. PPC: positively charged patch; PNC: negatively charged patch; SFvCSP: structural Fv charge symmetry parameter; PSH: surface hydrophobic patch. [Figure 9] Therapeutic Antibody Profiler analysis of SYLY. Scores calculated based on the five developability guidelines derived from clinical stage therapeutic values ​​are all within the favorable range. PPC: positively charged patch; PNC: negatively charged patch; SFvCSP: structural Fv charge symmetry parameter; PSH: surface hydrophobic patch. [Figure 10A] Overview of bispecific antibody types. Adapted from Brinkmann U, Kontermann RE. The making of bispecific antibodies. mAbs 2017; 9:182-212. [Figure 10B] This shows a continuation of Figure 10A. [Figure 11] SYLY-based ALPPL2 x CD3 DSDbodies were produced in HEK293A or ExpiCHO cells after transient transfection and purified by Ni-NTA. Purified DSDbodies were analyzed on reducing SDS-PAGE. MW: molecular weight marker. The two chains migrate at similar positions on reducing SDS-PAGE. VH_A: VH of anti-CD3. VL_B: VL of anti-ALPPL2 (e.g., VL of SYLY). VH_B: VH of anti-ALPPL2 (e.g., VH of SYLY). VL_A: VL of anti-CD3. [Figure 12]Binding of SYLY x CD3 DSDbody to human ALPP2 and ALPI measured by biolayer interferometry. Chips were loaded with human ALPPL2-Fc or human ALPI-Fc, followed by an association step with 100 nM SYLY x CD3 DSDbody, followed by a dissociation step. Calculated affinites are shown in the graph. ND: Not determined. [Figure 13] Binding of SYLY x CD3 DSDbody to human and cynomolgus CD3ε measured by biolayer interferometry. Chips were loaded with human or cynomolgus CD3ε followed by an association step with 100 nM SYLY x CD3 DSDbody, followed by a dissociation step. [Figure 14] Binding of SYLY-based bispecific antibodies (DSDbody) to live M28 cells. M28 cells were incubated with Fab for 1 h at RT and binding was analyzed by flow cytometry. The calculated affinity by curve fitting is 2.4 nM. There is no binding by the control bispecific YSC10 x CD3 DSDbody assembled on the non-binding isotype control antibody YSC10. [Figure 15] Binding of FYIA-based bispecific antibody (DSDbody) to live M28 cells. M28 cells were incubated with Fab for 1 h at RT and binding was analyzed by flow cytometry. The calculated affinity by curve fitting is 14.8 nM. There is no binding by the control bispecific YSC10 x CD3 DSDbody assembled on the non-binding isotype control antibody YSC10. [Figure 16] Binding of SYLY-based bispecific antibodies (DSDbodies) to live SKOV3 cells. SKOV3 cells were incubated with Fab for 1 h at RT and binding was analyzed by flow cytometry. The calculated affinity by curve fitting is 0.19 nM. There is no binding by the control bispecific YSC10 x CD3 DSDbody assembled on the non-binding isotype control antibody YSC10. [Figure 17]Binding of FYIA-based bispecific antibody (DSDbody) to live SKOV3 cells. SKOV3 cells were incubated with Fab for 1 h at RT and binding was analyzed by flow cytometry. The calculated affinity by curve fitting is 14.2 nM. There is no binding by the control bispecific YSC10 x CD3 DSDbody assembled on the non-binding isotype control antibody YSC10. [Figure 18] SYLY-based DSDbody BiTEs were incubated for 96 hours in the presence of SKOV3 (target) cells and human PBMCs (effector, E:T ratio 10:1). Cell viability was assessed by calcein AM. The calculated EC50 for the SYLY DSDbody is 0.3 pM. There is no killing by the control bispecific YSC10 x CD3 DSDbody assembled on the non-binding isotype control antibody YSC10. [Figure 19] FYIA-based DSDbody BiTEs were incubated for 96 hours in the presence of SKOV3 (target) cells and human PBMCs (effector, E:T ratio 10:1). Cell viability was assessed by calcein AM. The calculated EC50 for the FYIA DSDbody is 62.8 pM. There is no killing by the control bispecific YSC10 x CD3 DSDbody assembled on the non-binding isotype control antibody YSC10. [Figure 20] SYLY-based DSDbody BiTEs were incubated with HEK293 cells stably expressing ALPP (target) in the presence of human PBMCs (effector, E:T ratio 10:1) for 72 hours. Cell viability was assessed by calcein AM. The calculated EC50 for the SYLY DSDbody is 8.5 pM and the calculated EC50 for the control bispecific YSC10 x CD3 DSDbody is >100 nM. [Figure 21] SYLY-based DSDbody BiTEs were incubated with HEK293 cells (target) in the presence of human PBMCs (effector, E:T ratio 10:1) for 72 h. Cell viability was assessed by calcein AM. There was no apparent killing of HEK293 cells at antibody concentrations up to 100 nM. [Figure 22]Heat-induced aggregation assay. The aggregation temperature (Tagg) of the FYIA-based ALPPL2 x CD3 DSDbody is 65°C. [Diagram 23] Heat-induced aggregation assay. The aggregation temperature (Tagg) of the SYLY-based ALPPL2 x CD3 DSDbody is 70°C. [Figure 24] Binding to AsPC1 cells by flow cytometry. [Diagram 25] In vitro cytotoxicity in AsPC1-luc. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] definition As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" optionally includes a combination of two or more such molecules, and the like.

[0023] The term "antibody" as used herein refers to an isolated or recombinant binding agent that contains the necessary variable region sequence for specific binding to an antigen epitope. Thus, as used herein, "antibody" refers to any form of antibody of any class or subclass, or a fragment thereof, that exhibits the desired biological activity, such as binding to a specific target antigen. Thus, "antibody" is used in the broadest sense, and includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), human antibodies, chimeric antibodies, single domain antibodies, such as nanobodies, diabodies, camelid-derived antibodies, monovalent antibodies, bivalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, including, but not limited to, scFv, Fab, etc., so long as they exhibit the desired biological activity.

[0024] An "antibody fragment" includes a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific or multivalent antibodies formed from antibody fragments. A "Fab" fragment contains the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain. An F(ab')2 fragment generally has a pair of Fab fragments covalently linked by hinge cysteines near the carboxy terminus. Other chemical bonds of antibody fragments are also known. An "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site and is a dimer of one heavy chain variable domain and one light chain variable domain.

[0025] The "class" of an antibody refers to the type of constant domain or region possessed by its heavy chain. There are five major antibody classes: IgA, IgD, IgE, IgG, and IgM, which may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4. The antibodies described herein may be of any of these classes or subclasses.

[0026] As used herein, a "V region" refers to an antibody variable region domain comprising framework 1, CDR1, framework 2, framework 3 which contains CDR2 and CDR3, and framework 4 segments.

[0027] As used herein, "complementarity determining region (CDR)" refers to three hypervariable regions that interrupt the four "framework" regions of a variable domain. CDRs are primarily responsible for binding to an antigen epitope. The CDRs of each heavy or light chain are numbered consecutively from the N-terminus and are referred to as CDR1, CDR2, and CDR3.

[0028] The amino acid sequences of the CDR and framework regions can be determined using various definitions in the art, e.g., North, Kabat, Chothia, the international ImMunoGeneTics database (IMGT), and AbM (see, e.g., the North method) (see, e.g., North et al., J. Mol. Biol. 406(2):228-256, 2011; Johnson et al., supra; Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C. et al., 1989, Conformations of immunoglobulin hypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992, structural repertoire of the human VH segments J. Mol. Biol. 227, 799-817; see Al-Lazikani et al., J. Mol. Biol 1997, 273(4).The definition of CDR is as follows: Ruiz et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219-221 (2000); and Lefranc, M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan 1;29(1):207-9 (2001); MacCallum et al, Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); and Martin et al, Proc. Natl Acad. Sci. USA, 86, 9268-9272 (1989); Martin, et al, Methods Enzymol., 203, 121-153, (1991); Pedersen et al, Immunomethods, 1, 126, (1992); and Rees et al, In Sternberg MJE (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172 1996). References to CDRs herein refer to CDRs determined according to the method of North (see, e.g., North et al., J. Mol. Biol. 406(2):228-256, 2011), unless otherwise specified.

[0029] "Epitope" or "antigenic determinant" as used in this disclosure in the context of antibody binding refers to a portion on an antigen to which an antibody binds. Epitopes may be formed from contiguous amino acids and / or non-contiguous amino acids brought into close proximity by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically comprises at least 3 amino acids, more usually at least 5 or 8-10 amino acids, in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography and 2-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996). Binding of an antibody to an epitope may be affected by other environmental factors, such as the presence of calcium ions.

[0030] The term "bispecific antibody" as used herein refers to an antibody that binds to two or more different epitopes. In some embodiments, a bispecific antibody binds to epitopes of two different target antigens. In some embodiments, a bispecific antibody binds to two different epitopes of the same target antigen. Bispecific antibodies can be made in a number of ways. See, for example, Brinkmann U, Kontermann RE. The making of bispecific antibodies. mAbs 2017; 9:182-212 and Figures 10A-B. In some embodiments, the bispecific antibodies described herein are diabodies or knob-in-a-hole IgG antibodies, or otherwise use knob-in-a-hole technology. See, for example, Xu, et al., MAbs 7(1):231-42 (2015).

[0031] The phrase "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to polypeptides, including antibodies, bispecific antibodies, etc., having substantially identical amino acid sequences or derived from the same genetic source. The term also includes preparations of antibody molecules of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

[0032] As used herein, the term "specifically binds" to a target, e.g., human ALPP / ALPPL2, refers to a binding reaction in which the antibody binds to a target with greater affinity, greater avidity, and / or longer duration than an antibody binds to a different target. In some embodiments, a target binding protein has at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or more affinity for the target compared to an unrelated target when assayed under the same binding affinity assay conditions. As used herein, the terms "specific binding" to a particular target, "specifically binds" to a particular target, or "specific" to a particular target refer to, for example, the equilibrium dissociation constant K D For example, by a molecule (e.g., an antibody) having -2 M or less, e.g., 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 Equilibrium dissociation constant K for the target D In some embodiments, the antibody has a K of less than 100 nM or less than 10 nM. D has.

[0033] The terms "treat" and "treatment" refer to both therapeutic treatments and preventative or suppressive measures, in which the goal is to prevent or slow down undesirable physiological changes or disorders. For purposes of this disclosure, beneficial or desirable clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, improvement or mitigation of disease state, and remission (whether partial or complete). "Treatment" can also mean extending survival time compared to expected survival in the absence of treatment. In other aspects, the terms "treat", "treatment" and "treating" refer to inhibiting the progression of a proliferative disorder physically, e.g., by stabilization of discernible symptoms, physiologically, e.g., by stabilization of physical parameters, or both. In other aspects, the terms "treat", "treatment" and "treating" refer to reduction or stabilization of tumor size or cancer cell number.

[0034] The term "pharmaceutical acceptable carrier" as used herein refers to an excipient or diluent in a pharmaceutical composition. A pharmaceutical acceptable carrier must be compatible with other ingredients of the formulation and not deleterious to the recipient. In the present invention, a pharmaceutical acceptable carrier must provide sufficient pharmaceutical stability to the active ingredient. The nature of the carrier depends on the method of administration. For example, for intravenous administration, an aqueous carrier is generally used, while for oral administration, a solid carrier is preferred.

[0035] As used herein, the phrase "inhibiting proliferation of cells expressing ALPP and / or ALPPL2" refers to an anti-ALPP / ALPPL2 antibody or immunoconjugate described herein capable of reducing, preferably statistically significantly reducing, proliferation of cells expressing ALPP and / or ALPPL2 or a fragment thereof, compared to proliferation in the absence of said antibody or immunoconjugate. In one embodiment, when a cell expressing ALPP / ALPPL2 or a fragment thereof (e.g., a cancer cell) is contacted with an antibody or antigen-binding portion thereof or immunoconjugate described herein, proliferation of the cell may be reduced by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or 100% compared to proliferation measured in the absence of the antibody or antigen-binding portion thereof or immunoconjugate (control). Cell proliferation can be assayed using art-recognized techniques that measure the rate of cell division (e.g., using a cell titer glow assay or thymidine incorporation), the fraction of cells within a cell population undergoing cell division, and / or the rate of cell loss from a cell population due to terminal differentiation or cell death.

[0036] An "isolated antibody" as used herein is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to ALPP and / or ALPL2 is substantially free of antibodies that specifically bind to antigens other than ALPP and / or ALPPL2). Furthermore, an isolated antibody is typically substantially free of other cellular material and / or chemicals. In one embodiment, a combination of "isolated" monoclonal antibodies with different ALPP / ALPPL2 binding specificities are combined in a specifically disclosed composition. Any of the antibodies provided herein can be provided as isolated antibodies.

