Antigen binding molecules and uses thereof

Abstract

To provide tumor-associated antigens suitable for targeted antibody therapy against cancer.SOLUTION: Antigen binding molecules that specifically bind ALPPL2 and ALPP, but not ALPL and ALPI are provided. Also provided are chimeric molecules and pharmaceutical compositions comprising the antigen-binding molecules, methods of reducing the expression or activity of ALPPL2 in cancer cells, and methods of treating cancers in a subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates generally to the field of oncology. In particular, the present invention relates to ALPPL2 and / or Antigen-binding molecules that specifically bind to ALPP but not to ALPL or ALPI Concerning children. [Background technology]

[0002] Antibodies are attractive therapeutic agents due to their ability to bind to cell surface antigens and eliminate cancer cells. Clinically approved antibody therapeutics include Herceptin and Rituxan, which They are highly successful drugs for treating a variety of cancers, including hematological and solid cancers. Antibody therapy, for example, involves the activation of effector cells (such as natural killer cells or T cells) They act by recruiting receptors or by modulating signaling pathways in cancer cells. Antibodies can also be used to bind to toxins or radioisotopes to help eliminate cancer cells. The development of successful antibody therapy requires the use of antibodies that are preferentially expressed on cancer cells. This requires targeting of the cell surface antigens that are present on normal, healthy cells. This is because the current situation may result in unwanted side effects. Summary of the Invention [Problem to be solved by the invention]

[0003] In general, there are no tumor-associated antigens suitable for targeted antibody therapy against cancer. Therefore, developing effective treatments against such antigens is a significant challenge.

[0004] It is therefore generally desirable to overcome or ameliorate one or more of the above difficulties. I wish. [Means for solving the problem]

[0005] Specific binding to ALPPL2 and / or ALPP, but not to ALPL and ALPI an antigen-binding molecule that does not bind to (a) a heavy chain variable region comprising VHCDR1, VHCDR2, and VHCDR3 amino acid sequences; Area(V H ); and (b) a light chain variable region comprising the VLCDR1, VLCDR2, and VLCDR3 amino acid sequences; Area(V L ) Includes VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and The combination of VLCDR3 and VLCDR3 amino acid sequences is shown in one of the rows of Table 1. The combined molecules are disclosed herein.

[0006] Chimeric molecules comprising an antigen-binding molecule as defined herein and a heterologous moiety are disclosed herein. is shown.

[0007] An isolated molecule comprising a nucleic acid sequence encoding an antigen-binding molecule or chimeric molecule as defined herein. The resulting polynucleotides are disclosed herein.

[0008] an antigen-binding molecule as defined herein operably linked to one or more regulatory sequences; Alternatively, constructs comprising nucleic acid sequences encoding the chimeric molecules are disclosed herein.

[0009] Disclosed herein are host cells containing the constructs defined herein.

[0010] The antigen-binding molecule or chimeric molecule defined herein and a pharmaceutically acceptable carrier are Disclosed herein is a pharmaceutical composition comprising:

[0011] Disclosed herein are methods for reducing ALPPL2 expression or activity in cancer cells. The method includes contacting cancer cells with an antigen-binding molecule or chimeric molecule as defined herein. This includes making someone do something.

[0012] Methods for reducing or inhibiting tumor growth, survival and viability in a subject are provided herein. The present invention relates to a method for producing an antigen-binding molecule or chimeric molecule as defined herein. This includes administering it to elephants.

[0013] Disclosed herein are methods of treating cancer in a subject, the methods comprising administering to a subject a cancer-modifying antibody as defined herein. The method includes administering the antigen-binding molecule or chimeric molecule to a subject.

[0014] An antigen-binding molecule or chimeric molecule as defined herein for use in the treatment of cancer. Disclosed herein are:

[0015] The use of an antigen-binding molecule or antigen-binding protein as defined herein in the manufacture of a medicament for the treatment of cancer. The use of mela molecules is disclosed herein.

[0016] Methods for treating diseases or conditions associated with undesired expression of ALPPL2 in a subject A method is disclosed herein, the method comprising administering to a subject an antigen-binding molecule or chimera as defined herein. This involves administering the molecule to the subject.

[0017] Disclosed herein is a kit for detecting cancer, the kit comprising: The present invention also includes antigen-binding molecules or chimeric molecules.

[0018] Disclosed herein is a method for determining the likelihood of cancer in a subject, the method comprising: detecting ALPPL2 in a sample obtained from the An elevated level of PL2 indicates a possible cancer in the subject.

[0019] Disclosed herein are methods for treating cancer in a subject, the methods comprising: a) administering to the subject a therapeutic agent obtained from the subject; Detecting ALPPL2 in a sample (wherein the level of ALPPL2 in the sample compared to a reference an elevated level indicates an increased likelihood of cancer in the subject; and b) an increased likelihood of cancer. This includes treating subjects found to have elevated blood cholesterol levels.

[0020] A method for identifying subjects who may be responsive to treatment with anti-ALPPL2 antibodies is provided herein. Disclosed herein is a method for detecting ALPPL2 in a sample obtained from a subject. and an increase in the level of ALPPL2 indicates that the subject is responsive to treatment with an ALPPL2 antibody. Indicates that there is a possibility that

[0021] Identifying and Treating Subjects Who May Be Responsive to Treatment with Anti-ALPPL2 Antibodies A method is disclosed herein, the method comprising: a) detecting ALPPL2 in a sample obtained from a subject; detecting an increase in the level of ALPPL2 in response to treatment with an ALPPL2 antibody; and b) respond to treatment with an ALPPL2 antibody. This includes treating a subject found to be potentially susceptible to the disease.

[0022] We investigated antigen-binding molecules that specifically bind to ALPPL2, but not to ALPL and ALPI. Disclosed herein is a method for producing a hydroxybenzoate comprising the steps of: a) immunizing an animal, preferably a rabbit, with ALPPL2; b) from animals, specifically binds to ALPPL2 but not to ALPL and ALPI isolating non-specific B cells; and c) determining the amino acid sequence of the antibody expressed by the B cell Includes:

[0023] Embodiments of the present invention will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which: . [Brief explanation of the drawings]

[0024] [Figure 1] Figure 1 shows that a list of criteria was established to select candidate genes encoding plasma membrane proteins. ALPPL2, a placental-like alkaline phosphatase, emerged as one of the leading candidates for subsequent target validation. [Figure 2] Figure 2 shows immunohistochemical staining of gastric cancer cell lines (top) and gastric tumor microarrays (bottom). [Figure 3] Figure 3 shows the identification of ALPPL2 / ALPP-specific clones produced from rabbit B cell supernatants by ELISA and FACS (top), and affinity measurements of selected clones at a single concentration by biolayer interferometry (bottom). [Figure 4A] Figure 4 shows a comparison of ALPPL2 and ALPI reactivities of our humanized antibodies and equivalent humanized antibodies disclosed in the prior art, as measured by ELISA (Figure 4A) and surface plasmon resonance (Figure 4B). [Figure 4B] See [Figure 4A] above. [Figure 5A] FIG. 5A shows IHC staining of formalin-fixed paraffin-embedded (FFPE) sections with various gastric cancer cell lines C36, C45, and C130. [Figure 5B] FIG. 5B shows IHC staining of FFPE human gastric, ovarian, colorectal, pancreatic, testicular, mesothelioma and endometrial tumor microarrays with C36 at different H-scores of IHC 1+, IHC 2+ and IHC 3+. [Figure 5C] Figure 5C shows IHC staining of FFPE normal tissues with C36. All normal tissues show no C36 staining (negative), except for placental tissue, which shows positive IHC 2+ staining. [Figure 6A] FIG. 6A shows the cross-reactivity of selected clones produced from rabbit B cell supernatants to rhesus monkey orthologues identified by FACS screening. [Figure 6B] FIG. 6B shows that recombinant humanized C4 and C36 clones bind to CHO cells overexpressing the rhesus orthologues but not to WT CHO by FACS analysis. [Figure 7] FIG. 7 shows retention of high ALPP / ALPPL2 affinity after humanization of selected clones, as measured by surface plasmon resonance (SPR). [Figure 8] Figure 8 shows ADCC induction by humanized clones. ADCC induction measured in cocultures of the high-expressing gastric cancer cell line MKN1 with Jurkats CD16A reporter cells (top, left) and the low-expressing gastric cancer cell line MKN74 with Jurkats CD16A reporter cells (top, right). Enhancement of ADCC by C4 measured by CellTiter-Glo assay of gastric cancer cell lines in cocultures with primary NK cells (middle, left) and ovarian and pancreatic cancer cell lines with Jurkats CD16A reporter cells (middle, right). Enhancement of ADCC by Fc engineering of humanized C4 measured in cocultures of MKN74 with Jurkats CD16A reporter cells (bottom). [Figure 9]Figure 9 shows ADC killing of gastric cancer cell lines as measured by the CellTiter-Glo assay. Killing of a high-expressing gastric cancer cell line by a humanized clone via vc-MMAF conjugated to a secondary antibody (top row). Killing of gastric cancer cell lines by humanized C4 (middle row, left) and C12 (middle row, right) conjugated to vc-MMAE. Killing of gastric cancer cell lines by humanized C4 (bottom row, left) and C12 (bottom row, right) via vc-MMAF conjugated to a secondary antibody. [Figure 10] Figure 10 shows potent killing of various cancer cell lines by T cell engagers derived from humanized clones, as measured by xCELLigence real-time cell analysis of cancer cells in co-culture with expanded human T cells. C4 consistently demonstrated pM killing of gastric, ovarian, and pancreatic cancer cell lines, regardless of target expression level. [Figure 11] Figure 11 shows the potent killing of various cancer cell lines by T cell engagers with different anti-CD3 variants in different formats. Humanized Fab fragments are suitable for generating potent T cell engagers with different anti-CD3 pairings and different formats. [Figure 12A] Figure 12A shows the reactivity of selected clones to ALPPL2 but not ALPP, and the cancer cell-killing efficacy of these clones after humanization. FACS shows that chimerized C53 and C78 bind to ALPPL2 but not to ALPP, while rabbit C4 binds to both. [Figure 12B] Figure 12B shows the reactivity of selected clones to ALPPL2 but not ALPP, and the cancer cell-killing efficacy of these clones after humanization. ELISA shows that chimerized C53 and C78 bind to ALPPL2 but not to ALPP. [Figure 12C]Figure 12C shows the reactivity of selected clones to ALPPL2 but not ALPP, and the cancer cell-killing efficacy of these clones after humanization. ADCC induction by the chimeric clones is shown, as measured by co-culture of MKN74 with Jurkats CD16A reporter cells. [Figure 12D] Figure 12D shows the reactivity of selected clones to ALPPL2 but not ALPP, and the cancer cell-killing efficacy of these clones after humanization. ELISA shows that chimerized C53 and C78 cross-reacted with CHO cells overexpressing the rhesus ortholog identified by FACS. [Figure 12E] Figure 12E shows the reactivity of selected clones to ALPPL2 but not ALPP, and the cancer cell-killing efficacy of these clones after humanization. Humanized C53 demonstrates ALPPL2 specificity by ELISA. [Figure 12F] Figure 12F shows the reactivity of selected clones to ALPPL2 but not ALPP, and the cancer cell-killing efficacy of these clones after humanization. Co-culture of N87 with Jurkats CD16A reporter cells demonstrates that ADCC-inducing activity is maintained after humanization. [Figure 12G] Figure 12G shows the reactivity of selected clones to ALPPL2 but not to ALPP, and the cancer cell-killing efficacy of these clones after humanization. Humanized C53 showed nM binding affinity to ALPPL but not to ALPP by biolayer interferometry, whereas humanized C36 showed similar binding affinity to ALPPL2 and ALPP. [Figure 12H]Figure 12H shows the reactivity of selected clones to ALPPL2 but not ALPP, and the cancer cell-killing efficacy of these clones after humanization. Humanized C53 T cell engagers demonstrate potent killing of N87 gastric cancer cells as measured by xCELLigence real-time cell analysis of N87 cells co-cultured with expanded human T cells. Humanized C53 T cell engagers demonstrated pM killing similar to C36 T cell engagers. [Figure 13A] Figure 13A shows the binding profiles of humanized C4, C36, and C53 to different isoforms of the human alkaline phosphatase family, cancer cell lines, and normal immune cells. Humanized C4 and C36 show binding to human ALPPL2 and ALPP, but not to human ALPI and ALPL, while humanized C53 specifically binds to human ALPPL2 by FACS analysis, but does not bind to human ALPP, ALPI, and ALPL transiently expressed in 293T cells. [Figure 13B] Figure 13B shows the binding profiles of humanized C4, C36, and C53 to different isoforms of the human alkaline phosphatase family, cancer cell lines, and normal immune cells. Different doses of humanized C36, C4, and C53 show binding to the ALPPL2 / ALPP-positive cell line (NCI-N87), but not to the negative cell line (MIAPaca-2). Humanized C53 has binding affinity comparable to that of a commercially available antibody (catalog number: eBioScience #14-9870-82), and has weaker binding affinity than humanized C4 and C36. [Figure 13C] Figure 13C shows the binding profiles of humanized C4, C36, and C53 to different isoforms of the human alkaline phosphatase family, cancer cell lines, and normal immune cells. Humanized C36, C4, and C53 show no binding to naive and CD3 / CD28 bead + IL-2-activated human PBMCs (CD4+ / CD8+ T cells, B cells, CD11b+ myeloid Mφ cells). [Figure 13D]Figure 13D shows the binding profiles of humanized C4, C36, and C53 to different isoforms of the human alkaline phosphatase family, cancer cell lines, and normal immune cells. Humanized C36, C4, and C53 show no binding to T cells, B cells, or myeloid Mφ cells. DETAILED DESCRIPTION OF THE INVENTION

[0025] The present disclosure specifically binds to ALPPL2 and / or ALPP, but not to ALPL and The present invention teaches an antigen-binding molecule that does not bind to ALPPL2 and ALPI. and / or ALPP, or ALPPL2 and / or ALPP-expressing cells , with an affinity between about 14 pm and about 10 nM.

