ALPP-specific variant antigen-binding molecule

Abstract

The present invention relates to placental alkaline phosphatase (ALPP) and / or germline alkaline phosphatase (ALPPL2)-specific antigen-binding molecules, related fusion proteins, conjugates, and methods for producing the same. In a further aspect, the present invention relates to conjugated single domain antibodies (VHH domains).

Description

[Technical Field]

[0001] The present invention relates to placental alkaline phosphatase (ALPP) and / or germline alkaline phosphatase (ALPPL2)-specific antigen-binding molecules, related fusion proteins, conjugates, and methods for producing the same. In a further aspect, the present invention relates to conjugated single domain antibodies (VHH domains). [Background technology]

[0002] Alkaline phosphatase is a dimeric metalloenzyme found in a wide range of organisms that catalyzes the hydrolysis of phosphomonoesters (Le Du et al., J. Bio Chem. 2001, 276, 9158-9165). In humans, there are four isozymes: placental (referred to herein as ALPP, but also known as PLAP), germline (referred to herein as ALPPL2, but also known as ALPG or GCAP), intestinal (ALPI), and tissue-nonspecific (referred to herein as ALPL, but also known as TNAP).

[0003] These four isozymes are all glycoproteins anchored to the cell surface via a C-terminal glycophosphatidylinositol post-translational modification. ALPP, ALPPL2, and ALPI are all located on chromosome 2, while ALPL is located on chromosome 1. ALPP and ALPPL2 share a high sequence identity of 98% and have similar expression profiles. They are expressed in the placenta but are rarely present in normal adult tissues. ALPI shares a relatively high identity of approximately 87% with ALPP / ALPPL2. This isoform is widely expressed throughout the intestinal tract and plays an important role in gastrointestinal mucosal defense. The fourth isozyme, ALPL, is widely expressed in many tissues, including bone, liver, and kidney. ALPL shares a lower identity of approximately 57% with ALPP / ALPPL2.

[0004] Upregulation of ALPP and ALPPL2 has been observed at the mRNA and protein levels in a wide range of cancers, including ovarian, endometrial, cervical, gastric, esophageal, bladder, lung, pancreatic, and testicular cancers (Reiswich et al., J of Pathol. Clin. Res., 2021, 7, 577-589). High levels of ALPP / ALPPL2 have also been linked to poor prognosis in gastric and ovarian cancers (Orsaria et al., Cancer Biomarkers, 2016, 17, 479-486; Liu et al., Human Pathol., 2019, 86, 49-56). Given their expression levels in cancer cells compared with their relative absence in normal tissues, ALPP / ALPPL2 is an attractive target for cancer therapy.

[0005] Antibodies targeting ALPP or ALPPL2 have been previously described in the literature (Ravenni et al., MAbs, 2014, 6(1), 86-94), and anti-ALPPL2 and anti-ALPP antibody-drug conjugates (ADCs) have been reported (Su et al., Cancer Res., 2020, 80, 4552-4564; WO 2017095823; WO 2022197890 (Seagen)). ALPP / ALPPL2 CAR-T therapy has also been described for use as immunotherapy for the treatment of colorectal and cervical cancer (Li et al., Front. Biosci. 2020, 25, 1765-1786; WO 2019240934 (Promab); Yekehfallah et al., Biomolecules, 2022, 12, 1296).

[0006] Single domain antibodies (sdAbs), derived from camelid heavy chain-only antibodies (HCAbs), are small proteins consisting of variable domains that can selectively bind antigens with high specificity and affinity. These VHHs have been the genesis of many therapeutic agents and have several advantages over classical antibodies.

[0007] The small size of VHHs compared to full-length mAbs (12-15 kDa compared to approximately 150 kDa) allows for greater tissue and tumor penetration, potentially resulting in greater efficacy. Another advantage of these small single domains is their relative simplicity. Unlike mAbs, VHH domains do not rely on heavy-light chain pairing. The resulting modularity and ease of manufacture make VHH domains an attractive platform for engineering proteins and protein-drug conjugates with improved therapeutic properties.

[0008] Conjugates of specific antigen-binding molecules are described in PCT / EP2020 / 067210, filed June 19, 2020, which is incorporated herein by reference in its entirety. PCT / EP2020 / 067210 describes anthracycline (PNU) derivatives suitable for use in drug conjugates. Specifically, derivatives of PNU159682 are provided that lack the C14 carbon and attached hydroxyl functionality, and an ethylenediamino (EDA) group forms part of the linker region between the C13 carbonyl of PNU159682 and the maleimide group. Alternatively, the same molecule can be described as EDA-PNU as a "warhead" so that the EDA group is not considered part of the linker region. When the linker contains val-cit-PAB, the maleimide group can be replaced with any reactive group suitable for conjugation reactions. Such payloads can react with free thiol groups on another molecule. When free thiols are present on a protein, protein-drug conjugates (PDCs) can be formed.

[0009] The anthracycline derivative PNU-159682 has been described as a metabolite of nemorubicin (Quintieri et al. (2005) Clin. Cancer Res. 11, 1608-1617) and has been reported to exhibit extremely high potency in vitro cell killing in the picomolar to femtomolar range using one ovarian (A2780) and one breast cancer (MCF7) cell line (WO 2012 / 073217 A1). Derivatives of PNU-159682 are also described in WO 2016 / 102679.

[0010] Conjugation of PNU-159682 derivatives to antibodies is described in WO 2009 / 099741, WO 2016 / 127081 and WO 2016 / 102679, Yu et al, Clin. Cancer Res 2015, 21, 3298 and Stefan et al, Mol. Cancer. Ther., 2017, 16, 879.

[0011] Auristatin E (AE) and monomethylauristatin E (MMAE) are synthetic analogs of dolastatins, a unique group of linear pseudopeptides originally isolated from marine sources, some of which have highly potent cytotoxic activity against tumor cells. However, MMAE has the disadvantage of relatively high systemic toxicity. To improve tumor selectivity, MMAE is used in combination with an enzymatically cleavable valine-citrulline linker, particularly in ADC settings for more targeted tumor therapy (see, e.g., WO 2005 / 081711). After proteolytic cleavage, MMAE is preferably released intracellularly from the corresponding ADC. Monomethylauristatin F (MMAF) is an auristatin derivative with a C-terminal phenylalanine moiety. MMAF and various ester and amide derivatives thereof are disclosed in WO 2005 / 081711. Additional auristatin analogs having a C-terminal amide-substituted phenylalanine unit are described in WO 01 / 18032. WO 02 / 088172 and WO 2007 / 008603 claim MMAF analogs involving side chain modifications of phenylalanine, and WO 2007 / 008848 claims modifications of the carboxyl group of phenylalanine. Auristatin conjugates linked via the C-terminus are described in WO 2009 / 117531, and further conjugates are described in WO 2013 / 087716.

[0012] Described herein are ALPP and / or ALPPL2-specific variant antigen-binding molecules and their conjugates to MMAE and PNU-159682 derivatives that have advantageous properties. Summary of the Invention [Means for solving the problem]

[0013] The present invention relates generally to specific antigen-binding molecules.

[0014] According to one aspect, the present invention provides a compound of formula (I): FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4 (I) (In the formula, FW1 is the framework region, CDR1 is the CDR sequence, FW2 is the framework region, CDR2 is a CDR sequence, FW3 is the framework area, CDR3 is a CDR sequence, FW4 is the framework area) The present invention provides an antigen-binding molecule specific for placental alkaline phosphatase (ALPP) and / or germline alkaline phosphatase (ALPPL2), comprising an amino acid sequence represented by:

[0015] According to a further aspect, the present invention provides a compound of formula (I): FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4 (I) (In the formula, FW1 is the framework region, CDR1 is a CDR sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 15, FW2 is the framework region, CDR2 is a CDR sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 10, FW3 is the framework area, CDR3 is a CDR sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5, FW4 is the framework area) The present invention provides an antigen-binding molecule specific for placental alkaline phosphatase (ALPP) and / or germline alkaline phosphatase (ALPPL2), comprising an amino acid sequence represented by:

[0016] According to a further aspect, the present invention provides a recombinant fusion protein comprising a specific antigen-binding molecule disclosed herein.

[0017] According to a further aspect, the present invention provides a compound of formula (I): FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4 (I) (In the formula, FW1 is the framework region, CDR1 is the CDR sequence, FW2 is the framework region, CDR2 is a CDR sequence, FW3 is the framework area, CDR3 is a CDR sequence, FW4 is the framework area) or a functional variant thereof, wherein the antigen-binding molecule is fused to a fragment of an immunoglobulin Fc region, and the fragment of the immunoglobulin Fc region is engineered to dimerize with a second fragment of the immunoglobulin Fc region.

[0018] According to a further aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) a first recombinant fusion protein, the first recombinant fusion protein being a recombinant fusion protein disclosed herein; and (b) a second recombinant fusion protein comprising a second antigen-binding molecule fused to a second fragment of an immunoglobulin Fc region that is engineered to dimerize with a first fragment of an immunoglobulin Fc region. The present invention provides a recombinant fusion protein dimer comprising:

[0019] According to a further aspect, the present invention provides an ALPP- and / or ALPPL2-specific chimeric antigen receptor (CAR) comprising at least one ALPP- and / or ALPPL2-specific antigen binding molecule disclosed herein fused or conjugated to at least one transmembrane region and at least one intracellular domain.

[0020] The present invention also provides a cell comprising a chimeric antigen receptor disclosed herein, which is preferably an engineered T cell.

[0021] In a further aspect of the present invention, there is provided a nucleic acid sequence comprising a polynucleotide sequence encoding a specific antigen-binding molecule, recombinant fusion protein, recombinant fusion protein dimer, or chimeric antigen receptor disclosed herein.

[0022] Also provided are vectors containing the nucleic acid sequences disclosed herein and host cells containing such nucleic acids.

[0023] Provided is a method for preparing a specific antigen-binding molecule, recombinant fusion protein, recombinant fusion protein dimer, or chimeric antigen receptor disclosed herein, comprising culturing or maintaining a host cell containing the polynucleotide or vector under conditions such that the host cell produces the specific antigen-binding molecule, recombinant fusion protein, or chimeric antigen receptor, and optionally further comprising isolating the specific antigen-binding molecule, recombinant fusion protein, recombinant fusion protein dimer, or chimeric antigen receptor.

[0024] In a further aspect of the present invention, there is provided a pharmaceutical composition comprising the specific antigen-binding molecule, fusion protein, recombinant fusion protein dimer, or chimeric antigen receptor disclosed herein. The pharmaceutical composition may contain various pharmaceutically acceptable carriers. The pharmaceutical composition of the present invention may be for administration by any suitable method known in the art, including, but not limited to, intravenous, intramuscular, oral, intraperitoneal, or topical administration. In a preferred embodiment, the pharmaceutical composition may be prepared in the form of a liquid, gel, powder, tablet, capsule, or foam.

[0025] The specific antigen-binding molecules, recombinant fusion proteins, recombinant fusion protein dimers, and chimeric antigen receptors disclosed herein may be used in therapeutic methods. More specifically, the specific antigen-binding molecules, recombinant fusion proteins, recombinant fusion protein dimers, and chimeric antigen receptors disclosed herein may be used in the treatment of cancer. Preferably, the cancer is an ALPP- and / or ALPPL2-positive cancer. More preferably, the cancer is selected from the group consisting of ovarian cancer, endometrial cancer, cervical cancer, gastric cancer, esophageal cancer, bladder cancer, lung cancer, pancreatic cancer, and testicular cancer.

[0026] Also provided herein is the use of a specific antigen-binding molecule, recombinant fusion protein, recombinant fusion protein dimer, or chimeric antigen receptor disclosed herein in the manufacture of a medicament for treating a disease in a patient in need thereof.

[0027] The specific antigen-binding molecules, recombinant fusion proteins, recombinant fusion protein dimers, or chimeric antigen receptors disclosed herein, or pharmaceutical compositions disclosed herein, can be administered in a single dose. As used herein, "single dose" refers to a dosage regimen consisting of one dose. Alternatively, a multiple-dose regimen can be used. Without being bound by theory, the advantages of the specific binding molecules, recombinant fusion proteins, recombinant fusion protein dimers, or chimeric antigen receptors, or pharmaceutical compositions disclosed herein can be particularly apparent when administered in a single dose.

[0028] Furthermore, the present invention provides a method of treating a disease in a patient in need thereof, comprising administering to the patient a therapeutically effective dose of a specific antigen-binding molecule, recombinant fusion protein, recombinant fusion protein dimer, or chimeric antigen receptor disclosed herein, or a pharmaceutical composition disclosed herein.

[0029] Preferably, the cancer is an ALPP and / or ALPPL2-positive cancer type. More preferably, the cancer is selected from the group consisting of ovarian cancer, endometrial cancer, cervical cancer, gastric cancer, esophageal cancer, bladder cancer, lung cancer, pancreatic cancer and testicular cancer.

[0030] Also provided herein is a method of assaying for the presence of a target analyte in a sample, the method comprising adding to the sample a detectably labeled specific antigen-binding molecule disclosed herein, or a recombinant fusion protein disclosed herein, or a recombinant fusion protein dimer disclosed herein, and detecting binding of the molecule to the target analyte.