[0037] The term "effective amount" as used herein refers to an amount of an anti-ALPP / ALPPL2 antibody or antigen-binding portion thereof and / or immunoconjugate thereof that, when administered to a subject, is sufficient to provide treatment, prognosis, or diagnosis of a disease associated with the growth and / or proliferation of ALPP / ALPPL2-positive cells (e.g., ALPP / ALPPL2-positive cancers) as described herein. A therapeutically effective amount will vary depending on the subject and disease state being treated, the weight and age of the subject, the severity of the disease state, the method of administration, etc., and can be readily determined by one of skill in the art.The dosage for administration is, for example, about 1 ng to about 10,000 mg, about 5 ng to about 9,500 mg, about 10 ng to about 9,000 mg, about 20 ng to about 8,500 mg, about 30 ng to about 7,500 mg, about 40 ng to about 7,000 mg, about 50 ng to about 6,500 mg, about 100 ng to about 6,000 mg, about 200 ng to about 5,500 mg, about 300 ng to about 5,000 mg, about 400 ng to about 4,500 mg, about 500 ng to about 6,000 mg, about 700 ng to about 8,500 mg, about 800 ng to about 9,000 mg, about 900 ng to about 10,000 mg, about 1000 ng to about 15,000 mg, about 1500 ng to about 20,000 mg, about 1000 ng to about 30,000 mg, about 1500 ng to about 40,000 mg, about 1500 ng to about 50,000 mg, about 1500 ng to about 60,000 mg, about 1500 ng to about 70,000 mg, about 1500 ng to about 80,000 mg, about 1500 ng to about 90,000 mg, about 1500 ng to about 100,000 mg, about 1500 ng to about 100,000 mg, about 1500 ng to about 200,000 mg, about 1500 ng to about 300,000 mg, about 1 g~about 4,000mg, about 1μg~about 3,500mg, about 5μg~about 3,000mg, about 10μg~about 2,600mg, about 20μg~about 2,575mg, about 30μg~about 2,550mg, about 40μg~about 2,5 00mg, about 50μg to about 2,475mg, about 100μg to about 2,450mg, about 200μg to about 2,425mg, about 300μg to about 2,000, about 400μg to about 1,175mg, about 500μg to about 1,150 mg, about 0.5 mg to about 1,125 mg, about 1 mg to about 1,100 mg, about 1.25 mg to about 1,075 mg, about 1.5 mg to about 1,050 mg, about 2.0 mg to about 1,025 mg, about 2.5 mg to about 1,00 0mg, about 3.0mg to about 975mg, about 3.5mg to about 950mg, about 4.0mg to about 925mg, about 4.5mg to about 900mg, about 5mg to about 875mg, about 10mg to about 850mg, about 20mg to about 825 The effective amount may be about 30 mg to about 800 mg, about 40 mg to about 775 mg, about 50 mg to about 750 mg, about 100 mg to about 725 mg, about 200 mg to about 700 mg, about 300 mg to about 675 mg, about 400 mg to about 650 mg, about 500 mg, or about 525 mg to about 625 mg of an anti-ALPP / ALPPL2 antibody and / or antigen-binding portion thereof described herein, and / or an immunoconjugate thereof described herein. Dosage regiments may be adjusted to provide an optimal therapeutic response. An effective amount is also an amount in which any toxic or adverse effects (i.e., side effects) of the antibody or antigen-binding portion thereof are minimized and / or are outweighed by beneficial effects.

[0038] "Effector" refers to any molecule or combination of molecules having an activity which is desirable to deliver to and / or localize to a target cell. Effectors include, but are not limited to, labels, cytotoxins, enzymes, growth factors, transcription factors, antibodies, drugs, etc.

[0039] For example, the phrase "inhibiting the growth and / or proliferation" of cancer cells includes, among other things, inducing cell apoptosis or other cell death mechanisms, reducing the invasiveness of the cell, arresting the cell at a point in the cell cycle, and the like.

[0040] The term "immunoconjugate" refers to an antibody attached to one or more effectors, or to multiple antibodies attached to one or more effectors. The term "immunoconjugate" is intended to include effectors chemically conjugated to an antibody, as well as antibodies expressed as fusion proteins in which an antibody (or portion thereof) is attached directly or via a linker to a peptide effector or a peptide-containing effector.

[0041] Detailed Description of the Invention The present inventors have developed improved anti-ALPPL2 / ALPP antibodies that can be used as monospecific or bispecific antibodies. The antibodies can be used to kill tumor cells in various ways. The antibodies are also useful for detecting tumor cells, and can be used, for example, as companion diagnostics. The antibodies described herein bind to cells that express or overexpress ALPPL / ALPPL2. Exemplary non-limiting cells that express ALPPL / ALPPL2 include, but are not limited to, mesothelioma cells, testicular cancer cells, endometrial cancer cells, pancreatic cancer cells, ovarian cancer cells, non-small cell lung cancer cells, gastric cancer cells, and colon cancer cells.

[0042] As discussed in the Examples, the previously described M25FYIA (also known as FYIA) has been significantly improved to increase monovalent binding affinity to ALPPL2 while improving developability characteristics (e.g., avoidance of charged or hydrophobic patches and removal of deamidation and isomerization motifs). In some aspects, the antibodies provided herein comprise a variable region that specifically binds to ALPPL2 and ALPP. The variable region may, for example, comprise a heavy chain variable region and a light chain variable region. In some embodiments, the complementarity determining regions (CDRs) 1, 2, and 3 of the heavy chain variable region are selected from the following set: SEQ ID NOs:20, 21, and 22; SEQ ID NOs:23, 24, and 25; SEQ ID NOs:26, 27, and 28; SEQ ID NOs:29, 30, and 31; SEQ ID NOs:32, 33, and 34; SEQ ID NOs:35, 36, and 37; SEQ ID NOs:38, 39, and 40; SEQ ID NOs:41, 42, and 43; SEQ ID NOs:44, 45, and 46; SEQ ID NOs:47, 48, and 49; or SEQ ID NOs:50, 51, and 52, and the CDRs 1, 2, and 3 of the light chain variable region are selected from the following set: SEQ ID NOs:53, 54, and 55; SEQ ID NOs:56, 57, and 58; SEQ ID NOs:59, 60, and 61; SEQ ID NOs:62, 63, and 64; SEQ ID NOs:65, 66, and 67; SEQ ID NOs:68, 69, and 70; SEQ ID NOs:71, 72, and 73; or SEQ ID NOs:74, 75, and 76.

[0043] In embodiments in which the heavy chain variable region CDRs comprise SEQ ID NOs:20, 21, and 22 and the light chain variable region CDRs comprise SEQ ID NOs:53, 54, and 55, the antibody comprises framework sequences that differ from those found in M25FYIA or other antibodies in PCT Publication No. WO2017 / 095823 having these CDR sequences. For example, the entire heavy chain variable region may comprise SEQ ID NO:1 and the light chain variable region may comprise SEQ ID NO:12.

[0044] Given the similarity of various heavy and light chain variable regions, it is believed that any heavy chain variable region (or its CDRs, in different frameworks) can be combined with any light chain variable region (or its CDRs, in different frameworks) to form an antibody variable region that binds to ALPPL2 and ALPP.

[0045] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000001.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000002.tif23150 or the entire light chain variable sequence.

[0046] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000003.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000004.tif23150 or the entire light chain variable sequence.

[0047] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000005.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000006.tif24150 or the entire light chain variable sequence.

[0048] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000007.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000008.tif23150 or the entire light chain variable sequence.

[0049] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000009.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000010.tif23150 or the entire light chain variable sequence.

[0050] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000011.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000012.tif23150 or the entire light chain variable sequence.

[0051] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000013.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000014.tif23150 or the entire light chain variable sequence.

[0052] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000015.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000016.tif23150 or the entire light chain variable sequence.

[0053] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000017.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000018.tif23150 or the entire light chain variable sequence.

[0054] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000019.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000020.tif24150 or the entire light chain variable sequence.

[0055] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000021.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000022.tif23150 or the entire light chain variable sequence.

[0056] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000023.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000024.tif23150 or the entire light chain variable sequence.

[0057] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000025.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000026.tif23150 or the entire light chain variable sequence.

[0058] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000027.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000028.tif23150 or the entire light chain variable sequence.

[0059] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000029.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000030.tif23150 or the entire light chain variable sequence.

[0060] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000031.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000032.tif23150 or the entire light chain variable sequence.

[0061] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000033.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000034.tif23150 or the entire light chain variable sequence.

[0062] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000035.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000036.tif23150 or the entire light chain variable sequence.

[0063] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000037.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000038.tif23150 or the entire light chain variable sequence.

[0064] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000039.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000040.tif23150 or the entire light chain variable sequence.

[0065] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000041.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000042.tif23150 or the entire light chain variable sequence.

[0066] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000043.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000044.tif23150 or the entire light chain variable sequence.

[0067] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000045.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000046.tif23150 or the entire light chain variable sequence.

[0068] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000047.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000048.tif23150 or the entire light chain variable sequence.

[0069] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000049.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000050.tif23150 or the entire light chain variable sequence.

[0070] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000051.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000052.tif23150 or the entire light chain variable sequence.

[0071] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000053.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000054.tif24150 or the entire light chain variable sequence.

[0072] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000055.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000056.tif23150 or the entire light chain variable sequence.

[0073] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000057.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000058.tif23150 or the entire light chain variable sequence.

[0074] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000059.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000060.tif23150 or the entire light chain variable sequence.

[0075] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000061.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000062.tif23150 or the entire light chain variable sequence.

[0076] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000063.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000064.tif23150 or the entire light chain variable sequence.

[0077] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000065.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000066.tif23150 or the entire light chain variable sequence.

[0078] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000067.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000068.tif23150 or the entire light chain variable sequence.

[0079] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000069.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000070.tif23150 or the entire light chain variable sequence.

[0080] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000071.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000072.tif23150 or the entire light chain variable sequence.

[0081] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000073.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000074.tif23150 or the entire light chain variable sequence.

[0082] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000075.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000076.tif23150 or the entire light chain variable sequence.

[0083] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000077.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000078.tif23150 or the entire light chain variable sequence.

[0084] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000079.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000080.tif23150 or the entire light chain variable sequence.

[0085] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000081.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000082.tif23150 or the entire light chain variable sequence.

[0086] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000083.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000084.tif23150 or the entire light chain variable sequence.

[0087] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000085.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000086.tif23150 or the entire light chain variable sequence.

[0088] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000087.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000088.tif23150 or the entire light chain variable sequence.

[0089] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000089.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000090.tif23150 or the entire light chain variable sequence.

[0090] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000091.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000092.tif23150 or the entire light chain variable sequence.

[0091] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000093.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000094.tif23150 or the entire light chain variable sequence.

[0092] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000095.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000096.tif23150 or the entire light chain variable sequence.

[0093] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000097.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000098.tif23150 or the entire light chain variable sequence.

[0094] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000099.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000100.tif23150 or the entire light chain variable sequence.

[0095] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000101.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000102.tif24150 or the entire light chain variable sequence.

[0096] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000103.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000104.tif23150 or the entire light chain variable sequence.

[0097] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000105.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000106.tif23150 or the entire light chain variable sequence.

[0098] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000107.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000108.tif23150 or the entire light chain variable sequence.

[0099] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000109.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000110.tif23150 or the entire light chain variable sequence.

[0100] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000111.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000112.tif23150 or the entire light chain variable sequence.

[0101] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000113.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000114.tif23150 or the entire light chain variable sequence.

[0102] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000115.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000116.tif23150 or the entire light chain variable sequence.

[0103] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000117.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000118.tif23150 or the entire light chain variable sequence.

[0104] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000119.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000120.tif23150 or the entire light chain variable sequence.

[0105] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000121.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000122.tif23150 or the entire light chain variable sequence.

[0106] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000123.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000124.tif23150 or the entire light chain variable sequence.

[0107] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000125.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000126.tif23150 or the entire light chain variable sequence.

[0108] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000127.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000128.tif23150 or the entire light chain variable sequence.

[0109] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000129.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000130.tif23150 or the entire light chain variable sequence.

[0110] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000131.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000132.tif23150 or the entire light chain variable sequence.

[0111] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000133.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000134.tif23150 or the entire light chain variable sequence.

[0112] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000135.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000136.tif23150 or the entire light chain variable sequence.

[0113] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000137.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000138.tif23150 or the entire light chain variable sequence.

[0114] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000139.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000140.tif23150 or the entire light chain variable sequence.

[0115] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000141.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000142.tif23150 or the entire light chain variable sequence.

[0116] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000143.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000144.tif23150 or the entire light chain variable sequence.

[0117] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000145.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000146.tif23150 or the entire light chain variable sequence.

[0118] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000147.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000148.tif23150 or the entire light chain variable sequence.

[0119] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000149.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000150.tif24150 or the entire light chain variable sequence.

[0120] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000151.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000152.tif23150 or the entire light chain variable sequence.

[0121] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000153.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000154.tif23150 or the entire light chain variable sequence.

[0122] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000155.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000156.tif23150 or the entire light chain variable sequence.

[0123] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000157.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000158.tif23150 or the entire light chain variable sequence.

[0124] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000159.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000160.tif23150 or the entire light chain variable sequence.

[0125] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000161.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000162.tif23150 or the entire light chain variable sequence.

[0126] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000163.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000164.tif23150 or the entire light chain variable sequence.

[0127] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000165.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000166.tif23150 or the entire light chain variable sequence.

[0128] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000167.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000168.tif23150 or the entire light chain variable sequence.

[0129] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000169.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000170.tif23150 or the entire light chain variable sequence.

[0130] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000171.tif25150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000172.tif23150 or the entire light chain variable sequence.

[0131] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000173.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000174.tif23150 or the entire light chain variable sequence.

[0132] In some aspects, the antibodies described herein comprise a variable region that specifically binds ALPPL2 and ALPP, wherein the heavy chain variable region comprises: The light chain variable region comprises the CDRs of the heavy chain variable sequence shown in TIFF2024534543000175.tif26150 or the entire heavy chain variable sequence, in combination with the following: It includes the CDRs of the light chain variable sequence shown in TIFF2024534543000176.tif23150 or the entire light chain variable sequence.