[0026] Specific binding to ALPPL2 and / or binding to ALPL and ALPI An antigen-binding molecule that does not bind to VHCDR1, VHCDR2, and VHCDR3, The heavy chain variable region (V H ); and (b) VLCDR1, VLCDR2 and a light chain variable region (VLCDR3) containing the amino acid sequence L ) (where VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 amino acids The combination of sequences is shown in any of the rows of Table 1), and the antigen-binding molecule is It is revealed in writing.

[0027] Alkaline phosphatase, placenta-like 2 (ALPPL2) is a phosphodiesterase (PPH) P) family and two closely related enzymes expressed in placental trophoblast cells. isoforms (ALPPL2 and ALPP), and two widely expressed members Consists of ALPL (tissue non-specific, liver / bone / kidney) and ALPI (intestine). In this example, the antigen-binding molecule specifically binds to ALPPL2 and / or ALPP. In one embodiment, the antigen-binding molecule is specific for human ALPPL2 and / or human ALPP. In one embodiment, the antigen-binding molecule binds to ALPPL2 and / or ALPP. It has enhanced efficacy due to its high affinity to

[0028] In one embodiment, the antigen-binding molecule is any detectable molecule for ALPL or ALPI. In one embodiment, the antigen-binding molecule does not bind to human ALPL or human ALPI. It also does not have any detectable binding to human ALPL or human AL Detectable binding to PI, >10 nM, >100 nM, >1 μM, >10 μM, 100 These may also be referred to as having a dissociation constant (Kd) of greater than μM or greater than 1 mM. The antigen-binding molecule has a desirable therapeutic window due to its lack of binding to ALPL or ALPI. In one embodiment, the antigen binding molecule does not induce (or at most induces) T cell killing of normal cells. (Induce the minimum limit).

[0029] Without being bound by theory, the present inventors have determined that the tumor-associated antigen human placenta-like antigen It has high affinity (sub-nM Kd) and specific activity against potassium phosphatase (ALPPL2). Heterosexual (non-reactive to the closely related ALPL or ALPI), immunohistochemical activity (useful for developing companion diagnostics), and non-human primate orthologues (useful for toxicology research) The present inventors isolated a monoclonal antibody having cross-reactivity against the complementarity-determining region of the IgG1 gene. Several clones were synthesized by grafting CDRs onto a human IgG1 framework. These humanized antibodies retain high affinity for ALPPL2. We also demonstrated that these naked humanized antibodies were able to bind to Jurkats reporter and and primary natural killer (NK) cells and gastric cancer cells Induce potent antibody-dependent cellular cytotoxicity (ADCC) in co-culture assays with the ADCC induction was also observed in ovarian cancer and pancreatic cancer cell lines. demonstrated the efficacy of these conjugates in cancer cell killing and secondary assays. The suitability of humanized antibodies for use as antibody-drug conjugates has been demonstrated. In addition, bispecific antibodies can be produced by heterodimerizing these humanized antibodies with anti-CD3 antibodies. These bispecific antibodies function as potent T cell engagers (TcEs). and achieve picomolar (pM) killing of gastric, ovarian, and pancreatic cancer cell lines Therefore, these antibodies are effective against tumors expressing ALPPL2 on the cell surface. It can be used as a targeted therapy.

[0030] Table 1 shows the possible combinations of CDRs that may be present in an antigen-binding molecule. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0031] Table 2 shows the V in antigen-binding molecules derived from 36 antibody clones.H and V L Sequence (CD The combination of R1, 2 and 3 (underlined) is shown. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8]

[0032] Table 3 shows the V of the humanized clones. H , V L Some example sequences are provided. [Table 3-1] [Table 3-2]

[0033] The antigen-binding molecules of the present invention may be in isolated, purified, synthetic or recombinant form. Suitable antigen-binding molecules include monoclonal antibodies (MAbs), chimeric antibodies, human antibodies, and the like. Antibodies and their related compounds, including humanized antibodies, human antibodies, and antigen-binding fragments of such antibodies The antigen-binding molecule may be selected from an antigen-binding fragment. The antigen-binding molecule may be multivalent (e.g., bivalent) or monovalent. In some embodiments, the antigen-binding molecule comprises an Fc domain. In some embodiments, the antigen-binding molecule lacks an Fc domain. Antigen-binding molecules (e.g., Fab, scFab, Fab', scFv, single-arm antibodies, etc.) is.

[0034] The term "antigen-binding molecule" refers to a molecule that has binding affinity for a target antigen. The term refers to immunoglobulins, immunoglobulin fragments and non-immunoglobulins that exhibit antigen-binding activity. It will be understood that the present invention encompasses the derived protein frameworks. Suitable antigen-binding molecules include antibodies and antigen-binding fragments thereof. The term includes antibodies and antigen-binding fragments of antibodies.

[0035] Antigen-binding molecules as defined herein may be naked or may be conjugated to, for example, toxins, radioisotopes, small The molecule may be conjugated to other molecules or moieties such as drugs, polypeptides, etc.

[0036] The term "antibody" as used herein refers to an antibody that specifically binds to a particular antigen. or any antigen that contains at least one complementarity-determining region (CDR) that interacts with it. The term "antibody" refers to a binding molecule or molecular complex that binds together via disulfide bonds. four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by and full-length immunoglobulin molecules, including multimers thereof (e.g., IgM). Each heavy chain , heavy chain variable region (HCVR or V H and heavy chain constant regions. The constant region consists of three domains: CH 1. C H 2 and C H 3. Each light chain comprises a light chain variable Region (LCVR or V L and a light chain constant region. , one domain (C L 1) V H and V L The region is the complementarity determining region (CDR) The nucleotide sequences are composed of hypervariable regions called nucleotide sequences and more conserved interspersed regions called framework regions (FR). Each V H and V L The amino-terminus to carboxyl It consists of three CDRs and four FRs arranged towards the C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Different implementations of the invention In this embodiment, the FRs of the antibody (or antigen-binding portion thereof) of the present invention are identical to human germline sequences. The amino acid sequence may be unique or may be naturally or artificially modified. can be defined based on a parallel analysis of two or more CDRs.

[0037] Antibodies may be of any class, such as IgG, IgA, or IgM (or subclasses thereof). The antibody does not have to be of a particular class. Depending on the amino acid sequence, immunoglobulins can be assigned to different classes. There are five major classes of globulins: IgA, IgD, IgE, IgG, and IgM. Some of these are subclasses (isotypes), e.g., IgG1, IgG2, I These can be further divided into IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to globulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0038] As used herein, "complementarity determining regions" (CDRs; i.e., CDR1, CD The terms CDR1, CDR2, and CDR3) refer to antibody variable regions whose presence is necessary for antigen binding. Each variable domain typically comprises CDR1, CDR2 and Each CDR has three CDR regions identified as CDR1, CDR2, and CDR3. Amino acid residues from the "complementarity determining regions" defined by Kabat (i.e., light chain Residues 24-34 (L1), 50-56 (L2), and 89-97 in the variable domain (L3), and positions 31-35 (H1), 50-65 (H2) and and around 95-102 (H3); Kabat et al., Sequences of Protein ins of Immunological Interest, 5th ed., Public Health Service, National Institutes of H. health, Bethesda, Md. (1991)) and / or "hypermutable loops" Residues derived from (i.e., residues 26-32 (L1), 50-52 in the light chain variable domain) (L2) and positions 91-96 (L3) and residues 26-32 in the heavy chain variable domain ( H1), positions 53-55 (H2) and 96-101 (H3); Chothia and and Lesk J. Mol. Biol. 196:901-917 (1987)). In some instances, the complementarity determining regions include the CDR regions and The fragment may contain amino acids from both the nucleotide and hypervariable loops.

[0039] A "humanized" antibody is one in which the amino acid residues from the non-human CDRs and the amino acid residues from the human FRs are In certain embodiments, a humanized antibody comprises at least one typical The antibody may comprise substantially all of the two variable domains, including all or substantially all of the CDRs. The CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally comprises at least one portion of an antibody constant region derived from a human antibody. It may include a portion.

[0040] As used herein, a "chimeric" molecule is a molecule that is composed of one or more unrelated species. elements or are conjugated, fused, linked, translated, or linked to one another. Two or more compounds that can be linked via a linker, chemically synthesized, expressed from a nucleic acid sequence, etc. For example, peptide and nucleic acid sequences, peptide and detectable labels, unrelated peptide sequences, etc. The chimeric molecule may contain amino acids of different origins. In embodiments comprising sequences, the chimeric molecule comprises: (1) polypeptides that are not found together in nature; sequence (i.e., at least one of the amino acid sequences has at least one amino acid sequence that is identical to the other amino acid sequence) one that is heterologous to the other), or (2) contains amino acid sequences that are not naturally contiguous. For example, a "chimeric" antibody, as used herein, refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chains are derived from a different source or species It refers to antibodies derived from a specific species.

[0041] As used herein, "antigen" and its grammatical equivalents (e.g., "antigen"). The term "immunity" refers to specific humoral or cellular immune responses, such as antibody molecules or T-cell receptors. An antigen refers to a compound, composition, or substance that can be specifically bound by a product of a given antibody, e.g., For example, haptens, simple intermediate metabolites, sugars (e.g., oligosaccharides), lipids, and hormones. and macromolecules such as complex carbohydrates (e.g., polysaccharides), phospholipids, and proteins. General categories of antigens include viral antigens, bacterial antigens, and Antigens, fungal antigens, protozoan and other parasitic antigens, tumor antigens, antigens involved in autoimmune diseases, These include, but are not limited to, allergies and transplant rejection, toxins, and other miscellaneous antigens. Not limited.

[0042] An "antigen-binding site" is the site of an antigen-binding molecule that interacts with an antigen, i.e., one For example, the antigen-binding site of an antibody is composed of complementarity-determining regions (C Native immunoglobulin molecules typically contain two amino acid residues derived from the Fab molecules typically have a single antigen-binding site. The antigen-binding site of the described antigen-binding molecule typically binds to an antigen, more specifically to an epitope of the antigen. It specifically binds to the peptide.

[0043] "Antigen-binding fragment," "antigen-binding portion," "antigen-binding domain," and "antigen-binding site" The terms are used interchangeably herein and refer to the portion of an antigen-binding molecule that is involved in antigen binding. These terms refer to any naturally occurring antigen that specifically binds to an antigen to form a complex. Enzymatically derived, synthetic, or genetically engineered polypeptides or glycoproteins Contains lactic acid bacteria.

[0044] An antigen-binding fragment of an antibody, for example, encodes an antibody variable and optionally a constant domain. Any technique, such as protein digestion or recombinant genetic engineering, including DNA manipulation and expression Intact antibody molecules can be derived using any suitable standard technique. Such DNA can be prepared by known methods. and / or may be obtained from, for example, a commercial source, a DNA library (e.g., phage antigen These are readily available from libraries (including libraries of dinucleotides) or can be synthesized. A may, for example, be a sequence of one or more variable and / or constant domains arranged in an appropriate configuration. or to introduce codons, create cysteine ​​residues, modify or add amino acids. sequenced and manipulated using chemical or molecular biology techniques, for example, deletions That's fine.

[0045] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F( ab')2 fragment; (iii) Fd fragment; (iv) Fv fragment; (v) single-chain Fv (scFv (vi) dAb fragments; and (vii) antibody hypervariable regions (e.g., CDR3 fragments). isolated complementarity determining regions (CDRs) such as peptides or restrictive FR3-CDR3-F The minimum recognition unit consisting of amino acid residues that mimic the R4 peptide. Examples include domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR graphs, etc. Footing antibodies, one-armed antibodies, diabodies, triabodies, tetrabodies, minibodies antibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immune As used herein, SMIPs and shark variable IgNAR domains are also used. are encompassed within the expression "antigen-binding fragment".

