Antigen binding molecules directed against alppl2 and / or alpp and uses thereof

Abstract

This invention relates to antigen-binding molecules that specifically bind to ALPPL2 and / or ALPP but not ALPL or ALPI. The invention also relates to pharmaceutical compositions, immunoconjugates, and chimeric antigen receptors comprising said antigen-binding molecules. Furthermore, the invention relates to methods for reducing the expression or activity of ALPPL2 in cancer cells, and methods for treating cancer in subjects.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202180017750.8, filed on February 5, 2021, entitled "Antigen-binding molecules targeting ALPPL2 and / or ALPP and their uses therein". Technical Field

[0002] This invention generally relates to the field of oncology. Specifically, this invention relates to antigen-binding molecules that specifically bind to ALPPL2 and / or ALPP but not to ALPL or ALPI. Background Technology

[0003] Antibodies are attractive therapeutic agents because they can bind to cell surface antigens and eliminate cancer cells. Clinically approved antibody therapies include Herceptin and Rituxan, which are highly successful drugs for treating a variety of cancers, including blood cancers and solid tumors. Antibody therapies work by, for example, recruiting effector cells (such as natural killer cells or T cells) or by modulating signaling pathways in cancer cells. Antibodies can also be conjugated to toxins or radioisotopes to help eliminate cancer cells. The development of successful antibody therapies requires targeting cell surface antigens that are preferentially expressed on cancer cells. This is because the same surface antigens expressed on normal healthy cells can lead to undesirable side effects.

[0004] Generally, there is a lack of suitable tumor-associated antigens for targeted antibody therapies against cancer. Furthermore, developing effective therapies against such antigens to treat cancer remains a significant challenge.

[0005] Accordingly, there is usually a desire to overcome or improve one or more of the aforementioned problems. Summary of the Invention

[0006] This article discloses antigen-binding molecules that specifically bind to ALPPL2 and / or ALPP but not to ALPL or ALPI, which include:

[0007] (a) Heavy chain variable region (V H ), which contains the amino acid sequences of VHCDR1, VHCDR2, and VHCDR3; and

[0008] (b) Light chain variable region (V) L ), which contains the amino acid sequences VLCDR1, VLCDR2 and VLCDR3; where combinations of the amino acid sequences VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 are shown in any row of Table 1.

[0009] This article discloses chimeric molecules that include antigen-binding molecules and heterologous moieties as defined herein.

[0010] This article discloses an isolated polynucleotide comprising a nucleic acid sequence encoding an antigen-binding molecule or chimeric molecule as defined herein.

[0011] This paper discloses a construct comprising a nucleic acid sequence encoding an antigen-binding molecule or chimeric molecule as defined herein, the nucleic acid sequence being operatively linked to one or more regulatory sequences.

[0012] This paper discloses a host cell that contains constructs as defined herein.

[0013] This article discloses a pharmaceutical composition comprising an antigen-binding molecule or chimeric molecule as defined herein and a pharmaceutically acceptable carrier.

[0014] This article discloses a method for reducing the expression or activity of ALPPL2 in cancer cells, which involves contacting cancer cells with an antigen-binding molecule or chimeric molecule as defined herein.

[0015] This article discloses a method for reducing or inhibiting the proliferation, survival, and viability of tumors in subjects, which includes administering an antigen-binding molecule or chimeric molecule as defined herein to the subject.

[0016] This article discloses a method for treating cancer in a subject, wherein the method includes administering an antigen-binding molecule or chimeric molecule, as defined herein, to the subject.

[0017] This article discloses antigen-binding molecules or chimeric molecules, as defined herein, for the treatment of cancer.

[0018] This article discloses the use of antigen-binding molecules or chimeric molecules, as defined herein, in the preparation of medicaments for the treatment of cancer.

[0019] This article discloses a method for treating a disease or condition in a subject that is associated with undesirable ALPPL2 expression, wherein the method includes administering an antigen-binding molecule or chimeric molecule, as defined herein, to the subject.

[0020] This article discloses a kit for detecting cancer, which contains an antigen-binding molecule or chimeric molecule as defined herein.

[0021] This article discloses a method for determining the likelihood of a subject having cancer, wherein the method includes detecting ALPPL2 in a sample obtained from the subject, wherein an increase in the level of ALPPL2 in the sample compared to a reference indicates the likelihood of the subject having cancer.

[0022] This article discloses a method for treating cancer in a subject, wherein the method includes a) detecting ALPPL2 in a sample obtained from the subject, wherein an increase in the ALPPL2 level in the sample compared to a reference indicates an increased likelihood of the subject having cancer; and b) treating the subject for whom an increased likelihood of having cancer has been found.

[0023] This article discloses a method for identifying subjects who may respond to treatment with an anti-ALPPL2 antibody, the method comprising detecting ALPPL2 in a sample obtained from the subject, wherein an increase in ALPPL2 levels indicates that the subject may respond to treatment with an ALPPL2 antibody.

[0024] This article discloses a method for identifying and treating subjects who may respond to treatment with an ALPPL2 antibody, the method comprising a) detecting ALPPL2 in a sample obtained from the subject, wherein an increase in ALPPL2 levels indicates that the subject may respond to treatment with an ALPPL2 antibody; and b) treating the subject found to be likely to respond to treatment with an ALPPL2 antibody.

[0025] This article discloses a method for preparing antigen-binding molecules that specifically bind to ALPPL2 but not to ALPL or ALPI, the method comprising:

[0026] a) Immunize animals with ALPPL2, preferably rabbits.

[0027] b) Isolate B cells from the animals that specifically bind ALPPL2 but not ALPL or ALPI, and

[0028] c) Determine the amino acid sequence of the antibody expressed by the B cells.

[0029] Specifically, this disclosure relates to the following implementation schemes:

[0030] 1. An antigen-binding molecule that specifically binds to ALPPL2 and / or ALPP but not ALPL or ALPI, comprising:

[0031] a) Heavy chain variable region (V) H ), which contains the amino acid sequences of VHCDR1, VHCDR2, and VHCDR3; and

[0032] b) Light chain variable region (V) L ), which contains the amino acid sequences VLCDR1, VLCDR2 and VLCDR3; where combinations of the amino acid sequences VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 are shown in any row of Table 1.

[0033] 2. The antigen-binding molecule of embodiment 1, wherein the antigen-binding molecule specifically binds to ALPPL2 and / or ALPP or cells expressing ALPPL2 and / or ALPP with an affinity of about 14 pM to about 10 nM.

[0034] 3. The antigen-binding molecule described in Implementation Scheme 1, wherein the antigen-binding molecule specifically binds to rhesus monkey ALPPL2 / ALPP orthologs.

[0035] 4. The antigen-binding molecule according to embodiment 1, wherein the antigen-binding molecule comprises: a) a heavy chain variable region (V) comprising SEQ ID NO: 115, SEQ ID NO: 116 and SEQ ID NO: 117. H b) contains the light chain variable region (V) of SEQ ID NO:118, SEQ ID NO:119 and SEQ ID NO:120. L ).

[0036] 5. The antigen-binding molecule according to embodiment 1, wherein the antigen-binding molecule comprises:

[0037] a) V H The amino acid sequence, which is consistent with the V shown in any row of Table 2 or Table 3 H The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages in between) sequence identity, and

[0038] b) V L The amino acid sequence, which is consistent with that in Table 2 or Table 3 and V H The amino acid sequence is shown in the same row as V. L The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages in between) sequence identity.

[0039] 6. The antigen-binding molecule according to embodiment 1, wherein the antigen-binding molecule comprises:

[0040] a) V H The amino acid sequence that has at least 90% (including at least 91% to 100% and all integer percentages therein) sequence identity with SEQ ID NO: 291, and

[0041] b) V L The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages in between) sequence identity with SEQ ID NO: 292.

[0042] 7. The antigen-binding molecule described in Implementation Scheme 1, wherein the antigen-binding molecule does not bind to ALPP.

[0043] 8. The antigen-binding molecule described in Embodiment 1, wherein the antigen-binding molecule is an antibody or its antigen-binding fragment or a chimeric antigen receptor (CAR).

[0044] 9. The antigen-binding molecule of embodiment 8, wherein the antibody or its antigen-binding fragment is humanized or chimeric.

[0045] 10. The antigen-binding molecule of embodiment 8, wherein the antibody or its antigen-binding fragment is a full-length antibody, a substantially complete antibody, a Fab fragment, scFab, Fab', a single-chain variable fragment (scFv), or a single-arm antibody.

[0046] 11. The antigen-binding molecule described in Implementation Scheme 8, wherein the antibody is a bispecific or trispecific antibody.

[0047] 12. The antigen-binding molecule of embodiment 11, wherein the bispecific antibody comprises a first antigen-binding site that specifically binds to ALPPL2 and a second antigen-binding site that specifically binds to CD3.

[0048] 13. A chimeric molecule comprising an antigen-binding molecule according to any one of embodiments 1 to 12 and a heterologous portion.

[0049] 14. The chimeric molecule according to embodiment 13, wherein the heterologous portion is a detectable portion, a half-life extended portion, or a therapeutic portion.

[0050] 15. The chimeric molecule of embodiment 14, wherein the therapeutic portion is monomethylolpropamine F (MMAF) or monomethylolpropamine E (MMAE).

[0051] 16. An isolated polynucleotide comprising a nucleic acid sequence encoding an antigen-binding molecule according to any one of embodiments 1 to 10 or encoding a chimeric molecule according to any one of embodiments 13 to 15.

[0052] 17. A construct comprising a nucleic acid sequence encoding an antigen-binding molecule according to any one of embodiments 1 to 12 or a chimeric molecule according to any one of embodiments 13 to 15, said nucleic acid sequence being operatively linked to one or more regulatory sequences.

[0053] 18. A host cell comprising the construct described in embodiment 17.

[0054] 19. A pharmaceutical composition comprising an antigen-binding molecule according to any one of embodiments 1 to 12, or a chimeric molecule according to any one of embodiments 13 to 15, and a pharmaceutically acceptable carrier.

[0055] 20. A method for reducing the expression or activity of ALPPL2 in cancer cells, the method comprising contacting the cancer cells with an antigen-binding molecule according to any one of embodiments 1 to 12 or a chimeric molecule according to any one of embodiments 13 to 15.

[0056] 21. A method for reducing or inhibiting the proliferation, survival, and viability of tumors in a subject, the method comprising administering to the subject an antigen-binding molecule according to any one of embodiments 1 to 12 or a chimeric molecule according to any one of embodiments 13 to 15.

[0057] 22. A method of treating cancer in a subject, wherein the method comprises administering to the subject an antigen-binding molecule according to any one of embodiments 1 to 12 or a chimeric molecule according to any one of embodiments 13 to 15.

[0058] 23. The method described in implementation scheme 22, wherein the cancer is colorectal cancer, endometrial cancer, gastric cancer, mesothelioma, ovarian cancer, pancreatic cancer, or testicular cancer.

[0059] 24. An antigen-binding molecule according to any one of embodiments 1 to 12 or a chimeric molecule according to any one of embodiments 13 to 15, for the treatment of cancer.

[0060] 25. Use of the antigen-binding molecule according to any one of embodiments 1 to 12 or the chimeric molecule according to any one of embodiments 13 to 15 in the preparation of a medicament for treating cancer.

[0061] 26. A method for treating a disease or condition in a subject associated with undesirable ALPPL2 expression, wherein the method comprises administering to the subject an antigen-binding molecule according to any one of embodiments 1 to 12 or a chimeric molecule according to any one of embodiments 13 to 15.

[0062] 27. A kit for detecting cancer, the kit comprising an antigen-binding molecule according to any one of embodiments 1 to 12 or a chimeric molecule according to any one of embodiments 13 to 15.

[0063] 28. A method for determining the likelihood of a subject having cancer, wherein the method includes detecting ALPPL2 in a sample obtained from the subject, wherein an increase in the level of ALPPL2 in the sample compared to a reference indicates the likelihood of the subject having cancer.

[0064] 29. The method of embodiment 28, wherein the method includes detecting ALPPL2 with an antigen-binding molecule according to any one of embodiments 1 to 12 or a chimeric molecule according to any one of embodiments 13 to 15.

[0065] 30. A method for treating cancer in a subject, wherein the method comprises: a) detecting ALPPL2 in a sample obtained from the subject, wherein an increase in the level of ALPPL2 in the sample compared to a reference indicates an increased likelihood of the subject having cancer; and b) treating the subject for whom an increased likelihood of having cancer has been found.

[0066] 31. The method of embodiment 30, wherein the method includes detecting ALPPL2 with an antigen-binding molecule according to any one of embodiments 1 to 12 or a chimeric molecule according to any one of embodiments 13 to 15.

[0067] 32. The method of embodiment 31, wherein the method comprises treating the subject with an antigen-binding molecule according to any one of embodiments 1 to 12 or a chimeric molecule according to any one of embodiments 13 to 15.

[0068] 33. A method for identifying a subject who may respond to treatment with an anti-ALPPL2 antibody, the method comprising detecting ALPPL2 in a sample obtained from the subject, wherein an increase in ALPPL2 levels indicates that the subject may respond to treatment with an ALPPL2 antibody.