[0133] In some embodiments, the antibody comprises a heavy chain variable region having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence of any one of SEQ ID NOs:1-11. In some embodiments, the antibody comprises a light chain variable region having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence of any one of SEQ ID NOs:12-19.

[0134] In some embodiments, modifications such as pegylation or incorporation of long-chain polyethylene glycol polymers (PEG) can be optionally introduced into the antibody (e.g., either internally in the polypeptide chain or at the N-terminus or C-terminus), for example, to increase in vivo half-life. Introduction of PEG or long-chain polymers of PEG increases the effective molecular weight of the polypeptide, for example, to prevent rapid filtration into urine. In some embodiments, lysine residues in the sequence are conjugated to PEG directly or via a linker. Such linkers can be, for example, Glu residues or acyl residues that contain a thiol functional group to link to an appropriately modified PEG chain. Another method for introducing a PEG chain is to first introduce a Cys residue at the C-terminus or in place of a solvent-exposed residue, for example, an Arg or Lys residue. This Cys residue is then site-specifically attached to a PEG chain that contains, for example, a maleimide functional group. Methods for incorporating PEG or long chain polymers of PEG are known in the art (e.g., Veronese, FM, et al., Drug Disc. Today 10: 1451-8 (2005); Greenwald, RB, et al., Adv. Drug Deliv. Rev. 55: 217-50 (2003); Roberts, MJ, et al., Adv. Drug Deliv. Rev., 54: 459-76 (2002)), the contents of which are incorporated herein by reference.

[0135] In certain embodiments, specific mutations of an antibody can be made to alter the glycosylation of the polypeptide. Such mutations may be selected to introduce or eliminate one or more glycosylation sites, including, but not limited to, O-linked or N-linked glycosylation sites. In certain embodiments, the glycosylation sites and glycosylation pattern of the protein are unchanged compared to the native protein. In certain embodiments, protein variants include glycosylation variants in which the number and / or type of glycosylation sites are altered compared to the native protein. In certain embodiments, polypeptide variants include more or fewer N-linked glycosylation sites compared to the native polypeptide. N-linked glycosylation sites are characterized by the sequence: Asn-X-Ser or Asn-X-Thr, where the amino acid residue designated as X can be any amino acid residue. Amino acid residue substitutions that create this sequence provide potential new sites for the addition of N-linked carbohydrate chains. Alternatively, substitutions that eliminate this sequence remove existing N-linked carbohydrate chains. In certain embodiments, a rearrangement of the N-linked carbohydrate chain is provided in which one or more N-linked glycosylation sites (typically a naturally occurring N-linked glycosylation site) are eliminated and one or more new N-linked sites are created.

[0136] In some embodiments, the antibody is a tetramer or a fragment thereof. In some embodiments, the antibody is a single chain antibody, and the VH and VL domains that make up the antibody may be directly linked or may be linked by a peptide linker. Exemplary peptide linkers include: This includes, but is not limited to, TIFF2024534543000177.tif38148.

[0137] In various embodiments, the antibodies described herein may be produced by chemical synthesis or recombinantly expressed. For example, the sequence information provided herein can be used to chemically synthesize the anti-ALPPL2 specific antibodies or variants thereof described herein using well-known peptide synthesis methods. Solid phase synthesis is a preferred method for chemically synthesizing single-chain antibodies, in which the C-terminal amino acid of the sequence is attached to an insoluble support, followed by the sequential addition of the remaining amino acids in the sequence. The technique of solid phase synthesis is described in Barany and Merrifield, Solid Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield et al. (1963) J. Am. Chem. Soc, 85: 2149-2156, and Stewart et al. (1984) Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, 111.

[0138] In certain embodiments, the anti-ALPPL2 / ALPPL specific antibody or variant thereof described herein is recombinantly expressed using methods well known to those skilled in the art. For example, using the sequence information provided herein, nucleic acid encoding the desired antibody can be prepared according to a number of standard methods known to those skilled in the art. The nucleic acid is introduced into a host cell by transfection, which then expresses the desired antibody or its chain.

[0139] Molecular cloning methods to achieve these goals are known in the art. A wide variety of cloning and in vitro amplification methods are suitable for constructing recombinant nucleic acids. Examples of these techniques and instructions sufficient to guide the skilled artisan through many cloning exercises can be found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al. (1989) Molecular Cloning - A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook); and Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel). Methods for producing recombinant immunoglobulins are also known in the art. See Cabilly, U.S. Patent No. 4,816,567; and Queen et al. (1989) Proc. Natl Acad. Sci. USA 86: 10029-10033. Further, detailed protocols for antibody expression are provided in Liu et al. (2004) Cancer Res. 64: 704-710, Poul et al. (2000) J. Mol. Biol. 301: 1149-1161, etc.

[0140] Using the known and / or specified sequences (e.g., VH and / or VL sequences) of the antibodies provided herein, other antibody forms can be easily produced. Such forms include, but are not limited to, multivalent antibodies, complete antibodies, scFvs, (scFv')2, Fabs, (Fab')2, chimeric antibodies, and the like. For example, to produce (scFv')2 antibodies, two anti-ALPP / ALPPL2 antibodies are connected via a linker {e.g., carbon linker, peptide, etc.) or via a disulfide bond, e.g., a disulfide bond between two cysteines. Thus, for example, to produce disulfide-linked scFvs, a cysteine ​​residue can be introduced at the carboxy terminus of the antibodies described herein by site-directed mutagenesis. The scFvs can be expressed from this construct, purified by EVIAC, and analyzed by gel filtration. To produce (scFv')2 dimers, cysteines are reduced by incubation with 1 mM 3-mercaptoethanol, and half of the scFv is blocked by addition of DTB. Blocked and unblocked scFvs can be incubated together to form (scFv')2, and the resulting material can be analyzed by gel filtration. The affinity of the resulting dimers can be determined using standard methods, for example, by BIAcore. In an exemplary embodiment, (scFv')2 dimers are created by connecting scFv' fragments via a linker, for example, a peptide linker. This can be accomplished by a variety of means well known to those skilled in the art. For example, one approach is described in Holliger et al. (1993) Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (see also WO 94 / 13804).

[0141] Using the VH and / or VL sequences provided herein, Fab and (Fab')2 dimers can also be easily prepared. Fab is a light chain connected to VH-CH1 by disulfide bonds and can be easily produced using standard methods known to those skilled in the art. F(ab)'2 can be generated, for example, by dimerizing Fab as described above for (scFv')2 dimers.

[0142] Antibodies as contemplated herein include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as such fragments exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567; Morrison et al. (1984) Proc. Natl. Acad. Sci. 81: 6851-6855, etc.). Chimeric antibodies are antibodies that contain portions derived from two different species (e.g., human and non-human portions). Typically, the antigen-binding region (or variable region) of a chimeric antibody is derived from one source, and the constant region (which confers biological effector functions to the immunoglobulin) of the chimeric antibody is derived from another source. Numerous methods for making chimeric antibodies are known to those of skill in the art (see, e.g., U.S. Pat. Nos. 5,502,167, 5,500,362, 5,491,088, 5,482,856, 5,472,693, 5,354,847, 5,292,867, 5,231,026, 5,204,244, 5,202,238, 5,169,939, 5,081,235, 5,075,431, and 4,975,369, and PCT application WO 91 / 0996).

[0143] In another aspect, the present invention provides fully intact human anti-ALPP / ALPPL2 antibodies. Such antibodies can be readily produced in a manner similar to the generation of chimeric human antibodies, where the VH and VL domains described herein are fully human and can be readily engineered into substantially complete antibodies (e.g., IgG, IgA, IgM, etc.).

[0144] In certain embodiments, using the sequence information provided herein, anti-ALPP / ALPPL2 antibodies can be constructed as unibodies. Unibodies are an antibody technology that produces stable small antibody formats with a predicted therapeutic window longer than certain small antibody formats. In certain embodiments, unibodies are produced by removing the antibody hinge region from IgG4 antibodies. Unlike full-sized IgG4 antibodies, half molecule fragments are very stable and are called unibodies. When an IgG4 molecule is halved, only one region remains on the unibody that can bind to the target. Methods for producing unibodies are detailed in PCT publication WO2007 / 059782, which is incorporated herein by reference in its entirety {see also Kolfschoten et al. (2007) Science 31': 1554-1557).

[0145] In certain embodiments, the sequence information provided herein is used to construct affibody molecules that bind to ALPP / ALPPL2. Affibody molecules are a class of affinity proteins based on a 58 amino acid residue protein domain derived from one of the IgG binding domains of Staphylococcus aureus protein A. This three-helix bundle domain has been used as a scaffold for constructing combinatorial phagemid libraries from which affibody variants targeting desired molecules can be selected using phage display technology {see, for example, Nord et al. (1997) Nat. Biotechnol. 15: 772-777; Ronmark et al. (2002) Eur. J. Biochem., 269: 2647-2655.). Details of affibodies and production methods are known to those skilled in the art {see, for example, U.S. Patent No. 5,831,012, which is incorporated herein by reference in its entirety).

[0146] It will be appreciated that the antibody can be provided as an intact whole antibody {e.g., IgG), an antibody fragment, or a single chain antibody using methods well known to those of skill in the art. Furthermore, the antibody can be derived from essentially any mammalian species, although to reduce immunogenicity, it is desirable to use antibodies of the species in which the antibody and / or immunoconjugate will be used. In other words, for use in humans, it is desirable to use human antibodies, humanized antibodies, or chimeric human antibodies.

[0147] Any of the antibodies described herein, particularly the monospecific antibodies described herein in some embodiments, can be linked to an effector molecule. Anti-ALPP / ALPPL2 immunoconjugates can be formed by conjugating the antibodies described herein or antigen-binding portions thereof to an effector (e.g., a detectable label, another therapeutic agent, etc.). Exemplary therapeutic agents include, but are not limited to, for example, a cytotoxic or cytostatic agent (e.g., a chemotherapeutic agent), a toxin (e.g., an enzymatically active toxin from bacteria, fungi, plants, or animals, or fragments thereof), a radioisotope (e.g., a radioconjugate), or a second antibody.

[0148] In certain embodiments, anti-ALPP / ALPPL2 immunoconjugates can be used to target detectable labels to tumor sites or to otherwise detect the presence of cancer cells. This can facilitate tumor detection and / or localization. This can be useful for detecting primary tumors or, in certain embodiments, secondary tumors caused by cancers that express ALPPL2 (e.g., cancers including, but not limited to, mesothelioma, testicular cancer, endometrial cancer, ovarian cancer, pancreatic cancer, and non-small cell lung cancer).

[0149] Thus, in certain embodiments, the effector comprises detectable label.Suitable detectable labels include, but are not limited to, radiopaque label, nanoparticle, PET label, MRI label, radiolabel, etc.Among the radionuclides and useful in various embodiments, gamma emitters, positron emitters, X-ray emitters, and fluorescent emitters are suitable for localization, diagnosis and / or staging, and / or therapy, whereas beta emitters and alpha emitters, as well as electron emitters and neutron capture agents, such as boron and uranium, can also be used for therapy.

[0150] In various embodiments, detectable labels can be used in combination with external and / or internal detectors to provide a means for effectively localizing and / or visualizing cancer cells expressing ALPPL2. Such detection / visualization may be useful in various situations, including but not limited to pre-operative and intra-operative settings. Thus, in certain embodiments, the present invention relates to methods for detecting cancers expressing ALPPL2 in a mammalian body during surgery. These methods typically involve administering to the mammal a composition comprising an anti-ALPPL2 antibody labeled with a detectable label as described herein in an amount sufficient for detection by a detector (e.g., a gamma-detecting probe), and subjecting the mammal to radioimmunodetection in the relevant area of ​​the body, for example, using a gamma-detecting probe, after the active substance is taken up into the target tissue, preferably after blood clearance of the label.

[0151] In certain aspects, the labeled conjugated antibodies can be used in radio-guided surgical techniques, whereby relevant tissues within the subject's body can be detected and localized during surgery by a detector, e.g., a gamma detection probe.

[0152] Surgeons can use this probe during surgery to find tissues where uptake of radioisotopes, i.e., compounds labeled with, for example, low-energy gamma photon emitters, has occurred. In certain embodiments, such methods are particularly useful for localizing and removing secondary cancers caused by metastatic cells derived from a primary tumor.

[0153] The anti-ALPP / ALPPL2 antibodies described herein may be directly conjugated to a radiopaque moiety (e.g., at an available cysteine) or may be attached to a "package" (e.g., a chelate, liposome, polymeric microbead, nanoparticle, etc.) that carries, contains or includes a radiopaque material, e.g., as described below.

[0154] In addition to radiopaque labels, other labels are suitable for use. Detectable labels suitable for use in immunoconjugates include any composition that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Useful labels include magnetic beads (e.g., DYNABEADS™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, green fluorescent protein, etc.), radiolabels (e.g., H, I, S, C, or P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and other enzymes used in ELISA), and colorimetric labels, such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, nanoparticles, quantum dots, etc.