[0031] Further provided herein is a method of imaging a site of disease in a subject, the method comprising administering a detectably labeled specific antigen-binding molecule disclosed herein, or a detectably labeled recombinant fusion protein disclosed herein, or a recombinant fusion protein dimer disclosed herein.

[0032] Also provided herein is a method of diagnosing a disease or medical condition in a subject, the method comprising administering a specific antigen-binding molecule disclosed herein, or a recombinant fusion protein disclosed herein, or a recombinant fusion protein dimer disclosed herein.

[0033] Also contemplated herein are antibodies, antibody fragments, or antigen-binding molecules that compete with the ALPP- and / or ALPPL2-specific antigen-binding molecules disclosed herein for binding to ALPP and / or ALPPL2. The term "compete" when used in connection with an antigen-binding protein (e.g., a neutralizing antigen-binding protein or neutralizing antibody) means competition between antigen-binding proteins as determined by an assay in which the antigen-binding protein under test (e.g., an antibody or functional fragment thereof) interferes with or inhibits the specific binding of an antigen-binding molecule defined herein (e.g., the specific antigen-binding molecule of the first aspect) to a common antigen (e.g., ALPP and / or ALPPL2 in the case of the specific antigen-binding molecules disclosed herein).

[0034] Also described herein is a kit for diagnosing a subject suffering from or susceptible to cancer or providing a prognosis for a subject's condition, the kit comprising a detection means for detecting the concentration of an antigen present in a sample from a test subject, the detection means comprising an ALPP- and / or ALPPL2-specific antigen-binding molecule disclosed herein, a recombinant fusion protein disclosed herein, a recombinant fusion protein dimer disclosed herein, a chimeric antigen receptor disclosed herein, or a nucleic acid sequence disclosed herein, each optionally derivatized, wherein the presence of the antigen in the sample indicates that the subject is suffering from cancer. Preferably, the antigen comprises an ALPP- and / or ALPPL2 protein, more preferably an extracellular domain thereof. More preferably, the kit is used to identify the presence or absence of ALPP- and / or ALPPL2-positive cells in a sample or to determine their concentration in a sample. The kit may also include a positive control and / or a negative control against which the assay is compared and / or a detectable label.

[0035] The present invention also provides a method for diagnosing a subject suffering from or susceptibility to cancer or for providing a prognosis of a condition in a subject, the method comprising detecting the concentration of an antigen present in a sample obtained from the subject, wherein the detection is achieved using the ALPP- and / or ALPPL2-specific antigen binding molecule of the first or second aspect, the recombinant fusion protein of the third or fourth aspect, or the recombinant fusion protein dimer of the fifth aspect, the chimeric antigen receptor of the sixth aspect, or the nucleic acid sequence of the seventh aspect, each optionally derivatized, and wherein the presence of the antigen in the sample is indicative of the subject being suffering from cancer.

[0036] Also contemplated herein is a method for killing or inhibiting the growth of cells expressing ALPP and / or ALPPL2 in vitro or in a patient, comprising administering to the cells a pharmaceutically effective amount or dose of (i) an ALPP- and / or ALPPL2-specific antigen-binding molecule disclosed herein, a recombinant fusion protein disclosed herein, or a recombinant fusion protein dimer disclosed herein, a nucleic acid sequence disclosed herein, or a CAR or cell disclosed herein, or (ii) a pharmaceutical composition disclosed herein. Preferably, the cells expressing ALPP and / or ALPPL2 are cancer cells. More preferably, the ALPP and / or ALPPL2 are human ALPP and / or ALPPL2.

[0037] According to a further aspect, the present invention provides a compound of formula (II): X-FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4-Y (II) (In the formula, FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4 is an ALPP and / or ALPPL2-specific antigen-binding molecule disclosed herein, X and Y are optional amino acid sequences. wherein the specific antigen-binding molecule comprises an amino acid sequence represented by:

[0038] According to a further aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) an ALPP and / or ALPPL2-specific antigen-binding molecule, recombinant fusion protein, or recombinant fusion protein dimer disclosed herein; (b) at least one cytotoxic or cytostatic agent; The present invention provides a target binding molecule-drug conjugate comprising:

[0039] According to a further embodiment, (b) is an MMAE derivative; The target binding molecule-drug conjugate has the formula (VI): [ka] [X] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof; [L1] and [L2] are optional linkers selected from the group consisting of valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), peptide, dipeptide, tripeptide, -(CH2)n-, -(CH2CHO)n-, p-aminobenzyloxycarbonyl (PAB), Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, Val-Ala, Asn-Ala, any amino acid, a D-amino acid, Phe-Lys-PAB, and combinations thereof; and Y comprises an ALPP and / or ALPPL2-specific antigen-binding molecule or recombinant fusion protein disclosed herein. It has the following structure.

[0040] According to a further aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) an ALPP and / or ALPPL2-specific antigen-binding molecule, recombinant fusion protein, or recombinant fusion protein dimer disclosed herein; (b) anthracycline (PNU) derivatives and 1. A target binding molecule-drug conjugate comprising the formula (III): [ka] wherein [X] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof; [L1] and [L2] are valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), peptide, -(CH2)n -, -(CH2CH2O) n an optional linker selected from the group consisting of -, p-aminobenzyloxycarbonyl (PAB), Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, Val-Ala, Asn-Ala, any amino acid except glycine, and combinations thereof; and Y comprises an ALPP and / or ALPPL2-specific antigen-binding molecule, recombinant fusion protein, or recombinant fusion protein dimer disclosed herein. The present invention provides a target-binding molecule-drug conjugate having the structure:

[0041] According to a further aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) an ALPP and / or ALPPL2-specific antigen-binding molecule, recombinant fusion protein, or recombinant fusion protein dimer disclosed herein; (b) anthracycline (PNU) derivatives and 1. A target binding molecule-drug conjugate comprising formula (IV): [ka] wherein [X] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof; [Z] is a linker derived from a reactive group used to conjugate the anthracycline (PNU) derivative with the target binding molecule, and Y comprises an ALPP and / or ALPPL2-specific antigen-binding molecule, recombinant fusion protein, or recombinant fusion protein dimer disclosed herein. The present invention provides a target-binding molecule-drug conjugate having the structure:

[0042] According to a further aspect, the present invention provides an engineered ALPP and / or ALPPL2 monomer that has been engineered so as to be capable of forming a covalent bond with a further engineered ALPP and / or ALPPL2 monomer.

[0043] According to a further aspect, the present invention provides an artificially engineered ALPP and / or ALPPL2 homodimer comprising a first and a second artificially engineered ALPP and / or ALPPL2 monomer as disclosed herein.

[0044] According to a further aspect, the present invention provides a method for producing an ALPP and / or ALPPL2 specific binding molecule comprising the steps of: (a) immunizing an animal with an engineered ALPP and / or ALPPL2 homodimer disclosed herein; (b) isolating peripheral blood mononuclear cells (PBMCs) from the animal; (c) cloning RNA or cDNA sequences obtained from PBMCs into a vector; (d) transforming a prokaryotic cell with the vector described above under conditions that allow the production of the specific binding molecule; (e) selecting specific binding molecules by subjecting the specific binding molecules to antigen affinity selection; (f) recovering specific binding molecules having the desired specificity. The present invention provides a method comprising:

[0045] In a further aspect, the present invention provides biparatopic placental alkaline phosphatase (ALPP) and / or germline alkaline phosphatase (ALPPL2)-specific antigen-binding molecules.

[0046] According to a further aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) a biparatopic placental alkaline phosphatase (ALPP) and / or germline alkaline phosphatase (ALPPL2)-specific antigen-binding molecule; (b) at least one toxin, cytotoxic agent, or cytostatic agent; The present invention provides a biparatopic target binding molecule-drug conjugate comprising: [Brief explanation of the drawings]

[0047] [Figure 1] Cell surface binding of ALPP / ALPPL2 VHH domain (His6 tag) to CHO cells stably transfected with ALPP, ALPPL2, ALPI, or ALPL by flow cytometry. [Figure 2] Human IgG sequence used in IgG Fc fusion proteins. Additionally, the engineered hIgG1 Fc fusion proteins incorporate engineered cysteine ​​substitutions in the hIgG1 Fc sequence, for example, at positions S239C or S442C or both (EU numbering), to allow for site-specific labeling. [Figure 3] Cell surface binding of ALPP / ALPPL2 VHH-hFc fusion proteins to CHO cells stably transfected with ALPP, ALPPL2, ALPI, or ALPL by flow cytometry. [Figure 4] Structure of MC-vc-PAB-MMAE and structures of PNU linker payloads MA-PEG-vc-PAB-EDA-PNU159682 and MA-PEG-va-EDA-PNU159682. [Figure 5] In vitro efficacy of ALPP / ALPPL2 hFc PNU drug conjugates in killing CHO cells stably transfected with ALPP, ALPPL2, ALPI or ALPL. [Figure 6] In vitro efficacy of ALPP / ALPPL2 hFc PNU and ALPP / ALPPL2 hFc MMAE drug conjugates in killing ALPP / ALPPL2-expressing cancer cell lines. [Figure 7] Cell surface binding of biparatopic ALPP / ALPPL2 VHH-hFc drug conjugate (vc PAB-MMAE) to CHO cells stably transfected with ALPP, ALPPL2, ALPI, or ALPL by flow cytometry. [Figure 8]In vitro efficacy of biparatopic ALPP / ALPPL2 hFc MMAE drug conjugates in killing CHO cells stably transfected with ALPP, ALPPL2, ALPI, or ALPL. [Figure 9] In vitro efficacy of biparatopic ALPP / ALPPL2 hFc MMAE drug conjugates and MMAE payloads in killing ALPP / ALPPL2-expressing cancer cell lines and HeLa cancer cells that express little to no ALPP / ALPPL2. [Figure 10] In vitro efficacy of biparatopic ALPP / ALPPL2 hFc PNU drug conjugates in killing CHO cells stably transfected with ALPP, ALPPL2, ALPI, or ALPL. [Figure 11] In vitro efficacy of biparatopic ALPP / ALPPL2 hFc PNU drug conjugates in killing ALPP / ALPPL2-expressing cancer cell lines. [Figure 12] ALPP / ALPPL2 IHC staining of NCI-N87 (gastric cancer), HPAC (pancreatic adenocarcinoma), and Caov-3 (ovarian adenocarcinoma) mouse xenograft models. [Figure 13] In vivo efficacy of biparatopic VHH-hFc MMAE conjugates targeting ALPP / ALPPL2 in an NCI-N87 gastric cancer cell line-derived xenograft model. Vehicle data plotted up to the time the first animal in the vehicle group reached humane tumor burden. [Figure 14] In vivo efficacy of biparatopic VHH-hFc MMAE conjugates targeting ALPP / ALPPL2 in an HPAC pancreatic cancer cell line-derived xenograft model. Vehicle data plotted up to the time the first animal in the vehicle group reached humane tumor burden. [Figure 15] Internalization of monoparatopic proteins targeted to ALPP / ALPPL2. [Figure 16] Internalization of biparatopic proteins targeting ALPP / ALPPL2 and ALPP / ALPPL2 mAb h12F3. [Figure 17] In vitro efficacy of biparatopic versus monoparatopic drug-protein conjugates targeting ALPP / ALPPL2 in killing ALPP / ALPPL2-expressing cancer cells (NSCLC cell line NCI-H1651). [Figure 18] In vitro efficacy of ALPP / ALPPL2-targeting biparatopic drug-protein conjugates versus ALPP / ALPPL2 mAb h12F3 in killing CHO cells stably transfected with ALPP or ALPPL2. [Figure 19] In vivo efficacy of ALPP / ALPPL2-targeting biparatopic VHH-hFc MMAE conjugates and the ALPP / ALPPL2 ADC h12F3-MMAE in an NCI-N87 gastric cancer cell line-derived xenograft model. [Figure 20] In vivo efficacy of ALPP / ALPPL2-targeting biparatopic VHH-hFc-MMAE conjugates and the ALPP / ALPPL2 ADC h12F3-MMAE in a HPAC pancreatic cancer cell line-derived xenograft model. [Figure 21] In vivo efficacy of ALPP / ALPPL2-targeting biparatopic VHH-hFc MMAE conjugates and the ALPP / ALPPL2 ADC h12F3-MMAE in a Caov-3 ovarian cancer cell line-derived xenograft model. DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention relates generally to specific antigen-binding molecules. Specifically, the present invention provides single-domain antibodies (VVHs) specific for ALPP and / or ALPPL2, as well as related fusion proteins, chimeric antigen receptors, conjugates, and nucleic acids, and methods associated therewith. ALPP and / or ALPPL2-specific VHH domains are referred to herein as ALPP and / or ALPPL2-specific antigen-binding molecules.

[0049] Although antibodies against ALPP and / or ALPPL2 have been reported in the literature, the high sequence identity between ALPP, ALPPL2, ALPI, and ALPL means that high-affinity ALPP-specific binding agents are not readily generated. Furthermore, the large size of antibodies impairs their ability to penetrate solid tumors and renders regions of target proteins inaccessible due to steric factors, which can be particularly severe for cell surface proteins where oligomerization or receptor clustering is observed.