[0069] 34. The method of embodiment 33, wherein the method includes detecting ALPPL2 with an antigen-binding molecule according to any one of embodiments 1 to 12 or a chimeric molecule according to any one of embodiments 13 to 15.

[0070] 35. A method for identifying and treating a subject who may respond to treatment with an antibody against ALPPL2, the method comprising: a) detecting ALPPL2 in a sample obtained from the subject, wherein an increase in ALPPL2 levels indicates that the subject may respond to treatment with an antibody against ALPPL2; and b) treating the subject who is found to be likely to respond to treatment with an antibody against ALPPL2.

[0071] 36. A method for preparing an antigen-binding molecule that specifically binds to ALPPL2 but not ALPL or ALPI, the method comprising:

[0072] a) Immunize animals with ALPPL2, preferably rabbits.

[0073] b) Isolate B cells from the animals that specifically bind ALPPL2 but not ALPL or ALPI, and

[0074] c) Determine the amino acid sequence of the antibody expressed by the B cells. Attached Figure Description

[0075] The embodiments of the invention are described below by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0076] Figure 1 This study demonstrates the establishment of a set of criteria for selecting candidate genes encoding cell membrane proteins. Placental-like alkaline phosphatase ALPPL2 emerged as one of the best candidates for subsequent target validation.

[0077] Figure 2 Immunohistochemical staining of gastric cancer cell lines (top) and gastric tumor microarrays (bottom) is shown.

[0078] Figure 3 The image shows the identification of ALPPL2 / ALPP-specific clones generated from rabbit B cell supernatant by ELISA and FACS (top), and the affinity measurement of selected clones at a single concentration by biomembrane interference technique (bottom).

[0079] Figure 4 shows a comparison of ALPPL2 reactivity and ALPI reactivity between our humanized antibody and comparable humanized antibodies disclosed in the prior art, as measured by ELISA (top) and surface plasmon resonance (bottom).

[0080] Figure 5 shows IHC staining of formalin-fixed, paraffin-embedded (FFPE) sections of different gastric cancer cell lines by C36, C45, and C130 (A). IHC staining of FFPE-human gastric cancer, ovarian cancer, colorectal cancer, pancreatic cancer, testicular cancer, mesothelioma, and endometrial tumor microarrays by C36 yielded different H scores of IHC 1+, IHC 2+, and IHC 3+ (B). IHC staining of FFPE-normal tissues by C36 was also shown. Except for placental tissue, which showed positive IHC 2+ staining, all normal tissues showed no C36 staining (negative) (C).

[0081] Figure 6The cross-reactivity of selected clones generated from rabbit B cell supernatant with rhesus monkey orthologs is shown by FACS screening (A). FACS analysis showed that recombinant humanized C4 and C36 clones bound to CHO cells overexpressing rhesus monkey orthologs, but not to WT CHO cells (B).

[0082] Figure 7 shows that the selected clones retained high ALPP / ALPPL2 affinity after humanization, as measured by surface plasmon resonance (SPR).

[0083] Figure 8 The induction of ADCC by humanized clones is shown. ADCC induction was measured in co-cultures of gastric cancer cell line MKN1 with Jurkats CD16A reporter cells (top row, left) and in co-cultures of gastric cancer cell line MKN74 with Jurkats CD16A reporter cells (top row, right). The enhancement of ADCC by C4 was measured by CellTiter-Glo assay in gastric cancer cell lines co-cultured with primary NK cells (middle row, left) and in co-cultures of ovarian and pancreatic cancer cell lines with Jurkats CD16A reporter cells (middle row, right). The enhancement of ADCC by Fc engineering of humanized C4 was also shown (bottom row), as measured in co-cultures of MKN74 with Jurkats CD16A reporter cells.

[0084] Figure 9 The killing effect of ADCs on gastric cancer cell lines was measured using the CellTiter-Glo assay. The killing effect of humanized clones of vc-MMAF conjugated with secondary antibodies on gastric cancer cell lines expressing high levels of cancer cells is shown in the top row. The killing effect of humanized C4 (middle row, left) and C12 (middle row, right) conjugated with vc-MMAF on gastric cancer cell lines is shown in the bottom row. The killing effect of humanized C4 (bottom row, left) and C12 (bottom row, right) conjugated with vc-MMAF on gastric cancer cell lines is also shown in the bottom row.

[0085] Figure 10 This study demonstrates the potent killing effect of a humanized clone-derived T-cell engager on different cancer cell lines, as determined by xCELLigence real-time cell analysis of cancer cells co-cultured with expanded human T cells. Regardless of target expression levels, C4 consistently exhibited pM-level killing activity against gastric, ovarian, and pancreatic cancer cell lines.

[0086] Figure 11The potent killing effect of T-cell adaptors with different anti-CD3 variants against different cancer cell lines was demonstrated. Humanized Fab fragments were adapted to generate potent T-cell adaptors by using different anti-CD3 pairings and in different forms.

[0087] Figure 12 illustrates the responsiveness of selected clones to ALPPL2 but not ALPP, and the killing efficacy of these clones against cancer cells after humanization. FACS shows that chimeric C53 and C78 bind to ALPPL2 but not ALPP, while rabbit C4 binds to both (A). ELISA shows that chimeric C53 and C78 bind to ALPPL2 but not ALPP (B). The induction of ADCC by chimeric clones as measured by co-culturing MKN74 with Jurkats CD16A reporter cells is shown (C). ELISA shows that chimeric C53 and C78 cross-react with CHO cells overexpressing rhesus monkey orthologs identified by FACS (D). Humanized C53 showed ALPPL2 specificity by ELISA (E), and co-culturing N87 with Jurkats CD16A reporter cells showed that ADCC-inducing activity was maintained after humanization (F). Using biomembrane interference technology, humanized C53 exhibited nM-level binding affinity for ALPPL, but not for ALPP, while humanized C36 showed similar binding affinity for both ALPPL2 and ALPP (G). Measured by xCELLigence real-time cell analysis of N87 gastric cancer cells co-cultured with expanded human T cells, humanized C53 showed potent cytotoxic activity against N87 cells (H). The humanized C53 T cell adaptor exhibited pM-level cytotoxic activity similar to that of the C36 T cell adaptor.

[0088] Figure 13The binding profiles of humanized C4, C36, and C53 with different isotypes of the human alkaline phosphatase family, cancer cell lines, and normal immune cells are shown. FACS analysis revealed that humanized C4 and C36 bound to human ALPPL2 and ALPP, but not to human ALPI and ALPL, while humanized C53 specifically bound to human ALPPL2, but not to human ALPP, ALPI, and ALPL transiently expressed in 293T cells (A). Different doses of humanized C36, C4, and C53 showed binding to the ALPPL2 / ALPP positive cell line (NCI-N87), but not to the negative cell line (MIAPaca-2). Humanized C53 exhibited similar binding affinity to a commercially available antibody (product code: eBioScience #14-9870-82), but weaker affinity compared to humanized C4 and C36 (B). Humanized C36, C4, and C53 showed similarity to immature and CD3 / CD28 microbeads + IL-2 activated human PBMCs (CD4+ / CD8+ T cells, B cells, CD11b+ myeloid M cells). Cells) did not bind (C). Humanized C36, C4, and C53 showed binding with T cells, B cells, and myeloid M cells. Cells are not bound (D). Invention Details

[0089] This disclosure teaches antigen-binding molecules that specifically bind to ALPPL2 and / or ALPP, but not ALPL or ALPI. The antigen-binding molecules can bind to ALPPL2 and / or ALPP or cells expressing ALPPL2 and / or ALPP with an affinity of about 14 pM to about 10 nM.

[0090] This article discloses antigen-binding molecules that specifically bind to ALPPL2 and / or ALPP but not to ALPL or ALPI, which contain: (a) a heavy chain variable region (V H (a) contains the amino acid sequences of VHCDR1, VHCDR2, and VHCDR3; and (b) the light chain variable region (V L ), which contains the amino acid sequences VLCDR1, VLCDR2 and VLCDR3; where combinations of the amino acid sequences VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 are shown in any row of Table 1.

[0091] Alkaline phosphatase placental-like protein 2 (ALPPL2) is a member of the alkaline phosphatase (AP) family, consisting of two closely related isoforms (ALPPL2 and ALPP) expressed in the placental trophoblast, as well as two widely expressed members, ALPL (tissue-nonspecific, liver / bone / kidney) and ALPI (intestine). In one embodiment, the antigen-binding molecule specifically binds to ALPPL2 and / or ALPP. In one embodiment, the antigen-binding molecule specifically binds to human ALPPL2 and / or human ALPP. In one embodiment, the antigen-binding molecule has enhanced efficacy due to its high affinity for ALPPL2 and / or ALPP.

[0092] In one embodiment, the antigen-binding molecule does not have any detectable binding to ALPL or ALPI. In another embodiment, the antigen-binding molecule does not have any detectable binding to human ALPL or human ALPI. This may also be described as having a dissociation constant (Kd) greater than 10 nM, greater than 100 nM, greater than 1 µM, greater than 10 µM, greater than 100 µM, or greater than 1 mM for detectable binding to human ALPL or human ALPI. In one embodiment, the antigen-binding molecule has a desired therapeutic window due to the lack of binding to ALPL or ALPI. In one embodiment, the antigen-binding molecule does not induce (or induces very little) T-cell killing activity against normal cells.

[0093] Unbound by theoretical constraints, the inventors have isolated monoclonal antibodies against a tumor-associated antigen, human placental-like alkaline phosphatase 2 (ALPPL2), which possess high affinity (sub-nM Kd), specificity (unresponsive to closely related ALPL or ALPI), immunohistochemical activity (for developing complementary diagnostics), and cross-reactivity with non-human primate orthologs (for toxicological studies). The inventors have humanized several clones by transplanting complementarity-determining regions (CDRs) onto the human IgG1 backbone and shown that these humanized antibodies retain high affinity for ALPPL2. The inventors have also demonstrated that these naked humanized antibodies induce potent antibody-dependent cytotoxicity (ADCC) in co-culture assays of Jurkats reporter cells and primary natural killer (NK) cells with gastric cancer cell lines. ADCC induction has also been observed in ovarian and pancreatic cancer cell lines. By demonstrating the killing effect of primary conjugates on cancer cells in a secondary assay, the inventors have proven the suitability of using these humanized antibodies as antibody-drug conjugates. The inventors have also generated bispecific antibodies through the heterodimerization of these humanized antibodies with anti-CD3 antibodies. These bispecific antibodies function as potent T-cell adaptors (TcEs) and achieve picomolar (pM) killing of gastric, ovarian, and pancreatic cancer cell lines. Therefore, these antibodies can be used as targeted therapies against tumors expressing ALPPL2 on their cell surface.

[0094] Table 1 shows the possible combinations of CDRs that can exist on antigen-binding molecules.

[0095]

[0096]

[0097]

[0098] Table 2 shows the V antigen-binding molecules derived from 36 antibody clones. H and V L Combinations of sequences (CDR1, 2, and 3 with underscores).

[0099]

[0100]

[0101]

[0102]

[0103] Table 3 provides the V-type humanized clones.H V L Some examples of sequences

[0104]

[0105] The antigen-binding molecules of the present invention can be in isolated, purified, synthetic, or recombinant form. Suitable antigen-binding molecules can be selected from antibodies and their antigen-binding fragments, including monoclonal antibodies (MAbs), chimeric antibodies, humanized antibodies, human antibodies, and antigen-binding fragments of such antibodies. The antigen-binding molecules can be multivalent (e.g., bivalent) or monovalent. In some embodiments, the antigen-binding molecule includes an Fc domain. In other embodiments, the antigen-binding molecule lacks an Fc domain. In some embodiments, the antigen-binding molecule is a monovalent antigen-binding molecule (e.g., Fab, scFab, Fab', scFv, single-arm antibody, etc.).

[0106] The term "antigen-binding molecule" refers to a molecule that has a binding affinity for a target antigen. It should be understood that this term is extended to immunoglobulins, immunoglobulin fragments, and non-immunoglobulin-derived protein backbones that exhibit antigen-binding activity. Representative antigen-binding molecules that can be used to carry out this invention include antibodies and their antigen-binding fragments. The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies.

[0107] As defined in this article, antigen-binding molecules can be naked or conjugated with other molecules or parts (e.g., toxins, radioisotopes, small molecule drugs, peptides, etc.).

[0108] As used herein, the term "antibody" refers to any antigen-binding molecule or molecular complex containing at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen. The term "antibody" includes full-length immunoglobulin molecules and their multimers (e.g., IgM), which comprise four polypeptide chains: two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain contains a heavy chain variable region (which may be abbreviated as HCVR or V). H The heavy-chain constant region contains three structural domains, namely C H 1. C H 2 and C H 3. Each light chain contains a light chain variable region (which may be abbreviated as LCVR or V). L The light chain constant region contains one structural domain (C) and a light chain constant region. L 1). V H and V L The region can be further subdivided into high-variability regions, called complementary determinant regions (CDRs), interspersed with more conservative regions, called framing regions (FRs). Each VH and V L It consists of 3 CDRs and 4 FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present invention, the FRs (or their antigen-binding portions) of the antibody may be identical to the human germline sequence, or may be natural or artificially modified. Based on the side-by-side analysis of two or more CDRs, a common amino acid sequence can be defined.