[0155] In certain embodiments, suitable radiolabels include: 99 Tc, 99 Tc, 97 Ru, 95 Ru, 94 Tc, 90 Y, 90 Y, 89 Zr, 86 Y, 77 Br, 77 As, 76 Br, 75 Se, 72 As, 68 Ga, 68 Ga, 67 Ga, 67 Ga, 67 Cu, 67 Cu, 64 Cu, 62 Cu, 62 Cu, 59 Fe, 58 Co, 57 Co, 52 Mn, 52 Fe, 51 Cr, 47 Sc, 3 H, 35 S, 33 P, 32 P, 225 Ac, 224Ac、 223 Ra、 213 Bi、 212 Pb、 212 Bi、 211 At、 203 Pb、 203 Hg、 201 T1、 199 Au、 198 Au、 198 Au、 197 Pt、 18 F、 189 Re、 188 Re、 188 Re、 186 Re、 186 Re、 177 Lu、 177 Lu、 175 Yb、 172 Tm、 169 Yb、 169 Yb、 169 Er、 168 Tm、 167 Tm、 166 Ho、 166 Dy、 165 Tm、 165 Dy、 161 Tb、 15 0、 15 N、 159 Gd、 157 Gd、 153 Sm、 153 Pb、 151 Pm、 14 C、 149 Pm、 143 Pr、 142 Pr、 13 N、 133 I、 131 In、 131 I、 127 Te、 126 I、 125 Te、 125 I、 124 I、 123 I、 122 Te、 121 Te、 121 Sn、 U C、 113 In、 U1 ln、 U1 ln、 lu Ag、 lu Ag、 109 Pd、109 Pd, 107 Hg, 105 Ru, 105 Rh, 105 Rh, and 103 This includes, but is not limited to, Ru.

[0156] Means for detecting such labels are well known to those skilled in the art. Thus, for example, certain radiolabels can be detected using photographic film, scintillation detectors, PET imaging, MRI, etc. Fluorescent markers can be detected using a photodetector to detect emitted light. Enzyme labels are typically detected by providing a substrate for the enzyme and detecting the reaction product generated by the action of the enzyme on the substrate, and colorimetric labels are detected simply by visualizing the colored label.

[0157] In another embodiment, the effector is a polypeptide that acts on a cell (e.g., 60 The therapeutic agent may include a radiosensitizer that enhances the cytotoxic effect of ionizing radiation (which may be emitted by Co or X-ray sources). Numerous radiosensitizers are known, including, but not limited to, benzoporphyrin derivative compounds (see, for example, U.S. Pat. No. 5,945,439), 1,2,4-benzotriazine oxides (see, for example, U.S. Pat. No. 5,849,738), compounds containing certain diamines (see, for example, U.S. Pat. No. 5,700,825), BCNT (see, for example, U.S. Pat. No. 5,872,107), radiosensitizing nitrobenzoic acid amide derivatives (see, for example, U.S. Pat. No. 4,474,814), various heterocyclic derivatives (see, for example, U.S. Pat. No. 5,064,849), platinum complexes (see, for example, U.S. Pat. No. 4,921,963), and the like.

[0158] In certain embodiments, the effector may comprise an alpha emitter, i.e., a radioisotope that emits alpha particles. Recently, alpha emitters have been shown to be useful in the treatment of cancer (see, e.g., McDevitt et al. (2001) Science 294: 1537-1540; Ballangrud et al. (2001) Cancer Res. 61: 2008-2014; Borchardt et al. (2003) Cancer Res. 63: 5084-50). Suitable alpha emitters include: 212 Pb, 225 Ac, 227 Th, Bi, 213 Bi, 211 These include, but are not limited to, At.

[0159] Many of the drugs and / or radiolabels described herein can be provided as a chelate. The chelating molecule is typically conjugated to a molecule (e.g., biotin, avidin, streptavidin, etc.) that specifically binds to the epitope tag attached to the anti-ALPP / ALPPL2 antibody described herein.

[0160] Chelating groups are well known to those of skill in the art. In certain embodiments, the chelating group is derived from ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), cyclohexyl 1,2-diaminetetraacetic acid (CDTA), ethyleneglycol-0,0'-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), triethylenetetraminehexaacetic acid (TTHA), 1,4,7,10-tetraazacyclododecane-N,N'-,N'',N'''-tetraacetic acid (DOTA), hydroxyethyldiaminetriacetic acid (HEDTA), 1,4,8,11-tetra-azacyclotetradecane-N,N',N'',N'''-tetraacetic acid (TETA), substituted DTPA, substituted EDTA, and the like.

[0161] Examples of certain chelating agents include unsubstituted or substituted 2-iminothiolane and 2-iminothiacyclohexane, particularly 2-imino-4-mercaptomethylthiolane. One chelating agent, 1,4,7,10-tetraazacyclododecane-N,N,N'',N'''-tetraacetic acid (DOTA), is of particular interest because it can chelate a number of diagnostically and therapeutically important metals, such as radionuclides and radiolabels. Conjugates of DOTA with proteins, such as antibodies, have been described previously. For example, U.S. Patent No. 5,428,156 discloses methods for conjugating DOTA to antibodies and antibody fragments. To make these conjugates, one carboxylic acid group of DOTA is converted to an active ester that can react with amine or sulfhydryl groups on the antibody or antibody fragment. Lewis et al. (1994) Bioconjugate Chem. 5: 565-576 describe a similar method in which one carboxyl group of DOTA is converted to an active ester, the activated DOTA is mixed with an antibody, and the antibody is linked to DOTA via the ε-amino group of a lysine residue on the antibody, thereby converting one carboxyl group of DOTA to an amide moiety.

[0162] In certain embodiments, the chelator can be directly or via a linker to the epitope tag or the moiety that binds to the epitope tag.Conjugates of DOTA and biotin have been described (see, for example, Su (1995) J. N cl. Med., 36 (5 Suppl): 154P, which discloses the linkage of DOTA and biotin via available amino side chain biotin derivatives such as DOTA-LC-biotin or DOTA-benzyl-4-(6-amino-caproamide)-biotin).Yau et al, WO95 / 15335 discloses a method for producing nitro-benzyl-DOTA compounds that can be conjugated to biotin.The method includes cyclization reaction via transient projection of hydroxyl group; tosylation of amine; deprotection of transiently protected hydroxyl group; tosylation of deprotected hydroxyl group; and intramolecular tosylate cyclization. Wu et al. (1992) Nucl. Med. Biol., 19(2): 239-244, 111 IN and 90 discloses the synthesis of a macrocyclic chelator for radiolabeling with Y. Wu et al. prepare a labeled DOTA-biotin conjugate to study the stability and biodistribution of the conjugate with a model protein for research, avidin. The conjugate was prepared using biotin hydrazide, which contains a free amino group to react with an activated DOTA derivative prepared in situ.

[0163] The anti-ALPP / ALPPL2 antibodies described herein can be used to deliver a variety of cytotoxic and / or cytostatic agents, including therapeutic drugs, radioactive compounds, cytotoxic molecules derived from plants, fungi, or bacteria, biological proteins, and mixtures thereof. In certain embodiments, the cytotoxic agents may include intracellularly acting cytotoxic agents, such as small organic molecules, cytotoxic proteins or peptides, and radiation emitters, including the short-range high-energy a-emitters described above. Further representative therapeutic agents include radioisotopes, chemotherapeutic agents, immunomodulatory agents, antiangiogenic agents, antiproliferative agents, proapoptotic agents, and cytolytic enzymes (e.g., RNases). Agents may also include therapeutic nucleic acids, such as genes encoding immunomodulatory agents, antiangiogenic agents, antiproliferative agents, or proapoptotic agents. These drug descriptors are not mutually exclusive, and thus a therapeutic agent may be described using one or more of the above terms. For example, the radioisotope selected is also a cytotoxin. In various aspects, the therapeutic agent may be formulated as a pharma- ceutically acceptable salt, acid, or derivative of any of the above.

[0164] In certain embodiments, anti-ALPP / ALPPL2 antibodies are attached to therapeutic cytotoxic / cytostatic drugs. In various embodiments, drugs used to construct ADCs include, but are not limited to, microtubule inhibitors and DNA damaging agents, polymerase inhibitors (e.g., polymerase II inhibitors, a-amanitin), and the like. In certain embodiments, the antibody is conjugated to the drug directly or via a linker, while in other embodiments, the antibody is conjugated to a drug carrier {e.g., liposomes containing the drug, polymeric drug carriers, nanoparticle drug carriers, lipid drug carriers, dendrimer drug carriers, etc.).

[0165] In certain embodiments, the drug comprises a tubulin inhibitor, including, but not limited to, auristatin, dolastatin-10, synthetic derivatives of the natural product dolastatin-10, and maytansine or maytansine derivatives. In certain embodiments, the drug comprises an auristatin. Certain auristatins are selected from the group consisting of auristatin E (AE), auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin D (MMAD) or monomethyl dolastatin 10, monomethyl auristatin F (MMAF) or N-methylvaline-valine-dolaisoloiin-dolaproine-phenylalanine), monomethyl auristatin E (MMAE) or N-methylvaline-valine-dolaisoloiin-dolaproine-norephedrine, 5-benzoylvaleric acid-AE ester (AEVB), vcMMAE, and vcMMAF.

[0166] In certain embodiments, the drug comprises an enediyne. An enediyne is a type of antitumor bacterial product characterized by a 9-membered ring and a 10-membered ring, or by the presence of a ring structure with a triple-double-triple bond. Exemplary enediynes include, but are not limited to, calicheamicin, esperamicin, and dynemicin.

[0167] Calicheamicin is an enediyne antibiotic that was first isolated as a natural product from the soil organism Micromonospora echinospora ssp. calichensis (Zein et al. Science 27; 240(4856): 1198-1201, 1988). Calicheamicin generates double-stranded DNA breaks and subsequently induces apoptosis in target cells (Zein et al. Science 27; 240(4856): 1198-1201, 1988; Nicolaou et al. Chem. Biol. September; l(l):57-66, 1994; Prokop et al. Oncogene 22:9107-9120, 2003). In certain embodiments, the drug comprises calicheamicin or a calicheamicin analog. Examples of calicheamicin and its analogs suitable for use in anti-ALPPL2 immunoconjugates are disclosed, for example, in U.S. Patent Nos. 4,671,958, 4,970,198, 5,053,394, 5,037,651, 5,079,233, 5,264,586, and 5,108,912, the entireties of which are incorporated herein by reference. In certain embodiments, these compounds contain a functional group, such as a methyl trisulfide, which can react with a suitable thiol to form a disulfide while simultaneously introducing a hydrazide or other functional group useful for conjugating calicheamicin to an anti-ALPPL2 antibody. Disulfide analogs of calicheamicin can also be used, such as those described in U.S. Patent Nos. 5,606,040 and 5,770,710, the entireties of which are incorporated herein by reference. In one particular embodiment, the disulfide analog is N-acetyl-gamma-calicheamicin dimethylhydrazide.

[0168] In certain embodiments, the drug comprises geldanamycin. Geldanamycin is a benzoquinone ansamycin antibiotic that binds to Hsp90 (heat shock protein 90) and has been used as an antitumor drug. Exemplary geldanamycins include, but are not limited to, 17-AAG (17-N-allylamino-17-demethoxygeldanamycin) and 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin). In certain embodiments, the drug comprises maytansine. Maytansine or its derivative maytansinoids inhibits cell proliferation by inhibiting microtubule formation through inhibition of tubulin polymerization during mitosis (see, e.g., Remillard et al. 91975) Science 189: 1002-1005). Exemplary maytansines include, but are not limited to, mertansine (DM1); and maytansine analogs, e.g., DM3 or DM4, as well as ansamitocins.

[0169] In certain embodiments, the drug comprises a taxane. Taxanes are diterpenes that act as antitubulin or mitotic inhibitors. Exemplary taxanes include, but are not limited to, paclitaxel and docetaxel.

[0170] In certain embodiments, the drug comprises a DNA interacting agent, including, but not limited to, calicheamicin, duocarmycin, pyrrolobenzodiazepines (PBDs), and the like.

[0171] In another exemplary but non-limiting embodiment, the drug comprises a duocarmycin. A duocarmycin is a DNA damaging agent that can exert its mode of action at any phase of the cell cycle. Agents that are part of this class of duocarmycins typically have efficacy in the low picomolar range. Exemplary duocarmycins {e.g., duocarmycin analogs) that can be used as effectors in the chimeric constructs contemplated herein include, but are not limited to, duocarmycin A, duocarmycin Bl, duocarmycin B2, duocarmycin CI, duocarmycin C2, duocarmycin D, duocarmycin SA, cyclopropylbenzoindole duocarmycin (CC-1065), centanamycin, and rachelmycin.

[0172] In another exemplary but non-limiting embodiment, the drug comprises a pyrrolobenzodiazepine. In a particular embodiment, the drug comprises a synthetic derivative of two pyrrolobenzodiazepines linked by a flexible polymethylene tether. Pyrrolobenzodiazepines (PBDs) and PBD dimers are described in U.S. Patent No. 7,528,126 B2. U.S. Patent No. 7,528,126 B2 is incorporated herein by reference for the pyrrolobenzodiazepines and PBD dimers described therein. In certain embodiments, the pyrrolobenzodiazepine is selected from the group consisting of Anthramycin (and its dimer), Mazethramycin (and its dimer), Tomaymycin (and its dimer), Prothracarcin (and its dimer), Tikamycin (and its dimer), Neothramycin A (and its dimer), Neothramycin B (and its dimer), DC-81 (and its dimer), Sibiromycin (and its dimer), Polothramycin A (and its dimer), Polothramycin B (and its dimer), Sibanomycin (and its dimer), Abeymycin (and its dimer), SG2000, and SG2285.