[0050] As a result, there is a need in the art for the development of improved anti-ALPP and / or ALPPL2 binding protein agents, as well as therapeutic and diagnostic agents for malignancies associated with ALPP and / or ALPPL2 expression, that have functional or physical characteristics or properties distinct from those of antibodies. The present invention provides such agents in the form of the ALPP- and / or ALPPL2-specific antigen-binding molecules described herein.

[0051] Without being bound by theory, the ALPP- and / or ALPPL2-specific antigen-binding molecules described herein are believed to bind to both ALPP and ALPPL2, with the exception of D10, which preferentially binds to ALPP. Several variants, including A06, E06, E10, and F05, have been experimentally confirmed to bind to both ALPP and ALPPL2. Furthermore, the ALPP- and / or ALPPL2-specific antigen-binding molecules of the present invention may not bind to several related targets, such as ALPI and ALPL. The variants disclosed herein bind to several different, non-competing epitopes. Therefore, several biparatopic ALPP- and / or ALPPL2-specific antigen-binding molecules have been developed.

[0052] Binding of the ALPP- and / or ALPPL2-specific antigen-binding molecules of the present invention to transfected CHO cell lines was demonstrated, supporting the potential use of such molecules in the treatment of cancer, particularly cancers expressing ALPP and / or ALPPL2.

[0053] Various forms of ALPP and / or ALPPL2-specific antigen-binding molecules are described, including several types of fusion proteins. Fusion proteins containing immunoglobulin Fc regions, as well as both homo- and heterodimers, are also described. Fusing a protein to an Fc domain can improve protein solubility and stability, significantly increase plasma half-life, and improve overall therapeutic efficacy.

[0054] The present inventors have also produced VHH molecules conjugated to various moieties and payloads. Thus, the present invention also provides chemically conjugated VHHs. More specifically, several conjugated formats of ALPP and / or ALPPL2-specific antigen-binding molecules are provided.

[0055] According to one aspect, the present invention provides a compound of formula (I): FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4 (I) (In the formula, FW1 is the framework region, CDR1 is the CDR sequence, FW2 is the framework region, CDR2 is a CDR sequence, FW3 is the framework area, CDR3 is a CDR sequence, FW4 is the framework area) The present invention provides an antigen-binding molecule specific for placental alkaline phosphatase (ALPP) and / or germline alkaline phosphatase (ALPPL2), comprising an amino acid sequence represented by:

[0056] According to a further aspect, the present invention provides a compound of formula (I): FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4 (I) (In the formula, FW1 is the framework region, CDR1 is a CDR sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 15, FW2 is the framework region, CDR2 is a CDR sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 10, FW3 is the framework area, CDR3 is a CDR sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5, FW4 is the framework area) The present invention provides an antigen-binding molecule specific for placental alkaline phosphatase (ALPP) and / or germline alkaline phosphatase (ALPPL2), comprising an amino acid sequence represented by:

[0057] In one embodiment of the ALPP and / or ALPPL2-specific antigen binding molecule, CDR1 is a CDR sequence having an amino acid sequence selected from the group consisting of GRTFLSMG (SEQ ID NO: 11), GSIFSVNTMG (SEQ ID NO: 12), RRSFSEYAMG (SEQ ID NO: 13), GRALSDYNIG (SEQ ID NO: 14), and GRAFGDYNIG (SEQ ID NO: 15), CDR2 is a CDR sequence having an amino acid sequence selected from the group consisting of AAISWTGGSTGYADS (SEQ ID NO: 6), AVATSGGTIHYADS (SEQ ID NO: 7), AAIGWSGVIEYADS (SEQ ID NO: 8), ARKTWDGSSTRYDDS (SEQ ID NO: 9) and ARRTWDGSSTRYDDS (SEQ ID NO: 10); and / or CDR3 is a CDR sequence having an amino acid sequence selected from the group consisting of AARGDVYYGGDDATRYKY (SEQ ID NO: 1), NLVVPYGMIAGDHDY (SEQ ID NO: 2), AAGLGIRVVSQTPAY (SEQ ID NO: 3), AAGRYSRARGWEYDY (SEQ ID NO: 4), and AAGRYSRVRGWEYDY (SEQ ID NO: 5).

[0058] In one embodiment, the ALPP and / or ALPPL2-specific antigen binding molecule is Herein referred to as A05 QVQLVESGGGLVQAGASLTLSCAASGFTFNTRSIVWFRQAPGKEREAVSCISKVAGSTYYGDSVQGRFSSSTDNAKNTVSLQMNSLKPEDTAVYTCAASFAPNCYGWSVAANMEYWGEGTMVTVSS (SEQ ID NO: 16),

[0059] Herein referred to as A06 QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSS (SEQ ID NO: 17),

[0060] Herein referred to as A08 QVQLVESGGGLVQAGESLRLACAASGTIFSGNAVGWYRQAPEKQREWVSSITDYGDTDYAEAVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCYVYQRGESEEYWGQGTKVTVSS (SEQ ID NO: 18),

[0061] Herein referred to as A12 QVHLVECGGGSVQAGGFLRLSCAASGPSLSNYAMGRFRQAPGKEREFVAGISWGGGRTSYTDSVKGRFTISRDNAKNTVYLQMNNLEPEDTGVYYCAGRYGRGRNVEYEYDYWGQGTKVTVSS (SEQ ID NO: 19),

[0062] Herein referred to as B01 QVQLVESGGGLVQAGGSLRLSCEVTRSPMPIDTMAWYRQAPGRQRELVAIISRSGSTNYADSVKGRFTISRNDTKSTMYKSTMYLKMNTLELEDTGVYYCNAVGLWNGAEYWGQGTQVTVSS (SEQ ID NO: 20),

[0063] Herein referred to as B09 QVQLVESGGGSVQAGGSLRLSCAASGPSLSHYAMGWFRQAPGKEREFVAGISWGGGRTSYTDSVKGRFTISRDNAKNTVYLQMNNLKPEDTGVYYCAGRYGRGRNVEYEYDYWGQGTQVTVSS (SEQ ID NO: 21),

[0064] Herein referred to as B11 QVHLVESGGGSVQAGGSLRLSCAASGPSLSNYAMGWFRQAPGKEREFVAGISWGGGRTSYTDSVKGRFTISRDNAKNTVYLQMNNLKPEDTGVYYCAGRYGRGRNVEYEYDYWGQGTQVTVSS (SEQ ID NO: 22),

[0065] Herein referred to as C05 QVQLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSS (SEQ ID NO: 23),

[0066] Herein referred to as C06 QVQLVESGGGLVQPGGSLRVSCAASGPSLSNYAMGWFRQAPGKEREFVAGISWGGGRTSYTDSVKGRFTISRDNAKNTVYLQMNNLKPEDTGVYYCAGRYGRGRNVEYEYDYWGQGTQVTVSS (SEQ ID NO: 24),

[0067] Herein referred to as C07 QVQLVESGGGLVQAGESLRLACAASGTIFSGNAVGWYRQAPEKQREWVSSITDYGDTDYAEAVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCYVYQRGESEEYWGQGTQVTVSS (SEQ ID NO: 25),

[0068] Herein referred to as D04 QVQLVESGGGLVQPGGSLRLSCAASESTFSINVMGWYRQAPGKQRELVATITSGDITNYADSVKGRFTISRDNAENTVYLRTNSLKPEDTAVYYCNARGEYHSGNEYDYWGQGTQVTVSS (SEQ ID NO: 26),

[0069] Herein referred to as D08 QVHLVESGGGSVQAGGSLRLSCAASGPSLSNYAMGWFRQAPGKEREFVAGISWGGGRTSYTDSVKGRFTISRDNAKNTVYLQMNNLKPEDTGVYYCAGRYGRGRNVEYEYDYWGQGTKVTVSS (SEQ ID NO: 27),

[0070] Herein referred to as D10 QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSS (SEQ ID NO: 28),

[0071] Herein referred to as D11 QVQLVESGGGSVQAGGSLRLSCAASGPSLSNYAMGWFRQAPGKEREFVAGISWGGGRTSYTDSVKGRFTISRDNAKNTVYLQMNNLKPEDTGVYYCAGRYGRGRNVEYEYDYWGQGTMVTVSS (SEQ ID NO: 29),

[0072] Herein referred to as E06 QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSS (SEQ ID NO: 30),

[0073] Herein referred to as E10 QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSS (SEQ ID NO: 31),

[0074] Herein referred to as E11 QVQLVESGGGSVQAGGSLRLSCAASGPSLSNFAMGWFRQPPGKEREFVAGISWGGGRTSYTDSVKGRFTISRDNAKNTVYLQMNNLKPEDTGVYYCAGRYGRGRNVEYEYDYWGQGTQVTVSS (SEQ ID NO: 32),

[0075] Herein referred to as E12 QVQLVESGGGSVQAGGSLRLSCAASGPSISNFAMGWFRQAPGKEREFVAGISWGGGRTNYLDSVKGRFTISRDNAKNMVHLQMNSLKPEDTTVYYCAGRYGRGRDVEYEYDYWGQGTQVTVSS (SEQ ID NO: 33),

[0076] Herein referred to as F05 QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSS (SEQ ID NO: 34),

[0077] Herein referred to as F08 QVQLVESGGGSVQAGGSLRLSCAASGPSLSNYAMGWFRQAPGKEREFVAGISWGGGRTSYTDSVKGRFTISRDNAKNTVYLQMNNLKPEDTGVYYCAGRYGRGRNVEYEYDYWGQGTQVTVSS (SEQ ID NO: 35),

[0078] Herein referred to as F09 QVQLVESGGGSVQAGGSLRLSCAASGPSLSNYAMGWFRQAPGKEREFVAGISWGGGRTSCTDSVKGRFTISRDNAKNTVYLQMNNLKPEDTGVYYCAGRYGRGRNVEYEYDYWGQGTQVTVSS (SEQ ID NO: 36),

[0079] Herein referred to as F12 QVQLVESGGGLVEAGGSLRLSCAVSGSALSDYNIGWFRQAPGKEREFVARKTWDGSSTKYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQRTQVTVSS (SEQ ID NO: 37),

[0080] Herein referred to as G03 QVQLVESGGGTAQAGGSLRLACAASGRSVTMGWLGWVPRAERDDLEFITWFNWGTYYANSVNGRFTISTDNADNTVYPQMNRLKPENPAVYYCASFTGYRPLINRLNDVSDCGHGALVAVSS (SEQ ID NO: 38),

[0081] Herein referred to as H09 QVQLVESGGGSVQTGGSLRLSCAASGPSLSNYAMGWFRQAPGKEREFVAGISWGGGRTNYLDSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAGRYGRGRDVEYEYDYWGQGTQVTVSS (SEQ ID NO: 39),

[0082] Herein referred to as H10 QVQLVESGGGSVQTGGSLRLSCAASGPSLSNYAMGWFRQAPGKEREFVAGISWGGGRTNYLDSVKGRFTISRDNAKNTMYLQMNSLKPEDAAVYYCAGRYGRGRDVEYEYDYWGQGTQVTVSS (SEQ ID NO: 40) or functional variants having CDR1, CDR2 and CDR3 sequences according to any of them, and having FW1, FW2, FW3 and FW4 sequences that have at least 45%, at least 55%, at least 75%, at least 85%, at least 90%, at least 95% or at least 99% combined sequence identity to any combination FW1, FW2, FW3 and FW4 sequence thereof.

[0083] In a particularly preferred embodiment, the ALPP and / or ALPPL2-specific antigen-binding molecule is QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSS (SEQ ID NO: 17) It may comprise an amino acid sequence according to

[0084] An ALPP and / or ALPPL2-specific antigen-binding molecule may comprise an amino acid sequence according to SEQ ID NO: 17 ("A06") or a functional variant thereof having CDR1, CDR2 and CDR3 sequences according to SEQ ID NO: 17 and having FW1, FW2, FW3 and FW4 sequences that have at least 45%, at least 55%, at least 75%, at least 85%, at least 90%, at least 95% or at least 99% combined sequence identity to the combined FW1, FW2, FW3 and FW4 sequences of SEQ ID NO: 17.

[0085] A06-hFc exhibits high affinity binding to ALPP and ALPPL2, and A06-hFc drug conjugates show potent killing of ALPP / APPL2-expressing cells.

[0086] In a particularly preferred embodiment, the ALPP and / or ALPPL2-specific antigen-binding molecule is QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSS (SEQ ID NO: 28) It may comprise an amino acid sequence according to

[0087] An ALPP and / or ALPPL2-specific antigen-binding molecule may comprise an amino acid sequence according to SEQ ID NO: 28 ("D10") or a functional variant thereof having CDR1, CDR2 and CDR3 sequences according to SEQ ID NO: 28 and having FW1, FW2, FW3 and FW4 sequences that have at least 45%, at least 55%, at least 75%, at least 85%, at least 90%, at least 95% or at least 99% combined sequence identity to the combined FW1, FW2, FW3 and FW4 sequences of SEQ ID NO: 28.

[0088] D10-hFc exhibits high affinity binding to ALPP, and D10-hFc drug conjugates show potent killing of ALPP-expressing cells.