[0046] An antigen-binding fragment of an antibody will typically contain at least one variable domain. Domains can be of any size or amino acid composition and generally consist of one or more at least one C adjacent to or in frame with the framework sequences of DR may be included. L V associated with the domain H For antigen-binding fragments containing domains, V H Domains and V L The domains may be positioned relative to each other in any suitable arrangement, for example: The variable region may be dimeric, V H -V H , V H -V L or V L -V L May contain dimers Alternatively, the antigen-binding fragment of an antibody may be a monomeric V H or V L It may include a domain.

[0047] In certain embodiments, the antigen-binding fragment of an antibody contains at least one constant domain. The antigen-binding fragment of an antibody of the present invention may comprise at least one variable domain covalently linked to the antibody. Non-limiting exemplary configurations of variable and constant domains that may be found include: i)V H -C H 1;(ii)V H -C H 2;(iii)V H -C H 3;(iv)V H -C H 1-C H 2;(v)V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3; (vii)V H -C L ;(viii)VL -C H 1;(ix)V L -C H 2. (x)V L -C H 3;(xi)V L -C H 1-C H 2;(xii)V L -C H 1-C H 2-C H 3; (xiii) VL-C H 2-C H 3; and (xiv) V L -C L The exemplary methods listed above The variable and constant domains may be in any configuration, including any of the above configurations. The common domains may be directly linked to each other or may be separated by a full or partial hinge. Alternatively, they may be connected by a linker region. and providing flexible or semi-flexible linkages between adjacent variable and / or constant domains within a domain molecule. At least two (e.g., 5, 10, 15, 20, 40, 60 or more) Furthermore, antigen-binding fragments of antibodies of the present disclosure may be composed of amino acids of each other and / or is one or more monomers V H Or V L domain and non-covalently (e.g., disulfide of the variable and constant domain configurations listed above, associated (by peptide bond(s)) It may comprise any homodimer or heterodimer (or other multimer). An antigen-binding molecule typically comprises at least two different variable domains, each of which The domains can specifically bind to distinct antigens or different epitopes of the same antigen. Any multispecific antigen-binding molecule format, including bispecific antigen-binding molecule formats, The antibody can be used to generate antigen-binding fragments of the antibodies of the present disclosure using routine techniques available in the art. can be adapted for use in the context of

[0048] The term "variable region" or "variable domain" refers to the components of an antigen-binding molecule that are involved in binding to an antigen. The variable domains of the heavy and light chains of a native antibody domain (V H and V L ) generally have a similar structure, with each domain consisting of four It contains three conserved framework regions (FR) and three hypervariable regions (HVR). For example, Kindt et al., Immunology, 6th Edition, W.H. Freeman and Co., p. 91 (2007). H or V L domain may be sufficient to confer antigen-binding specificity.

[0049] As used herein, the term "constant domain" or "constant region" refers to a region other than the variable region. The constant region represents the sum of the domains of an antibody that are not directly involved in antigen binding but are involved in various Exhibits immune effector functions.

[0050] The term "bispecific antigen-binding molecule" refers to two antigen-binding molecules of the same antigen or two different antigens. It refers to a multispecific antigen-binding molecule that has the ability to bind to different epitopes. The original binding molecule can be bivalent, trivalent, or tetravalent. As used herein, "valent" refers to a valent), "valence", "valencies" or other grammatical variations thereof refers to the number of antigen-binding sites in an antigen-binding molecule These antigen recognition sites may recognize the same epitope or different epitopes. and bispecific molecules are described, for example, in Kostelny et al., 1992. J Immunol 148:1547; Pack and Pluckthun, 1992. Biochemis try 31:1579, Gruber et al. 1994. J lmmunol 5368, Zhu et al., 1997. Protein Sci 6:781, Hu et al., 1996. Can cer Res.56:3055, Adams et al., 1993.Cancer Res.5 3:4026, and McCartney et al., 1995. Protein Eng. 8 :301. Trivalent bispecific antigen-binding molecules and tetravalent bispecific antigen-binding molecules Molecules are also known in the art. See, e.g., Kontermann RE (ed.), Springer nger Heidelberg Dordrecht London New York See, e.g., J. Am. Chem. Soc., 199-216 (2011). Bispecific antigen-binding molecules can also be used in combination with other antigen-binding molecules. Higher valencies may also be present and are within the scope of the present invention. , can be produced, for example, by dock and lock conjugation methods. g, C.-H. et al. In: Bispecific Antibodies. Konterm ann RE (2011), supra).

[0051] The phrase "specifically binds" or "specific binding" refers to the binding of a specific molecule to a specific target molecule under physiological conditions. At least 2x the background, more typically 10x to 1x the background molecular association It refers to a binding reaction between two molecules that is more than 100 times stronger than the other two. When using a suitable binding agent, specific binding can be achieved even in heterogeneous populations of proteins and other biological materials. The presence of a protein in a given population is therefore definitive. Under certain conditions, a given antigen-binding molecule binds to a specific antigenic determinant, thereby identifying its presence. Specific binding to an antigenic determinant under such conditions identifies the antigenic determinant. It requires antigen-binding molecules that are selected for their specificity, and that do not cross-react with other molecules. This may be achieved by subtracting the antigen-binding molecules that bind to the antibody. Using the antibody mat, antigen binding proteins are identified so that they are specifically immunoreactive with a particular antigen. For example, solid-phase ELISA can be used to detect the presence of a synthetic molecule (e.g., an immunoglobulin). Immunoassays are routinely used to select antibodies specifically immunoreactive with proteins. (immunoassay functions that can be used to determine specific immune reactivity) For a description of the format and terms, see, for example, Harlow & Lane, Antib (See odies, A Laboratory Manual (1988)). Binding affinity Methods for determining sex and specificity are also well known in the art (e.g., Harlow and Lane, supra); Friefelder, "Physical Biochem istry:Applications to biochemistry and m olecular biology”(WHFreeman and Co.197 6).

[0052] In one embodiment, the antigen-binding molecule is transfected into cells expressing ALPPL2 at a concentration of about 14 pm to about 1 It specifically binds with an affinity between 0 nM and 10 nM.

[0053] "Affinity" or "binding affinity" refers to the affinity of a single binding site of a molecule (e.g., an antigen-binding molecule). refers to the strength of the sum of non-covalent interactions between a molecule and its binding partner (e.g., an antigen) Unless otherwise specified, as used herein, "binding affinity" refers to the affinity of a member of a binding pair. The term refers to the intrinsic binding affinity, which reflects a 1:1 interaction between a target molecule (e.g., an antigen-binding molecule). The affinity of X for its partner Y is generally given by the ratio of the dissociation rate constant to the association rate constant ( k respectively off and k on It can be expressed by the dissociation constant (Kd), which is Thus, equivalent affinities can be achieved with different rate constants as long as the ratio of the rate constants remains the same. Affinity can be determined by common methods known in the art, including those described herein. The specific method for measuring affinity is surface plasmon resonance ( SPR).

[0054] The terms "polypeptide," "peptide," or "protein" are used interchangeably herein. used interchangeably to form peptide bonds between the alpha-amino and carboxy groups of adjacent residues It refers to a linear series of amino acid residues linked together by bonds. However, residues in the "D" isomeric form may be present in the desired functional group. Any L-amino acid residue can be substituted, as long as the properties are retained by the polypeptide.

[0055] As used herein, the term "modified antibody" refers to a non-naturally occurring Synthetic forms of antibodies that have been altered to include at least two heavy chain portions, but not two complete Antibodies that do not contain heavy chains (e.g., domain-deleted antibodies or minibodies); Multispecific forms of antibodies engineered to bind to different epitopes of an antigen or a single antigen heavy chain molecules linked to scFv molecules, etc. ScFv molecules are known in the art and are described, for example, in U.S. Pat. Additionally, the term "modified antibody" refers to multivalent forms of antibodies (e.g., Antibodies include antibodies that bind to three or more copies of the same antigen (e.g., trivalent, tetravalent, etc. antibodies).

[0056] In one embodiment, the antigen binding molecule specifically binds to rhesus monkey ALPPL2. Ghezal ALPPL2 is shown in Genbank ID XP_011726419.1 It may have a sequence

[0057] In one embodiment, the antigen-binding molecule comprises (a) a V H At least 90% (at least 91% to 100% and their V has a sequence identity of H Amino acid sequence, and and (b) V in Table 2 or Table 3 H V shown on the same line as the amino acid sequence L For amino acid sequences At least 90% (at least 91% to 100% and all integer percentages in between) V with sequence identity of 100% (including percentage) L Contains the amino acid sequence.

[0058] In one embodiment, the antigen binding molecule specifically binds to ALPPL2 and ALPP. , but does not specifically bind to ALPL and ALPI.

[0059] The antigen-binding molecule may comprise, for example: a) a VH amino acid sequence having at least 90% sequence identity to SEQ ID NO: 281 and a VL amino acid sequence having at least 90% sequence identity to SEQ ID NO: 282. , b) V having at least 90% sequence identity to SEQ ID NO: 283 H Amino acid sequence and V having at least 90% sequence identity to SEQ ID NO: 284 L Amino acid sequence , c) V having at least 90% sequence identity to SEQ ID NO: 285 H Amino acid sequence and V having at least 90% sequence identity to SEQ ID NO: 286 L Amino acid sequence , d) V having at least 90% sequence identity to SEQ ID NO: 287 H Amino acid sequence and V having at least 90% sequence identity to SEQ ID NO: 288 L Amino acid sequence , e) V having at least 90% sequence identity to SEQ ID NO: 289 H Amino acid sequence and V having at least 90% sequence identity to SEQ ID NO: 290 L Amino acid sequence , f) V having at least 90% sequence identity to SEQ ID NO: 291 H Amino acid sequence and V having at least 90% sequence identity to SEQ ID NO: 292 L Amino acid sequence ,or g) V having at least 90% sequence identity to SEQ ID NO: 293 H Amino acid sequence and V having at least 90% sequence identity to SEQ ID NO: 294 L Amino acid sequence .

[0060] The phrase "at least 90% sequence identity" referred to herein means at least 9 It may include 1% to 100% and all integer percentages therebetween.

[0061] In one embodiment, the antigen-binding molecule does not bind to ALPP. In one embodiment, the antigen-binding molecule can bind to ALPP but not to ALPP. The antigen-binding molecule binds to L2 but not to ALPP, ALPL, or ALPI. , which may include: a) a heavy chain variable region comprising the amino acid sequences of SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33; Area(V H ) and the amino acid sequences of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36. a light chain variable region (VL) containing b) a heavy chain variable region comprising the amino acid sequences of SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69 Area(V H ) and the amino acid sequences of SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72. a light chain variable region (VL) containing c) a heavy chain variable region comprising the amino acid sequences of SEQ ID NO: 85, SEQ ID NO: 86 and SEQ ID NO: 87 Area(V H ) and the amino acid sequences of SEQ ID NO: 88, SEQ ID NO: 89 and SEQ ID NO: 90. a light chain variable region (VL) containing d) a heavy chain comprising the amino acid sequences of SEQ ID NO: 127, SEQ ID NO: 128 and SEQ ID NO: 129 Variable region (V H ) and the amino acids of SEQ ID NO: 130, SEQ ID NO: 131 and SEQ ID NO: 132 a light chain variable region (VL) comprising the amino acid sequence, e) a heavy chain comprising the amino acid sequences of SEQ ID NO: 133, SEQ ID NO: 134 and SEQ ID NO: 135 Variable region (V H ) and the amino acids of SEQ ID NO: 136, SEQ ID NO: 137 and SEQ ID NO: 138 a light chain variable region (VL) comprising the amino acid sequence, f) a heavy chain comprising the amino acid sequences of SEQ ID NO: 169, SEQ ID NO: 170 and SEQ ID NO: 171 Variable region (V H ) and the amino acids of SEQ ID NO: 172, SEQ ID NO: 173 and SEQ ID NO: 174 a light chain variable region (VL) comprising the amino acid sequence; or g) a heavy chain comprising the amino acid sequences of SEQ ID NO: 205, SEQ ID NO: 206 and SEQ ID NO: 207 Variable region (V H ) and the amino acids of SEQ ID NO: 208, SEQ ID NO: 209 and SEQ ID NO: 210 The light chain variable region (VL) contains the amino acid sequence.

[0062] In one embodiment, the antibody comprises: a) V having at least 90% sequence identity to SEQ ID NO: 221 H Amino acid sequence and V having at least 90% sequence identity to SEQ ID NO: 222 L Amino acid sequence , b) V having at least 90% sequence identity to SEQ ID NO: 223 H Amino acid sequence and V having at least 90% sequence identity to SEQ ID NO: 224 L Amino acid sequence , c) V having at least 90% sequence identity to SEQ ID NO: 239 H Amino acid sequence and V having at least 90% sequence identity to SEQ ID NO: 240 L Amino acid sequence , d) V having at least 90% sequence identity to SEQ ID NO: 253 H Amino acid sequence and V having at least 90% sequence identity to SEQ ID NO: 254 L Amino acid sequence , e) V having at least 90% sequence identity to SEQ ID NO: 255 H Amino acid sequence and V having at least 90% sequence identity to SEQ ID NO: 256 L Amino acid sequence , f) V having at least 90% sequence identity to SEQ ID NO: 267 H Amino acid sequence and V having at least 90% sequence identity to SEQ ID NO: 268 L Amino acid sequence ,or g) V having at least 90% sequence identity to SEQ ID NO: 279 H Amino acid sequence and V having at least 90% sequence identity to SEQ ID NO: 280 L Amino acid sequence .