[0173] In certain embodiments, the drug comprises a polymerase inhibitor, including but not limited to a polymerase II inhibitor, such as α-amanitin, and poly(ADP-ribose) polymerase (PARP) inhibitor.Exemplary PARP inhibitors include but are not limited to iniparib (BSI 201), talazoparib (BMN-673), olaparib (AZD-2281), olaparib, rucaparib (AG014699, PF-01367338), veliparib (ABT-888), CEP 9722, MK 4827, BGB-290, 3-aminobenzamide, etc.

[0174] In certain embodiments, the drug comprises a vinca alkyloid. Vinca alkyloids are also antitubulin agents. Exemplary vinca alkyloids include, but are not limited to, vincristine, vinblastine, vindesine, and vinorelbine.

[0175] The aforementioned drugs are exemplary but not limiting. In various embodiments, other anti-cancer drugs can be utilized, including, but not limited to, anti-cancer antibodies (e.g., HERCEPTIN®), antimetabolites, alkylating agents, topoisomerase inhibitors, microtubule targeting agents, kinase inhibitors, protein synthesis inhibitors, somatostatin analogs, glucocorticoids, aromatose inhibitors, mTOR inhibitors, protein kinase B (PKB) inhibitors, phosphatidylinositol, 3-kinase (PI3K) inhibitors, cyclin-dependent kinase inhibitors, anti-TRAIL molecules, MEK inhibitors, and the like. In certain embodiments, the anti-cancer compounds include fluorouracil (5-FU), capecitabine / XELODA, 5-trifluoromethyl-2'-deoxyuridine, methotrexate sodium, raltitrexed / Tomudex, pemetrexed / Alimta®, cytosine arabinoside (cytarabine, Ara-C) / thioguanine, 6-mercaptopurine (mercaptopurine, 6-MP), azathioprine / Azasan, 6-thioguanine (6-TG) / Prinetol (TEVA), pentostatin / Nipent, fludarabine phosphate / Fludara®, cladribine (2-CdA, 2-chlorodeoxyadenosine) / Leustatin, floxuridine (5-fluoro-2) / FUDR (Hospira, Inc.), ribonucleotide reductase inhibitors (RNR), cyclophosphamide / Cytoxan (BMS), neosar, ifosfamide / Mitoxana, thiotepa, BCNU - l,3-bis(2-chloroethyl)-l-nitrosourea, l,-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea, methyl CCNU, hexamethylmelamine, busulfan / Myleran, procarbazine HCL / Matulane, dacarbazine (DTIC), chlorambucil / Leukaran®, melphalan / Alkeran, cisplatinum (CDDP) / Platinol, carboplatin / Paraplatin, oxaliplatin / Eloxitan, bendamustine, carmustine, chloromethine, dacarbazine (DTIC), fotemustine,Lomustine, Mannosulfan, Nedaplatin, Nimustine, Prednimustine, Ranimustine, Satraplatin, Semustine, Streptozocin, Temozolomide, Treosulfan, Triaziquone, Triethylenemelamine, Thiotepa, Triplatin Tetranitrate, Trofosfamide, Uramustine, Doxorubicin HCL / Doxil, Daunorubicin Citrate / Daunoxome®, Mitoxantrone HCL / Novantrone, Actinomycin D, Etoposide / Vepesid, Topote These include, but are not limited to, vintothecin HCL / Hycamtin, teniposide (VM-26), irinotecan HCL (CPT-ll) / , camptosar®, camptothecin, belotecan, rubitecan, vincristine, vinblastine sulfate, vinorelbine tartrate, vindesine sulfate, paclitaxel / Taxol, docetaxel / Taxotere, nanoparticle paclitaxel, abraxane, ixabepilone, larotaxel, ortaxel, tesetaxel, vinflunine, and the like. In certain embodiments, the anti-cancer drug is carboplatin (e.g., PARAPLATIN®), cisplatin (e.g., PLATINOL®, PLATINOL-AQ®), cyclophosphamide (e.g., CYTOXAN®, NEOSAR®), docetaxel (e.g., TAXOTERE®), doxorubicin (e.g., ADRIAMYCIN®), erlotinib (e.g., TARCEVA®), etoposide (e.g., VEPESID®), fluorouracil (e.g., 5-FU®), Gemcitabine (e.g., GEMZAR®), imatinib mesylate (e.g., GLEEVEC®), irinotecan (e.g., CAMPTOSAR®), methotrexate (e.g., FOLEX®, MEXATE®, AMETHOPTERIN®), paclitaxel (e.g., TAXOL®, ABRAXANE®), sorafmib (e.g., NEXAVAR®), sunitinib (e.g., SUTENT®), topotecan (e.g., HYCAMTIN®),The anti-cancer drug comprises one or more drugs selected from the group consisting of vinblastine (e.g., VELBAN®), vincristine (e.g., ONCOVIN®, VINCASAR PFS®). In certain embodiments, the anti-cancer drug comprises one or more drugs selected from the group consisting of retinoic acid, retinoic acid derivatives, doxirubicin, vinblastine, vincristine, cyclophosphamide, ifosfamide, cisplatin, 5-fluorouracil, camptothecin derivatives, interferon, tamoxifen, and taxol. In certain embodiments, the anti-cancer compound is selected from the group consisting of Abraxane, doxorubicin, pamidronate disodium, anastrozole, exemestane, cyclophosphamide, epirubicin, toremifene, letrozole, trastuzumab, megestrol tamoxifen, paclitaxel, docetaxel, capecitabine, goserelin acetate, zoledronic acid, vinblastine, etc.), antisense molecules, siRNA, etc.

[0176] In certain embodiments, the cytotoxic / cytostatic agent comprises a protein or peptide toxin or fragment thereof. Enzymatically active toxins and fragments thereof are exemplified by, for example, diphtheria toxin A fragment, nonbinding active fragment of diphtheria toxin, exotoxin A (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, a-cyclin, certain Aleurites fordii proteins, certain dianthin proteins, Phytolacca americana proteins (PAP, PAPII, and PAP-S), Morodica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogillin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0177] In certain embodiments, cytotoxins may include, but are not limited to, Pseudomonas exotoxin, diphtheria toxin, ricin, abrin and its derivatives. Pseudomonas exotoxin A (PE) is a highly active monomeric protein (molecular weight 66 kD) secreted by Pseudomonas aeruginosa, which inhibits protein synthesis in eukaryotic cells by inactivating elongation factor 2 (EF-2) by catalyzing the ADP-ribosylation of EF-2 (catalyzing the transfer of the ADP-ribosyl moiety of oxidized NAD to EF-2).

[0178] The toxin contains three structural domains that act in concert to cause cytotoxicity: domain la (amino acids 1-252) mediates cell binding; domain II (amino acids 253-364) is responsible for translocation to the cytosol, and domain III (amino acids 400-613) mediates ADP-ribosylation of elongation factor 2, which inactivates the protein and causes cell death. The function of domain lb (amino acids 365-399) remains undetermined, but most of it, amino acids 365-380, can be deleted without loss of cytotoxicity. See Siegall et al. (1989) J. Biol. Chem. 264: 14256-14261.

[0179] In one particular embodiment, the antibody is attached to a preferred molecule in which domain la (amino acids 1-252) has been deleted and amino acids 365-380 have been deleted from domain lb. In one particular embodiment, all of domain lb and part of domain II (amino acids 350-394) can be deleted, particularly if the deleted sequences are replaced with a connecting peptide.

[0180] In addition, PE and other cytotoxic proteins can be further modified using site-directed mutagenesis or other techniques known in the art to alter the molecule for a particular desired use. For example, means can be used to alter the PE molecule in a manner that does not substantially affect the functional benefits provided by the PE molecules described herein, and such resulting molecules are intended to be included herein.

[0181] Methods for cloning genes encoding PE fused to various ligands are well known to those of skill in the art (see, e.g., Siegall et al. (1989) FASEB J., 3: 2647-2652; and Chaudhary et al. (1987) Proc. Natl. Acad. Sci. USA, 84: 4538-4542).

[0182] Like PE, diphtheria toxin (DT) kills cells by ADP-ribosylating elongation factor 2, thereby inhibiting protein synthesis. However, diphtheria toxin is divided into two chains, A and B, linked by a disulfide bridge. In contrast to PE, chain B of DT is at the carboxyl terminus and is responsible for receptor binding, while chain A is at the amino terminus and contains the enzymatic activity (Uchida et al. (1972) Science, 175: 901-903; Uchida et a / . (1973) J. Biol. Chem., 248: 3838-3844).

[0183] In certain embodiments, the antibody-diphtheria toxin immunoconjugate has the native receptor binding domain removed by truncation of the diphtheria toxin B chain. One exemplary modified diphtheria toxin is DT388, a DT with the carboxyl-terminal sequence beginning at residue 389 removed (see, e.g., Chaudhary et al. (1991) Bioch. Biophys. Res. Comm., 180: 545-551). Like the PE chimeric cytotoxin, the DT molecule can be chemically conjugated to the anti-ALPP antibody. However, in certain preferred embodiments, the antibody is fused to the diphtheria toxin by recombinant means (see, e.g., Williams et al. (1990) J. Biol. Chem. 265: 11885-11889).

[0184] In certain embodiments, anti-ALPP / ALPPL2 antibodies are attached to immunomodulators and function to localize the immunomodulators to cancer cells / tumor sites. Numerous immunomodulators capable of activating immune responses are known to those skilled in the art. In one exemplary but non-limiting embodiment, the immunomodulator comprises an anti-CD3 antibody. Anti-CD3 monoclonal antibodies induce human T cell proliferation in vitro and activate specific and non-specific cytolysis by human T cell clones and human peripheral blood lymphocytes. In vivo administration of anti-CD3 inhibits UV-induced mouse fibrosarcoma tumor growth.

[0185] In certain embodiments, the immunomodulator comprises agents that block immune checkpoints.Immune checkpoints refer to numerous inhibitory pathways built into the immune system that are important in maintaining self-tolerance and regulating the duration and amplitude of physiological immune responses in peripheral tissues to minimize collateral tissue damage.It is now clear that tumors exploit certain immune checkpoint pathways as a major immune resistance mechanism, particularly against tumor antigen-specific T cells.Many immune checkpoints are caused by ligand-receptor interactions, and can therefore be easily blocked by antibodies or regulated by recombinant ligands or receptors.

[0186] Cytotoxic T-lymphocyte antigen 4 (CTLA4) antibodies were the first of this class of immunotherapeutic agents to receive U.S. Food and Drug Administration (FDA) approval. Ipilimumab (Yervoy®), the first such drug approved for the treatment of advanced melanoma, blocks the activity of a checkpoint protein known as CTLA4, which is expressed on the surface of activated immune cells called cytotoxic T-lymphocytes. CTLA4 acts as a "switch" to inactivate these T cells, thereby weakening the strength of the immune response. Ipilimumab binds to CTLA4 and prevents it from sending its inhibitory signal. Two other FDA-approved checkpoint inhibitors, nivolumab (Opdivo®) and pembrolizumab (Keytruda®), work in a similar way but target a different checkpoint protein on activated T cells, known as PD-1. Nivolumab is approved to treat some patients with advanced melanoma or advanced lung cancer, and pembrolizumab is approved to treat some patients with advanced melanoma. Thus, in certain embodiments, the immunomodulatory agent comprises an antibody directed against CTLA4 (e.g., ipilimumab), and / or an antibody directed against PD-L1 (e.g., nivolumab, pembrolizumab), and / or an antibody directed against PD-L2.

[0187] Other examples of immune modulators that can be attached to anti-ALPP / ALPPL2 antibodies include ganciclovir, etanercept, tacrolimus, sirolimus, voclosporin, cyclosporin, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil (MYC). mofetil), methotrexate, glucocorticoids and analogs thereof, cytokines, xanthines, stem cell growth factors, lymphotoxins, tumor necrosis factor (TNF), hematopoietic factors, interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21), colony stimulating factors (e.g., granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF)), interferons (e.g., interferon-α, interferon-β, interferon-γ), stem cell growth factors referred to as "S1 factors," erythropoietin and thrombopoietin, or combinations thereof.

[0188] Useful immunomodulators also include antihormones that block hormone action on tumors, and immunosuppressants that suppress cytokine production, downregulate self-antigen expression, or mask MHC antigens. Representative antihormones include antiestrogens, including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxyphene, keoxyphene, LY117018, onapnstone, and toremifene; and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and antiadrenal agents. Exemplary immunosuppressants include, but are not limited to, 2-amino-6-aryl-5 substituted pyrimidines, azathioprine, cyclophosphamide, bromocriptine, danazol, dapsone, glutaraldehyde, anti-idiotypic antibodies against MHC antigens and MHC fragments, cyclosporin A, steroids, e.g., glucocorticosteroids, cytokine or cytokine receptor antagonists (e.g., anti-interferon antibodies, anti-IL1O antibodies, anti-TNFa antibodies, anti-IL2 antibodies), streptokinase, TGFP, rapamycin, T cell receptors, T cell receptor fragments, and T cell receptor antibodies.

[0189] In certain embodiments, the effector comprises a viral particle (e.g., filamentous phage, adeno-associated virus (AAV), lentivirus, etc.). The antibody can be conjugated to the viral particle and / or expressed on the surface of the viral particle (e.g., filamentous phage). Furthermore, the viral particle can comprise a nucleic acid that is delivered to target cells (e.g., cancer cells that express ALPPL2). The use of viral particles to deliver nucleic acids to cells is described in detail in WO99 / 55720, US6,670,188, US6,642,051, and US6,669,936.