[0109] Antibodies include any class of antibodies, such as IgG, IgA, or IgM (or their subclasses), and antibodies do not necessarily belong to any specific class. Immunoglobulins can be classified into different classes based on the antibody's amino acid sequence in its heavy chain constant region. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known.

[0110] As used herein, the term “complementarity-determining region” (CDR; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of the antibody variable domain that are essential for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2, and CDR3. Each complementarity-determining region may contain amino acid residues from, for example, the “complementarity-determining region” defined by Kabat (i.e., residues approximately 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and residues approximately 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or residues from the “high-variability ring” (i.e., residues approximately 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and residues approximately 26-32 (H1), 53-55 (L2), and 94-95 (L3) in the heavy chain variable domain) and residues approximately 26-32 (H1), 53-55 (L2), and 94-95 (L3) in the heavy chain variable domain). (H2) and residues 96-101 (H3); Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some cases, the complementarity-determining region may include amino acids from both the CDR region defined by Kabat and the hypervariable ring.

[0111] "Humanized" antibodies refer to antibodies containing amino acid residues from a non-human CDR and amino acid residues from a human FR. In some embodiments, a humanized antibody will substantially contain all of at least one (typically two) variable domains, wherein all or substantially all of the CDRs correspond to the CDRs of a non-human antibody, and all or substantially all of the FRs correspond to the FRs of a human antibody. Optionally, a humanized antibody may contain at least a portion of the antibody constant region derived from a human antibody.

[0112] As used herein, a “chimeric” molecule refers to a molecule comprising one or more unrelated types of components or containing two or more chemically distinct regions that can be conjugated, fused, linked, translated, attached via a linker, chemically synthesized, expressed by a nucleic acid sequence, etc. Examples include peptides and nucleic acid sequences, peptides and detectable markers, unrelated peptide sequences, etc. In embodiments where a chimeric molecule comprises amino acid sequences from different sources, the chimeric molecule includes (1) polypeptide sequences not found together in nature (i.e., at least one amino acid sequence is heterologous relative to at least one other amino acid sequence), or (2) non-naturally adjacent amino acid sequences. For example, as used herein, a “chimeric antibody” refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a specific source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0113] As used herein, the term “antigen” and its grammatical equivalents (e.g., “of antigen”) refer to a compound, composition, or substance that can be specifically bound to a particular humoral or cellular immune product (e.g., an antibody molecule or a T-cell receptor). Antigens can be any type of molecule, including, for example, haptens, simple intermediate metabolites, carbohydrates (e.g., oligosaccharides), lipids, and hormones, as well as macromolecules such as complex carbohydrates (e.g., polysaccharides), phospholipids, and proteins. Common categories of antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoan and other parasitic antigens, tumor antigens, antigens involved in autoimmune diseases, allergic reactions, and transplant rejection, toxins, and other confounding antigens.

[0114] An "antigen binding site" refers to a site in an antigen-binding molecule that provides for interaction with an antigen, namely one or more amino acid residues. For example, the antigen binding site of an antibody contains amino acid residues from the complementarity-determining region (CDR). Natural immunoglobulin molecules typically have two antigen binding sites, while Fab molecules typically have a single antigen binding site. The antigen binding sites of the antigen-binding molecules described herein typically bind specifically to antigens, and more specifically, to the epitopes of that antigen.

[0115] The terms “antigen-binding fragment,” “antigen-binding portion,” “antigen-binding domain,” and “antigen-binding site” are used interchangeably herein to refer to a portion of an antigen-binding molecule involved in antigen binding. These terms include any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex.

[0116] Antigen-binding fragments of antibodies can be obtained from, for example, intact antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques, including manipulating and expressing DNA encoding variable and optionally constant domains of the antibody. This DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or using molecular biology techniques to, for example, arrange one or more variable and / or constant domains into suitable conformations, or introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0117] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable region of an antibody (e.g., isolated complementarity-determining regions (CDRs), such as CDR3 peptides), or restricted FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-transplanted antibodies, single-arm antibodies, biantibodies, triantibodies, tetraantibodies, microantibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and variable IgNAR domains from sharks, are also included in the term "antigen-binding fragments" as used herein.

[0118] Antigen-binding fragments of antibodies typically contain at least one variable domain. Variable domains can be of any size or amino acid composition and generally contain at least one CDR (concurrent reading domain) that is adjacent to or conforms to one or more frame sequences. In the presence of V... L V associated with structural domains H In the antigen-binding fragment of the domain, V H and V L Domains can be arranged relative to each other in any suitable arrangement. For example, variable regions can be dimerized and contain V. H -V H V H -V L or V L -V L Dimer. Alternatively, the antigen-binding fragment of the antibody may contain monomer V. H or V L Structural domain.

[0119] In some embodiments, the antigen-binding fragment of the antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of the variable and constant domains that may be present in the antigen-binding fragment of the antibody of the present invention 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) V L -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) V L -C H 2-C H 3; and (xiv) V L -C L In any configuration of the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly interconnected or connected via all or part of hinge regions or linker regions. The hinge region may consist of at least two amino acids (e.g., 5, 10, 15, 20, 40, 60, or more), which causes flexible or semi-flexible connections between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment of the antibody of this disclosure may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above, which are interconnected with each other and / or with one or more monomers V. H or V LThe domains are associated non-covalently (e.g., via disulfide bonds). Multispecific antigen-binding molecules typically contain at least two different variable domains, each capable of specifically binding to a separate antigen or a different epitope on the same antigen. Using conventional techniques existing in the art, any form of multispecific antigen-binding molecule, including bispecific antigen-binding molecule forms, can be adapted for use in the case of the antigen-binding fragment of the antibody disclosed herein.

[0120] The term "variable region" or "variable domain" refers to the structural domain of the antibody heavy or light chain involved in the binding of antigen-binding molecules to antigens. The variable domains of the heavy and light chains of natural antibodies (V1 and V2, respectively) H and V L These typically have similar structures, with each domain containing four conserved frame regions (FRs) and three highly variable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single V H or V L The domain may be sufficient to confer antigen binding specificity.

[0121] As used in this article, the term "constant domain" or "constant region" refers to the sum of antibody domains excluding variable regions. Constant regions do not directly participate in antigen binding but exhibit various immune effector functions.

[0122] The term "bispecific antigen-binding molecule" refers to a multispecific antigen-binding molecule that has the ability to bind to the same antigen or two different epitopes on two different antigens. Bispecific antigen-binding molecules can be bivalent, trivalent, or tetravalent. As used herein, "valence," "potency," "binding valence," or other grammatical variations thereof refer to the number of antigen-binding sites in an antigen-binding molecule. These antigen recognition sites can recognize the same epitope or different epitopes. Bivalent and bispecific molecules are described, for example, by Kostelny et al., 1992. J Immunol 148:1547; Pack and Plückthun, 1992. Biochemistry 31:1579; Gruber et al. , 1994. J lmmunol 5368; Zhu et al., 1997. Protein Sci 6:781; Hu et al., 1996. Cancer Res. 56:3055; Adams et al., 1993. Cancer Res. 53:4026; and McCartney et al., 1995. Protein Eng.8:301. Trivalent and tetravalent bispecific antigen-binding molecules are also known in the art. See, for example, Kontermann RE (ed.), Springer Heidelberg Dordrecht London New York, pp. 199-216 (2011). Bispecific antigen-binding molecules can also have a higher valence than four and are also within the scope of this invention. Such antigen-binding molecules can be produced by, for example, docking and locking conjugation methods. (Chang, C.-H. et al., in Bispecific Antibodies. Kontermann RE (2011), see above).

[0123] The phrase “specific binding” or “specific binding” refers to a binding reaction between two molecules that, under physiological conditions, is at least twice the background level, and more typically, 10 to 100 times greater than the association of background molecules. When one or more detectable binders used are proteins, specific binding identifies the presence of a protein within a heterogeneous population of proteins and other biological products. Therefore, under specified immunoassay conditions, prescribed antigen-binding molecules bind to specific antigenic determinants, thereby identifying their presence. Under these conditions, specific binding to antigenic determinants requires selection of antigen-binding molecules based on their specificity to that determinant. This selection can be achieved by excluding antigen-binding molecules that cross-react with other molecules. Various immunoassay formats can be used to select antigen-binding molecules (e.g., immunoglobulins) to induce specific immune responses to specific antigens. For example, solid-phase ELISA immunoassays are commonly used to select antibodies that induce specific immune responses to proteins (see, for example, Harlow and Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immune reactivity). Methods for determining binding affinity and specificity are also well known in the field (see, for example, Harlow and Lane, above; Friefelder, “Physical Biochemistry: Applications to biochemistry and molecular biology” (WH Freeman and Co. 1976)).

[0124] In one embodiment, the antigen-binding molecule binds specifically to cells expressing ALPPL2 with an affinity of about 14 pM to about 10 nM.

[0125] “Affinity” or “binding affinity” refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antigen-binding molecule) and its binding partner (e.g., an antigen). Unless otherwise stated, “binding affinity” as used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., antigen-binding molecules). The affinity of molecule X for its partner Y is typically expressed using the dissociation constant (Kd), which is the dissociation and binding rate constant (k, k, and k) respectively. off and k on The ratio of the rate constants. Therefore, as long as the ratio of the rate constants remains constant, the equivalent affinity can include different rate constants. Affinity can be measured by methods commonly known in the art, including those described herein. One specific method for measuring affinity is surface plasmon resonance (SPR).

[0126] The terms “polypeptide,” “peptide,” or “protein” are used interchangeably herein to refer to a linear series of amino acid residues linked together by peptide bonds between adjacent α-amino and carboxyl groups. The amino acid residues are typically in their native “L” isomer form. However, residues in the “D” isomer form can substitute for any L-amino acid residue, provided the polypeptide retains the desired functional properties.

[0127] As used herein, the term "modified antibody" includes antibodies in synthetic form that have been altered to prevent them from being naturally occurring, for example, antibodies containing at least two heavy chain moieties but not two complete heavy chains (such as domain-deficient antibodies or microantibodies); antibodies in multispecific form (e.g., bispecific, trispecific, etc.) that have been altered to bind two or more different antigens or to different epitopes on 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. Patent No. 5,892,019. Additionally, the term "modified antibody" includes antibodies in multivalent form (e.g., trivalent, tetravalent, etc. antibodies that bind to three or more copies of the same antigen).

[0128] In one embodiment, the antigen-binding molecule specifically binds to rhesus monkey ALPPL2. Rhesus monkey ALPPL2 may have a sequence as shown in Genbank ID XP_011726419.1.

[0129] In one embodiment, the antigen-binding molecule includes: (a) V H The amino acid sequence, which is consistent with the V shown in any row of Table 2 or Table 3 H The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages in between) sequence identity, and (b) V LThe amino acid sequence, which is consistent with that in Table 2 or Table 3 and the V H The amino acid sequence is shown in the same row as V. L The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages in between) sequence identity.

[0130] In one implementation, the antigen-binding molecule specifically binds to ALPPL2 and ALPP, but not specifically to ALPL or ALPI.

[0131] For example, the antigen-binding molecule may include:

[0132] a) The VH amino acid sequence having at least 90% sequence identity with SEQ ID NO: 281 and the VL amino acid sequence having at least 90% sequence identity with SEQ ID NO: 282.

[0133] b) V that has at least 90% sequence identity with SEQ ID NO: 283 H The amino acid sequence and V have at least 90% sequence identity with SEQ ID NO:284 L amino acid sequence,

[0134] c) V that has at least 90% sequence identity with SEQ ID NO: 285 H The amino acid sequence and V have at least 90% sequence identity with SEQ ID NO:286 L amino acid sequence,

[0135] d) V that has at least 90% sequence identity with SEQ ID NO: 287 H The amino acid sequence and V have at least 90% sequence identity with SEQ ID NO:288 L amino acid sequence,

[0136] e) V that has at least 90% sequence identity with SEQ ID NO: 289 H The amino acid sequence and V have at least 90% sequence identity with SEQ ID NO:290 L amino acid sequence,

[0137] f) V that has at least 90% sequence identity with SEQ ID NO: 291 H The amino acid sequence and V have at least 90% sequence identity with SEQ ID NO:292. L amino acid sequence, or

[0138] g) V that has at least 90% sequence identity with SEQ ID NO: 293 HThe amino acid sequence and V have at least 90% sequence identity with SEQ ID NO:294 L Amino acid sequence.

[0139] The phrase “at least 90% sequence identity” mentioned in this specification can include at least 91% to 100% and all integer percentages in between.