[0063] In one embodiment, the antigen-binding molecule is an antibody or antigen-binding fragment thereof, or a chimeric antigen receptor. The condition is (CAR).

[0064] In one embodiment, the antibody or antigen-binding fragment thereof is humanized or chimerized.

[0065] In one embodiment, the antibody or antigen-binding fragment thereof is a humanized antibody comprising: a) a heavy chain variable region, i) EVQLVESGGGLVQPGGSLRLSCAASG (SEQ ID NO: 295) V having at least 90% sequence identity with H FR1, ii) at least 90% sequence identity to WVRQAPGKGLE (SEQ ID NO: 296) V with unity H FR2, iii)ASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY V having at least 90% sequence identity to CA (SEQ ID NO: 297) H FR3, iv) at least 90% sequence identity to WGQGTLVTVSS (SEQ ID NO: 298) V with unity H FR4 a heavy chain variable region comprising: b) a light chain variable region, i) DIQMTQSPSSLSASVGDRVTITCQAG (SEQ ID NO: 299) V having at least 90% sequence identity with L FR1, ii) at least 90% sequence identity to WYQQKPGKVPK (SEQ ID NO: 300) V with unity H FR2, iii) GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC (sequence V having at least 90% sequence identity to H FR3, i) at least 90% sequence identity to FGQGTKVEIK (SEQ ID NO: 302) V with H FR4 a light chain variable region comprising:

[0066] In one embodiment, the antibody or antigen-binding fragment thereof has at least 90% homology to the following sequence: % (at least 91% to 100% and all integer percentages in between) ) with C H 1 amino acid sequence containing: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKK (SEQ ID NO: 319).

[0067] In one embodiment, the antibody or antigen-binding fragment thereof has at least 90% homology to the following sequence: % (at least 91% to 100% and all integer percentages in between) ) with C L The amino acid sequence includes: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 320).

[0068] Exemplary antigen-binding molecules contemplated by the present disclosure include full-length immunoglobulins and antigen-binding molecules. Binding fragments (recombinant antigen-binding molecules which can be monovalent or multivalent, monospecific or multispecific) (including

[0069] In one embodiment, the antibody or antigen-binding fragment thereof is a full-length antibody, a substantially intact antibody, or a antibody, Fab fragment, scFab, Fab', single chain variable fragment (scFv) or single arm antibody It is the body.

[0070] In one embodiment, the antibody is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In one embodiment, the antibody is an IgG1 antibody. The body may have antibody-dependent cell-mediated cytotoxicity (ADCC) activity and induce NK cell killing. The heavy chain constant region may be a wild-type human Fc region or a region containing one or more amino acid substitutions. The antibody may be a human Fc region containing an immunoglobulin G (IgE) or a human Fc region containing an Fc region containing an IgE or a human ... Mutations that stabilize the disulfide bond between the two heavy chains of IgG, e.g., the hinge region of IgG4 (e.g., Angal et al., 1993. Mol. Immunol. 30: 105-08). See also, for example, U.S. Patent Application Publication No. 2005 / 0037000. The heavy chain constant region also modifies the properties of the antigen-binding molecule (e.g., Fc receptor binding, antibody binding). glycosylation, deamidation, complement binding, or methionine oxidation of the original binding molecule In some examples, the antigen-binding molecule may have a substitution (reducing or reducing the number of substitutions) as described in U.S. Pat. Mutations such as those described in US Pat. Nos. 5,624,821 and 5,648,260 In some embodiments, the antigen binding molecule may have reduced or modified to eliminate

[0071] In one embodiment, the antigen-binding molecule of the present invention is a monovalent antigen-binding molecule. Prototype binding molecules include: L , V H , C L and C H Fa consisting of one domain b fragment;V L , V H , C L and C H 1 domain and C H Part of the 2 domain Fab' fragment; V H and C H Fd fragment consisting of one domain; V of a single arm of an antibody L and V H Fv fragments consisting of domains; single-chain antibody molecules (e.g., scFab and sc Fv);V H Single domain antibody (dAb) fragments consisting of domains (Ward et al., 1989 , Nature, 341:544-546); and U.S. Patent Application Publication No. 200800 63641 (Genentech) or other single-arm antibodies such as those described in Other monovalent antibodies such as those described in WO2007048037 (Amgen) .

[0072] In one embodiment, the monovalent antigen-binding molecule comprises an Fv fragment. The Fv fragment retains the function of antigen-binding activity. The smallest unit of an immunoglobulin molecule with sufficient antibody activity is the scFv (single-chain fragment variable) format. The antigen-binding molecules of the kit are expressed in expression hosts such as E. coli and mammalian cells. The peptides are linked to each other by flexible peptide linkers that can be easily expressed in a functional form. Tied Weight (V H ) and light (V L ) chain variable region, and affinity can be controlled by protein engineering. The properties of scFvs can be improved, such as increased saturation and altered specificity (Ahmed et al., 2012. Clin Dev Immunol. 2012:980250). Linker Representative examples of sequences are described in Section 4.5 below. For scFv construction, The domain order is V H -Linker-V L or V L -Linker-V H It can be either , both orientations can be applied.

[0073] In some embodiments, the linker sequence used in the scFv is the pentapeptide GG GGS [SEQ ID NO: 66] (or G4S or Gly4Ser) multimers. is a 15-mer (G4S)3 (Huston et al., 1988, Proc Natl Acad Sci USA.85(16), 5879~83), 18-mer GGSSRSSSSGG GGSGGGG [SEQ ID NO: 67] (Andris-Widhopf et al., "Generat ion of human scFv antibody libraries: PCR amplification and assembly of light-and heavy-chain coding sequences.”Cold Spri ng Harbor Protocols, 2011(9)) and 20-mer (G4S) 4 (Schaefer et al., ''Construction of scFv Fragm ents from Hybridoma or Spleen Cells by P CR Assembly.''In: Antibody Engineering, R. Kontermann and S. Dubel, Springer Verlag, Hei delberg, Germany (2010) pp. 21-44). In the context of the antibody sequence, sequences with added functionality, such as epitope tags or or coding sequences containing Cre-Lox recombination sites, or sequences that improve scFv properties. Many other sequences have been proposed, including

[0074] Cloning of scFvs has been generally described (Schaefer et al., 2010, supra). As shown in the figure, two-step overlap PCR (splicing by overlap extension) (Splicing by Overlap Extension) or SOE-PC This is done by V H and V L The domains are first amplified and gel The linkers are then purified and subsequently assembled in a single step of assembly PCR. by overlapping the internal primers of the By adding a linker primer covering the fragment length, the fragment is generated in either of the three fragment lengths. fragment assembly PCR).

[0075] Single-chain Fv (scFv) antigen-binding molecules are prepared by incorporating appropriate translation, transcription initiation sites, and mammalian For expression of the scFv, the tag is prepared in the context of an appropriate expression vector containing a signal peptide sequence. The protein coding sequence can be cloned to transform e.g., E. coli, insect cells, The polypeptides may be recombinantly produced in a mammalian host cell or in a mammalian host cell.

[0076] In one embodiment, the monovalent antigen-binding molecule comprises a Fab fragment. A monovalent antigen-binding molecule consists of or consists essentially of a single antigen-binding fragment (Fab) and an Fc region. a single-arm antibody targeting the Fc region, the Fc region comprising a first and a second Fc polypeptide, The first and second Fc polypeptides are present in a complex.

[0077] Recombinant expression of Fc-containing monovalent antigen-binding molecules often results in the formation of undesired bivalent homodimers. Strategies for inhibiting homodimer formation are known, including , supporting unfavorable interactions between polypeptide chains and unwanted Fc homodimer formation Mutations are introduced into the immunoglobulin constant region to create altered structures that suppress the Non-limiting examples of this strategy for promoting heterodimerization include methods of forming two poly(A)-Cyclohexyl 2-(2-methyl-2-propanediol)-2-ones with 2-methyl-2-propanediol. Introduction of knob-into-hole (KIH) structures into peptides, as well as C L and C H 1 This includes taking advantage of the naturally occurring heterodimerization of domains (Kontermann, supra). pp. 1-28 (2011) Ridgway et al., 1996. Protein Eng. 9 (7):617-21; Atwell et al., 1997. J Mol Biol. 270(1 ):26-35; see those described in WO 2005 / 063816). These KIH mutations result in heterodimers of the Fc-containing knob and the heavy chain-containing hole. Enhanced chromatin synthesis, improved assembly of monovalent antibodies, and reduced levels of undesired bivalent antibodies do.

[0078] Modification of the Fc domain of an antigen-binding molecule also reduces Fc receptor binding and therefore reduces blood It may be desirable to reduce the likelihood of FcγRIIa-mediated activation of platelets. For example, the so-called "LALA" double mutation (Leu23 4Ala together with Leu235Ala) inhibits Fc receptor binding and effector function. It is known that the effect of ATP on the immune system is significantly impaired (Lund et al., 1991, J. Immunol. 147, 2657-2662; Lund et al., 1992, Mol. Immunol. 29:53-5 9) For human IgG4, the mutation S228P / L235E variant (SPLE) Engineering the IgG1-dependent cleavage domain has previously been shown to minimize FcγR binding ( Newman et al., 2001 , Clin. Immunol. 98, 164-174). IgG1 or IgG4 Fc Mutations in domains can be combined, for example, the LALA mutation in human IgG1 can be combined with the P3 or combining the SPLE mutation of human IgG4 with the mutation at P329G. When combined with the mutation, FcγR and C1q interactions were completely abolished (Schlot hauer et al., 2016, Protein Eng Des.Sel.29, 457~4 66).

[0079] In one embodiment, the desired affinity of an antigen-binding molecule (e.g., a MAb or antigen-binding fragment thereof) is No cross-linking, platelet activation, or immune effector function (e.g., antibody-dependent cell-mediated Antibody-mediated cytotoxicity (ADCC), phagocytosis (ADCP) and complement-dependent cytotoxicity (C Each of the IgG1 Fc chains of the antibody is P329 G, L235A, L234A (P329G LALA) mutations or IgG4 An antigen-binding molecule in which each of the Fc chains has the mutations P329G, S228P, and L235E.

[0080] In one embodiment, each of the IgG1 Fc chains of the antigen-binding molecule (or antibody) comprises: a) S239D, A330L and I332E or b) F243L, R292P, Y300 L, V305I, and P396L, which affect the immune response of antigen-binding molecules. Enhances effector function (e.g., ADCC).

[0081] In one embodiment, the antigen-binding molecule (or antibody) is selected from the group consisting of SEQ ID NO: 321, SEQ ID NO: 322, and SEQ ID NO: 323. or a CH having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 323 Contains a 2-CH3 sequence.

[0082] The antigen-binding molecule may comprise a heavy chain sequence. The heavy chain sequence may be, for example, C H 1 sequence (e.g., SEQ ID NO: 319) and C H 2-C H 3 sequences (e.g., SEQ ID NO: 321, SEQ ID NO: 322 or SEQ ID NO: 33), H It may contain or For example, the heavy chain sequence may consist of SEQ ID NO: 326, SEQ ID NO: 327, SEQ ID NO: 329, A sequence at least 70% similar to SEQ ID NO: 330, SEQ ID NO: 332 or SEQ ID NO: 333 It may contain amino acid sequences that have identity.

[0083] The antigen-binding molecule may comprise a light chain sequence. The light chain sequence may be, for example, a CL sequence (e.g., sequence 320) or from a VL sequence listed in Table 3 For example, the light chain sequence can be SEQ ID NO: 328, SEQ ID NO: 331, or SEQ ID NO: 334. The amino acid sequence may have at least 70% sequence identity to

[0084] In one embodiment, the present invention provides a method for producing a recombinant human Fc-like protein by co-expression of a light chain, a heavy chain, and a truncated Fc domain. Preferably, the heavy chain contains the hole mutation and the P329G mutation. The truncated Fc domain incorporates the knob mutation and P329G LAL Incorporate the A mutation.

[0085] The expression of the antigen-binding molecules disclosed herein can be achieved, for example, by expression in bacteria (e.g., Escherichia coli). E. coli), yeast, insect or mammalian host cells, suitable translation a suitable expression vector containing a translation, a transcription initiation site, and optionally a signal peptide sequence This can be achieved by cloning the protein coding sequence of the construct in the context of do.