[0089] In a particularly preferred embodiment, the ALPP and / or ALPPL2-specific antigen-binding molecule is QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSS (SEQ ID NO: 30) It may comprise an amino acid sequence according to

[0090] An ALPP and / or ALPPL2-specific antigen-binding molecule may comprise an amino acid sequence according to SEQ ID NO: 30 ("E06") or a functional variant thereof having CDR1, CDR2 and CDR3 sequences according to SEQ ID NO: 30 and having FW1, FW2, FW3 and FW4 sequences that have at least 45%, at least 55%, at least 75%, at least 85%, at least 90%, at least 95% or at least 99% combined sequence identity to the combined FW1, FW2, FW3 and FW4 sequence of SEQ ID NO: 30.

[0091] E06-hFc exhibits high affinity binding to ALPP and ALPPL2, and E06-hFc drug conjugates exhibit potent killing of ALPP-expressing cells.

[0092] In a particularly preferred embodiment, the ALPP and / or ALPPL2-specific antigen-binding molecule is QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSS (SEQ ID NO: 31) It may comprise an amino acid sequence according to

[0093] An ALPP and / or ALPPL2-specific antigen-binding molecule may comprise an amino acid sequence according to SEQ ID NO: 31 ("E10") or a functional variant thereof having CDR1, CDR2 and CDR3 sequences according to SEQ ID NO: 31 and having FW1, FW2, FW3 and FW4 sequences that have at least 45%, at least 55%, at least 75%, at least 85%, at least 90%, at least 95% or at least 99% combined sequence identity to the combined FW1, FW2, FW3 and FW4 sequence of SEQ ID NO: 31.

[0094] E10-hFc exhibits high affinity binding to ALPP and ALPPL2, and E10-hFc drug conjugates show potent killing of ALPP / ALPPL2-expressing cells.

[0095] In a particularly preferred embodiment, the ALPP and / or ALPPL2-specific antigen-binding molecule is QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSS (SEQ ID NO: 34) It may comprise an amino acid sequence according to

[0096] An ALPP and / or ALPPL2-specific antigen-binding molecule may comprise an amino acid sequence according to SEQ ID NO: 34 ("F05") or a functional variant thereof having CDR1, CDR2 and CDR3 sequences according to SEQ ID NO: 34 and having FW1, FW2, FW3 and FW4 sequences that have at least 45%, at least 55%, at least 75%, at least 85%, at least 90%, at least 95% or at least 99% combined sequence identity to the combined FW1, FW2, FW3 and FW4 sequences of SEQ ID NO: 34.

[0097] F05-hFc exhibits high affinity binding to ALPP and ALPPL2, and F05-hFc drug conjugates show potent killing of ALPP / ALPPL2-expressing cells.

[0098] ALPP and / or ALPPL2-specific antigen-binding molecules may comprise the CDR sequences of the clones shown in Table 1 below. In a preferred embodiment, ALPP and / or ALPPL2-specific antigen-binding molecules have a combined sequence of any of the clones shown in Table 1 below.

[0099] [Table 1] JPEG2025542294000005.jpg255170JPEG2025542294000006.jpg255170

[0100] All possible combinations and permutations of the framework regions, complementarity determining regions and hypervariable regions listed herein are expressly contemplated herein.

[0101] The sequence identity referred to in relation to the molecules of the invention can be determined at the level of individual CDRs or FWs, combined CDRs or FWs, or it can be determined over the length of the entire molecule. The CDR and FW sequences described may be longer or shorter, whether due to additions or deletions of amino acids at the N- or C-terminus of the sequence, or insertions or deletions of amino acids in the sequence.

[0102] The framework region FW1 is preferably 20 to 30 amino acids in length, more preferably 22 to 28 amino acids in length, and even more preferably 24 to 26 amino acids in length. In a preferred embodiment, FW1 is 24 amino acids in length. In a preferred embodiment, FW1 is 25 amino acids in length.

[0103] In an alternative definition, the CDR region CDR1 is preferably 8 to 10 amino acids in length. In one preferred embodiment, CDR1 is 10 amino acids in length. In another preferred embodiment, CDR1 is 8 amino acids in length.

[0104] The framework region FW2 is preferably 10 to 15 amino acids in length, more preferably 12 to 14 amino acids in length. In a preferred embodiment, FW2 is 13 amino acids in length.

[0105] In an alternative definition, the CDR region CDR2 is preferably 10 to 20 amino acids in length, more preferably 12 to 15 amino acids in length. In one preferred embodiment, CDR2 is 12 amino acids in length. In another preferred embodiment, CDR2 is 14 amino acids in length. In another preferred embodiment, CDR2 is 15 amino acids in length.

[0106] The framework region FW3 is preferably 30 to 40 amino acids in length, more preferably 33 to 38 amino acids in length. In one preferred embodiment, FW3 is 33 amino acids in length. In another preferred embodiment, FW3 is 38 amino acids in length.

[0107] In an alternative definition, the CDR region CDR3 is preferably 10 to 20 amino acids in length, more preferably 11 to 19 amino acids in length. In one preferred embodiment, the CDR3 is 11 amino acids in length. In another preferred embodiment, the CDR3 is 14 amino acids in length. In another preferred embodiment, the CDR3 is 15 amino acids in length. In another preferred embodiment, the CDR3 is 16 amino acids in length. In another preferred embodiment, the CDR3 is 18 amino acids in length. In another preferred embodiment, the CDR3 is 19 amino acids in length.

[0108] The framework region FW4 is preferably 8 to 12 amino acids in length, more preferably 10 to 11 amino acids in length. In a preferred embodiment, FW4 is 11 amino acids in length.

[0109] In one embodiment of the ALPP and / or ALPPL2-specific antigen binding molecule, FW1 is a framework region of 24 to 25 amino acids, FW2 is a 13 amino acid framework region, FW3 is a 33 amino acid framework region, and / or FW4 is an 11 amino acid framework region.

[0110] In one embodiment of the ALPP and / or ALPPL2-specific antigen binding molecule, FW1 has an amino acid sequence selected from the group consisting of QVHLVESGGGLVQAGGSLRLSCAAS (SEQ ID NO: 53), VQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 54), QVQLVESGGGLVQAGGSLRLSCAVS (SEQ ID NO: 55) and QVQLVESGGGLVQAGGSLRLSCAAS (SEQ ID NO: 56), or a functional variant of any thereof having at least 45%, at least 55%, at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity; FW2 has an amino acid sequence selected from the group consisting of WFRQAPGKEREFV (SEQ ID NO: 64) and WYRQAPGKQRDLV (SEQ ID NO: 65), or a functional variant of any thereof having at least 45%, at least 55%, at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity; FW3 has an amino acid sequence selected from the group consisting of VKGRFTISRESDKETMYLQMNSLKPEDTAVYYC (SEQ ID NO: 76), VKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC (SEQ ID NO: 77), VKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYC (SEQ ID NO: 78), VKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYC (SEQ ID NO: 79) and VKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYC (SEQ ID NO: 80), or a functional variant of any of them having at least 45%, at least 55%, at least 75%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity; and / or FW4 has an amino acid sequence selected from the group consisting of WGQGTQVTVSS (SEQ ID NO: 84) and WGQGTKVTVSS (SEQ ID NO: 85), or any functional variant thereof having at least 45%, at least 55%, at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity.

[0111] The ALPP- and / or ALPPL2-specific antigen-binding molecules of the present invention may be humanized. The ALPP- and / or ALPPL2-specific antigen-binding molecules of the present invention may be deimmunized.

[0112] It will be appreciated by those skilled in the art that the humanized ALPP and / or ALPPL2-specific antigen-binding molecules described herein can be further humanized, for example, by substituting FW region amino acids with corresponding amino acids from a human VH or VL sequence.

[0113] The ALPP- and / or ALPPL2-specific antigen-binding molecules of the present invention may also be conjugated to a detectable label, dye, toxin, drug, prodrug, radionuclide or biologically active molecule.

[0114] Preferably, an ALPP- and / or ALPPL2-specific antigen-binding molecule binds to ALPP and ALPPL2. More preferably, an ALPP-specific antigen-binding molecule does not bind to ALPI or ALPL.

[0115] Preferably, ALPP and / or ALPPL2-specific antigen-binding molecules selectively interact with ALPP proteins with an affinity constant of approximately 0.001 to 50 nM, preferably 0.1 to 30 nM, and even more preferably 0.1 to 10 nM. The affinity constant can be measured by biolayer interferometry (BLI). In monomers, the interaction is 1:1. In the VHH-hFc format, the inventors used two approaches. One is to immobilize ALPP, thus increasing the apparent K of bivalent VHH-hFc due to the avidity effect. D The other approach is a 1:1 format, where VHH-hFc is immobilized and ALPP is flowed over the surface, thus achieving the K for "true" 1:1 binding. D Typically, as used herein, affinity constants refer to those measured by biolayer interferometry (BLI) using a 1:1 binding format. By this method, for example, E06 and E10 are in the range of 0.1 to 10 nM.

[0116] Furthermore, ALPP- and / or ALPPL2-specific antigen-binding molecules are preferably capable of mediating the killing of ALPP- and / or ALPPL2-expressing tumor cells or inhibiting cancer cell proliferation.

[0117] An ALPP- and / or ALPPL2-specific antigen-binding molecule can be endocytosed upon binding to ALPP and / or ALPPL2. In other embodiments, an ALPP- and / or ALPPL2-specific antigen-binding molecule cannot be endocytosed upon binding to ALPP and / or ALPPL2.

[0118] In a further aspect of the present invention, a recombinant fusion protein comprising the specific antigen-binding molecule disclosed herein is provided. Preferably, in the recombinant fusion protein, the specific antigen-binding molecule is fused to one or more biologically active proteins. The specific antigen-binding molecule may be fused to one or more biologically active proteins via one or more linker domains. Preferred linkers include, but are not limited to, [G4S] x (wherein x is 1, 2, 3, 4, 5, or 6). Particularly preferred linkers are G4S (SEQ ID NO: 236), referred to herein as the "short linker," and [G4S]3 (SEQ ID NO: 237), referred to herein as the "long linker." These linkers may be particularly useful when the recombinant fusion protein is expressed in different expression systems with different glycosylation patterns, such as CHO and insect, as well as expression systems that do not glycosylate the expressed protein (e.g., E. coli). Any recombinant fusion protein sequence disclosed herein that includes a [G4S]3 linker may alternatively have any other linker sequence disclosed herein.

[0119] It is also recognized that the fusion proteins of the present invention can be constructed in any order, i.e., the ALPP and / or ALPPL2-specific antigen-binding molecule may be present at the N-terminus, C-terminus, or not at either terminus (e.g., within a longer amino acid sequence).

[0120] Preferred biologically active proteins include, but are not limited to, immunoglobulins, immunoglobulin Fc regions, fragments of immunoglobulin Fc regions, Fc heavy chains, CH2 regions, CH3 regions, immunoglobulin Fab regions, Fab', Fv, Fv-Fc, single-chain Fv (scFv), scFv-Fc, (scFv)2, diabodies, triabodies, tetrabodies, bispecific T cell engagers, inteins, VNAR domains, single-domain antibodies (sdAbs), VH domains, or scaffold proteins (such as affibodies, centyrins, and darpins). Another preferred biologically active protein is an immunoglobulin Fc region. Other preferred fusion proteins include VHH-VHH and VHH-VHH-VHH.

[0121] In one embodiment, the at least one biologically active protein is an immunoglobulin Fc region. Thus, the recombinant fusion protein may comprise a sequence according to SEQ ID NO: 87 to SEQ ID NO: 91.

[0122] A06-hFc (long linker) QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 87)

[0123] D10-hFc (long linker) QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 88)

[0124] E06-hFc (long linker) QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 89)

[0125] E10-hFc (long linker) QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 90)

[0126] F05-hFc (long linker) QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 91)

[0127] SEQ ID NOs: 87-91 each contain a (G4S)3 linker. Corresponding sequences in which the (G4S)3 linker is replaced with a (G4S)1 linker are also expressly contemplated herein. For example, a recombinant fusion protein may contain one or more of the following SEQ ID NOs: 92-96.

[0128] A06-hFc (short linker) QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 92)

[0129] D10-hFc (short linker) QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 93)

[0130] E06-hFc (short linker) QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 94)

[0131] E10-hFc (short linker) QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 95)

[0132] F05-hFc (short linker) QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 96)

[0133] In a further embodiment, the at least one biologically active protein is an immunoglobulin Fc region that has been further modified to include a Cys(C) mutation (ie, mutation of an Fc amino acid to a cysteine ​​residue).

[0134] The C mutation may be at position S239 (EU numbering). The recombinant fusion protein may therefore comprise a sequence according to SEQ ID NO: 97 or SEQ ID NO: 101.

[0135] A06-hFc(long linker)S239C QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 97)

[0136] D10-hFc (long linker) S239C QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 98)

[0137] E06-hFc(long linker)S239C QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 99)

[0138] E10-hFc (long linker) S239C QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 100)

[0139] F05-hFc(long linker)S239C QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 101)

[0140] SEQ ID NOs: 102-106 each contain a (G4S)3 linker. Corresponding sequences in which the (G4S)3 linker is replaced with a (G4S)1 linker are also expressly contemplated herein. For example, a recombinant fusion protein may contain one or more of the following SEQ ID NOs: 102-106.