[0140] In one embodiment, the antigen-binding molecule does not bind to ALPP. The antigen-binding molecule may bind to ALPPL2 but not to ALPP. In one embodiment, the antigen-binding molecule binds to ALPPL2 but not to ALPP, ALPL, or ALPI. The antigen-binding molecule may include:

[0141] a) Heavy chain variable region (V) containing the amino acid sequences of SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33 H ), and the light chain variable region (V) containing the amino acid sequences of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36. L ),

[0142] b) The heavy chain variable region (V) containing the amino acid sequences of SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69. H ), and the light chain variable region (V) containing the amino acid sequences of SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72. L ),

[0143] c) The heavy chain variable region (V) containing the amino acid sequences of SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87. H ), and the light chain variable region (V) containing the amino acid sequences of SEQ ID NO: 88, SEQ ID NO: 89 and SEQ ID NO: 90. L ),

[0144] d) Heavy chain variable region (V) containing the amino acid sequences of SEQ ID NO: 127, SEQ ID NO: 128 and SEQ ID NO: 129 H ), and the light chain variable region (V) containing the amino acid sequences of SEQ ID NO: 130, SEQ ID NO: 131 and SEQ ID NO: 132. L ),

[0145] e) Heavy chain variable region (V) containing the amino acid sequences of SEQ ID NO: 133, SEQ ID NO: 134 and SEQ ID NO: 135 H ), and the light chain variable region (V) containing the amino acid sequences of SEQ ID NO: 136, SEQ ID NO: 137 and SEQ ID NO: 138. L ),

[0146] f) Heavy chain variable region (V) containing the amino acid sequences of SEQ ID NO: 169, SEQ ID NO: 170, and SEQ ID NO: 171 H ), and the light chain variable region (V) containing the amino acid sequences of SEQ ID NO: 172, SEQ ID NO: 173 and SEQ ID NO: 174. L );or

[0147] g) The heavy chain variable region (V) containing the amino acid sequences of SEQ ID NO: 205, SEQ ID NO: 206, and SEQ ID NO: 207. H ), and the light chain variable region (V) containing the amino acid sequences of SEQ ID NO: 208, SEQ ID NO: 209 and SEQ ID NO: 210. L ).

[0148] In one implementation, the antibody includes:

[0149] a) V that has at least 90% sequence identity with SEQ ID NO: 221 H The amino acid sequence and V have at least 90% sequence identity with SEQ ID NO:222 L amino acid sequence,

[0150] b) V that has at least 90% sequence identity with SEQ ID NO: 223 H The amino acid sequence and V have at least 90% sequence identity with SEQ ID NO:224 L amino acid sequence,

[0151] c) V that has at least 90% sequence identity with SEQ ID NO: 239 H The amino acid sequence and V have at least 90% sequence identity with SEQ ID NO:240 L amino acid sequence,

[0152] d) V that has at least 90% sequence identity with SEQ ID NO: 253 HThe amino acid sequence and V have at least 90% sequence identity with SEQ ID NO:254 L amino acid sequence,

[0153] e) V that has at least 90% sequence identity with SEQ ID NO: 255 H The amino acid sequence and V have at least 90% sequence identity with SEQ ID NO:256 L amino acid sequence,

[0154] f) V that has at least 90% sequence identity with SEQ ID NO: 267 H The amino acid sequence and V have at least 90% sequence identity with SEQ ID NO:268 L amino acid sequence, or

[0155] g) V that has at least 90% sequence identity with SEQ ID NO: 279 H The amino acid sequence and V have at least 90% sequence identity with SEQ ID NO:280 L Amino acid sequence.

[0156] In one implementation, the antigen-binding molecule is an antibody or its antigen-binding fragment, or a chimeric antigen receptor (CAR).

[0157] In one implementation, the antibody or its antigen-binding fragment is humanized or chimeric.

[0158] In one embodiment, the antibody or its antigen-binding fragment is a humanized antibody, comprising:

[0159] a) Heavy chain variable region, which includes:

[0160] i) V H FR1, which shares at least 90% sequence identity with EVQLVESGGGLVQPGGSLRLSCAASG (SEQ ID NO: 295),

[0161] ii) V H FR2, which shares at least 90% sequence identity with WVRQAPGKGLE (SEQ ID NO: 296),

[0162] iii) V H FR3, which has at least 90% sequence identity with the following sequences:

[0163] ASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA (SEQ ID NO: 297),

[0164] iv) V H FR4, which shares at least 90% sequence identity with WGQGTLVTVSS (SEQ ID NO: 298), and

[0165] b) Light chain variable region, which includes:

[0166] i) V L FR1, which has at least 90% sequence identity with the following sequences:

[0167] DIQMTQSPSSSLSASVGDRVTITCQAG (SEQ ID NO: 299),

[0168] ii) V H FR2, which shares at least 90% sequence identity with WYQQKPGKVPK (SEQ ID NO: 300),

[0169] iii) V H FR3, which shares at least 90% sequence identity with GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC (SEQ ID NO: 301),

[0170] i)V H FR4 has at least 90% sequence identity with FGQGTKVEIK (SEQ ID NO: 302).

[0171] In one embodiment, the antibody or its antigen-binding fragment comprises a C sequence having at least 90% (including at least 91% to 100% and all integer percentages therebetween) sequence identity with the following sequences. H 1. Amino acid sequence:

[0172] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK (SEQ ID NO: 319).

[0173] In one embodiment, the antibody or its antigen-binding fragment comprises a C sequence having at least 90% (including at least 91% to 100% and all integer percentages therebetween) sequence identity with the following sequences. L amino acid sequence:

[0174] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 320).

[0175] Representative antigen-binding molecules covered by this disclosure include full-length immunoglobulins and antigen-binding fragments, including recombinant antigen-binding molecules, which may be monovalent or multivalent, and monospecific or multispecific.

[0176] In one embodiment, the antibody or its antigen-binding fragment is a full-length antibody, a substantially complete antibody, a Fab fragment, scFab, Fab', a single-chain variable fragment (scFv), or a single-arm antibody.

[0177] In one embodiment, the antibody has an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In one embodiment, the antibody is an IgG1 antibody. The antibody may have antibody-dependent cell-mediated cytotoxicity (ADCC) activity and may induce NK cell killing. The heavy chain constant region may be a wild-type human Fc region or a human Fc region containing one or more amino acid substitutions. The antibody may have mutations that stabilize the disulfide bonds between the two heavy chains of the immunoglobulin, such as mutations in the IgG4 hinge region, as disclosed in the prior art (e.g., Angal et al., 1993. Mol. Immunol., 30:105-08). See also, for example, US 2005 / 0037000. The heavy chain constant region may also have substitutions that alter the properties of the antigen-binding molecule (e.g., reducing one or more of the following: Fc receptor binding, antigen-binding molecule glycosylation, deamidation, complement binding, or methionine oxidation). In some cases, antigen-binding molecules may have mutations, such as those described in U.S. Patents 5,624,821 and 5,648,260. In some embodiments, antigen-binding molecules are modified to reduce or eliminate effector function.

[0178] In one embodiment, the antigen-binding molecule of the present invention is a monovalent antigen-binding molecule. A non-limiting monovalent antigen-binding molecule comprises: V L V H C L and C H Fab fragments composed of 1 structural domain; composed of V L V H C L and C H 1. Structural domains and C HFab' fragments, consisting of a portion of a 2-structural domain; composed of V H and C H Fd fragments composed of 1 structural domain; V-arms of the antibody L and V H Fv fragments composed of structural domains; single-chain antibody molecules (e.g., scFab and scFv); single-domain antibody (dAb) fragments (Ward et al., 1989 Nature 341:544-546), which consist of V H Domain composition; and single-arm antibodies (e.g., described in US20080063641 (Genentech)) or other monovalent antibodies (e.g., described in WO2007048037 (Amgen)).

[0179] In one embodiment, the monovalent antigen-binding molecule comprises an Fv fragment. The Fv fragment is the smallest unit of an immunoglobulin molecule with antigen-binding activity. The antigen-binding molecule in the form of scFv (single-chain variable fragment) consists of a heavy (V) H ) chain and light (V L The variable regions of the chain are linked together by flexible peptide linkers, which can be easily expressed in functional form in expression hosts such as E. coli and mammalian cells, allowing protein engineering to improve scFv properties, such as increasing affinity and altering specificity (Ahmed et al., 2012). Clin Dev Immunol. 2012:980250). Representative examples of connector sequences are described in Section 4.5 below. In scFv construction, the order of domains can be V H -Connector-V L or V L -Connector-V H And it can be applied in both directions.

[0180] In some implementations, the linker sequence used in scFv is a multimer of the pentapeptide GGGGS [SEQ ID NO: 66] (or G4S or Gly4Ser). These linkers include the 15-mer (G4S)3 (Huston et al., 1988). Proc Natl Acad Sci USA.85(16), 5879–83), 18-mer GGSSRSSSSGGGGSGGGG [SEQ ID NO: 67] (Andris-Widhopf et al., “Generation of human scFv antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences.” ColdSpring Harbor Protocols, 2011(9)) and 20-mer (G4S)4 (Schaefer et al., “Construction of scFv Fragments from Hybridoma or Spleen Cells by PCR Assembly.” In Antibody Engineering, R. Kontermann and S. Dübel, Springer Verlag, Heidelberg, Germany (2010) pp. 21–44). Many other sequences have been proposed (often in the context of a specific antibody sequence), including sequences with additional functionality, such as epitope tags or coding sequences containing Cre-Lox recombination sites or sequences that improve scFv properties.

[0181] As described (Schaefer et al., 2010, see above), cloning of scFv is typically accomplished using a two-step overlap PCR method (also known as overlap extension splicing or SOE-PCR). First, the V... H and V L The domains are amplified and gel purified, and then a secondary assembly is performed in a single step of assembly PCR. Adapters are generated by overlapping two inner primers or by adding adapter primers whose sequences cover the entire adapter (three-fragment assembly PCR).

[0182] With a suitable expression vector containing appropriate translation and transcription start sites and signal peptide sequences (in the case of mammalian expression), cloning the protein-coding sequence of scFv can recombinantly generate single-chain Fv (scFv) antigen-binding molecules in, for example, E. coli, insect cells, or mammalian host cells.

[0183] In one embodiment, the monovalent antigen-binding molecule comprises a Fab fragment. In an illustrative example of this type, the monovalent antigen-binding molecule is a single-arm antibody consisting of, or substantially consisting of, a single antigen-binding fragment (Fab) and an Fc region, wherein the Fc region comprises first and second Fc polypeptides, and wherein the first and second Fc polypeptides are present in the complex.

[0184] Recombinant expression of Fc-containing monovalent antigen-binding molecules often leads to undesirable divalent homodimer contamination. Strategies to inhibit homodimer formation are known, including methods that introduce mutations into the immunoglobulin constant region to produce structural alterations that support unfavorable interactions between polypeptide chains and inhibit unwanted Fc homodimer formation. Non-limiting examples of such strategies promoting heterodimerization include introducing knock-in-hole (KIH) structures into both polypeptides, and utilizing C... L and C H 1. The naturally occurring heterodimerization of the structural domain (see, Kontermann, above, 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; as described in WO 2005 / 063816). These KIH mutations promote heterodimerization of handle-containing Fc and pore-containing heavy chains, thereby improving the assembly of monovalent antibodies and reducing the level of unwanted divalent antibodies.

[0185] It may also be necessary to modify the Fc domain of antigen-binding molecules to reduce Fc receptor binding and thus decrease the likelihood of FcγRIIa-mediated platelet activation. For example, the so-called “LALA” double mutation (Leu234Ala and Leu235Ala together) in human IgG (including IgG1) is known to significantly impair Fc receptor binding and effector function (Lund et al., 1991). J. Immunol 147, 2657–2662; Lund et al. , 1992, Mol. Immunol. 29:53–59). For human IgG4, the engineered mutant S228P / L235E variant (SPLE) has previously been shown to have minimal FcγR binding (Newman et al., 2001). Clin. Immunol.(98, 164–174). Mutations in the Fc domain of IgG1 or IgG4 can be combined, for example, combining the LALA mutation in human IgG1 with a mutation at P329G, or combining the SPLE mutation in human IgG4 with a mutation at P329G, completely eliminating the FcγR and C1q interaction (Schlothauer et al., 2016). Protein Eng Des. Sel. 29, 457-466).

[0186] In one embodiment, each IgG1 Fc chain of the antibody in the antigen-binding molecule (e.g., MAb or its antigen-binding fragment) carries a P329G, L235A, L234A (P329G LALA) mutation or each IgG4 Fc chain carries a P329G, S228P, L235E mutation to reduce or eliminate any undesirable cross-linking, platelet activation, or immune effector functions of the antigen-binding molecule (e.g., antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC)).

[0187] In one embodiment, each IgG1 Fc chain of the antigen-binding molecule (or antibody) carries mutations, including a) S239D, A330L, and I332E, or b) F243L, R292P, Y300L, V305I, and P396L, which enhance the immune effector function of the antigen-binding molecule (e.g., ADCC).

[0188] In one embodiment, the antigen-binding molecule (or antibody) comprises a CH2-CH3 sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 321, SEQ ID NO: 322 or SEQ ID NO: 323.