[0190] In certain embodiments, the anti-ALPP / ALPPL2 antibodies described herein can be chemically conjugated to effector molecules (e.g., cytotoxins, labels, ligands, drugs, liposomes, etc.). Means for chemically conjugating molecules are well known to those skilled in the art. The procedure for attaching an effector to an antibody varies depending on the chemical structure of the effector and / or antibody. Polypeptides typically contain a variety of functional groups, such as carboxylic acid (COOH) groups or free amine (-NH2) groups, available for reaction with appropriate functional groups on the effector molecule for binding to the effector. Alternatively, the antibody and / or effector can be derivatized to expose or attach additional reactive functional groups. Derivatization can involve the attachment of any of a number of linker molecules, such as those available from Pierce Chemical Company, Rockford Illinois.

[0191] As used herein, a "linker" is a molecule that is used to connect a targeting molecule to an effector molecule. The linker can form a covalent bond with both the targeting molecule and the effector molecule. Suitable linkers are well known to those skilled in the art, and include, but are not limited to, straight or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the targeting molecule and the effector molecule are polypeptides, the linker may be connected to the component amino acids via their side chains (e.g., via disulfide bonds with cysteine). However, in a preferred embodiment, the linker is connected to the alpha carbon amino group and carboxyl group of the terminal amino acid.

[0192] Immunoconjugates can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCL), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutareldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., triene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al. (1987) Science 238: 1098. Carbon-14-labeled l-isocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary, but non-limiting, chelating agent for conjugating, e.g., radionucleotides to the antibody (see, e.g., WO1994 / 011026 (PCT / US1993 / 010953)).

[0193] In certain embodiments, conjugation of an antibody to an effector (e.g., a drug, liposome, etc.) or a linker attached to an effector is performed at a solvent accessible reactive amino acid, e.g., lysine or cysteine, that can be derived from reduction of interchain disulfide bonds present in the antibody. In certain embodiments, cysteine ​​conjugation can be performed after reduction of the four interchain disulfide bonds.

[0194] The disclosure also provides bispecific antibodies comprising a variable region described herein that specifically binds ALPPL2 and ALPP and a second variable region that specifically binds a surface marker on peripheral blood mononuclear cells (PBMCs), e.g., CD3. For example, bispecific antibodies can be constructed by combining anti-ALPPL2 and ALPP with anti-T cell (e.g., CD3) antibody fragments using any known bispecific antibody configuration. Examples include, but are not limited to, BiTEs (bispecific T cell engagers) (Harrington et al. (2015) PloS One 10: e0135945; Klinger et al. (2012) Blood, 119: 6226-6233; Molhoj et al. (2007) Mol. Immunol. 44: 1935-1943), diabodies, or DART (dual affinity retargeting) platforms (Chi chili et al. (2015) Sci. Transl. Med. 7: 289ra282; Moore et al. (2011) Blood, 117: 4542-4551). BiTE (bispecific T cell engager) molecules are very well characterized and have already shown promise in the clinic (reviewed in Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011)). BiTEs are tandem scFv molecules in which two scFv molecules are linked by a flexible linker. Further bispecific formats that have been evaluated for T cell binding include diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and their derivatives, such as tandem diabodies (Kipriyanov et al., J Mol Biol 293, 41-66 (1999)). More recently developed are the so-called DART (dual affinity retargeting) molecules, which are based on the diabody format but feature a C-terminal disulfide bridge for further stabilization (Moore et al., Blood 117, 4542-51 (2011)).An even larger size format is the so-called triomab, an all-hybrid mouse / rat IgG molecule currently being evaluated in clinical trials (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)).

[0195] In certain embodiments, diabodies are contemplated that comprise one or more of the VH and VL domains described herein. The term "diabody" refers to an antibody fragment that typically has two antigen binding sites. These fragments typically comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short for the two domains on the same chain to pair, these domains are forced to pair with complementary domains on another chain, creating two antigen binding sites. Diabodies are described in more detail in, for example, EP404,097; WO93 / 11161, and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448.

[0196] In some embodiments, the anti-CD3 variable region of the bispecific antibody comprises a heavy chain variable region CDR1, CDR2, and CDR3 comprising SEQ ID NO:79, SEQ ID NO:80, and SEQ ID NO:81, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 comprising SEQ ID NO:82, SEQ ID NO:83, and SEQ ID NO:84, respectively. In some embodiments, the bispecific antibody comprises a heavy chain variable region comprising SEQ ID NO:77, a light chain variable region comprising SEQ ID NO:78, or both. Non-limiting exemplary bispecific antibodies comprise one or both of SEQ ID NO:85 and SEQ ID NO:86, or one or both of SEQ ID NO:87 and SEQ ID NO:88.

[0197] Also provided are cells expressing CARs comprising ALPPL2 binding domains and ALPP binding domains derived from the antibodies described herein. Chimeric antigen receptors (CARs) are recombinant receptor constructs in which an extracellular antigen binding domain (e.g., ALPPL2 binding domains and ALPP binding domains derived from the antibodies described herein) is connected to a transmembrane domain and further linked to an intracellular signaling domain (e.g., the intracellular T cell signaling domain of a T cell receptor) that transmits a signal and induces a function. In certain embodiments, immune cells (e.g., T cells or natural killer (NK) cells or macrophages) are genetically engineered to express a CAR comprising one or more ALPPL2 binding domains and ALPP binding domains of the antibodies described herein and have effector cell function (e.g., T cell cytotoxic function).

[0198] In some standard CAR embodiments, the components include an extracellular targeting domain, including the ALPPL2 and ALPP variable regions described herein, a transmembrane domain, and an intracellular signaling / activation domain, which are typically assembled linearly as one fusion protein. The "transmembrane domain" is the part of the CAR that links the extracellular binding moiety and the intracellular signaling domain and anchors the CAR to the plasma membrane of a host cell engineered to express the CAR, for example, the plasma membrane of an immune effector cell. The intracellular region may contain the signaling domain of the TCR complex and / or one or more co-stimulatory signaling domains, for example, co-stimulatory signaling domains derived from CD28, 4-1BB (CD137), and OX-40 (CD134). For example, "first generation CARs" generally have a CD3-zeta signaling domain. Additional co-stimulatory intracellular domains may also be introduced (e.g., second and third generation CARs), and additional domains, including homing and suicide domains, may be included in the CAR construct. CAR components are further described below.

[0199] The CAR construct encoding the CAR may also include a sequence encoding a signal peptide to direct the extracellular domain to the cell surface.

[0200] In some embodiments, the CAR may contain one or more hinge domains that connect the antigen binding domain, including the anti-ALPPL2 binding domain and the ALPP binding domain, and the transmembrane domain for positioning the antigen binding domain. Such hinge domains may be derived from natural sources, synthetic sources, semi-synthetic sources, or recombinant sources. The hinge domain may comprise the amino acid sequence of a natural immunoglobulin hinge region, for example, a natural human immunoglobulin hinge region, or a modified immunoglobulin hinge region. Exemplary hinge domains suitable for use in the CAR described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins, for example, CD8α, CD4, CD28, PD1, CD152, and CD7, which may be wild-type hinge regions derived from these molecules or may be modified.

[0201] Any suitable transmembrane domain for use in CAR construction can be used. Such transmembrane domains include, but are not limited to, all or part of the transmembrane domains of the alpha, beta or zeta chains of the T cell receptor, CD28, CD27, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some aspects, the transmembrane domain comprises at least one of the following: e.g., KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rβ, IL2Rγ, IL7Ra, ITGAl, VLAl, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITG The transmembrane domains may include AM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100, (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME, (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, or NKG2C.

[0202] The transmembrane domain incorporated into the CAR construct may be derived from either natural, synthetic, semi-synthetic, or recombinant sources.

[0203] The CAR construct of the present disclosure comprises one or more intracellular signaling domains, also referred to herein as costimulatory domains, i.e., cytoplasmic domains that activate or otherwise regulate immune cells (e.g., T lymphocytes). The intracellular signaling domain is generally responsible for activating at least one of the normal effector functions of the immune cell into which the CAR is introduced. In one embodiment, a costimulatory domain that increases CAR immune T cell cytokine production is used. In another embodiment, a costimulatory domain that facilitates immune cell (e.g., T cell) replication is used. In yet another embodiment, a costimulatory domain that prevents CAR immune cell (e.g., T cell) exhaustion is used. In another embodiment, a costimulatory domain that increases immune cell (e.g., T cell) anti-tumor activity is used. In yet a further embodiment, a costimulatory domain that enhances CAR immune cell (e.g., T cell) survival (e.g., after infusion into a patient) is used.

[0204] Examples of intracellular signaling domains for use in CARs include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors, which act in concert to initiate signal transduction following antigen receptor binding, as well as any derivatives or variants of these sequences and any recombinant sequences that have the same functional capabilities.

[0205] The primary signaling domain regulates either stimulatory or inhibitory primary activation of the TCR complex. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs.

[0206] Examples of ITAM-containing primary intracellular signaling domains include the ITAM-containing primary intracellular signaling domains of CD3zeta, typically FcRgamma, FcγRlla, FcRβ (FcεRib), CD3gamma, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12. In one embodiment, the CAR comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3-zeta.

[0207] The intracellular signaling domain of the CAR may include only the primary intracellular signaling domain, or may include additional desired intracellular signaling domains useful in the context of the CAR of the present invention. For example, the intracellular signaling domain of the CAR may include a CD3 zeta chain portion and a costimulatory signaling domain. Costimulatory signaling domain refers to a CAR portion that includes the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for efficient response between lymphocytes and antigens. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that bind to CD83. For example, CD27 costimulation has been shown to enhance the expansion, effector function, and survival of human CART cells in vitro and to increase the persistence and antitumor activity of human T cells in vivo (Song et al. Blood. 2012; 119(3):696-706).Further examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, These include CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAMl, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM, (SLAMFl, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, and CD19a.

[0208] In some embodiments, CAR may be designed as an inducible CAR, or may otherwise include a mechanism for reversibly expressing CAR, or for controlling CAR activity so as to restrict CAR activity to a primarily desired environment.Thus, for example, in some embodiments, CAR-expressing cells use split CAR.Split CAR approaches are described in more detail in publications WO2014 / 055442 and WO2014 / 055657.

[0209] In some embodiments, a cell expressing a CAR comprising one or more ALPPL2 binding domains and an ALPP binding domain described herein also expresses a second CAR, e.g., a second CAR comprising a different antigen binding domain, e.g., a different antigen binding domain that binds to the same target or a different target.

[0210] In some embodiments, a host cell, e.g., a host T cell, is modified using a gene editing system, e.g., a Cas / CRISPR system, a transcription activator-like effector nuclease (TALEN) system, a homing endonuclease (HE) system, or a zinc finger nuclease (ZFN) system, to express an ALPPL2-binding domain and an ALPP-binding domain described herein, or a chimeric molecule, e.g., a chimeric receptor comprising such domains.

[0211] The nucleic acid and viral vector (e.g., viral particle) are delivered to a target cell (e.g., CD8 + Many methods are available for introducing the nucleic acid into a T cell (T cell). Non-limiting examples of suitable methods include electroporation (e.g., nucleofection), virus or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, calcium phosphate precipitation, polyethylenimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, microparticle- or nanoparticle-mediated nucleic acid delivery, and the like. In some embodiments, viral vectors, such as adenovirus, adeno-associated virus (AAV), lentivirus vectors, vaccinia virus vectors, or any of a number of different vectors, may be used. In some

[0212] The present invention is not limited by the type of immune cells that are genetically modified to express CAR. Exemplary immune cells include, but are not limited to, T cells, e.g., α / β T cells and γ / δ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, macrophages, and bone marrow-derived phagocytes. In some embodiments, the T cells are CD8+ T cells Treg cells. In some embodiments, the immune cells, e.g., T cells, are autologous cells from the patient receiving immunotherapy. In some embodiments, the immune cells are allogeneic. Methods for producing CAR-expressing cells are described, for example, in US2016 / 0185861 and US2019 / 0000880.

[0213] The antibody described herein and the cell expressing the CAR described herein can be used to treat cancer, i.e., cancer cells expressing ALPPL2 or ALPP or both.Cancers that can be treated include tumors that are not vascularized or are not yet substantially vascularized, as well as tumors that are vascularized.Cancers can include non-solid tumors, can include solid tumors, and can include cancer cells (e.g., cancer stem cells).Types of cancers that can be treated using the antibody or CAR described herein include, but are not limited to, mesothelioma, testicular cancer, endometrial cancer, and a subset of ovarian cancer, pancreatic cancer, and non-small cell lung cancer.

[0214] The antibodies described herein (including ALPPL2-binding and ALPP-binding fragments thereof, affinity matured variants, or scFvs) can be used in vivo for diagnosis or in vitro (e.g., using a biological sample obtained from an individual). In some embodiments, for example, the methods allow for the detection of mesothelioma, testicular cancer, endometrial cancer, and a subset of ovarian cancer, pancreatic cancer, and non-small cell lung cancer.

[0215] When used for detection or diagnosis, the antibodies are typically conjugated or otherwise associated with a detectable label, which may be direct, e.g., covalent, or indirect, e.g., using a secondary binding agent, chelator, or linker.

[0216] Labeled antibodies can be provided to individuals to confirm the applicability of the intended therapy. For example, labeled antibodies can be used to detect the expression or density of ALPPL2 and / or ALPP in diseased areas. For therapies intended to target ALPPL2 and ALPP, the density of βALPPL2 and / or ALPP is typically greater than that of non-disease tissue. Labeled antibodies can also indicate that the therapy is accessible to diseased areas. Thus, patients can be selected for therapy based on imaging results. Anatomical characterization, such as determining the exact borders of cancer, can be accomplished using standard imaging methods (e.g., CT scanning, MRI, PET scanning, etc.). Such in vivo methods can be performed using any of the antibodies disclosed herein.