[0141] A06-hFc (short linker) S239C QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 102)

[0142] D10-hFc (short linker) S239C QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 103)

[0143] E06-hFc (short linker) S239C QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 104)

[0144] E10-hFc (short linker) S239C QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 105)

[0145] F05-hFc (short linker) S239C QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 106)

[0146] The C mutation may be at position S442 (EU numbering). The recombinant fusion protein may therefore comprise a sequence according to SEQ ID NOs: 107-111.

[0147] A06-hFc(long linker)S442C QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 107)

[0148] D10-hFc(long linker)S442C QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 108)

[0149] E06-hFc(long linker)S442C QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 109)

[0150] E10-hFc(long linker)S442C QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 110)

[0151] F05-hFc(long linker)S442C QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 111)

[0152] SEQ ID NOs: 107-111 each contain a (G4S)3 linker. Corresponding sequences in which the (G4S)3 linker is replaced with a (G4S)1 linker are also expressly contemplated herein. For example, a recombinant fusion protein may contain one or more of the following SEQ ID NOs: 112-116.

[0153] A06-hFc (short linker) S442C QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 112)

[0154] D10-hFc (short linker) S442C QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 113)

[0155] E06-hFc (short linker) S442C QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 114)

[0156] E10-hFc (short linker) S442C QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 115)

[0157] F05-hFc (short linker) S442C QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 116)

[0158] The C mutation may be at both positions S239 and S442 (EU numbering). Thus, the recombinant fusion protein may comprise a sequence according to SEQ ID NOs: 117-121.

[0159] A06-hFc (long linker) S239C and S442C QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 117)

[0160] D10-hFc (long linker) S239C and S442C QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 118)

[0161] E06-hFc (long linker) S239C and S442C QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 119)

[0162] E10-hFc (long linker) S239C and S442C QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 120)

[0163] F05-hFc (long linker) S239C and S442C QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 121)

[0164] SEQ ID NOs: 117-121 each contain a (G4S)3 linker. Corresponding sequences in which the (G4S)3 linker is replaced with a (G4S)1 linker are also expressly contemplated herein. For example, a recombinant fusion protein may contain one or more of the following SEQ ID NOs: 122-126.

[0165] A06-hFc (short linker) S239C and S442C QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 122)

[0166] D10-hFc (short linker) S239C and S442C QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 123)

[0167] E06-hFc (short linker) S239C and S442C QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 124)

[0168] E10-hFc (short linker) S239C and S442C QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 125)

[0169] F05-hFc (short linker) S239C and S442C QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 126)

[0170] In one embodiment, the at least one biologically active protein is a fragment of an immunoglobulin Fc region selected from the group consisting of an Fc heavy chain, a CH2 region, and a CH3 region.

[0171] In one embodiment, the fragment of an immunoglobulin Fc region is an Fc heavy chain.

[0172] In one embodiment, a fragment of an immunoglobulin Fc region is engineered to dimerize with a second fragment of an immunoglobulin Fc region.

[0173] As used herein, an immunoglobulin Fc region that has been "engineered to dimerize" can contain at least one amino acid substitution. Typically, the at least one amino acid substitution promotes and / or makes more energetically favorable the interaction and / or association of the immunoglobulin Fc region with a second fragment, thereby promoting and / or making dimerization more energetically favorable. Such recombinant fusion proteins can have particular utility in the preparation of bispecific and / or biparatopic binders.

[0174] Methods for generating Fc-based bispecific and / or biparatopic binders through the pairing of two distinct Fc heavy chains that are engineered to dimerize are known in the art. These methods allow for the assembly of Fc regions from two different heavy chains, each fused to a target binding domain or sequence with different binding characteristics. The target binding domains or sequences can be directed to different targets to generate multispecific binders and / or to different regions or epitopes on the same target to generate biparatopic binding proteins. Multiple binding domains or sequences can be fused to an Fc region to create multispecific or multiparatopic binders, or both, within the same protein. Methods for generating these asymmetric bispecific and / or biparatopic binders via heterodimerization of two different Fc heavy chains or fragments thereof include, but are not limited to, knobs-into-holes (YT), knobs-into-holes (CW-CSAV), CH3 charge pairing, Fab arm exchange, SEED technology, BEAT technology, HA-TF, ZW1 approach, Biclonic approach, EW-RVT, and Triomab. See, e.g., Brinkman & Kontermann, (2017) mAbs, 9:2, 182-212; Klein et al. (2012) mAbs 4:6, 653-663; Wang et al. (2019) Antibodies, 8, 43; and Dietrich et al. (2020) BBA-Proteins and Proteomics 1868 140250, each of which is incorporated herein by reference in its entirety.

[0175] In one embodiment, a fragment of an immunoglobulin Fc region is engineered to dimerize with a second fragment of an immunoglobulin Fc region by a method selected from the group consisting of knobs-into-holes (YT), knobs-into-holes (CW-CSAV), CH3 charge pairing, Fab arm exchange, SEED technology, BEAT technology, HA-TF, ZW1 approach, Biclonic approach, EW-RVT, and Triomab.

[0176] In one embodiment, one or more residues of the fragment of an immunoglobulin Fc region comprise one or more amino acid substitutions suitable for heterodimerization with a second fragment of an immunoglobulin Fc region comprising one or more corresponding amino acid substitutions.

[0177] In one embodiment, the one or more amino acid substitutions are selected from the group consisting of T366Y, Y407T, S354C, T366W, Y349C, T366S, L368A and Y407V.

[0178] In one embodiment, the one or more amino acid substitutions are selected from the group consisting of T366Y and Y407T.

[0179] Any portion of the fusion proteins of the present invention can be engineered to enable conjugation. In a preferred example, an immunoglobulin Fc region is used, which can be engineered to contain a cysteine ​​residue as a conjugation site. Preferred introduced cysteine ​​residues include, but are not limited to, S252C and S473C (Kabat numbering), which correspond to S239C and S442C, respectively, in the EU numbering system. In some embodiments, any of the fusion proteins disclosed herein can contain a S239C point mutation. In some embodiments, any of the fusion proteins disclosed herein can contain a S442C point mutation. In some embodiments, any of the fusion proteins disclosed herein can contain both the S239C and S442C point mutations. It is expressly contemplated herein that the sequence of any of the fusion proteins disclosed herein can be modified to contain the S239C and / or S442C point mutations. Additionally, cysteine ​​residues may be incorporated into linker regions, including but not limited to, the linkers between VHH and hFc in VHH-hFc and hFc-VHH fusion proteins. Cysteine ​​residues may also be added to the C-terminus of the Fc region, either directly or as part of a C-terminal tag.

[0180] In one embodiment, a recombinant fusion comprising multiple VHH domains is provided. Thus, the recombinant fusion of the present invention may be a dimer, trimer, or higher multimer of VHHs. In such a recombinant fusion, the specificity of each VHH may be the same or different. Recombinant fusions of the present invention include, but are not limited to, bispecific or trispecific molecules in which each VHH domain binds to a different antigen or to different epitopes on a single antigen (biparatopic binders). The term "biparatopic" as used herein is intended to encompass molecules that bind to multiple epitopes on a given antigen. Molecules that bind to three or more epitopes on a given antigen are also contemplated herein, and it should be understood that when the term "biparatopic" is used, the possibility of triparatopic or multiparatopic molecules is also contemplated.

[0181] According to one aspect, there is also provided a recombinant fusion comprising an ALPP- and / or ALPPL2-specific antigen-binding molecule disclosed herein and a second ALPP- and / or ALPPL2-binding molecule, optionally a second ALPP- and / or ALPPL2-binding molecule disclosed herein. Also provided is a recombinant fusion comprising an ALPP- and / or ALPPL2-specific antigen-binding molecule disclosed herein and a peptide sequence, protein domain, or protein capable of extending the serum half-life of the ALPP- and / or ALPPL2-specific antigen-binding molecule, such as a fusion with an HSA-binding domain or an Fc protein.

[0182] Examples of biparatopic and multivalent fusion proteins include, but are not limited to: A06-D10 A06-E06 A06-E10 A06-F05 D10-E06 D10-E10 D10-F05 E06-E10 E06-F05 E10-F05 are listed,

[0183] A06 is QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSS (SEQ ID NO: 17) and

[0184] D10 is QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSS (SEQ ID NO: 28) and

[0185] E06 is QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSS (SEQ ID NO: 30) and

[0186] E10 is QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSS (SEQ ID NO: 31) and

[0187] F05 is QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSS (SEQ ID NO: 34) and

[0188] (-) corresponds to a linker as defined herein. In one embodiment, the linker can be a [G4S]x linker, such as G4S (SEQ ID NO: 236) and [G4S]3 (SEQ ID NO: 237). Optionally, the linker can contain a Cys residue. An example of a Cys-containing linker is GCGGS[G4S]2 (SEQ ID NO: 274).

[0189] Recombinant biparatopic fusion protein dimers can also be made by fusing any of the recombinant fusion proteins disclosed herein, particularly an ALPP and / or ALPPL2 binder disclosed herein, onto one arm of an Fc fusion and a binder to a different ALPP and / or ALPPL2 epitope onto the other.

[0190] In certain embodiments, the specific binding molecules or recombinant fusions of the present invention can be expressed with N- or C-terminal tags to aid in purification. Examples include, but are not limited to, His6 and / or Myc. Furthermore, N- or C-terminal tags can be further engineered to contain additional cysteine ​​residues to serve as conjugation points. Thus, it will be recognized that reference to specific binding molecules or recombinant fusions in all aspects of the present invention is intended to encompass such molecules with various N- or C-terminal tags, which may also contain additional cysteines for conjugation.

[0191] Additional recombinant fusions are listed below. It is recognized that not all combinations of linkers and VHHs or fusion partners are listed below. However, all such combinations are expressly encompassed by the present invention.

[0192] Biparatopic dimer A06-D10 A06-E06 A06-E10 A06-F05 D10-E06 D10-E10 D10-F05 E06-E10 E06-F05 E10-F05 D10-A06 E06-A06 E10-A06 F05-A06 E06-D10 E10-D10 F05-D10 E10-E06 F05-E06 F05-E10

[0193] The linker between the VHH domains is preferentially, but not exclusively, (G4S)5 (SEQ ID NO: 242), (G4S)3 (SEQ ID NO: 237), or (G4S)7 (SEQ ID NO: 243), and different combinations of different linkers can be combined within the same construct. The linker may contain a single cysteine ​​residue or multiple cysteine ​​residues to facilitate site-selective bioconjugation of payloads to proteins using thiol-mediated chemical coupling methods. Cys-containing linkers include, but are not limited to, GCGGS[G4S]2 (SEQ ID NO: 274).

[0194] In this case, an additional C-terminal (or N-terminal) tag sequence may or may not be present.

[0195] C-terminal tags include, but are not limited to, tags containing a polyhistidine sequence (e.g., His6) to facilitate purification, tags containing a c-Myc sequence (e.g., EQKLISEEDL (SEQ ID NO: 245)) to enable detection, and / or tags containing a cysteine ​​residue to allow labeling and bioconjugation using thiol-reactive payloads and probes, and combinations thereof. Preferential C-terminal tags include, but are not limited to, AAAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 249) ACAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 250) AAAHHHHHH (SEQ ID NO: 254) ACAHHHHHH (SEQ ID NO: 255) ACA (SEQ ID NO: 259) Examples include:

[0196] Here, A06 is QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSS (SEQ ID NO: 17) and

[0197] D10 is QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSS (SEQ ID NO: 28) and

[0198] E06 is QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSS (SEQ ID NO: 30) and

[0199] E10 is QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSS (SEQ ID NO: 31) and

[0200] F05 is QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSS (SEQ ID NO: 34) is.

[0201] As mentioned above, all combinations of VHHs and linkers are expressly encompassed herein. Humanized derivatives of VHHs are also encompassed herein.

[0202] According to one aspect, there is also provided a recombinant fusion comprising an ALPP- and / or ALPPL2-specific antigen-binding molecule disclosed herein and a recombinant toxin, including, but not limited to, Pseudomonas exotoxin PE38 and diphtheria toxin.

[0203] In one embodiment, there is also provided a recombinant fusion comprising an ALPP- and / or ALPPL2-specific antigen-binding molecule disclosed herein and a recombinant CD3-binding protein. Examples of recombinant ALPP- and / or ALPPL2- and CD3-binding agents include, but are not limited to: A06-CD3 D10-CD3 E06-CD3 E10-CD3 F05-CD3 CD3-A06 CD3-D10 CD3-E06 CD3-E10 CD3-F05 are listed,

[0204] A06 is QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSS (SEQ ID NO: 17) and

[0205] D10 is QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSS (SEQ ID NO: 28) and

[0206] E06 is QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSS (SEQ ID NO: 30) and

[0207] E10 is QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSS (SEQ ID NO: 31) and

[0208] F05 is QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSS (SEQ ID NO: 34) is.