[0189] Antigen-binding molecules may contain heavy chain sequences. Heavy chain sequences may, for example, include or constitute the V sequences listed in Table 3. H Sequence, the V H Sequence and C H 1 sequence (e.g., SEQ ID NO: 319) and C H 2-C H 3. Sequence fusion (e.g., SEQ ID NO: 321, SEQ ID NO: 322, or SEQ ID NO: 33). For example, the heavy chain sequence may contain an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 326, SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 330, SEQ ID NO: 332, or SEQ ID NO: 333.

[0190] Antigen-binding molecules may contain light chain sequences. These light chain sequences may, for example, include or constitute the V sequences listed in Table 3. L Sequence, the V L The sequence is directly fused with the CL sequence (e.g., SEQ ID NO: 320). For example, the light chain sequence may contain an amino acid sequence that has at least 70% sequence identity with SEQ ID NO: 328, SEQ ID NO: 331, or SEQ ID NO: 334.

[0191] In one embodiment, the invention covers a monovalent antigen-binding molecule generated by co-expressing a light chain, a heavy chain, and a truncated Fc domain. Suitably, the heavy chain contains a pore mutation and a P329G LALA mutation, while the truncated Fc domain contains a handle mutation and a P329G LALA mutation.

[0192] With a suitable expression vector containing appropriate translation and transcription start sites and (where appropriate) signal peptide sequences, the protein-coding sequence of the cloned construct can be used to express the antigen-binding molecules disclosed herein in host cells such as bacteria (e.g., Escherichia coli), yeast, insects, or mammals.

[0193] In one embodiment, the antigen-binding molecule is a multivalent antigen-binding molecule, and non-limiting examples include: immunoglobulins, F(ab')2, tandem scFv (taFv or scFv2), scFv-Fc, biantibodies, and dAb2 / V. H H2, microantibodies, ZIP microantibodies, barnase-barstar dimers, hand-access pore derivatives, SEED-IgG, heterologous Fc-scFv, Fab-scFv, (Fab)2 / sc(Fab)2, scFv-(TNFα)3, scFv-Jun / Fos, Fab′-Jun / Fos, tribody, trimerbody, tribi-minibody, barnase-barstar trimer, collabody, DNL-F(ab)3, scFv3-C H 1 / C L , Fab-scFv2, IgG-scFab, IgG-scFv, scFv-IgG, scFv2-Fc, F(ab′)2-scFv2, scDB-Fc, scDb-C H3. Db-Fc, scFv2-H / L, DVD-Ig, tandAb, scFv-dhlx-scFv, dAb2-IgG, dAb-IgG, dAb-Fc-dAb, tetrabody, streptabody (scFv-streptavidin)4, (scFv-p53)4, [sc(Fv)2]2, tandem double antibodies (tandab) and their combinations.

[0194] In one embodiment, the multivalent antigen-binding molecule is selected from the following molecules: IgG-like antibodies (e.g., triomab / tetra-hybridoma, Trion Pharma / Fresenius Biotech; hand-accessor, Genentech; CrossMAbs, Roche; electrostatic pairing antibody, AMGEN; LUZ-Y, Genentech; chain-exchange engineered domain (SEED) variant, EMD Serono; biolonic, Merus; and Fab exchange antibody, Genmab), symmetric IgG-like antibodies (e.g., dual-target (DT)-Ig, GSK / Domantis; dual-antibody, Genentech; cross-linked MAb, Karmanos Cancer Center; MAb2, F-star; and Coy X variant, Coy X / Pfizer), IgG fusions (e.g., dual variable domain (DVD)-Ig, Abbott; IgG-like bispecific antibody, Eli Lilly; Ts2Ab, Medimmune / AZ; BsAb, ZymoGenetics; HERCULES, Biogen Idec; TvAb (Roche), Fc fusions (e.g., scFv / Fc fusion, Academic Institution; SCORPION, Emergent BioSolutions / Trubion, ZymoGenetics / BMS; Fc-DART, MacroGenics; Bis(ScFv)2-Fab, National Research Center for Antibody Medicine), Fab fusions (e.g., F(ab)2, Medarex / AMGEN; Bis-acting or Bis-Fab, Genentech; Dock and Lock (DNL), ImmunoMedics; Bivalent Bispecific, Biotechnol; and Fab-Fv, UCB-Celltech), and ScFv- and bispecific antibodies (e.g., Bispecific T-cell adaptors (BiTEs), Micromet; Tandem bispecific antibodies (Tandab), Affimed; DARTs, MacroGenics; Single-chain bispecific antibodies, Academic; TCR-like antibodies, AIT, Receptor Logics; Human serum albumin scFv fusion, Merrimack;Combodies (Epogen Biotech), IgG / non-IgG fusions (e.g., immune cytokines, EMDSerono, Philogen, ImmunGene, ImmunoMedics; superantigen fusion proteins, Active Biotech; and immune-mobilizing anticancer mTCRs, ImmTAC), and oligoclonal antibodies (e.g., Symptomogen and Merus).

[0195] In one embodiment, the antibody is a bispecific or trispecific antibody. In one embodiment, the antibody is a bispecific antibody. The bispecific antibody may be an antibody comprising a first antigen-binding site that specifically binds to ALPPL2 and a second antigen-binding site that specifically binds to CD3. In one embodiment, the bispecific antibody is capable of binding to cancer cells and recruiting immune effector cells (e.g., T cells) to kill cancer cells. Antigen-binding peptides that specifically bind to CD3 are well known in the art. The second antigen-binding site may, for example, comprise a CD3-specific CDR sequence or a VH / VL sequence from muromonab (Orthoclone OKT3), foralumab, teplizumab, blinatumomab, or visilizumab. The bispecific antibody may, for example, comprise the VH CDR sequence of SEQ ID NO: 335-337 and the VL CDR sequence of SEQ ID NO: 338-340. Alternatively, the antibody may comprise the VH CDR sequence of SEQ ID NO: 341-343 and the VL CDR sequence of SEQ ID NO: 344-346.

[0196] In one embodiment, the bispecific antibody of the present invention is formed using a “bump-in-lumen” strategy, also known as a “handle-in-pore” strategy, which involves designing an interface between the first and second peptides for heteromeric oligomerization. The preferred interface comprises at least a portion of the CH3 domain of the antibody’s constant structural domain. It has been reported that “handle-in-pore” mutations in the CH3 domain of the Fc sequence significantly reduce homodimer formation (see, for example, Merchant et al., 1998, Nature Biotechnology, 16:677-681). The “bump” is constructed by replacing small amino acid side chains at the interface of the first peptide with larger amino acid side chains (e.g., tyrosine or tryptophan). Optionally, a compensatory “lumen” of the same or similar size as the bump is created at the interface of the second peptide by replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine). When a properly positioned and sized bump or lumen is present at the interface of the first or second peptide, it is only necessary to design the corresponding lumen or bump at adjacent interfaces, respectively. The protrusions and lumens can be created through synthetic means, such as altering the nucleic acid encoding the polypeptide or through peptide synthesis. For a further description of the hand access hole, see U.S. Patents 5,731,168; 5,807,706; and 5,821,333.

[0197] A general method for preparing heteropolymers using a "protrusion into lumen" strategy involves expressing a first polynucleotide encoding a first polypeptide and a second polynucleotide encoding a second polypeptide in one or separate host cells. The first polynucleotide has been modified from the original polynucleotide to encode a protrusion, and the second polynucleotide has been modified from the original polynucleotide to encode a lumen. The polypeptide is expressed in a common host cell, and the heteropolymer is recovered from the host cell culture, or expressed in separate host cells, recovered, and purified, and then the heteropolymer is formed. In some embodiments, the resulting heteropolymer is a multimeric antibody, such as a bispecific antibody.

[0198] chimeric molecules

[0199] This article discloses chimeric molecules that include antigen-binding molecules and heterologous portions as defined herein.

[0200] In one implementation, the heterogeneous portion is a detectable portion, a half-life extension portion, or a therapeutic portion.

[0201] The detectable portion covered by this invention includes any type known in the art suitable for diagnostic testing (including in vitro testing and in vivo imaging). For example, the detectable portion can be a fluorophore, a radionuclide reporter, metal-containing nanoparticles or microparticles, an ultrasound contrast agent (e.g., nanobubble-type or microbubble-type), or an optical imaging dye. This also includes contrast particles visible in magnetic resonance imaging (MRI) and magnetic particle imaging (MPI). For example, fluorophores can be detected and / or imaged by fluorescence polarization, fluorescence-activated cell sorting, and fluorescence microscopy, which may or may not be combined with electrospray ionization-mass spectrometry (ESI-MS) detection and fluorescence emission computed tomography (FLECT) imaging. Radionuclide reporters can be detected and imaged by radionuclide (nuclear) detection, such as single-photon emission computed tomography (SPECT), positron emission tomography (PET), or scintillation imaging. Optical imaging can be used to detect metal-containing nanoparticles or microparticles, including MRI, which is typically used with paramagnetic nanoparticles or microparticles, and MPI, which is typically used with superparamagnetic particles. Ultrasound imaging, including contrast-enhanced ultrasound (CEU), can be used to detect ultrasound contrast agents.

[0202] Detectable markers can also be enzyme-substrate markers. Enzymes typically catalyze chemical changes in chromogenic substrates, which can be measured using a variety of techniques. For example, the enzyme can catalyze a chemical change in a chromogenic substrate, which can be measured using a variety of techniques. For example, the enzyme can catalyze a color change in the substrate, which can be measured spectrophotometrically. Alternatively, the enzyme can alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying fluorescence changes are described above. Chemiluminescent substrates are converted to an electronically excited state through a chemical reaction, and can then emit measurable light (e.g., using a chemiluminescence meter) or provide energy to a fluorescent acceptor. Examples of enzymatic markers include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as unease and xanthine oxidase), lactoperoxidase, microperoxidase, etc.

[0203] Examples of enzyme-substrate combinations include, for example:

[0204] 1) Horseradish peroxidase (HRPO) uses hydrogen peroxide to oxidize dye precursors (e.g., o-phenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB));

[0205] 2) Alkaline phosphatase (AP), using p-nitrophenyl phosphate as the chromogenic substrate; and

[0206] 3) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-β-D-galactosidase) or a fluorescent substrate 4-methylumbelliferyl-β-D-galactoside.

[0207] In another embodiment of the invention, labeling of the antigen-binding molecule is not required; its presence can be detected using a labeled antibody bound to the antigen-binding molecule. The antigen-binding molecule of the present invention can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, immunohistochemistry, and immunoprecipitation assays.

[0208] In one embodiment, the chimeric molecule comprises at least one heterologous portion, namely a "half-life extension portion". The half-life extension portion may include, for example, (i) an XTEN polypeptide; (ii) an Fc region; (iii) albumin; (iv) an albumin-binding polypeptide or fatty acid; (v) the C-terminal peptide (CTP) of the 13 subunit of human chorionic gonadotropin; (vi) PAS; (vii) HAP; (viii) transferrin; (ix) polyethylene glycol (PEG); (x) hydroxyethyl starch (HES); (xi) polysialic acid (PSA); (xii) a scavenging receptor or fragment thereof that blocks the binding of the chimeric molecule to a scavenging receptor; (xiii) a low-complexity peptide; (xiv) or any combination thereof. In some embodiments, the half-life extension portion comprises an Fc region. In other embodiments, the half-life extension portion comprises two Fc regions fused together via a linker. Exemplary heterologous portions also include, for example, FcRn-binding portions (e.g., an intact Fc region or a portion thereof bound to FcRn), single-chain Fc regions (scFc regions, such as those described in U.S. Patent Publication No. 20080260738, WO 2008 / 012543, and WO 2008 / 1439545), or processable scFc regions. In some embodiments, the heterologous portion may include attachment sites for a non-peptide portion, such as polyethylene glycol (PEG), hydroxyethyl starch (HES), polysialic acid, or any derivative, variant, or combination of these portions.

[0209] In some embodiments, at least one heterologous component is a therapeutic component. In some embodiments, the therapeutic component is selected from anticancer components (e.g., cell-inhibiting / toxic, and / or antiproliferative drugs), immunotherapeutic components, and anti-inflammatory components. In some embodiments, the therapeutic agent can be used to treat cancer. Useful anticancer agents include chemotherapy agents, representative examples of which include anti-microtubule agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, mono-, bis-, and tri-platinum complexes, and carboplatin), anthracyclines, antibiotics, antifolate agents, antimetabolites, calmodulin inhibitors, chemosensitizers, pyromalacin, etoposide, fluorinated pyrimidines, ionophores, lexitropsin, maytansine alkaloids, nitrosourea, platinol, pore-forming compounds, purine antimetabolites, puromycin, radiosensitizers, rapamycin, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, etc.

[0210] In one implementation, the treatment component is olistatin, such as monomethylolistatin F (MMAF) or monomethylolistatin E (MMAE).

[0211] In one implementation, the antigen-binding molecule is linked to the treatment portion via a valine-citrulline (Vc) linker.