[0217] Any of the antibodies disclosed herein can be used in in vitro diagnostic or monitoring methods, for example, using cells or tissues derived from a patient sample, hi some aspects, the labeled antibodies described herein are used as they can bind to fixed as well as non-fixed cells.

[0218] Diagnostic agents comprising the antibodies described herein include, for example, those described in the following reference: Armstrong et al., Diagnostic Imaging, 5 thThe diagnostic agent may include any diagnostic agent known in the art as shown in Ed., Blackwell Publishing (2004); Torchilin, VP, Ed., Targeted Delivery of Imaging Agents, CRC Press (1995); Vallabhajosula, S., Molecular Imaging: Radiopharmaceuticals for PET and SPECT, Springer (2009). The terms "detectable agent", "detectable moiety", "label", "imaging agent" and similar terms are used interchangeably herein. Diagnostic agents can be detected in a variety of ways, including agents that provide and / or enhance detectable signals. Detectable signals include, but are not limited to, gamma-emitting signals, radioactive signals, echogenic signals, optical signals, fluorescent signals, absorption signals, magnetic signals, or tomographic signals. Techniques for imaging diagnostic agents may include, but are not limited to, single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, positron emission tomography (PET), computed tomography (CT), X-ray imaging, gamma imaging, etc. PET is particularly sensitive and quantitative, and therefore useful for characterizing processes in vivo (Olafsen et al. (2012) Tumour Biol. 33:669-77; Cai et al. (2007) J Nucl Med. 48:304-10). This is more useful than companion diagnostics, and will generally be useful for diagnosing, clinically staging, and following patients during any treatment regimen. Detection methods involving one or more antibodies as described herein may include, but are not limited to, for example, ELISA, electrochemiluminescence immunoassay (ECLIA), Western blot analysis, radioimmunoassay, immunofluorometry, immunoprecipitation, equilibrium dialysis, immunodiffusion, solution-phase assay, or immunohistochemical or other methods.

[0219] Pharmaceutical compositions comprising the antibody described herein or cells expressing the CAR described herein may include one or more pharma- ceutically acceptable carriers. Acceptable carriers and excipients in pharmaceutical compositions are non-toxic to recipients at the dosages and concentrations used. Acceptable carriers and excipients may include buffers, antioxidants, preservatives, polymers, amino acids, and carbohydrates. Pharmaceutical compositions may be administered parenterally in the form of injectable formulations. Pharmaceutical compositions for injection (i.e., intravenous injection) can be formulated using a sterile solution or any pharma- ceutically acceptable liquid as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, saline, and cell culture media (e.g., Dulbecco's Modified Eagle's Medium (DMEM), alpha-Modified Eagle's Medium (alpha-MEM), F-12 medium). Formulation methods are known in the art. See, e.g., Banga (ed.) Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (2nd ed.) Taylor & Francis Group, CRC Press (2006).

[0220] The pharmaceutical composition may be formed into a unit dosage form if desired. The amount of active ingredient, e.g., an antibody described herein, contained in the pharmaceutical preparation is such that an appropriate dosage within the specified range (e.g., a dosage within the range of 0.01 to 500 mg per kg of body weight) is provided.

[0221] The pharmaceutical compositions described herein may be formulated for subcutaneous, intramuscular, intravenous, parenteral, intraarterial, intrathecal, or intraperitoneal administration. The pharmaceutical compositions may also be formulated for oral, nasal, spray, aerosol, rectal, or vaginal administration, and may be administered by oral, nasal, spray, aerosol, rectal, or vaginal administration. For injection formulations, various effective pharmaceutical carriers are known in the art. In some embodiments, the pharmaceutical compositions may be administered locally or systemically (e.g., topically). In certain embodiments, the pharmaceutical compositions may be administered locally to affected areas, such as the skin or cancerous tissue.

[0222] The dosage of pharmaceutical composition depends on the route of administration, the disease to be treated, and the physical characteristics, such as the age, weight, and general health of the subject.In some embodiments, the amount of active ingredient (e.g., the antibody described herein) contained in a single dose is administered in an amount that effectively prevents, delays, or treats the disease without inducing significant toxicity.The dosage may be adjusted by the physician according to conventional factors, such as the degree of the disease and various parameters of the subject.

[0223] Pharmaceutical compositions may be administered in a manner compatible with the dosage formulation and in such amount as will be therapeutically effective to ameliorate or remedy the symptoms. Pharmaceutical compositions may be administered in a variety of dosage forms, such as subcutaneous, intravenous, and oral dosage forms (e.g., ingestible solutions, drug release capsules). Pharmaceutical compositions containing an active ingredient (e.g., an antibody described herein) may be administered to a subject in need thereof one or more times (e.g., 1-10 times or more), for example, daily, weekly, monthly, semi-annually, yearly, or as medically necessary. Dosages may be provided in one or more dosing regimens. The timing between administrations may decrease as the medical condition improves or increase as the patient's health condition declines.

[0224] In determining the effective amount of antibody to be administered, the physician may evaluate the circulating plasma level of the antibody and the toxicity of the antibody. In general, the dose equivalent of the antibody is about 1 ng / kg to 10 mg / kg for a typical subject. In some embodiments, the dose range for subcutaneous or intravenous administration is 0.1 g to 20, for example, 0.3 g to 3 mg / kg. EXAMPLES

[0225] Example 1 The high specificity of ALPPL2 allows the development of additional therapeutic agents far beyond ADCs, including bispecific immune effector cell engagers with different tumor killing mechanisms. Thus, we sought to develop and characterize a bispecific T cell engager targeted to ALPPL2. The first M25 antibody is fully human in sequence and binds to ALPPL2-expressing cells with an apparent binding affinity of sub-nM to low nM in the IgG1 format (Su et al, 2020). The monovalent binding of M25 single chain fragment variable (scFv) or Fab to ALPPL2 is moderate / low. We previously performed affinity maturation studies to identify a high affinity variant of M25, M25FYIA (also known as FYIA), with >100-fold improved monovalent binding to ALPPL2 compared to the parent M25 (Su et al, 2020; Liu et al, 2017). Most importantly, FYIA retains high specificity, binding only to ALPPL2 / ALPP, but not to the closely related ALPI (Su, Y., et al. Cancer Res., 2020 Aug 31; WO2017095823).

[0226] In this study, FYIA was further optimized to increase monovalent binding affinity to ALPPL2 while improving developability characteristics (avoidance of charged or hydrophobic patches and removal of deamidation and isomerization motifs) in the context of bispecific T cell engager development. The framework regions of FYIA were cloned from the germline sequence (IGHV3-23 * 04 and IGLV2-14 *FYIA_germ was modified to match human ALPPL2 (VH and VL sequences shown in Tables 1 and 2, respectively) to create FYIA_germ. Using FYIA_germ as a starting point, a combination of site-directed mutagenesis and error-prone PCR mutagenesis was used to generate clones that were selected or screened for improved binding to human ALPPL2 and lacking binding to human ALPI. A panel of affinity- and developability-optimized human antibodies was identified (VH and VL sequences shown in Tables 1 and 2, respectively). A lead antibody from this panel, FYIAgermopt, also known as SYLY, was selected for bispecific antibody construction.

[0227] Table 1: Heavy chain variable sequences TIFF2024534543000178.tif87150TIFF2024534543000179.tif205150

[0228] Table 2: Light chain variable sequence TIFF2024534543000180.tif214150

[0229] The following sections describe the optimized properties of this new panel of FYIA variants, including the lead antibody SYLY. With regard to the improved binding affinity, in a side-by-side study of biolayer interferometry measurements of the binding of monovalent Fabs to human ALPPL2, the calculated affinity is 8.6 nM for FYIA, 13.0 nM for FYIA_germ, 0.88 nM for FYIA_germ_6-6, and 0.19 nM for FYIA_germopt (also known as SYLY) (Figure 1). In a comparative study of the binding of monovalent Fabs to the mesothelioma cell line M28, the apparent binding affinity is 30.55 nM for FYIA, 69.41 nM for FYIA_germ, and 0.97 nM for FYIA_germopt (Figure 2). To assess binding specificity, the optimized FYIA variants were tested for binding to HEK293 transfected with human ALPI. As shown in Figure 3, the optimized variants, like the parent FYIA, do not bind to ALPI. Additional clones beyond those shown in Figure 1 (FYIA_germ_SY and FYIA_germ_6-6) were also studied for binding to M28 cells as monovalent Fabs. As shown in Figure 4, the apparent binding affinity is 1.27 nM for FYIA_germ_SY and 4.82 nM for FYIA_germ_6-6. Table 3 summarizes the results of the cell binding studies. In monovalent Fab form, the lead antibody FYIA_germopt (also known as SYLY) has a greater than 30-fold improvement over the parent FYIA.

[0230] (Table 3) TIFF2024534543000181.tif47128

[0231] Regarding developability, in a comparative study of heat-induced aggregation assays, SYLY showed improved thermal stability at a higher aggregation temperature (Tagg) than FYIA (76°C vs. 73°C, Figure 5). For reference, the parent FYIA (as a human IgG1 molecule) and the clinically used daratumumab (IgG1 form) were also comparatively studied. As shown in Figure 6, FYIA showed higher thermal stability than daratumumab. In a second set of analyses that analyzed the variable domain sequences of the M25 / FYIA / SYLY series against five developability guidelines derived from clinical stage therapeutic values ​​(Raybould, MIJ et al, 2019), the parent M25 and its derivatives (FYIA and SYLY) all showed favorable properties without any identified potential developability issues (Figure 7 for M25, Figure 8 for FYIA, Figure 9 for SYLY).

[0232] We then used SYLY to construct an ALPPL2 x CD3 bispecific T cell engager. We humanized the SP34 mouse anti-human CD3 antibody (Table 4).

[0233] Table 4: CD3 binding sequences TIFF2024534543000182.tif55150

[0234] There are over 80 different bispecific antibody constructions (Figures 10A and 10B, adapted from Brinkmann et al.) that can be used to construct ALPPL2 x CD3. Furthermore, we have developed bispecific antibodies using different molecular structures. We have constructed dual stabilized diabodies (DSDbodies) using interchain disulfide linkages and CH1 / CL pairing. CH1 / CL pairing forces heterodimer formation. The DSDbody sequences are shown in Table 5.

[0235] (Table 5) TIFF2024534543000183.tif236150

[0236] A hexahistidine tag was added to the C-terminus of CH1 for purification by metal affinity chromatography using nickel-loaded affinity resin (Ni-NTA agarose). DSDbodies were produced by transient transfection in HEK293A or ExpiCHO cells, purified by NI-NTA, and analyzed by reducing SDS-PAGE (Figure 11). Bispecific antibodies can also be constructed from DSDbody variants without interchain disulfide bonds (sequences shown in Table 6).

[0237] Biolayer interferometry analysis revealed that the SYLY-based bispecific ALPPL2 x CD3 DSDbody bound to human ALPPL2 with high affinity (KD ~0.2 nM) and specificity (no binding to human ALPI) (Figure 12). The DSDbody was also assessed for cross-species binding to human and cynomolgus CD3 epsilon chains by biolayer interferometry. As shown in Figure 13, the SYLY-based DSDbody showed similar binding affinity to human and cynomolgus CD3 molecules (20 nM and 18 nM, respectively).

[0238] The binding of the SYLY-based bispecific ALPPL2 x CD3 DSDbody to tumor cells was studied by flow cytometry using the mesothelioma cell line M28. As shown in Figure 14, the DSDbody binds to M28 cells with an apparent binding affinity of 2.4 nM. No binding was detected for the non-binding human antibody YSC10 and the control DSDbody constructed from the same anti-CD3 antibody. For comparison, a reference DSDbody constructed from the parent FYIA was also studied on M28 cells using flow cytometry. As shown in Figure 15, the FYIA-based DSDbody showed an apparent binding affinity of 14.8 nM to M28 cells.

[0239] In addition to mesothelioma cells, we also studied the binding of the SYLY-based bispecific ALPPL2 x CD3 DSDbody on the ovarian cancer cell line SKOV3. As shown in Figure 16, the SYLY-based ALPPL2 x CD3 bispecific DSDbody binds to SKOV3 cells with an apparent affinity of 0.19 nM. No binding was detected by the control bispecific YSC10 x CD3 assembled on the non-binding isotype control antibody YSC10. For comparison, a reference DSDbody constructed from the parent FYIA was also studied on SKOV3 cells using flow cytometry. As shown in Figure 17, the FYIA-based DSDbody showed an apparent binding affinity of 14.2 nM to SKOV3 cells.

[0240] Target-dependent cytotoxicity was studied using ALPPL2-expressing tumor cells and human PBMCs as a source of effector cells. SYLY-based bispecific DSDbodies were incubated with the ovarian cancer cell line SKOV3 (target) in the presence of human PBMCs (effector) at an E:T ratio of 10:1 for 96 hours. Cell viability was measured using calcein Am solution. As shown in Figure 18, SYLY-based DSDbodies kill SKOV3 cells with a calculated EC50 of 0.3 pM. No killing was observed with the control bispecific YSC10 x CD3 DSDbody assembled on the non-binding isotype control antibody YSC10. For comparison, a reference DSDbody constructed from the parent FYIA was also studied under the same conditions and E:T ratios on SKOV3 cells. As shown in Figure 19, the calculated EC50 of FYIA DSDbody is 62.8 pM.