[0086] In one embodiment, the antigen-binding molecule is a multivalent antigen-binding molecule, non-limiting examples of which include , immunoglobulin, F(ab')2, tandem scFv (taFv or scFv2), scFv-Fc, diabody, dAb2 / V H H2, minibody, ZIP mini antibody, Lunase-Barstar dimer, knob-into-hole derivative, SEED-IgG, Terror Fc-scFv, Fab-scFv, Fab)2 / sc(Fab)2, scFv-( TNFα)3, scFv-Jun / Fos, Fab'-Jun / Fos, tribody, Trimer body, tribiminibody, barnase-bar Star trimer, collabody, DNL-F(ab)3, scFv 3-C H 1 / C L , Fab-scFv2, IgG-scFab, IgG-scFv, sc Fv-IgG, scFv2-Fc, F(ab')2-scFv2, scDB-Fc, sc Db-C H 3, Db-Fc, scFv2-H / L, DVD-Ig, tandAb, scF v-dhlx-scFv, dAb2-IgG, dAb-IgG, dAb-Fc-dAb, Tetrabodies, streptabodies (scFv-streptabodies) vidin)4, (scFv-p53)4, [sc(Fv)2]2; tandem diabody ( tandab) and combinations thereof.

[0087] In one embodiment, the multivalent antigen-binding molecule is an IgG-like antibody (e.g., triomab / qu adroma, Trion Pharma / Fresenius Biotech; Nobu Into-Hole, Genentech; CrossMAb, Roche; electrostatically Electrostatically matched antibodies ies), AMGEN; LUZ-Y, Genentech; strand exchange engineering domain (SEE D) Body, EMD Serono; biolonic, Merus; and Fab exchange antibodies, Genmab), symmetric IgG-like antibodies (e.g., dual targeting (DT)-Ig, GSK / Domantis; Two-in-One Antibody, Genentech; Cross-Linking MAb, Karma nos cancer center; MAb2, F-star; and Coy Xb ody, Coy X / Pfizer), IgG fusions (e.g., dual variable domain (DV D)-Ig, Abbott; IgG-like bispecific antibody, Eli Lilly; Ts2Ab ,Medimmune / AZ;BsAb,ZymoGenetics;HERCULES , Biogen Idec; TvAb, Roche) Fc fusions (e.g., scFv / F cfusion, Academic Institution; SCORPION, Emerg ent BioSolutions / Trubion, ZymoGenetics / BM S; Dual Affinity Retargeting Technology (Fc-DART), MacroGenics; Heavy (ScFv)2-Fab, National Research Center fo r Antibody Medicine) Fab fusions (e.g., F(ab)2, Me darex / AMGEN; Dual Action or Bis-Fab, Genentech; Dock DNL, ImmunoMedics; bivalent bispecific ent bispecific), Biotechnol; and Fab-Fv, UCB -Celltech), ScFv and diabody-based antibodies (e.g., bispecific T cells Engager (BiTE), Micromet; Tandem Diabody (Tandab ), Affimed; DART, MacroGenics; Single-chain diabody, Aca demic; TCR-like antibodies, AIT, Receptor Logics; Human serum albumin Min scFv Fusions, Merrimack; and COMBODIES, Epigen Biotech), IgG / non-IgG fusions (e.g., immunocytokines, EMDSe rono, Philogen, ImmunGene, ImmunoMedics; Super -antigen fusion proteins, Active Biotech; and immune mobilization against cancer TCR(immune mobilizing mTCR Against Cancer) r), ImmTAC) and oligoclonal antibodies (e.g., Symphogen and and Merus).

[0088] In one embodiment, the antibody is a bispecific or trispecific antibody. The antibody is a bispecific antibody. A bispecific antibody is a first antibody that specifically binds to ALPPL2. and a second antigen-binding site that specifically binds to CD3. In one embodiment, the bispecific antibody binds to cancer cells and stimulates immune effector cells It can mobilize immune cells (e.g., T cells) to kill cancer cells. Antigen-binding polypeptides that can be used in combination with the second antigen-binding site are well known in the art. For example, muromonab (Orthoclone OKT3), foralumab, tep CD3-specific CDR sequences or VH from tafamidis, blinatumomab, or visilizumab The bispecific antibody may comprise, for example, the VH C / VL sequences of SEQ ID NOs: 335 to 337. The antibody may comprise a DR sequence and a VL CDR sequence of SEQ ID NOs: 338 to 340. The VH CDR sequences of SEQ ID NOs: 341 to 343 and the VL CDR sequences of SEQ ID NOs: 344 to 346 It may include CDR sequences.

[0089] In one embodiment, the bispecific antibody of the invention comprises a first and second nucleotide sequence for hetero-oligomerization. The second, also called "knob-into-hole" structure, helps to engineer the interface between the polypeptides. "Protuberance-into-cavity" The preferred interface is formed using a "" strategy. At least part of the "knob-into-hole" mutation in the CH3 domain of the Fc sequence It has been reported that the formation of homodimers is significantly reduced (e.g., Merch ant et al., 1998, Nature Biotechnology, 16:677-68 1). The "protrusions" are small amino acid side chains extending from the interface of the first polypeptide to larger by replacing the amino acid with a larger side chain (e.g., tyrosine or tryptophan). Compensatory "cavities" of the same or similar size as the protrusions may be used in some cases to accommodate larger arms. by replacing the amino acid side chain with a smaller side chain (e.g., alanine or threonine) Thus, appropriately positioned and dimensioned protrusions are created on the surface of the second polypeptide. Alternatively, if the cavity is present at the interface of either the first or second polypeptide, It is only necessary to manipulate the corresponding cavities or protrusions at the respective adjacent interfaces. The cavities may be engineered by synthetic means, such as by altering a nucleic acid encoding the polypeptide. Alternatively, they can be prepared by peptide synthesis. For explanations, see U.S. Patent Nos. 5,731,168, 5,807,706, and 5,807,706. See, 821,333.

[0090] A general method for preparing heteromultimers using the "protrusion-into-cavity" strategy is In one or a separate host cell, from the original polynucleotide to encode the protrusion A polynucleotide encoding the altered first polypeptide and a polynucleotide encoding the cavity. a second polynucleotide encoding a second polypeptide that is altered from the original polynucleotide to encode a second polypeptide; The polypeptide is obtained by cloning the polypeptide from a host cell culture. in a common host cell with recovery of heteromultimers or after recovery and purification In some embodiments, the polypeptides are expressed in separate host cells with the formation of a dimer. The heteromultimer is a multimeric antibody, e.g., a bispecific antibody.

[0091] chimeric molecules Chimeric molecules comprising an antigen-binding molecule as defined herein and a heterologous moiety are disclosed herein. is shown.

[0092] In one embodiment, the heterologous moiety is a detectable moiety, a half-life extending moiety, or a therapeutic moiety. be.

[0093] Detectable moieties contemplated by the present invention include, for example, in vitro detection and in vivo detection. Detectable species include any species known in the art that are suitable for diagnostic detection, including bioimaging. The functional moiety may be, for example, a fluorophore, a radionuclide reporter, a metal-containing nanoparticle or may be particles, ultrasound contrast agents (e.g., nanobubbles or microbubbles), or optical imaging agents. These can be used in magnetic resonance imaging (MRI) and magnetic particle imaging (MRI). Contrast particles that can be visualized by MPI (multi-photon spectroscopy) are also included. Fluorophores can be used, for example, Emission ionization mass spectrometry (ESI-MS) detection, and fluorescence emission computed tomography Fluorescence polarization, with or without FLECT imaging; They can be detected and / or imaged by fluorescence-activated cell sorting and fluorescence microscopy. Radionuclide reporters can be used for imaging, for example, single photon emission computed tomography (SPEC). Radionuclides (nuclear) such as T, positron emission tomography (PET) or scintigraphy imaging ) detection. Metal-containing nanoparticles or microparticles can be detected and imaged by: MRI is typically used with paramagnetic nanoparticles or microparticles, and with superparamagnetic particles. It can be detected using optical imaging, including MPI, which is commonly used for ultrasound. Contrast agents can be detected using ultrasound imaging, including contrast-enhanced ultrasound (CEU). This can be done.

[0094] The detectable label may also be an enzyme-substrate label. Enzymes are generally used in a variety of techniques. For example, the enzyme may catalyze a chemical alteration of a chromogenic substrate that can be measured using It can catalyze a chemical change in a chromogenic substrate that can be measured using a variety of techniques, e.g. For example, the enzyme may catalyze a color change in a substrate that can be measured spectrophotometrically. The dye may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantitating the change in fluorescence The chemiluminescent substrate is electronically excited by a chemical reaction and then reacts with the and may emit light that can be measured (e.g., using a chemiluminometer) or may be fluorescently Examples of enzyme labels include luciferase (e.g., firefly). luciferase and bacterial luciferase; U.S. Patent No. 4,737,456), luciferase Feline, 2,3-dihydrophthalazinedione, malate dehydrogenase, urease , peroxidases such as horseradish peroxidase (HRPO), alkaline phosphatase tase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidase enzymes (e.g., glucose oxidase, galactose oxidase, and glucose- 6-phosphate dehydrogenase), heterocyclic oxidases (e.g., unease e) and xanthine oxidase), lactoperoxidase, microperoxidase Examples include aze.

[0095] Examples of enzyme-substrate combinations include, for example: 1) Horseradish peroxidase (HRPO) utilizes hydrogen peroxide to produce dye precursors (e.g. For example, orthophenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzyl oxidizes tetracycline hydrochloride (TMB); 2) Alkaline phosphatase using para-nitrophenyl phosphate as the chromogenic substrate Ze (AP); and 3) chromogenic (e.g., p-nitrophenyl-β-D-galactosidase) or fluorescent substrates β-D-galactosidase using 4-methylumbelliferyl-β-D-galactosidase as a substrate ctosidase (β-D-Gal).

[0096] In another embodiment of the present invention, the antigen-binding molecule does not need to be labeled, and the antigen-binding molecule is Its presence can be detected using a labeled antibody that binds to the antigen. The study included competitive binding assays, direct and indirect sandwich assays, immunohistochemistry, and The antibody can be used in any known assay method, such as immunoprecipitation assays.

[0097] In one embodiment, the chimeric molecule contains at least one heterologous moiety that is a "half-life extending moiety." The half-life extending moiety can be, for example, (i) an XTEN polypeptide; (ii) an Fc; ii) albumin, (iv) albumin-binding polypeptide or fatty acid, (v) human chorionic villus C-terminal peptide (CTP) of the 13 subunit of sex gonadotropin, (vi) PAS; (vii) HAP; (viii) transferrin; (ix) polyethylene glycol (P EG); (x) hydroxyethyl starch (HES), (xi) polysialic acid (PSA) (xii) a clearance receptor or a clearance receptor that blocks the binding of the chimeric molecule to the clearance receptor; or a fragment thereof; (xiii) a low complexity peptide; (xiv) or any combination thereof. In some embodiments, the half-life extending moiety comprises an Fc region. In embodiments, the half-life extending moiety comprises two Fc regions fused by a linker. Exemplary heterologous moieties include, for example, FcRn binding moieties (e.g., complete FcRn binding moieties). Fc region or a portion thereof), single chain Fc region (scFc region, e.g., U.S. Pat. WO 2008 / 0260738, WO 2008 / 012543 and WO 2008 / 012544 2008 / 1439545) or a processable scFc region. In embodiments, the heterologous moiety is polyethylene glycol (PEG), hydroxyethyl starch, or the like. polysialic acid, or any derivative, variant, or moiety thereof may contain binding sites for non-polypeptide moieties, such as combinations.

[0098] In some embodiments, at least one heterologous moiety is a therapeutic moiety. In embodiments, the therapeutic moiety is an anti-cancer moiety (e.g., a cytostatic / toxic agent and / or or antiproliferative agents), immunotherapeutic moieties, and anti-inflammatory moieties. In some cases, the therapeutic agents are useful in the treatment of cancer. Useful classes of anti-cancer agents include chemotherapeutic agents. Representative examples include antitubulin agents, auristatins, and DNA minor groove binders. , DNA replication inhibitors, alkylating agents (e.g., platinum complexes, e.g., cisplatin, mono(platinum) gold), bis(platinum) and trinuclear platinum complexes and carboplatin), anthracyclines, Antibiotics, antifolates, antimetabolites, calmodulin inhibitors, chemotherapy sensitizers, Duocalma Isin, etoposide, fluorinated pyrimidines, ionophores, lexitropsin, maytan Synoids, nitrosoureas, platinol, pore-forming compounds, purine antimetabolites, puromycin Isin, radiosensitizers, rapamycin, steroids, taxanes, topoisomerase inhibitors , vinca alkaloids, etc.

[0099] In one embodiment, the therapeutic moiety is monomethylauristatin F (MMAF) or monomethylauristatin F (MMAF). and auristatins such as methylauristatin E (MMAE).

[0100] In one embodiment, the antigen binding molecule is linked to a therapeutic agent via a valine-citrulline (Vc) linker. It is connected to the use part.

[0101] Polynucleotides, constructs and host cells Encoding an antigen-binding molecule as defined herein, or a chimeric molecule as defined herein. Disclosed herein is an isolated polynucleotide comprising a nucleic acid sequence that

[0102] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein; A polymer of nucleotides, which may be mRNA, RNA, cRNA, cDNA, or DNA The term typically refers to ribonucleotides or deoxynucleotides, or or a modified form of any type of nucleotide, of at least 10 bases in length. This term refers to the polymeric form of DNA. The term includes single and double stranded forms of DNA.