[0212] Polynucleotides, constructs and host cells

[0213] This article discloses an isolated polynucleotide comprising a nucleic acid sequence encoding an antigen-binding molecule or a chimeric molecule as defined herein.

[0214] The terms “polynucleotide” or “nucleic acid” are used interchangeably herein and refer to a polymer of nucleotides, which may be mRNA, RNA, cRNA, cDNA, or DNA. The term generally refers to a polymer of nucleotides with a length of at least 10 bases, and nucleotides may be ribonucleotides, deoxynucleotides, or modified forms of any type of nucleotide. The term includes both single-stranded and double-stranded forms of DNA.

[0215] This article also discloses vectors containing nucleic acids encoding antigen-binding molecules as described herein.

[0216] The term "vector" refers to a nucleic acid molecule, preferably a DNA molecule derived from, for example, plasmids, bacteriophages, or viruses, into which a nucleic acid sequence can be inserted or cloned. The vector preferably contains one or more unique restriction sites and is capable of autonomous replication in a defined host cell (including target cells or their tissues, or progenitor cells or their tissues), or can integrate with the defined host genome such that the cloned sequence is reproducible. Accordingly, the vector can be a self-replicating vector, i.e., a vector existing as an extrachromosomal entity whose replication is independent of chromosome replication, such as a linear or closed circular plasmid, extrachromosomal element, microchromosome, or artificial chromosome. The vector can contain any components used to ensure self-replication. Alternatively, the vector can be one that, when introduced into a host cell, integrates into the genome and replicates along with the integrated chromosome. A vector system can contain a single vector or plasmid, two or more vectors or plasmids (which together contain the complete DNA of the host cell genome to be introduced), or transposons. The choice of vector typically depends on the compatibility of the vector with the host cell into which it will be introduced. The vector may also include selection markers, such as antibiotic resistance genes, which can be used to select suitable transformants. Examples of such resistance genes are well known to those skilled in the art.

[0217] This paper discloses a construct comprising a nucleic acid sequence encoding an antigen-binding molecule or a chimeric molecule as defined herein, the nucleic acid sequence being operatively linked to one or more regulatory sequences.

[0218] The term "construction" refers to a recombinant gene molecule comprising one or more isolated nucleic acid sequences from different sources. Thus, a construct is a chimeric molecule in which two or more nucleic acid sequences from different sources are assembled into a single nucleic acid molecule, and includes any construct containing: (1) nucleic acid sequences comprising regulatory and coding sequences not found together in nature (i.e., at least one nucleotide sequence is heterologous relative to at least one other nucleotide sequence); (2) sequences encoding non-naturally adjacent functional RNA molecules or protein portions; or (3) non-naturally adjacent promoter portions. Representative constructs include any recombinant nucleic acid molecule, such as plasmids, granules, viruses, autonomously replicating polynucleotide molecules, bacteriophages, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecules derived from any source, capable of genome integration or autonomous replication, comprising nucleic acid molecules in which one or more nucleic acid molecules have been operatively linked. The constructs of the present invention typically include the necessary elements for guiding the expression of a nucleic acid sequence of interest (e.g., a target nucleic acid sequence or a regulatory nucleic acid sequence) also contained in the construct. Such elements may include regulatory elements or regulatory sequences, such as a promoter operatively linked to a nucleic acid sequence of interest (to direct transcription of that nucleic acid sequence), and often also include a polyadenylation sequence. In some embodiments of the invention, the construct may be contained within a vector. In addition to the components of the construct, the vector may also include, for example, one or more selectable markers, one or more origins of replication (e.g., prokaryotic and eukaryotic origins), at least one multiple cloning site, and / or elements that facilitate stable integration of the construct into the host cell genome. Two or more constructs may be contained in a single nucleic acid molecule, such as a single vector, or may be contained in two or more separate nucleic acid molecules, such as two or more separate vectors. An “expression construct” typically includes at least one regulatory sequence operatively linked to a nucleotide sequence of interest. In this way, for example, a promoter operatively linked to the nucleotide sequence to be expressed is provided in the expression construct for expression in an organism or a portion thereof, including a host cell. For the purpose of carrying out this invention, conventional compositions and methods for preparing and using constructs and host cells are well known to those skilled in the art, see, for example, Molecular Cloning: A Laboratory Manual, 3rd Edition, Volumes 1, 2 and 3, JF Sambrook, DW Russell and N. Irwin, ColdSpring Harbor Laboratory Press, 2000.

[0219] As used in this article, "regulatory elements," "regulatory sequences," and "regulatory sequences" refer to nucleic acid sequences (such as DNA) necessary for the expression of operablely linked coding sequences in a specific host cell. For example, regulatory sequences applicable to prokaryotic cells include promoters and optional cis-acting sequences, such as operon sequences and ribosome binding sites. Regulatory sequences applicable to eukaryotic cells include transcriptional regulatory sequences such as promoters, polyadenylation signals, transcriptional enhancers, translational regulatory sequences such as translational enhancers and internal ribosome binding sites (IRES), nucleic acid sequences regulating mRNA stability, and guide sequences that direct the polynucleotide-encoded products of transcription into intracellular compartments or the extracellular environment.

[0220] This paper discloses a host cell that contains constructs as defined herein.

[0221] The terms “host,” “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including progeny cells of this type. Host cells include “transformants” and “transformed cells,” which include primary transformed cells and their derived progeny, regardless of passage number. The nucleic acid contents of the progeny may not be identical to those of the parent cells and may contain mutations. This document includes mutant progeny that have the same function or biological activity as those screened or selected in the original transformed cells. Host cells are any type of cell system that can be used to produce the antigen-binding molecules of this invention. Host cells include cultured cells, such as mammalian cultured cells (e.g., CHO cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells), yeast cells, insect cells, and plant cells, as well as cells contained in transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0222] Pharmaceutical Composition

[0223] This article discloses a pharmaceutical composition comprising an antigen-binding molecule or a chimeric molecule as defined herein, and a pharmaceutically acceptable carrier.

[0224] A "pharmaceutically acceptable carrier" refers to a drug delivery system composed of a non-biologically or otherwise undesirable substance that can be administered to a subject along with a selected active agent without causing any or substantial adverse reactions. Carriers can include excipients and other additives such as diluents, detergents, colorants, wetting agents or emulsifiers, pH buffers, preservatives, etc.

[0225] Representative pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delay agents, salts, preservatives, pharmaceuticals, pharmaceutical stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and similar substances and combinations thereof (see, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Unless any conventional carrier is incompatible with the active ingredient, its use in pharmaceutical compositions is covered.

[0226] Pharmaceutical compositions can be in various forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), dispersants or suspensions, liposomes, and suppositories. Preferred dosage forms depend on the intended route of administration and therapeutic application. Suitable pharmaceutical compositions can be administered intravenously, subcutaneously, or intramuscularly. In some embodiments, the composition is present in an injectable or infusionable solution dosage form. Preferred routes of administration are parenteral (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular). In certain embodiments, the pharmaceutical composition is administered by intravenous infusion or injection. In other embodiments, the pharmaceutical composition is administered by intramuscular or subcutaneous injection.

[0227] As used herein, the phrases “parenteral administration” and “administered via parenteral administration” mean administration by means other than enteral and local administration, usually by injection, and including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intra-bursal, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions.

[0228] Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffer media. In this invention, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1 M, preferably 0.05 M phosphate buffer or 0.8% saline. Other common parenteral media include sodium phosphate solutions, Ringer's glucose, glucose and sodium chloride, lactated Ringer's solution, or non-volatile oils. Intravenous media include fluids and nutritional supplements, electrolyte supplements, such as Ringer's glucose-based supplements. Preservatives and other adjuvants may also be present, such as antimicrobial agents, antioxidants, chelating agents, and inert gases.

[0229] More specifically, pharmaceutical compositions suitable for injection include sterile aqueous solutions (if water-soluble) or dispersants, and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersants. In this case, the composition must be sterile and should be fluid to achieve a degree of injectability. It should be stable under manufacturing and storage conditions and preferably preserved to prevent contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof. For example, suitable flowability can be maintained by using a coating such as lecithin and / or by maintaining the desired particle size. In certain embodiments, the pharmaceutical agents of this disclosure can be conjugated with a medium for cell delivery. In these embodiments, the pharmaceutical agent can be encapsulated in a suitable medium to aid in delivery to target cells, to improve the stability of the pharmaceutical agent, or to minimize the potential toxicity of the pharmaceutical agent. As those skilled in the art will understand, a variety of mediums are suitable for delivering the pharmaceutical agents of this disclosure. Non-limiting examples of suitable structured fluid delivery systems may include nanoparticles, liposomes, microemulsions, micelles, dendritic polymers, and other phospholipid-containing systems. Methods for incorporating the pharmaceutical agents of this disclosure into delivery media are known in the art. Although various embodiments are given below, it should be understood that other methods known in the art for incorporating antigen-binding molecules as described herein into delivery media are also included.

[0230] The dosing regimen can be adjusted to provide the best expected response (e.g., therapeutic response). For example, a single bolus injection can be administered, multiple fractionated doses can be administered over a period of time, or the dose can be proportionally reduced or increased as indicated by the urgency of the treatment situation. The antigen-binding molecule of this disclosure can be administered on various occasions. The interval between single doses can be daily, weekly, monthly, or yearly. The interval can also be irregular, as indicated by measuring the blood level of the modified peptide or antigen in the patient. Alternatively, the antigen-binding molecule can be administered as a sustained-release formulation, in which case the frequency of administration needs to be reduced. The dose and frequency vary based on the half-life of the peptide in the patient.

[0231] For ease of administration and uniformity of dosage, it may be advantageous to formulate the composition in unit dosage form. As used herein, “unit dosage form” refers to a physically discrete unit suitable as a unit dose for a subject to be treated; each unit contains a predetermined amount of the active compound, which, when combined with a pharmaceutically acceptable carrier, produces the desired therapeutic effect. The specifications of the unit dosage form of the present invention depend on and are directly dependent on: (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations of the technology for formulating such active compound, for treatment sensitivity in an individual.

[0232] The dosage and treatment regimen of the antigen-binding molecule can be determined by a skilled technician. In some embodiments, the antigen-binding molecule is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 0.01 to 50 mg / kg, such as 0.01 to 0.1 mg / kg, such as about 0.1 to 1 mg / kg, about 1 to 5 mg / kg, about 5 to 25 mg / kg, or about 10 to 50 mg / kg. Dosage regimens can range from, for example, once weekly to once every 2, 3, or 4 weeks.

[0233] It should be noted that dosage values ​​may vary depending on the type and severity of the condition to be alleviated. It should also be understood that, for any given subject, the specific dosage regimen should be adjusted over time based on individual needs and the professional judgment of the person administering or supervising the application of the composition, and the dosage ranges set forth herein are merely exemplary and not intended to limit the scope or practice of the claimed compositions.

[0234] Treatment

[0235] This article provides a method for reducing the expression or activity of ALPPL2 in cells, such as cancer cells. The method involves contacting the cancer cells with an antigen-binding molecule, chimeric molecule, polynucleotide, construct, vector, host cell, or pharmaceutical composition as defined herein.

[0236] This article discloses a method for reducing the expression or activity of ALPPL2 in cancer cells, the method comprising contacting cancer cells with an antigen-binding molecule as defined herein or a chimeric molecule as defined herein.

[0237] As used herein, the term "tumor" refers to any malignant or benign growth and proliferation of cellular cells, as well as all precancerous and cancerous cells and tissues. The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals, typically characterized in part by uncontrolled cell growth. As used herein, the term "cancer" refers to both non-metastatic and metastatic cancer, including early and late-stage cancer. The term "precancerous" refers to a condition or growth that typically precedes or develops into cancer. "Non-metastatic" means a benign cancer or cancer that remains at the primary site and has not spread to the lymphatic or vascular system or tissues outside the primary site. Generally, non-metastatic cancer refers to any stage 0, I, or II cancer, and sometimes stage III cancer. "Early-stage cancer" refers to non-invasive or non-metastatic cancer, or cancer classified as stage 0, I, or II cancer. The term "late-stage cancer" usually refers to stage III or IV cancer, but can also refer to stage II cancer or a substage of stage II cancer. Those skilled in the art will understand that classifying stage II cancer as early-stage or late-stage depends on the specific type of cancer. Illustrative examples of cancer include, but are not limited to, breast cancer, prostate 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, kidney cancer, carcinoma, melanoma, brain cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, endometrial cancer, multiple myeloma, rectal cancer, mesothelioma, endometrial cancer, and esophageal cancer. In one exemplary embodiment, the cancer is colorectal cancer, endometrial cancer, gastric cancer, mesothelioma, ovarian cancer, pancreatic cancer, or testicular cancer.

[0238] This article provides a method for reducing or inhibiting the proliferation, survival, and viability of tumors in a subject, the method comprising administering to the subject an antigen-binding molecule, chimeric molecule, polynucleotide, construct, vector, host cell, or pharmaceutical composition as defined herein.