[0209] Any of the CD3 binding sequences and variants thereof known in the art can be substituted for the above. For example: UCL OKT3 sequence (International Publication No. 2019008379) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTRYTMHWVRQAPGQGLEWMGYINPSRGYTNYNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARYYDDHYCLDYWGQGTMVTVSSVEGGSGGSGGSGGSGGVDDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQWSSNPFTFGQGTKVEIK (SEQ ID NO: 261) Harpoon ID20 (International Publication No. 2016187594 Brochure) DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO: 262) is.

[0210] According to a further aspect, the present invention provides a compound of formula (I): FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4 (I) (In the formula, FW1 is the framework region, CDR1 is the CDR sequence, FW2 is the framework region, CDR2 is a CDR sequence, FW3 is the framework area, CDR3 is a CDR sequence, FW4 is the framework area) or a functional variant thereof, wherein the antigen-binding molecule is fused to a fragment of an immunoglobulin Fc region, and the fragment of the immunoglobulin Fc region is engineered to dimerize with a second fragment of the immunoglobulin Fc region.

[0211] In one embodiment, the fragment of an immunoglobulin Fc region is selected from the group consisting of an Fc heavy chain, a CH2 region, and a CH3 region.

[0212] In one embodiment, the fragment of an immunoglobulin Fc region is an Fc heavy chain.

[0213] The Fc region can be engineered to reduce FcγR binding. Thus, the Fc regions disclosed herein can be engineered to reduce FcγR binding.

[0214] As used herein, an immunoglobulin Fc region that has been "engineered to dimerize" can contain at least one amino acid substitution. Typically, the at least one amino acid substitution promotes and / or makes more energetically favorable the interaction and / or association of the immunoglobulin Fc region with a second fragment, thereby promoting and / or making dimerization more energetically favorable. Such recombinant fusion proteins can have particular utility in the preparation of bispecific and / or biparatopic binders.

[0215] Methods for generating Fc-based bispecific and / or biparatopic binders through the pairing of two distinct Fc heavy chains that are engineered to dimerize are known in the art. These methods allow for the assembly of Fc regions from two different heavy chains, each fused to a target-binding domain or sequence with different binding characteristics. The target-binding domains or sequences can be directed to different targets to generate multispecific binders and / or to different regions or epitopes on the same target to generate biparatopic binding proteins. Multiple binding domains or sequences can be fused to an Fc region to create multispecific or multiparatopic binders, or both, within the same protein. Methods for generating these asymmetric bispecific and / or biparatopic binders via heterodimerization of two different Fc heavy chains or fragments thereof include, but are not limited to, knobs-into-holes (YT), knobs-into-holes (CW-CSAV), CH3 charge pairing, Fab arm exchange, SEED technology, BEAT technology, HA-TF, ZW1 approach, Biclonic approach, EW-RVT, and Triomab. See, e.g., Brinkman & Kontermann, (2017) mAbs, 9:2, 182-212; Klein et al. (2012) mAbs 4:6, 653-663; Wang et al. (2019) Antibodies, 8, 43; and Dietrich et al. (2020) BBA-Proteins and Proteomics 1868 140250, each of which is incorporated herein by reference in its entirety.

[0216] In one embodiment, a fragment of an immunoglobulin Fc region is engineered to dimerize with a second fragment of an immunoglobulin Fc region by a method selected from the group consisting of knobs-into-holes (YT), knobs-into-holes (CW-CSAV), CH3 charge pairing, Fab arm exchange, SEED technology, BEAT technology, HA-TF, ZW1 approach, Biclonic approach, EW-RVT, and Triomab.

[0217] Knobs-into-holes (YT) can comprise a T366Y substitution in the first CH3 domain and a Y407T substitution in the second CH3 domain.

[0218] Knobs-into-holes (CW-CSAV) may include one or more (preferably all) of the following substitutions in the first CH3 domain: S354C, T366W. Knobs-into-holes (CW-CSAV) may include one or more (preferably all) of the following substitutions in the second CH3 domain: Y349C, T366S, L368A, Y407V. Knobs-into-holes (CW-CSAV) may include a disulfide bond in CH3.

[0219] The CH3 charge pairing may include one or more (preferably all) of the following substitutions in the first CH3 domain: K392D, K409D. The CH3 charge pairing may include one or more (preferably all) of the following substitutions in the second CH3 domain: E356K, D399K.

[0220] Fab arm exchange can include a K409R substitution in the first CH3 domain and a F405L substitution in the second CH3 domain. Fab arm exchange and DuoBody capture the same Fc changes. Thus, DuoBody technology can include a K409R substitution in the first CH3 domain and a F405L substitution in the second CH3 domain.

[0221] SEED technology can incorporate known substitutions and / or result in IgG / A chimeras. Complementarity within the CH3 interface, which allows heterodimeric assembly of Fc chains, was developed by designing strand-exchange engineered domain (SEED) heterodimers. These SEED CH3 domains, composed of alternating segments derived from human IgA and IgG CH3 sequences (AG SEED CH3 and GA SEED CH3), were used to generate so-called SEED bodies (Davis et al. (2010) PEDS 23,4,195-202, incorporated herein by reference in its entirety). Molecular modeling suggested that interaction with FcRn was impaired in AG SEED CH3, so residues at the CH2-CH3 junction were reverted to the IgG sequence. Pharmacokinetic studies confirmed that the half-life of SEED bodies was comparable to that of other Fc fusion proteins and IgG1.

[0222] BEAT technology engineered the constant α and β domains of the human T cell receptor into the IgG1 CH3 dimer interface to drive heterodimerization (Skegro et al (2017) JBC 292(23)9745-9759). An additional D410Q mutation could further increase heterodimer formation in this system (Stutz & Blein 2020 JBC 295(28)9392-9408).

[0223] HA-TF may comprise one or more (preferably all) of the following substitutions in the first CH3 domain: S364H, F405A. HA-TF may comprise one or more (preferably all) of the following substitutions in the second CH3 domain: Y349T, T394F.

[0224] The ZW1 approach may include one or more (preferably all) of the following substitutions in the first CH3 domain: T350V, L351Y, F405A, Y407V. The ZW1 approach may include one or more (preferably all) of the following substitutions in the second CH3 domain: T350V, T366L, K392L, T394W.

[0225] The biclonic approach may include one or more (preferably all) of the following substitutions in the first CH3 domain: 366K (+351K). The biclonic approach may include one or more (preferably all) of the following substitutions in the second CH3 domain: 351D or E or D at 349, 368, 349, or 349+355.

[0226] EW-RVT may comprise one or more (preferably all) of the following substitutions in the first CH3 domain: K360E, K409W. EW-RVT may comprise one or more (preferably all) of the following substitutions in the second CH3 domain: Q347R, D399V, F405T. EW-RVT may comprise a disulfide bond in CH3. The disulfide bridge may be supported by the further incorporation of Y349C in the first CH3 domain and S354C in the second CH3 domain.

[0227] Triomabs can be formed by fusing a mouse hybridoma with a rat hybridoma, resulting in the production of a bispecific asymmetric hybrid IgG molecule. Preferential pairing of a light chain with its corresponding heavy chain can then occur.

[0228] In one embodiment, one or more residues of the fragment of an immunoglobulin Fc region contain one or more amino acid substitutions that are suitable for knobs-in-holes (KIH) dimerization with a second fragment of an immunoglobulin Fc region that contains one or more corresponding amino acid substitutions.

[0229] In one embodiment, the one or more amino acid substitutions are selected from the group consisting of T366Y, Y407T, S354C, T366W, Y349C, T366S, L368A and Y407V.

[0230] In one embodiment, the one or more amino acid substitutions are selected from the group consisting of T366Y and Y407T.

[0231] In one embodiment, the antigen-binding molecule is an ALPP- and / or ALPPL2-specific antigen-binding molecule.

[0232] The recombinant fusion protein may comprise the sequence according to SEQ ID NO: 127 to SEQ ID NO: 131.

[0233] A06-hFc (long linker) S239C+Y407T QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 127)

[0234] D10-hFc (long linker) S239C+Y407T QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 128)

[0235] E06-hFc (long linker) S239C+Y407T QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 129)

[0236] E10-hFc (long linker) S239C+Y407T QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 130)

[0237] F05-hFc (long linker) S239C+Y407T QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 131)

[0238] SEQ ID NOs: 127-131 each contain a (G4S)3 linker. Corresponding sequences in which the (G4S)3 linker is replaced with a (G4S)1 linker are also expressly contemplated herein. For example, a recombinant fusion protein may contain one or more of the following SEQ ID NOs: 132-136.

[0239] A06-hFc (short linker) S239C+Y407T QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 132)

[0240] D10-hFc (short linker) S239C+Y407T QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 133)

[0241] E06-hFc (short linker) S239C+Y407T QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 134)

[0242] E10-hFc (short linker) S239C+Y407T QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 135)

[0243] F05-hFc (short linker) S239C+Y407T QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 136)

[0244] The recombinant fusion protein may comprise a sequence according to SEQ ID NOs: 137-141. A06-hFc (long linker) S239C+T366Y QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 137)

[0245] D10-hFc (long linker) S239C+T366Y QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 138)

[0246] E06-hFc (long linker) S239C+T366Y QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 139)

[0247] E10-hFc (long linker) S239C+T366Y QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 140)

[0248] F05-hFc (long linker) S239C+T366Y QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 141)

[0249] SEQ ID NOs: 137-141 each contain a (G4S)3 linker. Corresponding sequences in which the (G4S)3 linker is replaced with a (G4S)1 linker are also expressly contemplated herein. For example, a recombinant fusion protein may contain one or more of the following SEQ ID NOs: 142-146.

[0250] A06-hFc (short linker) S239C+T366Y QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 142)

[0251] D10-hFc (short linker) S239C+T366Y QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 143)

[0252] E06-hFc (short linker) S239C+T366Y QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 144)

[0253] E10-hFc (short linker) S239C+T366Y QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 145)

[0254] F05-hFc (short linker) S239C+T366Y QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 146)

[0255] The recombinant fusion protein may comprise the sequence according to SEQ ID NO: 147 to SEQ ID NO: 151. A06-hFc (long linker) S442C+Y407T QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 147)

[0256] D10-hFc (long linker) S442C+Y407T QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 148)

[0257] E06-hFc (long linker) S442C+Y407T QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 149)

[0258] E10-hFc (long linker) S442C+Y407T QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 150)

[0259] F05-hFc (long linker) S442C+Y407T QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 151)

[0260] SEQ ID NOs: 147-151 each contain a (G4S)3 linker. Corresponding sequences in which the (G4S)3 linker is replaced with a (G4S)1 linker are also expressly contemplated herein. For example, a recombinant fusion protein may contain one or more of the following SEQ ID NOs: 152-156.

[0261] A06-hFc (short linker) S442C+Y407T QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 152)

[0262] D10-hFc (short linker) S442C+Y407T QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 153)

[0263] E06-hFc (short linker) S442C+Y407T QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 154)

[0264] E10-hFc (short linker) S442C+Y407T QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 155)

[0265] F05-hFc (short linker) S442C+Y407T QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 156)

[0266] The recombinant fusion protein may comprise a sequence according to SEQ ID NOs: 157-161.

[0267] A06-hFc (long linker) S442C+T366Y QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 157)

[0268] D10-hFc (long linker) S442C+T366Y QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 158)

[0269] E06-hFc (long linker) S442C+T366Y QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 159)

[0270] E10-hFc (long linker) S442C+T366Y QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 160)

[0271] F05-hFc (long linker) S442C+T366Y QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 161)

[0272] SEQ ID NOs: 157-161 each contain a (G4S)3 linker. Corresponding sequences in which the (G4S)3 linker is replaced with a (G4S)1 linker are also expressly contemplated herein. For example, a recombinant fusion protein may contain one or more of the following SEQ ID NOs: 162-166.

[0273] A06-hFc (short linker) S442C+T366Y QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 162)

[0274] D10-hFc (short linker) S442C+T366Y QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 163)

[0275] E06-hFc (short linker) S442C+T366Y QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 164)

[0276] E10-hFc (short linker) S442C+T366Y QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 165)

[0277] F05-hFc (short linker) S442C+T366Y QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 166)

[0278] The recombinant fusion protein may comprise a sequence according to SEQ ID NOs: 167-171. A06-hFc (long linker) S239C and S442C + Y407T QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 167)

[0279] D10-hFc (long linker) S239C and S442C + Y407T QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 168)

[0280] E06-hFc (long linker) S239C and S442C + Y407T QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 169)

[0281] E10-hFc (long linker) S239C and S442C + Y407T QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 170)

[0282] F05-hFc (long linker) S239C and S442C + Y407T QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 171)

[0283] SEQ ID NOs: 167-171 each contain a (G4S)3 linker. Corresponding sequences in which the (G4S)3 linker is replaced with a (G4S)1 linker are also expressly contemplated herein. For example, a recombinant fusion protein may contain one or more of the following SEQ ID NOs: 172-176.