[0103] Also disclosed herein are vectors containing nucleic acids encoding the antigen-binding molecules described herein. can be.

[0104] A "vector" refers to a nucleic acid molecule, preferably a vector into which a nucleic acid sequence may be inserted or cloned, e.g., DNA fragments derived from plasmids, bacteriophages, or viruses that can be cloned The vector preferably contains one or more unique restriction sites, In defined host cells, including target cells or tissues or their precursor cells or tissues The cloned sequence may be capable of autonomous replication or may be capable of reproduction. Thus, the vector may be capable of autonomous replication. They exist as vectors, i.e., extrachromosomal entities, the replication of which is independent of chromosomal replication Vectors, such as linear or closed circular plasmids, extrachromosomal elements, minichromosomes or It may be an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be integrated into the genome when introduced into a host cell, and the integrated plasmid may be transfected. The vector system may be a single vector or a is a plasmid, two or more vectors that together contain the total DNA to be introduced into the genome of a host cell Alternatively, the vector may comprise a plasmid, or a transposon. The choice of vector is typically The compatibility of the vector with the host cell into which the vector is to be introduced may depend on the vector's compatibility with the host cell into which the vector is to be introduced. A selection gene, such as an antibiotic resistance gene, can be used to select suitable transformants. Examples of such resistance genes are well known to those skilled in the art.

[0105] an antigen-binding molecule as defined herein operably linked to one or more regulatory sequences; or a construct comprising a nucleic acid sequence encoding a chimeric molecule as defined herein. It is revealed in writing.

[0106] The term "construct" refers to one or more isolated nucleic acid sequences from different sources. A construct therefore refers to a recombinant genetic molecule containing two or more nucleic acid sequences of different origins. are assembled into a single nucleic acid molecule and (1) are not found together in nature. regulatory and coding sequences (i.e., at least one of the nucleotide sequences is a nucleic acid sequence comprising a nucleotide sequence heterologous to at least one of the nucleotide sequences of ) a sequence that encodes a portion of a functional RNA molecule or protein that is not naturally adjacent to it or (3) any construct containing a portion of a promoter that is not naturally contiguous. Representative constructs include those capable of genomic integration or autonomous replication and containing one or more nucleic acid molecules. Plasmids, cosmids, and the like, which contain operably linked nucleic acid molecules and are derived from any source. , a virus, an autonomously replicating polynucleotide molecule, a phage, or a linear or circular Any recombinant nucleic acid molecule, such as a single- or double-stranded DNA or RNA nucleic acid molecule, is included. The constructs of the present invention generally comprise a nucleic acid sequence, such as a target nucleic acid sequence or a modulator nucleic acid sequence. It may contain elements necessary to direct the expression of a nucleic acid sequence of interest also contained in the construct. Such elements are operatively linked to (direct the transcription of) a nucleic acid sequence of interest. It may contain control elements or regulatory sequences such as linked promoters, polyadenylation In certain embodiments of the invention, the construct is contained in a vector. In addition to the components of the construct, the vector may contain, for example, one or more A selectable marker, one or more origins of replication, e.g., prokaryotic and eukaryotic origins, At least one multiple cloning site and / or genome of the construct's host cell The two or more constructs may contain elements to promote stable integration into a single It may be contained within a single nucleic acid molecule, such as a vector, or may be contained within two or more separate vectors, etc. An "expression construct" generally refers to a nucleic acid sequence that contains two or more distinct nucleic acid molecules of interest. The gene contains at least one regulatory sequence operably linked to the nucleotide sequence. For example, a promoter operably linked to the nucleotide sequence to be expressed may be used to induce expression of the host cell. The present invention is also provided in an expression construct for expression in an organism or part thereof comprising the vector. Conventional compositions and methods for preparing and using the constructs and host cells are , well known to those skilled in the art, see, for example, Molecular Cloning: A Laboratories atory Manual, 3rd Edition, Volumes 1, 2, and 3, J.F. Sambrook, D.W. Russell, and N. Irwin, Cold Spring Harbor See r Laboratory Press, 2000.

[0107] "Control element," "control sequence," "regulatory sequence," and the like, as used herein, The nucleic acid sequences necessary for the expression of an operably linked coding sequence in a particular host cell The control sequences suitable for prokaryotes include, for example, promoters, and optionally cis-acting sequences such as an operator sequence and a ribosome binding site. Control sequences suitable for eukaryotic cells include transcription control sequences, such as promoters, promoter sequences, and promoter sequences. Deoxyribonucleotides, transcriptional enhancers, translational regulatory sequences, e.g., translational enhancers and and internal ribosome binding sites (IRES), nucleic acid sequences that regulate mRNA stability, and The product encoded by the transcribed polynucleotide is delivered to a subcellular compartment or subcellular compartments within the cell. It contains a targeting sequence that targets it to the external environment.

[0108] Disclosed herein are host cells containing the constructs defined herein.

[0109] The terms "host," "host cell," "host cell line," and "host cell culture" are used interchangeably. is used generically to refer to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. The term "subject cells" includes "transformants" and "transformed cells," which include primary Transformed cells and progeny derived therefrom regardless of the number of passages are included. Progeny are those that are naturally related to the parent cell. The nucleic acid content of the transformed strain may not be completely identical to that of the transformed strain, and may contain mutations. The cells that have the same function or biological activity as those screened or selected for in the cells The host cells produce the antigen-binding molecules of the present invention, and their mutated progeny are included herein. Host cells include cultured cells, any type of cell line that can be used to produce the desired product. For example, mammalian cultured cells, such as CHO cells, BHK cells, NS0 cells, and SP2 / 0 cells , YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or These include hybridoma cells, yeast cells, insect cells, and plant cells, to name just a few. This results in a transgenic animal, a transgenic plant, or a cultured plant or animal tissue. This also includes cells contained in tissue.

[0110] Pharmaceutical Composition an antigen-binding molecule as defined herein, or a chimeric molecule as defined herein, and Disclosed herein are pharmaceutical compositions comprising a pharmaceutically acceptable carrier.

[0111] A "pharmaceutically acceptable carrier" is a carrier that is not biologically or otherwise undesirable. means a pharmaceutical vehicle composed of a substance that is free from any or It can be administered to a subject in conjunction with a selected active agent without causing substantial adverse reactions. The body may contain excipients and other additives, such as diluents, surfactants, colorants, wetting agents or milk. These may include antioxidants, pH buffering agents, preservatives, etc.

[0112] Representative pharmaceutically acceptable carriers include any of the above, as would be known to one of ordinary skill in the art. Any solvents, dispersion media, coating agents, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, Excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, such as similar substances and combinations thereof (e.g., Remington's Pharmaceutical Sciences, 18th edition, Mack Print (See ing Company, 1990, pp. 1289-1329). Any conventional carrier Unless incompatible with the active ingredient(s), its use in pharmaceutical compositions is not contemplated. can be.

[0113] Pharmaceutical compositions can be in a variety of forms, including, for example, liquid, semi-solid, and solid forms. Dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions, or suspensions The preferred form depends on the intended mode of administration and treatment. Depending on the application, suitable pharmaceutical compositions may be administered intravenously, subcutaneously, or intramuscularly. In some embodiments, the composition is in the form of an injectable or pourable solution. The mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the pharmaceutical composition is administered by intravenous infusion or injection. The compositions are administered by intramuscular or subcutaneous injection.

[0114] As used herein, the phrases "parenteral administration" and "parenterally administered" generally refer to means modes of administration other than enteral and topical administration by injection, including intravenous, intramuscular, intraarterial, Intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular , including but not limited to, intrathecal, intraspinal, epidural and intrasternal injections and infusions It will not be done.

[0115] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, oligosaccharides, and the like. Vegetable oils such as celery oil, and injectable organic esters such as ethyl oleate. The carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, saline In the present invention, the pharmaceutically acceptable carrier includes water and a buffer medium. M, preferably 0.05M phosphate buffer or 0.8% saline, Other common parenteral vehicles include, but are not limited to, sodium phosphate solution, Ringer's Contains a solution of dextrose, dextrose and sodium chloride, lactated Ringer's solution or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, Ringer's dextrose-based Preservatives and other additives, such as antibacterial agents, antifungal agents, etc. Oxidizing agents, chelating agents, inert gases, and the like may also be present.

[0116] More specifically, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or These include dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In such cases, the composition must be sterile and fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and is preferably The carrier may be, for example, water, air, or a mixture of water and air and may be protected from the contaminating action of microorganisms such as bacteria and fungi. alcohols, polyols (e.g., glycerol, propylene glycol, and liquid polyols) ethylene glycol, etc.) and suitable mixtures thereof. Proper fluidity can be achieved, for example, by the use of a coating such as lecithin and / or by the addition of any necessary This can be maintained by maintaining the desired particle size. In certain embodiments, the agents of the present disclosure include: It may be conjugated to a vehicle for cellular delivery. These are used to aid in the delivery of drugs to target cells, to increase drug stability, or to reduce drug latency. To minimize potential toxicity, the compound may be packaged in an appropriate vehicle. As can be appreciated, a variety of vehicles are suitable for delivering the agents of the present disclosure. Non-limiting examples of structured fluid delivery systems include nanoparticles, liposomes, microemulsions, and These may include phospholipid-containing systems, micelles, dendrimers, and other phospholipid-containing systems. Methods for incorporating into delivery vehicles are known in the art. Various embodiments are presented below. However, methods known in the art for incorporating the antigen-binding molecules described herein into delivery vehicles are also available. It will be appreciated that other methods are contemplated.

[0117] Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, or several divided doses may be administered over time. or proportionally reduce or increase the dose as indicated by the exigencies of the therapeutic situation. The antigen-binding molecules of the present disclosure may be administered multiple times. The interval between single administrations is The intervals may be daily, weekly, monthly, or yearly. Or it may be irregular as shown by measuring blood levels of the antigen. Alternatively, the antigen-binding molecule can be administered as a sustained release formulation, in which case it can be administered less frequently. The dosage and frequency of administration will depend on the half-life of the polypeptide in the patient. Change.

[0118] Compositions are formulated in dosage unit form for ease of administration and uniformity of dosage. As used herein, the dosage unit forms may be used in a variety of dosage forms, each of which may be administered in a single dose for the subject to be treated. Each unit represents a physically discrete unit suited as a unit dosage for the required pharmaceutical preparation. A predetermined amount of active ingredient calculated to produce a desired therapeutic effect in combination with a commercially acceptable carrier. The specifications of the dosage unit forms of the present invention are determined by (a) the specific characteristics of the active compound and (b) the specific therapeutic effect to be achieved, and (c) the treatment of susceptibility in the individual. The limitations inherent in the technology of formulating such active compounds are dictated by and directly depend on them. do.

[0119] The dosage and treatment regimen of the antigen-binding molecule can be determined by one skilled in the art. In certain embodiments, the antigen-binding molecule is administered at a dose of about 0.01 to 50 mg / kg, for example 0.0 1 to 0.1 mg / kg, for example, about 0.1 to 1 mg / kg, about 1 to 5 mg / kg, about 5 to 2 by injection (e.g., subcutaneous or intravenous) at a dose of 5 mg / kg, approximately 10–50 mg / kg. The dosing schedule may be, for example, once a week for 2, 3, or 4 weeks. can change up to once per

[0120] It is noted that dosage values ​​may vary depending on the type and severity of the symptoms to be alleviated. For any particular subject, the particular dosing regimen will depend on the individual needs and the administration of the composition. should be adjusted over time according to the professional judgment of those managing or supervising the provision of The dosage ranges set forth herein are exemplary only and are not intended to be limiting of the scope or practice of the claimed compositions. It should be further understood that this is not intended to be limiting.

[0121] Treatment method A method for reducing the expression or activity of ALPPL2 in cells (such as cancer cells) is disclosed. The present invention provides a method for reducing the expression or activity of ALPPL2 in cancer cells. provided herein is a method comprising treating cancer cells with an antigen-binding molecule as defined herein, contacted with the chimeric molecule, polynucleotide, construct, vector, host cell or pharmaceutical composition This includes making

[0122] Disclosed herein are methods for reducing ALPPL2 expression or activity in cancer cells. The method further comprises treating the cancer cells with an antigen-binding molecule as defined herein or an antigen-binding molecule as defined herein. The method includes contacting the chimeric molecule with a chimeric molecule that is

[0123] The term "tumor" as used herein refers to any tumor, whether malignant or benign. The growth and proliferation of any neoplastic cells, as well as all precancerous and cancerous cells and The terms "cancer" and "cancerous" typically refer to tissue that is affected by uncontrolled cell growth. The term "prostate cancer" refers to or describes a physiological condition in mammals that is characterized in part by a prostate cancer. When used herein, the term "cancer" refers to non-metastatic and metastatic cancers, including early and late stage cancers. The term "precancerous" refers to a condition that typically precedes or develops into a cancer. Refers to a progressive condition or growth. "Non-metastatic" means that the tumor is benign or remains at the primary site. This means that the cancer has not spread to the lymphatic or vascular systems or to tissues other than the primary site. Generally, non-metastatic cancers are classified as stage 0, I, or II cancers, and sometimes stage "Early stage cancer" refers to any cancer that is a stage III cancer, but is not invasive or metastatic. "means cancer that is not present or is classified as stage 0, I, or II cancer." The term "late-stage cancer" generally refers to stage III or stage IV cancer, but It may also refer to stage II cancer or substages of stage II cancer. The classification of stage II cancer as either early or late stage cancer is a specific It will be understood that the type of cancer will depend on the type. Illustrative examples of cancer include breast cancer, prostate cancer, or testicular cancer, ovarian cancer, cervical cancer, pancreatic cancer, colorectal cancer, lung cancer, hepatocellular carcinoma , gastric cancer, liver cancer, bladder cancer, urinary tract cancer, thyroid cancer Cancer, kidney cancer, carcinoma, melanoma, brain cancer, non-small cell lung cancer, squamous cell carcinoma of the head and neck, These include endometrial cancer, multiple myeloma, rectal cancer, mesothelioma, endometrial cancer, and esophageal cancer. In an exemplary embodiment, the cancer is selected from the group consisting of, but not limited to, colorectal cancer, endometrial cancer, and cancer, gastric, mesothelioma, ovarian, pancreatic or testicular cancer.