[0239] This article discloses a method for reducing or inhibiting the proliferation, survival, and viability of tumors in subjects, the method comprising administering to subjects an antigen-binding molecule or a chimeric molecule as defined herein.

[0240] The term "patient" includes human and other mammalian subjects receiving preventative or therapeutic treatment. As used herein, the term "subject" includes any human or non-human animal. For example, the method of the present invention can be used to treat a subject suffering from cancer. In one embodiment, the subject is a human. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cattle, chickens, amphibians, reptiles, etc. For example, the method of the present invention can be used to treat a subject suffering from cancer. In one embodiment, the subject is a human. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cattle, chickens, amphibians, reptiles, etc.

[0241] The methods disclosed herein may include administering a “therapeuticly effective amount” of a pharmaceutical agent (e.g., an antigen-binding molecule, chimeric molecule, polynucleotide, construct, vector, host cell, or pharmaceutical composition) to a subject. As used herein, the term “therapeuticly effective amount” includes, within its meaning, a non-toxic but sufficient amount of a pharmaceutical agent or compound to provide the desired therapeutic effect. The exact amount required will vary from subject to subject to a variety of factors, such as the species being treated, the subject’s age and general condition, the severity of the condition being treated, the specific pharmaceutical agent administered, and the manner of administration. Therefore, a precise “effective amount” cannot be specified. However, for any given situation, a person skilled in the art can determine the appropriate “effective amount” simply through routine experiments.

[0242] In one embodiment, a method for treating cancer in a subject is provided, wherein the method includes administering to the subject an antigen-binding molecule, chimeric molecule, polynucleotide, construct, vector, host cell, or pharmaceutical composition as defined herein.

[0243] This article discloses a method for treating cancer in a subject, wherein the method includes administering an antigen-binding molecule or a chimeric molecule as defined herein to the subject.

[0244] As used herein, the term “treatment / management” can refer to (1) preventing or delaying the onset of one or more symptoms of a disease; (2) suppressing the development of a disease or one or more symptoms of a disease; (3) alleviating a disease, i.e., the disappearance of at least one or more symptoms of a disease; and / or (4) reducing the severity of one or more symptoms of a disease.

[0245] In one implementation plan, the cancer is stomach cancer, ovarian cancer, or pancreatic cancer.

[0246] This article discloses antigen-binding molecules, chimeric molecules, polynucleotides, constructs, vectors, host cells, or pharmaceutical compositions as defined herein for use as medicines.

[0247] This article discloses antigen-binding molecules or chimeric molecules as defined herein for the treatment of cancer.

[0248] This article discloses the use of antigen-binding molecules, chimeric molecules, polynucleotides, constructs, vectors, host cells, or pharmaceutical compositions in the preparation of medicaments for the treatment of subjects in need. Subjects may be those with cancer.

[0249] This article discloses the use of antigen-binding molecules or chimeric molecules as defined herein in the preparation of pharmaceutical agents for the treatment of cancer.

[0250] This article discloses a method for treating a condition or disease associated with undesirable ALPPL2 expression in a subject, wherein the method includes administering to the subject an antigen-binding molecule, chimeric molecule, polynucleotide, construct, vector, host cell, or pharmaceutical composition as defined herein.

[0251] This article discloses a method for treating a condition or disease associated with undesirable ALPPL2 expression in a subject, wherein the method includes administering an antigen-binding molecule or a chimeric molecule as defined herein to the subject.

[0252] In one implementation, the symptom or condition associated with undesirable ALPPL2 expression is cancer.

[0253] In one implementation plan, the cancer is a solid tumor.

[0254] In one implementation plan, the cancer is cervical cancer, colon cancer, endometrial cancer, stomach cancer, ovarian cancer, or pancreatic cancer.

[0255] Reagent test kit

[0256] This article discloses a kit for detecting cancer, which contains an antigen-binding molecule or chimeric molecule as defined herein.

[0257] Diagnostic methods

[0258] This article discloses a method for determining the likelihood of a subject having cancer, wherein the method includes detecting ALPPL2 in a sample obtained from the subject, wherein an increase in the level of ALPPL2 in the sample compared to a reference indicates the likelihood of the subject having cancer.

[0259] In one implementation, the method includes detecting ALPPL2 with an antigen-binding molecule or a chimeric molecule as defined herein.

[0260] This article discloses a method for treating cancer in subjects, wherein the method includes a) detecting ALPPL2 in a sample obtained from the subject, wherein an increase in ALPPL2 level in the sample, compared to a reference, indicates an increased likelihood of the subject having cancer; and b) treating the subject for whom an increased likelihood of having cancer was found.

[0261] In one implementation, the method includes detecting ALPPL2 with an antigen-binding molecule or a chimeric molecule as defined herein.

[0262] In one implementation, the method includes treating a subject with an antigen-binding molecule or a chimeric molecule as defined herein.

[0263] This article discloses a method for identifying subjects who may respond to treatment with an anti-ALPPL2 antibody, the method comprising detecting ALPPL2 in a sample obtained from the subject, wherein an increase in ALPPL2 levels indicates that the subject may respond to treatment with an ALPPL2 antibody.

[0264] In one implementation, the method includes detecting ALPPL2 with an antigen-binding molecule or a chimeric molecule as defined herein.

[0265] This article discloses a method for identifying and treating subjects who may respond to treatment with an anti-ALPPL2 antibody, the method comprising a) detecting ALPPL2 in a sample obtained from the subject, wherein an increase in ALPPL2 levels indicates that the subject may respond to treatment with an ALPPL2 antibody; and b) treating the subject found to be likely to respond to treatment with an ALPPL2 antibody.

[0266] As used in this article, “and / or” refers to and covers any and all possible combinations of one or more of the related listed items, as well as any missing combinations when interpreted as alternatives (or).

[0267] As used herein, the singular forms “a,” “an,” and “the” include plural references unless explicitly stated in the context. For example, the term “a pharmaceutical agent” includes multiple pharmaceutical agents, including mixtures thereof.

[0268] The term "approximately" means that a quantity, level, value, number, frequency, percentage, size, size, quantity, weight, or length differs from a reference quantity, level, value, number, frequency, percentage, size, size, quantity, weight, or length by as much as 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0269] Unless the context otherwise requires, throughout this specification and in the following statements, the word “comprise” and some variations such as “comprises” and “comprising” shall be understood to mean including the said integer or step or group of integers or steps, but not excluding any other integer or step or group of integers or steps.

[0270] References to any prior publications (or information derived therefrom) or any known content in this specification are not and should not be construed as an endorsement or acknowledgment or in any way implying that such prior publications (or information derived therefrom) or known content constitute part of the general knowledge of the field of study covered by this specification.

[0271] Those skilled in the art will understand that, apart from the specific descriptions, the invention described herein is susceptible to variations and modifications. It should be understood that the invention includes all such variations and modifications falling within its spirit and scope. The invention also includes all steps, features, compositions, and compounds individually or collectively mentioned or indicated in this specification, as well as any and all combinations of any two or more of the stated steps or features.

[0272] Some embodiments of the present invention will now be described with reference to the following examples, which are for illustrative purposes only and are not intended to limit the general scope described above.

[0273] Example

[0274] Target ID and background

[0275] Gastric cancer is a common disease in East Asia, with 79% of patients diagnosed at stage IV and a five-year survival rate of less than 5%. A novel cell surface biomarker, ALPPL2, has been identified as a target for therapeutic antibodies and complementary diagnostics. Biomarker identification was performed on RNA sequencing data from 19 gastric cancer patients through rigorous bioinformatics analysis.

[0276] ALPPL2 protein expression was validated in six gastric cancer cell lines using a commercially available anti-ALPPL2 antibody via immunohistochemical staining. Strong membrane staining was observed in gastric cancer cell lines overexpressing ALPPL2 mRNA, while no significant staining was observed in cell lines not expressing ALPPL2 transcripts. Clinical prevalence was also assessed by immunohistochemical staining of two types of gastric tumor microarrays. A total of 198 tumor cores from different stages of the disease and different regions of the stomach were stained. The results showed that ALPPL2 membrane staining was observed in 32 of the 198 cases, accounting for 16%. No significant membrane staining was observed in adjacent matched or mismatched normal tissues.

[0277] Antibody production

[0278] Antibodies against human ALPPL2 were generated by immunizing rabbits with an antigen. Rabbit antibodies were isolated by directly cloning the antibody gene from a single rabbit B cell.

[0279] During the screening process, clones that bind to ALPPL2 but not to the associated ALPI expressed in normal intestinal tissue were selected. Figure 1 and 2 A total of 36 clones with high affinity for human ALPP / ALPPL2 were isolated. The amino acid sequences of the variable region and complementarity-determining region are shown in Tables 1 and 2.

[0280] Affinity and specificity

[0281] Screening and identification of specific clones using ELISA and high-throughput flow cytometry. Figure 3 Rabbit kidney cells were transfected with truncated (for ELISA screening) or full-length (for FACS screening) ALPI and ALPPL2.

[0282] Further screening identified a subset of clones specific to ALPPL2 / ALPP but not to ALPI, which were then used for affinity measurements using biomembrane interferometry. Figure 3 In this technique, single-concentration supernatant clones from rabbit B cells are immobilized on a protein A biosensor. The biosensor is then incubated with the analyte to measure affinity.

[0283] A comparable humanized monoclonal antibody disclosed in the prior art was engineered by grafting a CDR onto the same backbone, and its binding to both ALPPL2 and ALPI was evaluated by ELISA. This comparable humanized monoclonal antibody possesses the following V... H and V L sequence:

[0284]

[0285] The synthesized gene was cloned into an expression vector to produce recombinant antibodies. The surface plasmon resonance data in Figure 4 show that antibodies disclosed in the prior art exhibit non-specific binding to ALPI, while the antibodies of this invention do not.

[0286] Immunohistochemical (IHC) activity

[0287] To enable the development of complementary diagnostics, the IHC activity of the antibodies was evaluated (Figure 5). Detection was achieved via antigen retrieval through proteinase K digestion, rather than heat-mediated antigen retrieval. IHC detection of ALPPL2+ve cell line (SCH) and formalin-fixed paraffin-embedded (FFPE) human tumor tissues was achieved using C36, C45, and C130. This indicates that these antibodies have diagnostic applications in patient stratification and therapeutic applications in treating ALPPL2 / ALPP+ve tumors, including gastric cancer, ovarian cancer, colorectal cancer, pancreatic cancer, endometrial cancer, mesothelioma, and testicular cancer. C36 showed negative staining in all normal tissues except the placenta, indicating no / low expression of ALPPL / ALPP in normal tissues. This also suggests the optimal therapeutic window for these antibodies in clinical practice.

[0288] Cross-reactivity in non-human primates (NHP)

[0289] The cross-reactivity of the antibody with non-human primate orthologs was further evaluated. Figure 6 The selected clones showed responsiveness to rhesus monkey orthologs.

[0290] Humanized cloning (affinity, selectivity, and specificity)

[0291] Selected clones (C4, C15, C131, C12, C18, C36, and C53) were humanized by transplanting CDRs into the human IgG1 backbone. Surface plasmon resonance (SPR) analysis showed that these humanized clones retained high ALPP / ALPPL2 affinity (Figure 7). SPR was investigated using a Biacore T200. Ligands (e.g., ALPPL2 or ALPP) were immobilized on a CM5 biosensor chip and captured with streptavidin. The ligand-loaded sensor was then incubated with different concentrations of analytes (recombinantly expressed humanized antibody clones) to measure affinity.

[0292] Humanized clones (C4, C36, and C53) maintained selectivity for cancer-specific ALPPL2 and / or ALPP, but not for widely expressed ALPI and ALPL. Figure 13 Humanized clones are also specific for cancer cells, but not for normal, immature, or stimulated immune cells. Figure 13 ).

[0293] Humanized clone (ADCC)

[0294] Antibody-dependent cytotoxicity was first assessed by co-culturing primary NK cells with cancer cell lines using reporter drugs or primary NK cells, thereby testing the therapeutic efficacy of humanized clones. Figure 8Compared to other clones, C4 generated the most potent ADCC induction in two gastric cancer cell lines with high and low target expression. The potency of C4 was confirmed in co-culture assays of primary NK cells with different gastric cancer cell lines. C4 achieved near-complete killing of high-expressing cell lines and showed potency proportional to target expression levels (maximum % kill and EC50). Furthermore, C4 was tested in reporter assays and ADCC induction was confirmed in different ovarian and pancreatic cancer cell lines.

[0295] Further modification of the Fc region of humanized C4 was performed to enhance ADCC. The reported assays further confirmed that humanized C4 with engineered Fc more potently induced ADCC in gastric cancer cell lines.

[0296] Humanized clone (ADC)

[0297] The suitability of using these humanized antibodies as antibody-drug conjugates was evaluated. First, the killing effect of these antibodies on gastric cancer cell lines was tested in the presence of secondary antibodies conjugated with VC-MMAF. Figure 9 Secondary ADC assays revealed some potency differences between clones; cytotoxicity was observed only in selected gastric cancer cell lines with high target expression, but not in other cell lines. These results were confirmed using the same antibody conjugated to vc-MMAE. Primary conjugation of the antibody to vc-MMAE enhanced the cytotoxicity observed in the secondary ADC assays against the same gastric cancer cell lines.