[0241] Target-dependent cytotoxicity was further studied using HEK293 cells stably expressing ALPP (target). SYLY-based DSDbodies were incubated with HEK293-ALPP cells in the presence of human PBMCs at an E:T ratio of 10:1 for 72 hours, after which cell viability was assessed by calcein AM. As shown in Figure 20, the calculated EC50 of the SYLY DSDbody is 8.5 pM, while the calculated EC50 of the control bispecific YSC10 x CD3 DSDbody is >100 nM. No killing was observed in HEK293 cells not expressing the target antigen (ALPP or ALPPL2, Figure 21).

[0242] The DSDbodies showed excellent thermal stability: in a heat-induced aggregation assay, the FYIA-based ALPPl2 x CD3 DSDbody showed an aggregation temperature (Tagg) of 65°C (Figure 22), while the SYLY-based ALPPL2 x CD3 DSDbody showed an even higher Tagg of 70°C (Figure 23).

[0243] Example 2 This example demonstrates the effect of antibodies described herein on a reporter-expressing pancreatic cancer line, AsPC1.

[0244] The bispecific (ALPPL2 x CD3) SYLY DSDBody was incubated with AsPC1 cells for 1 h at room temperature, washed, and further incubated with anti-tag (hexahistidine) antibody for detection by flow cytometry. The MFI values ​​were curve-fitted and the K D was estimated to be 4.35±0.68 nM. See FIG.

[0245] In vitro cyto tox assays using reporter-expressing cancer cell lines were performed as follows: Firefly luciferase-expressing AsPC1-luc cells were plated at 3,000 cells / well in 96-well tissue culture plates and cultured overnight. 30,000 cells / well of PBMCs were added to the target cells (E:T ratio=10:1). Antibodies were serially diluted and incubated for 72 hours. Cell viability was measured by bioluminescence using a microtiter plate reader. Percentage of cell viability was derived from normalized luciferase signal and curve-fitted to obtain an estimated EC50 (<1 pM, approximately 0.245 pM). See Figure 25.

[0246] It is understood that the examples and embodiments described herein are illustrative only, and that various changes or modifications will be suggested to those skilled in the art in light of the examples and embodiments described herein, and are within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are incorporated by reference in their entirety for all purposes.

Claims

1. An antibody or a fragment thereof comprising a variable region that specifically binds to ALPPL2 and ALPP, the antibody comprising a heavy chain variable region and a light chain variable region, Complementarity determining regions (CDR) 1, CDR2, and CDR3 of the heavy chain variable region are SEQ ID NO: 44, 45, and 46; SEQ ID NO:20, 21, and 22; SEQ ID NO:23, 24, and 25; SEQ ID NO:26, 27, and 28; SEQ ID NO:29, 30, and 31; SEQ ID NO:32, 33, and 34; SEQ ID NO:35, 36, and 37; SEQ ID NO:38, 39, and 40; SEQ ID NO: 41, 42, and 43; SEQ ID NOs: 47, 48, and 49; or SEQ ID NO: 50, 51, and 52 and / or CDR1, CDR2, and CDR3 of the light chain variable region are SEQ ID NO:71, 72, and 73; SEQ ID NO:53, 54, and 55; SEQ ID NO:56, 57, and 58; SEQ ID NO:59, 60, and 61; SEQ ID NO:62, 63, and 64; SEQ ID NO:65, 66, and 67; SEQ ID NOs: 68, 69, and 70; or SEQ ID NO: 74, 75, and 76 selected from the group consisting of the antibody does not have the heavy chain variable region of M25FYIA and the light chain variable region of M25FYIA; The antibody or fragment thereof.

2. CDR1, CDR2, and CDR3 of the heavy chain variable region comprise SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, respectively; and CDR1, CDR2, and CDR3 of the light chain variable region comprise SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:73, respectively; or CDR1, CDR2, and CDR3 of the heavy chain variable region comprise SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25, respectively; and CDR1, CDR2, and CDR3 of the light chain variable region comprise SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58, respectively; or CDR1, CDR2, and CDR3 of the heavy chain variable region comprise SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28, respectively; and CDR1, CDR2, and CDR3 of the light chain variable region comprise SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58, respectively; or CDR1, CDR2, and CDR3 of the heavy chain variable region comprise SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively; and CDR1, CDR2, and CDR3 of the light chain variable region comprise SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67, respectively; or CDR1, CDR2, and CDR3 of the heavy chain variable region comprise SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, respectively; and CDR1, CDR2, and CDR3 of the light chain variable region comprise SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55, respectively; or CDR1, CDR2, and CDR3 of the heavy chain variable region comprise SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively; and CDR1, CDR2, and CDR3 of the light chain variable region comprise SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61, respectively; The antibody or fragment thereof according to claim 1. a) the heavy chain variable region is selected from the group consisting of SEQ ID NOs: 9, 1, 2, 3, 4, 5, 6, 7, 8, 10, and 11; and / or b) the light chain variable region is selected from the group consisting of SEQ ID NOs: 18, 12, 13, 14, 15, 16, 17, and 19; Optionally, the heavy chain variable region comprises SEQ ID NO:1 and the light chain variable region comprises SEQ ID NO:12; or the heavy chain variable region comprises SEQ ID NO:2 and the light chain variable region comprises SEQ ID NO:13; or the heavy chain variable region comprises SEQ ID NO:3 and the light chain variable region comprises SEQ ID NO:13; or the heavy chain variable region comprises SEQ ID NO:9 and the light chain variable region comprises SEQ ID NO:18; or the heavy chain variable region comprises SEQ ID NO:7 and the light chain variable region comprises SEQ ID NO:16; or the heavy chain variable region comprises SEQ ID NO:8 and the light chain variable region comprises SEQ ID NO:12; or the heavy chain variable region comprises SEQ ID NO:6 and the light chain variable region comprises SEQ ID NO:14; The antibody or fragment thereof according to claim 1. Claim 4: a) the antibody is an IgG, IgA, or IgE antibody; the IgG antibody is optionally an IgG1, IgG2, IgG3, or IgG4 antibody; and / or b) the antibody is a monospecific antibody; The antibody or fragment thereof according to claim 1.

5. the antibody is linked to a cytotoxic agent; The cytotoxic agent is selected from the group consisting of a radionucleotide, a microtubule inhibitor, a DNA damaging agent, a polymerase inhibitor, a tubulin inhibitor, an auristatin, an enediyne, geldanamycin, maytansine or a maytansine derivative, a taxane, a pyrrolobenzodiazepine (PBD) or a PBD dimer, a duocarmycin, a polymerase II inhibitor, a poly(ADP-ribose) polymerase (PARP) inhibitor, a vinca alkiloid (vinca alkyloid, antimetabolite, alkylating agent, topoisomerase inhibitor, microtubule targeting agent, kinase inhibitor, protein synthesis inhibitor, glucocorticoid, aromatose inhibitor, mTOR inhibitor, protein kinase B (PKB) inhibitor, phosphatidylinositol 3-kinase (PI3K) inhibitor, cyclin-dependent kinase inhibitor, anti-TRAIL molecule, MEK inhibitor, or camptothecin derivative, and optionally, the cytotoxic agent is a DNA damaging agent, auristatin, dolastatin-10, mertansine (DM1), DM3 or DM4, auristatin E (AE), auristatin B (AE), auristatin C (AE), auristatin D (AE), auristatin E (AE), auristatin F (AE), auristatin G (AE), auristatin H (AE), auristatin I ... dolastatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin D (MMAD) or monomethyl dolastatin 10, monomethyl auristatin F (MMAF), N-methylvaline-valine-dolaisoleucine-dolaproline-phenylalanine, monomethyl auristatin E (MMAE), N-methylvaline-valine-dolaisoleucine-dolaproline-norephedrine, 5-benzoylvaleric acid-AE ester (AEVB), vcMMAE, vcMMAF, polymerase II inhibitor, α-amanitin, poly(ADP-ribose) polymerase (PARP) inhibitor, iniparib (BSI) 201), talazoparib (BMN-673), olaparib (AZD-2281), olaparib, rucaparib (AG014699, PF-01367338), veliparib (ABT-888), CEP 9722, MK 4827, BGB-290, 3-aminobenzamide, a topoisomerase inhibitor, or a radioisotope, optionally 212 Pb, 225 Ac, 227 Th, Bi, 213 Bi, or 211 At, The antibody or fragment thereof according to claim 1.

6. the antibody is a bispecific antibody comprising a second variable region that specifically binds to a second target protein; the antibody comprises a second heavy chain variable region and a second light chain variable region; Optionally, the second target protein is expressed on the surface of a human immune effector cell or is human CD3. The antibody or fragment thereof according to claim 1.

7. the CDR1, CDR2, and CDR3 of the second heavy chain variable region comprise SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81, and the CDR1, CDR2, and CDR3 of the second light chain variable region comprise SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84; optionally, (i) the second heavy chain variable region comprises SEQ ID NO:77 and the second light chain variable region comprises SEQ ID NO:78; (ii) the antibody comprises SEQ ID NO:85 and SEQ ID NO:86; or (iii) the antibody comprises SEQ ID NO:87 and SEQ ID NO:88; The antibody or fragment thereof according to claim 6.

8. A pharmaceutical composition comprising the antibody of claim 1.

9. A nucleic acid encoding the antibody of any one of claims 1 to 4, 6, or 7, or a vector comprising said nucleic acid.

10. 10. A cell comprising the nucleic acid or vector of claim 9, optionally a mammalian cell.

11. 10. A method for producing an antibody, comprising culturing a cell comprising nucleic acid encoding the antibody of any one of claims 1 to 4, 6, or 7 under conditions that allow the production of the antibody.

12. A pharmaceutical composition for use in a method for killing cancer cells, comprising the antibody of any one of claims 1 to 7, The method includes contacting the antibody with cancer cells; Optionally, (i) the antibody is a bispecific antibody comprising a second variable region that specifically binds to human CD3, the antibody comprising a second heavy chain variable region and a second light chain variable region, and binding of the antibody brings CD3-expressing peripheral blood mononuclear cells (PBMCs) or T cells into close proximity with cancer cells; optionally, the CDR1, CDR2, and CDR3 of the second heavy chain variable region comprise SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81, and the CDR1, CDR2, and CDR3 of the second light chain variable region comprise SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84; optionally, the second heavy chain variable region comprises SEQ ID NO:77 and the second light chain variable region comprises SEQ ID NO:78; the antibody comprises SEQ ID NO:85 and SEQ ID NO:86, or the antibody comprises SEQ ID NO:87 and SEQ ID NO:88; or (ii) the antibody is linked to a cytotoxic agent; The cytotoxic agent is selected from the group consisting of a radionucleotide, a microtubule inhibitor, a DNA damaging agent, a polymerase inhibitor, a tubulin inhibitor, an auristatin, an enediyne, geldanamycin, maytansine or a maytansine derivative, a taxane, a pyrrolobenzodiazepine (PBD) or a PBD dimer, a duocarmycin, a polymerase II inhibitor, a poly(ADP-ribose) polymerase (PARP) inhibitor, a vinca alkiloid (vinca alkyloid, antimetabolite, alkylating agent, topoisomerase inhibitor, microtubule targeting agent, kinase inhibitor, protein synthesis inhibitor, glucocorticoid, aromatose inhibitor, mTOR inhibitor, protein kinase B (PKB) inhibitor, phosphatidylinositol 3-kinase (PI3K) inhibitor, cyclin-dependent kinase inhibitor, anti-TRAIL molecule, MEK inhibitor, or camptothecin derivative, and optionally, the cytotoxic agent is a DNA damaging agent, auristatin, dolastatin-10, mertansine (DM1), DM3 or DM4, auristatin E (AE), auristatin B (AE), auristatin C (AE), auristatin D (AE), auristatin E (AE), auristatin F (AE), auristatin G (AE), auristatin H (AE), auristatin I ... dolastatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin D (MMAD) or monomethyl dolastatin 10, monomethyl auristatin F (MMAF), N-methylvaline-valine-dolaisoleucine-dolaproline-phenylalanine, monomethyl auristatin E (MMAE), N-methylvaline-valine-dolaisoleucine-dolaproline-norephedrine, 5-benzoylvaleric acid-AE ester (AEVB), vcMMAE, vcMMAF, polymerase II inhibitor, α-amanitin, poly(ADP-ribose) polymerase (PARP) inhibitor, iniparib (BSI) 201), talazoparib (BMN-673), olaparib (AZD-2281), olaparib, rucaparib (AG014699, PF-01367338), veliparib (ABT-888), CEP 9722, MK 4827, BGB-290, 3-aminobenzamide, a topoisomerase inhibitor, or a radioisotope, optionally 212 Pb, 225 Ac, 227 Th, Bi, 213 Bi, or 211 At, The pharmaceutical composition.

13. 1. A chimeric antigen receptor (CAR)-expressing human cell, wherein the CAR comprises the heavy chain variable region and the light chain variable region of the antibody of claim 1, optionally wherein the human cell is a T cell, a natural killer cell, or a macrophage.

14. 1. A method for detecting tumor cells in a sample, comprising: contacting the sample with the antibody of any one of claims 1 to 3; and detecting specific binding between the antibody and the tumor cells. The method comprising:

15. A pharmaceutical composition for use in a method of treating a human with cancer, comprising an antibody of any one of claims 1-7, a pharmaceutical composition of claim 8, or a CAR-expressing human cell of claim 13.

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