[0124] Methods for reducing or inhibiting tumor growth, survival and viability in a subject are provided herein. The present invention provides a method for the production of an antigen-binding molecule, a chimeric molecule, a polynucleotide, or a polypeptide as defined herein. administering the nucleotide, construct, vector, host cell or pharmaceutical composition to a subject. .

[0125] Methods for reducing or inhibiting tumor growth, survival and viability in a subject are provided herein. Disclosed herein is a method comprising administering to a subject an antigen-binding molecule as defined herein or a The method includes administering to a subject a chimeric molecule that is

[0126] The term "patient" refers to humans and other mammals receiving either prophylactic or therapeutic treatment. As used herein, the term "subject" includes any human or mammalian subject. For example, the methods of the present invention can be used to treat subjects with cancer. In one embodiment, the subject is a human. The term "non-human animal" refers to All vertebrates, including mammals and non-mammals, such as non-human primates, sheep, For example, the method of the present invention can be used to treat cancer-related mammals, including mammals such as pigs, cattle, chickens, amphibians, and reptiles. The present invention can be used to treat a subject suffering from atopic dermatitis. In one embodiment, the subject is a human. The term "non-human animals" refers to all vertebrates, e.g., mammals and non-mammals, e.g., Examples include non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles.

[0127] The methods disclosed herein involve administering a "therapeutically effective amount" of an agent (e.g., an antigen-binding molecule, a chimeric administration of a compound (such as a molecule, polynucleotide, construct, vector, host cell, or pharmaceutical composition) to a subject As used herein, the term "therapeutically effective amount" is intended to include The pharmaceutical composition contains a non-toxic but sufficient amount of the drug or compound to produce the desired therapeutic effect. The precise amount to be administered will depend on the species being treated, the age and general condition of the subject, the severity of the condition being treated, and the The dose will vary from subject to subject depending on factors such as the specific drug administered and the mode of administration. For this reason, it is not possible to specify an exact "effective amount." However, for any given For any given case, an appropriate "effective amount" will be determined by one of ordinary skill in the art using only routine experimentation. It can be done.

[0128] In one embodiment, a method of treating cancer in a subject is provided, the method comprising administering to a subject a cancer treatment ... antigen-binding molecules, chimeric molecules, polynucleotides, constructs, vectors, host cells or administering the pharmaceutical composition to the subject.

[0129] Disclosed herein are methods of treating cancer in a subject, the methods comprising administering to a subject a cancer-modifying antibody as defined herein. The method comprises administering an antigen-binding molecule or a chimeric molecule as defined herein to a subject.

[0130] As used herein, the term "treat" means to (1) treat one or more symptoms of a disorder. (2) preventing or delaying the onset of a disorder or one or more symptoms of a disorder; (3) to alleviate the disorder, i.e., to reduce or at least reduce the severity of the disorder or impairment. (4) causing regression of one or more symptoms of the disorder; and / or Or it may refer to causing a decrease in the severity of multiple symptoms.

[0131] In one embodiment, the cancer is gastric, ovarian, or pancreatic cancer. .

[0132] An antigen-binding molecule, chimeric molecule, polynucleotide, or the like as defined herein for use as a pharmaceutical. The nucleotides, constructs, vectors, host cells or pharmaceutical compositions are disclosed herein.

[0133] An antigen-binding molecule as defined herein or a compound as defined herein for use in the treatment of cancer. Disclosed herein are chimeric molecules defined as:

[0134] The use of antigen-binding molecules, chimeric molecules, polynucleotides, or the like in the manufacture of a medicament for the treatment of a subject in need thereof Use of the nucleotides, constructs, vectors, host cells or pharmaceutical compositions disclosed herein The subject may be a subject suffering from cancer.

[0135] an antigen-binding molecule as defined herein or the present invention in the manufacture of a medicament for the treatment of cancer. Disclosed herein are uses of chimeric molecules as defined herein.

[0136] Methods for treating disorders or conditions associated with undesired expression of ALPPL2 in a subject A method is disclosed herein, which method comprises the step of: administering the polynucleotide, construct, vector, host cell or pharmaceutical composition to a subject Includes:

[0137] Methods for treating disorders or conditions associated with undesired expression of ALPPL2 in a subject A method is disclosed herein, the method comprising administering to a subject an antigen-binding molecule as defined herein or The method includes administering to a subject a chimeric molecule as defined herein.

[0138] In one embodiment, the disorder or condition associated with unwanted expression of ALPPL2 is cancer. be.

[0139] In one embodiment, the cancer is a solid tumor.

[0140] In one embodiment, the cancer is selected from the group consisting of cervical cancer, colon cancer, endometrial cancer, gastric cancer, and the like. ic), ovarian cancer, or pancreatic cancer.

[0141] kit Disclosed herein is a kit for detecting cancer, the kit comprising: The present invention also includes antigen-binding molecules or chimeric molecules.

[0142] Diagnostic methods Disclosed herein is a method for determining the likelihood of cancer in a subject, the method comprising: detecting ALPPL2 in a sample obtained from the An elevated level of PL2 indicates a possible cancer in the subject.

[0143] In one embodiment, the method comprises administering to a subject an antigen-binding molecule as defined herein or a compound as defined herein. The method includes detecting ALPPL2 with a chimeric molecule.

[0144] Disclosed herein are methods for treating cancer in a subject, the methods comprising: a) administering to the subject a therapeutic agent obtained from the subject; Detecting ALPPL2 in a sample (wherein the level of ALPPL2 in the sample compared to a reference an elevated level indicates an increased likelihood of cancer in the subject; and b) an increased likelihood of cancer. This includes treating subjects found to have elevated blood cholesterol levels.

[0145] In one embodiment, the method comprises administering to a subject an antigen-binding molecule as defined herein or a compound as defined herein. The method includes detecting ALPPL2 with a chimeric molecule.

[0146] In one embodiment, the method comprises administering to a subject an antigen-binding molecule as defined herein or a compound as defined herein. The method includes treating a subject with a chimeric molecule that is

[0147] A method for identifying subjects who may be responsive to treatment with anti-ALPPL2 antibodies is provided herein. Disclosed herein is a method for detecting ALPPL2 in a sample obtained from a subject. and an increase in the level of ALPPL2 indicates that the subject is responsive to treatment with an ALPPL2 antibody. Indicates that there is a possibility that

[0148] In one embodiment, the method comprises administering to a subject an antigen-binding molecule as defined herein or a compound as defined herein. The method includes detecting ALPPL2 with a chimeric molecule.

[0149] Identifying and Treating Subjects Who May Be Responsive to Treatment with Anti-ALPPL2 Antibodies A method is disclosed herein, the method comprising: a) detecting ALPPL2 in a sample obtained from a subject; detecting an increase in the level of ALPPL2 in response to treatment with an ALPPL2 antibody; and b) respond to treatment with an ALPPL2 antibody. This includes treating a subject found to be potentially susceptible to the disease.

[0150] As used herein, "and / or" means one or more of the associated listed items. Any and all possible combinations of or multiple, as well as combinations when interpreted as alternatives (or). Refers to and encompasses a lack of alignment.

[0151] As used in this application, the singular forms "a," "an," and "the" are used where the context otherwise requires. Unless expressly indicated, plural referents are included. For example, the term "drug" includes Contains multiple drugs, including mixtures.

[0152] "About" means a reference to a quantity, level, value, number, frequency, or percentage. 15, 14, 13, 12 for dimensions, size, amount, weight or length , 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% change in quantity y), level, value, number, frequency, percentage, dimension, size, amount, weight It means quantity or length.

[0153] Throughout this specification and the following description, unless the context otherwise requires, the term "including" will be used. prise" and the words "comprises" and "contains" Variations such as "comprising" are not necessarily related to the stated integer or step or integers. or a group of steps, but any other integer or step or integers or steps It will be understood to mean that the group is not excluded.

[0154] This disclosure does not incorporate any prior publication (or information derived therefrom) or any prior known matter. References in this specification to that earlier publication (or information derived therefrom) or any previously published The matter of knowledge forms part of the common general knowledge in the field to which this specification relates. shall not be construed or interpreted as a form of agreement or endorsement or any suggestion that should not be interpreted.

[0155] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It will be understood that the present invention is open to the It is to be understood that the present invention includes all such variations and modifications. All of the steps, features, compositions and compounds mentioned or shown in the document, individually or collectively, The present invention also includes any and all combinations of any two or more of the steps or features.

[0156] Certain specific embodiments of the present invention will now be described, by way of example only, to illustrate the scope of the general principles set forth above. The present invention is illustrated by reference to the following examples, which are not intended to be limiting.

[0157] Example Target ID and Background Gastric cancer is a disease that is widespread in East Asia, and 79% of patients are diagnosed at stage IV, with a 5-year survival rate of less than 5%. The marker ALPPL2 is commonly used as a target for therapeutic antibodies and companion diagnostics. RNA sequencing from 19 gastric cancer patients The data will undergo rigorous bioinformatics analysis to identify biomarkers. It was.

[0158] ALPPL2 protein expression was measured by immunohistochemical staining using a commercially available anti-ALPPL2 The antibody was tested in six gastric cancer cell lines. In gastric cancer cell lines overexpressing PPL2 mRNA, Strong membrane staining was observed, but no clear staining was observed in cell lines that did not overexpress ALPPL2 transcripts Clinical prevalence was also assessed using two gastric tumor microarrays. The results were evaluated by immunohistochemical staining of various stages of the disease and the stomach. h) A total of 198 tumor cores from various regions were stained. The results showed that 32 of the 198 cases The results show that 16% of the tumors showed ALPPL2 membrane staining. No obvious membrane staining was observed in either the IL-1 or non-matched normal tissues.

[0159] antibody generation Antibodies against human ALPPL2 were generated by immunizing rabbits with the antigen. Isolation of rabbit antibodies by direct cloning of the antibody gene from a single rabbit B cell did.

[0160] During the screening process, we identified a gene that binds to ALPPL2 but is expressed in normal intestinal tissue. Clones that did not bind to related ALPP / A were selected (Fig. 1 and Fig. 2). A total of 36 clones with high affinity for LPPL2 were isolated. The amino acid sequences of the complementarity determining regions are shown in Tables 1 and 2.

[0161] Affinity and specificity Screening specific clones and analyzing them using ELISA and high-throughput flow cytometry Rabbit kidney cells were used to identify the cleaved (ELISA screened) ALPI and ALPPL2 in either full-length (for FACS screening) or full-length (for FACS screening) forms were transfected with either

[0162] Specific for ALPPL2 / ALPP but not for ALPI A subset of clones that did not express the IgG was further analyzed for affinity determination using BioLayer Interferometry. In this technique, different concentrations of supernatant clones from rabbit B cells were used for further selection (Figure 3). The enzyme was immobilized on a Protein A biosensor. The biosensor was then incubated with the analyte. The affinity was measured by bating.

[0163] Equivalent humanized monoclonal antibodies disclosed in the prior art can be obtained by arranging the CDRs in the same framework. The results were analyzed by ELISA using a grafted antibody against ALPPL2 and ALPI. The equivalent humanized monoclonal antibodies were evaluated for binding to both V H and V L It has an array.

[0164] [Table 4]

[0165] The genes were synthesized and cloned into expression vectors for recombinant antibody production. Surface plasmon resonance data demonstrate that the antibodies disclosed in the prior art are ALP-like, but not the antibodies of the present invention. The results show non-specific binding to I.