[0298] Humanized clones (T-cell adaptors)

[0299] These humanized antibodies are further illustrated by their successful adaptation as T-cell adaptors to induce potent T-cell-mediated killing of cancer cells. Figure 10 , Figure 11 (See Figure 12). Through humanized cloning and heterodimerization of the anti-CD3 antibody, the bispecific antibody achieved potent and specific killing of gastric cancer, ovarian cancer, and pancreatic cancer cell lines, regardless of target expression levels. In particular, C4 achieved almost complete killing of multiple cancer cell lines at pM concentrations.

[0300] ALPPL2-specific clones

[0301] Chimeric and humanized C53 clones exhibited binding specificity for ALPPL2 but not ALPP (Figure 12). Chimeric C53 also showed cross-reactivity with rhesus monkey orthologs. The binding affinity of humanized C53 and humanized C36 for ALPPL and ALPP was compared using biomembrane interferometry. In this technique, biotinylated ligands (i.e., ALPPL2 or ALPP) were immobilized on an SA biosensor. The ligand-loaded sensor was then incubated with different concentrations of analytes (recombinantly expressed humanized antibody clones) in buffer. Biomembrane interferometry determined that the humanized C53 antibody exhibited nM-level binding affinity for ALPPL but not for ALPP, while the humanized C36 showed similar binding affinity for both ALPPL2 and ALPP.

[0302] This further illustrates that the humanized C53 antibody retains its cytotoxic efficacy in ADCC and is suitable as a T-cell adaptor to induce potent T-cell-mediated killing of cancer cells.

Claims

1. An antigen-binding molecule that specifically binds to ALPPL2 but not ALPP, ALPL, or ALPI, comprising: a) Heavy chain variable region (V) containing SEQ ID NO: 309, SEQ ID NO: 310 and SEQ ID NO: 311 H ), and the light chain variable region (V) containing SEQ ID NO: 312, SEQ ID NO: 313 and SEQ ID NO:

314. L ); b) The heavy chain variable region (VH) containing the amino acid sequences of SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33, and the light chain variable region (VL) containing the amino acid sequences of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36; c) The heavy chain variable region (VH) containing the amino acid sequences of SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69, and the light chain variable region (VL) containing the amino acid sequences of SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72; d) The heavy chain variable region (VH) containing the amino acid sequences of SEQ ID NO: 85, SEQ ID NO: 86 and SEQ ID NO: 87, and the light chain variable region (VL) containing the amino acid sequences of SEQ ID NO: 88, SEQ ID NO: 89 and SEQ ID NO: 90; e) The heavy chain variable region (VH) containing the amino acid sequences of SEQ ID NO: 127, SEQ ID NO: 128 and SEQ ID NO: 129, and the light chain variable region (VL) containing the amino acid sequences of SEQ ID NO: 130, SEQ ID NO: 131 and SEQ ID NO: 132; f) The heavy chain variable region (VH) containing the amino acid sequences of SEQ ID NO: 133, SEQ ID NO: 134 and SEQ ID NO: 135, and the light chain variable region (VL) containing the amino acid sequences of SEQ ID NO: 136, SEQ ID NO: 137 and SEQ ID NO: 138; g) The heavy chain variable region (VH) containing the amino acid sequences of SEQ ID NO: 169, SEQ ID NO: 170, and SEQ ID NO: 171, and the light chain variable region (VL) containing the amino acid sequences of SEQ ID NO: 172, SEQ ID NO: 173, and SEQ ID NO: 174; or h) The heavy chain variable region (VH) containing the amino acid sequences of SEQ ID NO: 205, SEQ ID NO: 206 and SEQ ID NO: 207, and the light chain variable region (VL) containing the amino acid sequences of SEQ ID NO: 208, SEQ ID NO: 209 and SEQ ID NO:

210.

2. The antigen-binding molecule of claim 1, wherein the antigen-binding molecule specifically binds to ALPPL2 or cells expressing ALPPL2 with an affinity of 14 pM to 10 nM.

3. The antigen-binding molecule of claim 1, wherein the antigen-binding molecule specifically binds to rhesus monkey ALPPL2 orthologs.

4. The antigen-binding molecule of claim 1, wherein the antigen-binding molecule comprises: a) a heavy chain variable region (V0) comprising SEQ ID NO:309, SEQ ID NO:310 and SEQ ID NO:

311. H b) contains the light chain variable region (V) of SEQ ID NO: 312, SEQ ID NO: 313 and SEQ ID NO:

314. L ).

5. The antigen-binding molecule of claim 1, wherein the antigen-binding molecule comprises: a) V H amino acid sequence and V L amino acid sequence, the V H The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages therein) sequence identity with SEQ ID NO: 317, said V L The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages in between) sequence identity with SEQ ID NO: 318; b) V H amino acid sequence and V L amino acid sequence, the V H The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages therein) sequence identity with SEQ ID NO: 221, said V L The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages in between) sequence identity with SEQ ID NO: 222; c) V H amino acid sequence and V L amino acid sequence, the V H The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages therein) sequence identity with SEQ ID NO: 233, wherein V L The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages therein) sequence identity with SEQ ID NO: 234; d) V H amino acid sequence and V L amino acid sequence, the V H The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages therein) sequence identity with SEQ ID NO: 239, wherein V L The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages in between) sequence identity with SEQ ID NO: 240; e) V H amino acid sequence and V L amino acid sequence, the V H The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages therein) sequence identity with SEQ ID NO: 253, wherein V L The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages therein) sequence identity with SEQ ID NO: 254; f) V H amino acid sequence and V L amino acid sequence, the V H The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages therein) sequence identity with SEQ ID NO: 255, wherein V L The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages in between) sequence identity with SEQ ID NO: 256; g) V H amino acid sequence and V L amino acid sequence, the V H The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages therein) sequence identity with SEQ ID NO: 267, said V L The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages therein) sequence identity with SEQ ID NO: 268; or h) V H amino acid sequence and V L amino acid sequence, the V H The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages therein) sequence identity with SEQ ID NO: 279, wherein V L The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages in between) sequence identity with SEQ ID NO:

280.

6. The antigen-binding molecule of claim 1, wherein the antigen-binding molecule comprises: a) The VH amino acid sequence, which has at least 90% (including at least 91% to 100% and all integer percentages therein) sequence identity with SEQ ID NO: 317, and b) The VL amino acid sequence having at least 90% (including at least 91% to 100% and all integer percentages in between) sequence identity with SEQ ID NO:

318.

7. An antigen-binding molecule that specifically binds to ALPPL2 and ALPP but not ALPL or ALPI, comprising: i) Heavy chain variable regions (V) containing SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 H ), and the light chain variable region (V) containing SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO:

6. L ); ii) Heavy chain variable region (V) containing SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21 H ), and the light chain variable region (V) containing SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO:

24. L ); iii) Heavy chain variable region (V) containing SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 H ), and the light chain variable region (V) containing SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO:

42. L ); iv) Heavy chain variable region (V) containing SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51 H ), and the light chain variable region (V) containing SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO:

54. L ); v) The heavy chain variable region (V) containing SEQ ID NO: 91, SEQ ID NO: 92 and SEQ ID NO: 93 H ), and the light chain variable region (V) containing SEQ ID NO: 94, SEQ ID NO: 95 and SEQ ID NO:

96. L ); vi) The heavy chain variable region (V) containing SEQ ID NO: 181, SEQ ID NO: 182 and SEQ ID NO: 183 H ), and the light chain variable region (V) containing SEQ ID NO: 184, SEQ ID NO: 185 and SEQ ID NO:

186. L ).

8. The antigen-binding molecule of claim 7, wherein the antigen-binding molecule specifically binds to ALPPL2 and ALPP or cells expressing ALPPL2 or ALPP with an affinity of about 14 pM to about 10 nM.

9. The antigen-binding molecule of claim 7, wherein the antigen-binding molecule specifically binds to rhesus monkey ALPPL2 / ALPP orthologs.

10. The antigen-binding molecule of claim 7, wherein the antigen-binding molecule comprises: a) V H amino acid sequence and V L amino acid sequence, the V H The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages therein) sequence identity with SEQ ID NO: 281, wherein V L The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages in between) sequence identity with SEQ ID NO: 282; b) V H amino acid sequence and V L amino acid sequence, the V H The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages therein) sequence identity with SEQ ID NO: 283, wherein V L The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages therein) sequence identity with SEQ ID NO: 284; c) V H amino acid sequence and V L amino acid sequence, the V H The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages therein) sequence identity with SEQ ID NO: 285, wherein V L The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages in between) sequence identity with SEQ ID NO: 286; d) V H amino acid sequence and V L amino acid sequence, the V H The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages therein) sequence identity with SEQ ID NO: 287, wherein V L The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages in between) sequence identity with SEQ ID NO: 288; e) V H amino acid sequence and V L amino acid sequence, the V H The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages therein) sequence identity with SEQ ID NO: 289, wherein V L The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages therein) sequence identity with SEQ ID NO: 290; f) V H amino acid sequence and V L amino acid sequence, the V H The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages therein) sequence identity with SEQ ID NO: 293, wherein V L The amino acid sequence has at least 90% (including at least 91% to 100% and all integer percentages in between) sequence identity with SEQ ID NO:

294.

11. The antigen-binding molecule of claim 7, wherein the antigen-binding molecule is an antibody or its antigen-binding fragment or a chimeric antigen receptor (CAR).

12. The antigen-binding molecule of claim 11, wherein the antibody or its antigen-binding fragment is humanized.

13. The antigen-binding molecule of claim 11, wherein the antibody or its antigen-binding fragment is a full-length antibody, a substantially complete antibody, a Fab fragment, scFab, Fab', a single-chain variable fragment (scFv), or a single-arm antibody.

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

15. The antigen-binding molecule of claim 14, wherein the bispecific antibody comprises a first antigen-binding site that specifically binds to ALPPL2 and a second antigen-binding site that specifically binds to CD3.

16. A chimeric molecule comprising an antigen-binding molecule according to any one of claims 1 to 15 and a heterologous portion.

17. The chimeric molecule of claim 16, wherein the heterologous portion is a detectable portion, a half-life extended portion, or a therapeutic portion.

18. The chimeric molecule of claim 17, wherein the therapeutic portion is monomethylolpropamine F (MMAF) or monomethylolpropamine E (MMAE).

19. An isolated polynucleotide comprising a nucleic acid sequence encoding an antigen-binding molecule according to any one of claims 1 to 15.

20. A construct comprising a nucleic acid sequence encoding an antigen-binding molecule according to any one of claims 1 to 15, said nucleic acid sequence being operatively linked to one or more regulatory sequences.

21. A host cell comprising the construct of claim 20.

22. A pharmaceutical composition comprising an antigen-binding molecule according to any one of claims 1 to 15, or a chimeric molecule according to any one of claims 16 to 18, and a pharmaceutically acceptable carrier.

23. The antigen-binding molecule according to any one of claims 1 to 15 or the chimeric molecule according to any one of claims 16 to 18, for the treatment of cancer.

24. Use of the antigen-binding molecule according to any one of claims 1 to 15 or the chimeric molecule according to any one of claims 16 to 18 in the preparation of a medicament for treating cancer.

25. The antigen-binding molecule used according to claim 23 or the use according to claim 24, wherein the cancer is colorectal cancer, endometrial cancer, gastric cancer, mesothelioma, ovarian cancer, pancreatic cancer, or testicular cancer.

26. Use of the antigen-binding molecule according to any one of claims 1 to 15 or the chimeric molecule according to any one of claims 16 to 18 in the preparation of a medicament for treating a disease or condition in a subject associated with undesirable ALPPL2 expression.

27. The use as claimed in claim 26, wherein the disease or condition is cancer.

28. The use according to claim 27, wherein the cancer is colorectal cancer, endometrial cancer, gastric cancer, mesothelioma, ovarian cancer, pancreatic cancer, or testicular cancer.

29. A kit for detecting cancer, the kit comprising an antigen-binding molecule according to any one of claims 1 to 15 or a chimeric molecule according to any one of claims 16 to 18.

30. A method for determining the likelihood of a subject having cancer, wherein the method includes detecting ALPPL2 in a sample obtained from the subject, wherein an increase in the level of ALPPL2 in the sample compared to a reference indicates the likelihood of the subject having cancer, wherein the method includes detecting ALPPL2 with an antigen-binding molecule according to any one of claims 1 to 15 or a chimeric molecule according to any one of claims 16 to 18.

31. A method for identifying a subject who may respond to treatment with an anti-ALPPL2 antibody, the method comprising detecting ALPPL2 in a sample obtained from the subject, wherein an increase in ALPPL2 levels indicates that the subject may respond to treatment with an ALPPL2 antibody, wherein the method comprises detecting ALPPL2 with an antigen-binding molecule according to any one of claims 1 to 15 or a chimeric molecule according to any one of claims 16 to 18.

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