[0284] A06-hFc (short linker) S239C and S442C + Y407T QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 172)

[0285] D10-hFc (short linker) S239C and S442C + Y407T QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 173)

[0286] E06-hFc (short linker) S239C and S442C + Y407T QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 174)

[0287] E10-hFc (short linker) S239C and S442C + Y407T QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 175)

[0288] F05-hFc (short linker) S239C and S442C + Y407T QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 176)

[0289] The recombinant fusion protein may comprise a sequence according to SEQ ID NOs: 177-181. A06-hFc (long linker) S239C and S442C + T366Y QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 177)

[0290] D10-hFc (long linker) S239C and S442C + T366Y QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 178)

[0291] E06-hFc (long linker) S239C and S442C + T366Y QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 179)

[0292] E10-hFc (long linker) S239C and S442C + T366Y QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 180)

[0293] F05-hFc (long linker) S239C and S442C + T366Y QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 181)

[0294] SEQ ID NOs: 177-181 each contain a (G4S)3 linker. Corresponding sequences in which the (G4S)3 linker is replaced with a (G4S)1 linker are also expressly contemplated herein. For example, a recombinant fusion protein may contain one or more of the following SEQ ID NOs: 182-186.

[0295] A06-hFc (short linker) S239C and S442C + T366Y QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 182)

[0296] D10-hFc (short linker) S239C and S442C + T366Y QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 183)

[0297] E06-hFc (short linker) S239C and S442C + T366Y QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 184)

[0298] E10-hFc (short linker) S239C and S442C + T366Y QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 185)

[0299] F05-hFc (short linker) S239C and S442C + T366Y QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 186)

[0300] The recombinant fusion protein may be a biparatopic dimer comprising any one or any two of SEQ ID NOs: 87-186. The biparatopic dimer may comprise one of SEQ ID NOs: 87-126. The biparatopic dimer may comprise one of SEQ ID NOs: 127-136, 147-156, or 167-176, which includes a Y407T point mutation. The biparatopic dimer may comprise one of SEQ ID NOs: 137-146, 157-166, or 177-186, which includes a T366Y point mutation.

[0301] In a preferred embodiment, the biparatopic dimer may comprise D10-hFc(long linker)S239C+T366Y (SEQ ID NO: 138) and E06-hFc(long linker)S239C Y407T (SEQ ID NO: 129).

[0302] In a preferred embodiment, the biparatopic dimer may comprise D10-hFc(long linker)S239C+T366Y (SEQ ID NO: 138) and E10-hFc(long linker)S239C Y407T (SEQ ID NO: 130).

[0303] In a preferred embodiment, the biparatopic dimer may comprise D10-hFc(long linker)S239C+T366Y (SEQ ID NO: 138) and F05-hFc(long linker)S239C Y407T (SEQ ID NO: 131).

[0304] In a preferred embodiment, the biparatopic dimer may comprise A06-hFc(long linker)S239C T366Y (SEQ ID NO: 137) and F05-hFc(long linker)S239C+Y407T (SEQ ID NO: 131).

[0305] In a preferred embodiment, the biparatopic dimer may comprise D10-hFc(short linker)S239C+T366Y (SEQ ID NO: 143) and E06-hFc(short linker)S239C+Y407T (SEQ ID NO: 154).

[0306] In a preferred embodiment, the biparatopic dimer may comprise D10-hFc(short linker)S239C+T366Y (SEQ ID NO: 143) and E10-hFc(short linker)S239C+Y407T (SEQ ID NO: 135).

[0307] In a preferred embodiment, the biparatopic dimer may comprise D10-hFc(short linker)S239C+T366Y (SEQ ID NO: 143) and F05-hFc(short linker)S239C+Y407T (SEQ ID NO: 136).

[0308] In a preferred embodiment, the biparatopic dimer may comprise A06-hFc(short linker)S239C+T366Y (SEQ ID NO: 137) and F05-hFc(short linker)S239C+Y407T (SEQ ID NO: 136).

[0309] In a preferred embodiment, the biparatopic dimer may comprise A06-hFc(short linker)S442C+T366Y (SEQ ID NO: 157) and F05-hFc(short linker)S442C+Y407T (SEQ ID NO: 156).

[0310] In a preferred embodiment, the biparatopic dimer may comprise A06-hFc (short linker) S239C and S442C + T366Y (SEQ ID NO: 177) and F05-hFc (short linker) S239C and S442C + Y407T (SEQ ID NO: 176).

[0311] In a preferred embodiment, the biparatopic dimer may comprise F05-hFc(short linker)S239C+T366Y (SEQ ID NO: 146) and A06-hFc(short linker)S239C+Y407T (SEQ ID NO: 132).

[0312] In a preferred embodiment, the biparatopic dimer may comprise F05-hFc(short linker)S442C+T366Y (SEQ ID NO: 166) and A06-hFc(short linker)S442C+Y407T (SEQ ID NO: 152).

[0313] In a preferred embodiment, the biparatopic dimer may comprise F05-hFc (short linker) S239C and S442C + T366Y (SEQ ID NO: 186) and A06-hFc (short linker) S239C and S442C + Y407T (SEQ ID NO: 172).

[0314] In a preferred embodiment, the biparatopic dimer may comprise D10-hFc (long linker) S239C and S442C + T366Y (SEQ ID NO: 178) and E06-hFc (long linker) S239C and 442C + Y407T (SEQ ID NO: 169).

[0315] In a preferred embodiment, the biparatopic dimer may comprise D10-hFc (long linker) S239C and S442C + T366Y (SEQ ID NO: 178) and E10-hFc (long linker) S239C and S442C + Y407T (SEQ ID NO: 170).

[0316] In a preferred embodiment, the biparatopic dimer may comprise D10-hFc (long linker) S239C and S442C + T366Y (SEQ ID NO: 178) and F05-hFc (long linker) S239C and S442C + Y407T (SEQ ID NO: 171).

[0317] In a preferred embodiment, the biparatopic dimer may comprise A06-hFc (long linker) S239C and S442C + T366Y (SEQ ID NO: 177) and F05-hFc (long linker) S239C and S442C + Y407T (SEQ ID NO: 171).

[0318] In a preferred embodiment, the biparatopic dimer may comprise D10-hFc(short linker) S239 / 442C T366Y (SEQ ID NO: 183) and E06-hFc(short linker) S239 / 442C Y407T (SEQ ID NO: 174).

[0319] In a preferred embodiment, the biparatopic dimer may comprise D10-hFc (short linker) S239C and S442C + T366Y (SEQ ID NO: 183) and E10-hFc (short linker) S239C and S442C + Y407T (SEQ ID NO: 175).

[0320] In a preferred embodiment, the biparatopic dimer may comprise D10-hFc (short linker) S239C and S442C + T366Y (SEQ ID NO: 183) and F05-hFc (short linker) S239C and S442C + Y407T (SEQ ID NO: 176).

[0321] Any recombinant fusion protein disclosed herein can be associated with any linker and payload disclosed herein. Any biparatopic dimer disclosed herein can be associated with any linker and payload disclosed herein. Conjugation can be via any one or more of the S239C and / or S442C residues in the biparatopic dimer. Preferably, the biparatopic dimer can be associated with the linker and payload vc-MMAE. Preferably, the biparatopic dimer comprises the conjugate disclosed in Example 6, which has been shown to be highly effective in vivo.

[0322] SEQ ID NOs: 97-186 include the S239C mutation, the S442C mutation, or both the S239C and S442C mutations for use in conjugation reactions. If the recombinant fusion protein is not to be conjugated (e.g., to an anthracycline (PNU) derivative or an auristatin derivative (MMAE)), the S239C and S442C mutations are not required, and positions 239 and 442 may be S instead of C. Thus, in alternative embodiments, a recombinant fusion protein or biparatopic dimer may include a sequence according to any one of SEQ ID NOs: 97-186, except that each sequence does not include the S239C and S442C mutations.

[0323] Thus, the recombinant fusion protein may comprise the sequence according to SEQ ID NO: 188 to SEQ ID NO: 192. A06-hFc (long linker) + Y407T QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 188)

[0324] D10-hFc (long linker) + Y407T QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 189)

[0325] E06-hFc (long linker) + Y407T QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 190)

[0326] E10-hFc (long linker) + Y407T QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 191)

[0327] F05-hFc (long linker) + Y407T QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 192)

[0328] SEQ ID NOs: 188-192 each contain a (G4S)3 linker. Corresponding sequences in which the (G4S)3 linker is replaced with a (G4S)1 linker are also expressly contemplated herein. For example, a recombinant fusion protein may contain one or more of the following SEQ ID NOs: 193-197.

[0329] A06-hFc (short linker) + Y407T QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 193)

[0330] D10-hFc (short linker) + Y407T QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 194)

[0331] E06-hFc (short linker) + Y407T QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 195)

[0332] E10-hFc (short linker) + Y407T QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 196)

[0333] F05-hFc (short linker) + Y407T QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 197)

[0334] The recombinant fusion protein may comprise a sequence according to SEQ ID NO: 199 to SEQ ID NO: 203. A06-hFc (long linker) + T366Y QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 199)

[0335] D10-hFc (long linker) + T366Y QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 200)

[0336] E06-hFc (long linker) + T366Y QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 201)

[0337] E10-hFc (long linker) + T366Y QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 202)

[0338] F05-hFc (long linker) + T366Y QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 203)

[0339] SEQ ID NOs: 199-203 each contain a (G4S)3 linker. Corresponding sequences in which the (G4S)3 linker is replaced with a (G4S)1 linker are also expressly contemplated herein. For example, a recombinant fusion protein may contain one or more of the following SEQ ID NOs: 204-208.

[0340] A06-hFc (short linker) + T366Y QVHLVESGGGLVQAGGSLRLSCAASGRTFLSMGWFRQAPGKEREFVAAISWTGGSTGYADSVKGRFTISRESDKETMYLQMNSLKPEDTAVYYCAARGDVYYGGDDATRYKYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 204)

[0341] D10-hFc (short linker) + T366Y QVQLVESGGGLVQPGGSLRLSCAASGSIFSVNTMGWYRQAPGKQRDLVAVATSGGTIHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLVVPYGMIAGDHDYWGQGTKVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 205)

[0342] E06-hFc (short linker) + T366Y QVHLVESGGGLVQAGGSLRLSCAASRRSFSEYAMGWFRQAPGKEREFVAAIGWSGVIEYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAGLGIRVVSQTPAYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 206)

[0343] E10-hFc (short linker) + T366Y QVQLVESGGGLVQAGGSLRLSCAVSGRALSDYNIGWFRQAPGKEREFVARKTWDGSSTRYDDSVKGRFTVSIEDAKNTAYLQMSSLKTEDTAVYYCAAGRYSRARGWEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 207)

[0344] F05-hFc (short linker) + T366Y QVQLVESGGGLVQAGGSLRLSCAASGRAFGDYNIGWFRQAPGKEREFVARRTWDGSSTRYDDSVKGRFTVSIDNAKNTAYLQMNSLKTEDTAVYYCAAGRYSRVRGWEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 208)

[0345] According to a further aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) a first recombinant fusion protein, wherein the first recombinant fusion protein is a recombinant fusion protein disclosed herein; (b) a second recombinant fusion protein comprising a second antigen-binding molecule fused to a second fragment of an immunoglobulin Fc region that is engineered to dimerize with a first fragment of an immunoglobulin Fc region; and The present invention provides a recombinant fusion protein dimer comprising:

[0346] In one embodiment, the second fragment of an immunoglobulin Fc region is selected from the group consisting of an Fc heavy chain, a CH2 region, and a CH3 region.

[0347] In one embodiment, the second fragment of an immunoglobulin Fc region is an Fc heavy chain.

[0348] In one embodiment, the second fragment of an immunoglobulin Fc region is engineered to dimerize with a second fragment of an immunoglobulin Fc region by a method selected from the group consisting of knobs-into-holes (YT), knobs-into-holes (CW-CSAV), CH3 charge pairing, Fab arm exchange, SEED technology, BEAT technology, HA-TF, ZW1 approach, Biclonic approach, EW-RVT, and Triomab.

[0349] In one embodiment, one or more residues of the fragment of an immunoglobulin Fc region comprise one or more amino acid substitutions suitable for knobs-in-holes (KIH) dimerization with a second fragment of an immunoglobulin Fc region comprising one or more corresponding amino acid mutations.

[0350] In one embodiment, the one or more amino acid substitutions are selected from the group consisting of T366Y, Y407T, S354C, T366W, Y349C, T366S, L368A and Y407V.

[0351] In one embodiment, the one or more amino acid substitutions are selected from the group consisting of T366Y and Y407T.

[0352] Any sequence of the recombinant fusion proteins disclosed herein can include any one or more amino acid substitutions selected from the group consisting of T366Y, Y407T, S354C, T366W, Y349C, T366S, L368A, and Y407V. Thus, SEQ ID NO: 209 can be modified by the incorporation of any one or more amino acid substitutions selected from the group consisting of T366Y, Y407T, S354C, T366W, Y349C, T366S, L368A, and Y407V and incorporated into the recombinant fusion proteins described herein in place of the human Fc region sequence. SEQ ID NO: 209 (human Fc region) EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0353] In one embodiment, the second antigen binding molecule is an ALPP and / or ALPPL2-specific antigen binding molecule.