[0166] Immunohistochemistry (IHC) activity To enable the development of a companion diagnostic, the IHC activity of the antibodies was assessed (Figure 5). Antigen retrieval by proteinase-K digestion enabled detection, whereas heat-mediated antigen retrieval did not. C36, C45, and C130 were ALPPL2+ve cell lines by IHC. Detection in (SCH) and formalin-fixed paraffin-embedded (FFPE) human tumor tissues This makes it possible to develop antibodies that can treat gastric cancer, ovarian cancer, ALPPL2 / A, including colorectal cancer, pancreatic cancer, endometrial cancer, mesothelioma, and testicular cancer May have diagnostic and therapeutic applications in patient stratification for the treatment of LPP+ve tumors. C36 showed negative staining in all normal tissues except for the placenta. This suggests that ALPPL / ALPP is not expressed / expressed at low levels in normal tissues. This also indicates the optimal therapeutic window for these antibodies in the clinic.

[0167] Non-human primate (NHP) cross-reactivity The antibodies were further evaluated for cross-reactivity to non-human primate orthologues (Figure 6) Selected clones showed reactivity to the rhesus monkey orthologue.

[0168] Humanized clones (affinity, selectivity and specificity) Selected clones (C4, C15, C131, C12, C18, C36, and C53) were The CDRs were humanized by grafting them onto a human IgG1 framework. The humanized clones were identified using surface plasmon resonance to have high ALPP / ALPPL2 affinity. Surface plasmon resonance analysis of the Biacore T200 demonstrated that the Biacore T200 retains the ATP-dependent ATP content (Figure 7). The test was performed using a biosensor CM5 chip with a ligand (e.g., ALPPL2 or The ALPP was captured and immobilized with streptavidin. The sensor was incubated with different concentrations of the analyte (a recombinantly expressed humanized antibody clone). The affinity was measured.

[0169] Humanized clones (C4, C36, and C53) express cancer-specific ALPPL2 and / or maintained selectivity for ALPP, but was selective for the widely expressed ALPI and ALPL. The humanized clones were also specific to cancer cells, but did not maintain their selectivity (Figure 13). However, it was not specific for normal naive and stimulated immune cells (Fig. 13).

[0170] Humanized clone (ADCC) The therapeutic efficacy of humanized clones was assessed by co-culturing reporter or primary NK cells with cancer cell lines. Antibody-dependent cellular cytotoxicity was first assessed by administering the antibody to the mice (Figure 8). C4 was identified in both gastric cancers with high and low target expression when compared with other clones ( This resulted in the most potent ADCC induction in gastric cancer cell lines. The efficacy of C4 was confirmed in primary NK cells and different gastric cancer cell lines. C4 achieved near-complete killing of the high-expressing cell line, demonstrating its ability to target The efficacy (maximum killing % and EC50) was proportional to the expression level of C4. Induction of ADCC in different ovarian and pancreatic cancer cell lines was confirmed by transporter assay. was done.

[0171] The Fc region of humanized C4 was further engineered to enhance ADCC. Therefore, humanized C4 with an engineered Fc has been shown to be effective against gastric cancer. It was confirmed that it induces ADCC more potently in cell lines.

[0172] Humanized clone (ADC) These humanized antibodies were evaluated for their suitability for use as antibody-drug conjugates. First, we investigated the killing of gastric cancer cell lines by these antibodies. The secondary ADC antibody was tested in the presence of a secondary antibody conjugated to c-MMAF (Figure 9). Assays revealed some differences in potency between clones; killing was high In selected gastric cancer cell lines with low target expression These results were observed only in vc-MMAE and control groups, but not in the others. The primary conjugate of the antibody with vc-MMAE was confirmed using the same antibody. The gating was consistent with the same gastric cancer observed in the second-line ADC assay. er) enhanced killing of cell lines.

[0173] Humanized clone (T cell engager) These humanized antibodies are T cell endogenous antibodies for inducing potent T cell-mediated killing of cancer cells. It was further demonstrated that the device can be successfully adapted for use as a gauger (Fig. 10, 11 and 12). Heterodimerization of the humanized clone with anti-CD3 antibody Bispecific antibodies have been shown to be effective against gastric and ovarian cancers, regardless of the expression level of the target. In particular, C4 achieved potent and specific killing of multiple cell lines, including pancreatic cancer, at pM concentrations. achieved near-complete killing of several cancer cell lines.

[0174] ALPPL2-specific clones Chimeric and humanized C53 clones showed binding specificity for ALPPL2, but However, the chimeric C53 did not show binding specificity to ALPP (Fig. 12). Cross-reactive to Ghezal orthologues. Humanized to ALPPL and ALPP The binding affinity of humanized C53 compared to C36 was performed using biolayer interferometry. In this technique, biotinylated ligands (i.e., ALPPL2 or ALPP) are ligated to SA. The ligand-loaded sensor was then immobilized on a biosensor at different concentrations in a buffer solution. The antibodies were incubated with the analyte (a recombinantly expressed humanized antibody clone). The antibody has a nM binding affinity for ALPPL as determined by biolayer interferometry. Humanized C36 showed no binding affinity to ALPP, whereas humanized C36 showed no binding affinity to ALPP. It showed similar binding affinities for L2 and ALPP.

[0175] The humanized C53 antibody maintained its killing efficacy in both ADCCs, demonstrating potent suppression of cancer cells. and are suitable for use as T cell engagers to induce efficient T cell-mediated killing. Further exemplified.

Claims

1. Specific binding to ALPPL2 and / or ALPP, but not to ALPL and ALPI an antigen-binding molecule that does not bind to a) a heavy chain variable region comprising the VHCDR1, VHCDR2 and VHCDR3 amino acid sequences (V Ｈ ); and b) a light chain variable region comprising the VLCDR1, VLCDR2, and VLCDR3 amino acid sequences; (V) Ｌ ); VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and and VLCDR3 amino acid sequence combinations shown in any of the rows of Table 1. Combined molecules.

2. ALPPL2 and / or ALPP, or ALPPL2 and / or ALP specifically binds to cells expressing P with an affinity between about 14 pm and about 10 nM.

1. An antigen-binding molecule described in 1.

3. 10. The antibody of claim 1, which specifically binds to a rhesus monkey ALPPL2 / ALPP ortholog. Antigen-binding molecules.

4. a) a heavy chain variable region (V) comprising SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117 Ｈ ); and b) a light chain variable fragment comprising SEQ ID NO:118, SEQ ID NO:119, and SEQ ID NO:

120. Area (V Ｌ The antigen-binding molecule of claim 1 , comprising:

5. a) V shown in either the row of Table 2 or Table 3 Ｈ At least 9 for the amino acid sequence 0% (including at least 91% to 100% and all integer percentages therebetween) V having sequence identity to Ｈ the amino acid sequence, and b) V in Table 2 or Table 3 Ｈ V shown on the same line as the amino acid sequence Ｌ For amino acid sequences At least 90% (at least 91% to 100% and all integer percentages therebetween) V having sequence identity (including Ｌ Amino acid sequence The antigen-binding molecule of claim 1, comprising:

6. a) at least 90% (at least 91% to 100% and above) to SEQ ID NO: 291 a VH amino acid sequence having a sequence identity of , and b) at least 90% (at least 91% to 100% and above) to SEQ ID NO: 292 VL amino acid sequence having sequence identity with The antigen-binding molecule of claim 1, comprising:

7. The antigen-binding molecule of claim 1, which does not bind to ALPP.

8. The antibody of claim 1, which is an antibody or an antigen-binding fragment thereof, or a chimeric antigen receptor (CAR). The antigen-binding molecule described.

9. The antibody or antigen-binding fragment thereof according to claim 8, wherein the antibody or antigen-binding fragment thereof is humanized or chimerized. The antigen-binding molecule described above.

10. The antibody or antigen-binding fragment thereof may be a full-length antibody, a substantially intact antibody, a Fab a fragment, scFab, Fab', single chain variable fragment (scFv) or one-arm antibody, The antigen-binding molecule of claim 8.

11. The antigen-binding molecule of claim 8, wherein the antibody is a bispecific antibody or a trispecific antibody. child.

12. the bispecific antibody comprises a first antigen-binding site that specifically binds to ALPPL2; and The antigen-binding molecule of claim 11, comprising a second antigen-binding site that specifically binds to CD3. 。

13. A chimeric molecule comprising the antigen-binding molecule of any one of claims 1 to 12 and a heterologous moiety. child.

14. wherein the heterologous moiety is a detectable moiety, a half-life extending moiety, or a therapeutic moiety.

14. The chimeric molecule according to 13.

15. The therapeutic moiety is monomethyl auristatin F (MMAF) or monomethyl auristatin The chimeric molecule of claim 14, which is statin E (MMAE).

16. The antigen-binding molecule of any one of claims 1 to 10 or any one of claims 13 to 15. An isolated polynucleotide comprising a nucleic acid sequence encoding any one of the chimeric molecules. 。

17. 13. Any one of claims 1 to 12, operably linked to one or more regulatory sequences. or the chimeric molecule of any one of claims 13 to 15. A construct comprising a nucleic acid sequence that encodes a nucleotide sequence encoding ...

18. A host cell comprising the construct of claim 17.

19. An antigen-binding molecule according to any one of claims 1 to 12 or claims 13 to 15. A pharmaceutical composition comprising the chimeric molecule of any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

20. A method for reducing the expression or activity of ALPPL2 in cancer cells, comprising: The cells are treated with an antigen-binding molecule according to any one of claims 1 to 12 or any one of claims 13 to 16.

6. A method comprising contacting a subject with a chimeric molecule described in any one of claims 5.

21. A method for reducing or inhibiting tumor growth, survival, and viability in a subject. and an antigen-binding molecule according to any one of claims 1 to 12 or any one of claims 13 to 15. A method comprising administering to the subject the chimeric molecule of any one of claims 1 to 4.

22. 13. A method of treating cancer in a subject, comprising administering to a subject an antigen binding agent according to any one of claims 1 to 12. administering to the subject a chimeric molecule or a chimeric molecule of any one of claims 13 to 15 The method includes:

23. The cancer is colorectal cancer, endometrial cancer, gastric cancer, mesothelioma, ovarian cancer 23. The method of claim 22, wherein the cancer is pancreatic cancer or testicular cancer.

24. 13. An antigen binding molecule according to any one of claims 1 to 12 for use in the treatment of cancer. A molecule or a chimeric molecule according to any one of claims 13 to 15.

25. 13. The use of an antibody according to any one of claims 1 to 12 in the manufacture of a medicament for the treatment of cancer. Use of a prototypic binding molecule or a chimeric molecule according to any one of claims 13 to 15.

26. Methods for treating diseases or conditions associated with undesired expression of ALPPL2 in a subject A method for producing an antigen-binding molecule according to any one of claims 1 to 12 or claim 13.

16. A method comprising administering to the subject a chimeric molecule described in any one of claims 1 to 15.

27. A kit for detecting cancer, comprising an antigen according to any one of claims 1 to 12. A kit comprising a binding molecule or a chimeric molecule according to any one of claims 13 to 15.

28. 1. A method for determining the possibility of cancer in a subject, comprising: detecting LPPL2, and determining the level of ALPPL2 in the sample compared to a reference. wherein an elevation indicates a likelihood of cancer in said subject.

29. The antigen-binding molecule of any one of claims 1 to 12 or any one of claims 13 to 15.

29. The method according to claim 28, comprising detecting ALPPL2 with the chimeric molecule according to any one of claims 28 to 28. How to do it.

30. 1. A method of treating cancer in a subject, comprising: a) detecting ALPPL2 in a sample obtained from said subject; wherein an increase in the level of ALPPL2 in the sample compared to a reference is and b) detecting an increased likelihood of cancer in said subject; and treating a subject found to have the condition.

31. The antigen-binding molecule of any one of claims 1 to 12 or any one of claims 13 to 15.

31. The method according to claim 30, comprising detecting ALPPL2 with the chimeric molecule according to any one of claims 30 to 31. How to do it.

32. The antigen-binding molecule of any one of claims 1 to 12 or any one of claims 13 to 15.

32. The method of claim 31, comprising treating the subject with any one of the chimeric molecules of claim 31. Law.

33. A method for identifying subjects who may be responsive to treatment with an anti-ALPPL2 antibody. and detecting ALPPL2 in a sample obtained from said subject, An increase in the level indicates that the subject may be responsive to treatment with the ALPPL2 antibody. A method to show that

34. The antigen-binding molecule of any one of claims 1 to 12 or any one of claims 13 to 15.

34. The method according to claim 33, comprising detecting ALPPL2 with the chimeric molecule according to any one of claims 33 to 34. How to do it.

35. Identifying and Treating Subjects Who May Be Responsive to Treatment with Anti-ALPPL2 Antibodies a) detecting ALPPL2 in a sample obtained from said subject, The increased level of ALPPL2 may be responsive to treatment with the ALPPL2 antibody. and b) detecting a gene that is responsive to treatment with said ALPPL2 antibody. treating said subject found to be likely to be a candidate for the disease.

36. An antigen-binding molecule that specifically binds to ALPPL2, but not to ALPL or ALPI, was prepared. A method for producing a fluororesin comprising the steps of: a) immunizing an animal, preferably a rabbit, with ALPPL2; b) from said animals, a protein that specifically binds to ALPPL2 but not to ALPL and ALPI is isolated; isolating B cells that are c) determining the amino acid sequence of the antibody expressed by said B cells. A method comprising:

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