[0354] In one embodiment, the second specific antigen binding molecule is an immunoglobin, an immunoglobulin Fab region, a Fab', an Fv, an Fv-Fc, a single chain Fv (scFv), an scFv-Fc, an (scFv)2, a diabody, a triabody, a tetrabody, a bispecific t cell engager (BiTE), an intein, a VNAR domain, a single domain antibody (sdAb), or a VH domain.

[0355] Preferably, the recombinant fusion protein dimer is a biparatopic dimer comprising a first recombinant fusion protein and a second recombinant fusion protein specific for different regions or epitopes on the same target (e.g., ALPP and / or ALPPL2). The biparatopic dimer may comprise any ALPP- and / or ALPPL2-specific antigen-binding molecule or fusion protein, such as those disclosed herein.

[0356] Epitope binning was performed on the ALPP and / or ALPPL2-specific antigen-binding molecules disclosed herein. A05, B09, B11, E06, E10, F05, F08, and G03 are in epitope bin 1. C07 and D04 are in epitope bin 4. A06 is in epitope bin 2. B01 is in epitope bin 3. D10 is in epitope bin 5. An ALPP and / or ALPPL2-specific antigen-binding molecule or a fusion protein thereof from one epitope bin can be combined with an ALPP and / or ALPPL2-specific antigen-binding molecule or a fusion protein thereof from another epitope bin to form a biparatopic dimer. Preferred pairings are given below. D10-E06 D10-E10 D10-F05 A06-F05

[0357] In one embodiment, (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 138 (D10-hFc(long linker)S239C+T366Y), and (b) The second recombinant fusion protein comprises a sequence according to SEQ ID NO: 129 (E06-hFc(long linker)S239C+Y407T).

[0358] In one embodiment, (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 138 (D10-hFc(long linker)S239C+T366Y), and (b) The second recombinant fusion protein comprises a sequence according to SEQ ID NO: 130 (E10-hFc (long linker) S239C+Y407T).

[0359] In one embodiment, (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 138 (D10-hFc(long linker)S239C+T366Y), and (b) The second recombinant fusion protein comprises a sequence according to SEQ ID NO: 131 (F05-hFc(long linker)S239C+Y407T).

[0360] In one embodiment, (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 137 (A06-hFc S239C T366Y), and (b) The second recombinant fusion protein comprises a sequence according to SEQ ID NO: 131 (F05-hFc(long linker)S239C+Y407T).

[0361] In one embodiment, (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 143 (D10-hFc(short linker)S239C+T366Y), and (b) The second recombinant fusion protein comprises a sequence according to SEQ ID NO: 134 (E06-hFc (short linker) S239C+Y407T).

[0362] In one embodiment, (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 143 (D10-hFc(short linker)S239C+T366Y), and (b) The second recombinant fusion protein comprises a sequence according to SEQ ID NO: 135 (E10-hFc (short linker) S239C Y407T).

[0363] In one embodiment, (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 143 (D10-hFc(short linker)S239C+T366Y), and (b) The second recombinant fusion protein comprises a sequence according to SEQ ID NO: 136 (F05-hFc (short linker) S239C+Y407T).

[0364] In one embodiment, (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 178 (D10-hFc S239C and S442C T366Y), and (b) The second recombinant fusion protein comprises a sequence according to SEQ ID NO: 169 (E06-hFc S239C and S442C+Y407T).

[0365] In one embodiment, (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 178 (D10-hFc S239C and S442C+T366Y), and (b) The second recombinant fusion protein comprises a sequence according to SEQ ID NO: 170 (E10-hFc S239C and S442C+Y407T).

[0366] In one embodiment, (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 178 (D10-hFc S239C and S442C+T366Y), and (b) The second recombinant fusion protein comprises a sequence according to SEQ ID NO: 171 (F05-hFc S239C and S442C+Y407T).

[0367] In one embodiment, (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 177 (A06-hFc S239C and S442C+T366Y), and (b) The second recombinant fusion protein comprises a sequence according to SEQ ID NO: 171 (F05-hFc S239C and S442C+Y407T).

[0368] In one embodiment, (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 183 (D10-hFc (short linker) S239C and S442C+T366Y); and (b) The second recombinant fusion protein comprises a sequence according to SEQ ID NO: 174 (E06-hFc (short linker) S239C and S442C+Y407T).

[0369] In one embodiment, (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 183 (D10-hFc (short linker) S239C and S442C+T366Y); and (b) The second recombinant fusion protein comprises the sequence according to SEQ ID NO: 175 (E10-hFc (short linker) S239C and S442C+Y407T).

[0370] In one embodiment, (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 183 (D10-hFc (short linker) S239C and S442C+T366Y); and (b) The second recombinant fusion protein comprises a sequence according to SEQ ID NO: 176 (F05-hFc (short linker) S239C and S442C+Y407T).

[0371] According to a further aspect, the present invention provides an ALPP- and / or ALPPL2-specific chimeric antigen receptor (CAR) comprising at least one ALPP- and / or ALPPL2-specific antigen binding molecule disclosed herein fused or conjugated to at least one transmembrane region and at least one intracellular domain.

[0372] The present invention also provides a cell comprising a chimeric antigen receptor disclosed herein, which cell is preferably an engineered T cell.

[0373] In a further aspect of the present invention, there is provided a nucleic acid sequence comprising a polynucleotide sequence encoding the specific antigen-binding molecule, recombinant fusion protein, recombinant fusion protein dimer, or chimeric antigen receptor disclosed herein.

[0374] Also provided are vectors containing the nucleic acid sequences disclosed herein and host cells containing such nucleic acids.

[0375] Provided is a method for preparing a specific antigen-binding molecule, recombinant fusion protein, recombinant fusion protein dimer, or chimeric antigen receptor disclosed herein, comprising culturing or maintaining a host cell containing the polynucleotide or vector under conditions such that the host cell produces the specific antigen-binding molecule, recombinant fusion protein, or chimeric antigen receptor, and optionally further comprising isolating the specific antigen-binding molecule, recombinant fusion protein, or chimeric antigen receptor.

[0376] In a further aspect of the present invention, there is provided a pharmaceutical composition comprising the specific antigen-binding molecule, recombinant fusion protein, recombinant fusion protein dimer, or chimeric antigen receptor disclosed herein. The pharmaceutical composition may contain various pharmaceutically acceptable carriers. The pharmaceutical composition of the present invention may be administered by any suitable method known in the art, including, but not limited to, intravenous, intramuscular, oral, intraperitoneal, or topical administration. In a preferred embodiment, the pharmaceutical composition may be prepared in the form of a liquid, gel, powder, tablet, capsule, or foam.

[0377] The specific antigen-binding molecules, recombinant fusion proteins, or chimeric antigen receptors disclosed herein can be used in therapeutic methods. More specifically, the specific antigen-binding molecules, recombinant fusion proteins, recombinant fusion protein dimers, or chimeric antigen receptors disclosed herein can be used in the treatment of cancer. Preferably, the cancer is an ALPP- and / or ALPPL2-positive cancer. More preferably, the cancer is selected from the group consisting of ovarian cancer, endometrial cancer, cervical cancer, gastric cancer, esophageal cancer, bladder cancer, lung cancer, pancreatic cancer, and testicular cancer.

[0378] Also provided herein is the use of a specific antigen-binding molecule, recombinant fusion protein, recombinant fusion protein dimer, or chimeric antigen receptor disclosed herein in the manufacture of a medicament for treating a disease in a patient in need thereof.

[0379] Furthermore, the present invention provides a method of treating a disease in a patient in need thereof, comprising administering to the patient a therapeutically effective dose of a specific antigen-binding molecule, recombinant fusion protein, recombinant fusion protein dimer, or chimeric antigen receptor disclosed herein, or a pharmaceutical composition disclosed herein.

[0380] Preferably, the cancer is an ALPP and / or ALPPL2-positive cancer type. More preferably, the cancer is selected from the group consisting of ovarian cancer, endometrial cancer, cervical cancer, gastric cancer, esophageal cancer, bladder cancer, lung cancer, pancreatic cancer and testicular cancer.

[0381] Also provided herein is a method of assaying for the presence of a target analyte in a sample, the method comprising adding to the sample a detectably labeled specific antigen-binding molecule disclosed herein, or a recombinant fusion protein disclosed herein, or a recombinant fusion protein dimer disclosed herein, and detecting binding of the molecule to the target analyte.

[0382] Further provided herein is a method for imaging a site of disease in a subject, the method comprising administering to the subject a detectably labeled specific antigen-binding molecule disclosed herein, or a detectably labeled recombinant fusion protein disclosed herein, or a recombinant fusion protein dimer disclosed herein.

[0383] Also provided herein is a method of diagnosing a disease or medical condition in a subject, the method comprising administering a specific antigen-binding molecule disclosed herein, or a recombinant fusion protein disclosed herein, or a recombinant fusion protein dimer disclosed herein.

[0384] Also contemplated herein are antibodies, antibody fragments, or antigen-binding molecules that compete with the ALPP- and / or ALPPL2-specific antigen-binding molecules disclosed herein for binding to ALPP and / or ALPPL2. The term "compete" when used in connection with an antigen-binding protein (e.g., a neutralizing antigen-binding protein or neutralizing antibody) means competition between antigen-binding proteins as determined by an assay in which the antigen-binding protein under test (e.g., an antibody or functional fragment thereof) interferes with or inhibits the specific binding of an antigen-binding molecule defined herein (e.g., the specific antigen-binding molecule of the first aspect) to a common antigen (e.g., ALPP and / or ALPPL2 in the case of the specific antigen-binding molecules disclosed herein).

[0385] Also described herein is a kit for diagnosing a subject suffering from or susceptible to cancer or providing a prognosis for a subject's condition, the kit comprising a detection means for detecting the concentration of an antigen present in a sample from a test subject, the detection means comprising an ALPP- and / or ALPPL2-specific antigen-binding molecule disclosed herein, a recombinant fusion protein or recombinant fusion protein dimer disclosed herein, a chimeric antigen receptor disclosed herein, or a nucleic acid sequence disclosed herein, each optionally derivatized, wherein the presence of the antigen in the sample indicates that the subject is suffering from cancer. Preferably, the antigen comprises an ALPP- and / or ALPPL2 protein. More preferably, the kit is used to identify the presence or absence of ALPP- and / or ALPPL2-positive cells in a sample or to determine their concentration in a sample. The kit may also include a positive control and / or a negative control against which the assay is compared and / or a detectable label.

[0386] The present invention also provides a method for diagnosing a subject suffering from or susceptibility to cancer or providing a prognosis of a condition in a subject, comprising detecting the concentration of an antigen present in a sample obtained from the subject, wherein the detection is achieved using an ALPP- and / or ALPPL2-specific antigen-binding molecule disclosed herein, a recombinant fusion protein or a recombinant fusion protein dimer disclosed herein, a chimeric antigen receptor disclosed herein, or a nucleic acid sequence disclosed herein, each optionally derivatized, and wherein the presence of the antigen in the sample indicates that the subject is suffering from cancer.

[0387] Also contemplated herein is a method for killing or inhibiting the growth of cells expressing ALPP and / or ALPPL2 in vitro or in a patient, comprising administering to the cells a pharmaceutically effective amount or dose of (i) an ALPP- and / or ALPPL2-specific antigen-binding molecule disclosed herein, a recombinant fusion protein or recombinant fusion protein dimer disclosed herein, a nucleic acid sequence disclosed herein, or a CAR or cell disclosed herein, or (ii) a pharmaceutical composition disclosed herein. Preferably, the cells expressing ALPP and / or ALPPL2 are cancer cells. More preferably, the ALPP and / or ALPPL2 are human ALPP and / or ALPPL2.

[0388] According to a further aspect, the present invention provides a compound of formula (II): X-FW1-CDR1-FW2-CDR2-FW3-CDR3-Y (II) (In the formula, FW1-CDR1-FW2-CDR2-FW3-CDR3 is an ALPP and / or ALPPL2-specific antigen-binding molecule disclosed herein, X and Y are optional amino acid sequences. wherein the specific antigen-binding molecule comprises an amino acid sequence represented by:

[0389] In certain preferred embodiments, the specific antigen-binding molecule according to this aspect of the invention may be further conjugated to a third, fourth, or fifth moiety. Conjugation of additional moieties is also contemplated. In some cases, the third, fourth, or fifth moiety may be conjugated to the second moiety. It is therefore understood that any moiety according to this aspect of the invention may have additional moieties conjugated thereto. The description of preferred features of the second moiety below also applies mutatis mutandis to the third, fourth, fifth, or higher moieties.

[0390] Preferably, X or Y, individually, are either absent or selected from the group including an immunoglobulin, an immunoglobulin Fc region, a fragment of an immunoglobulin Fc region, an Fc heavy chain, a CH2 region, a CH3 region, an immunoglobulin Fab region, Fab', Fv, Fv-Fc, single chain Fv (scFv), scFv-Fc, (scFv)2, diabody, triabody, tetrabody, bispecific T cell engager, an intein, a VNAR domain, a single domain antibody (sdAb), a VH domain, a scaffold protein (such as an affibody, centyrin, darpin) or a toxin, including, but not limited to, Pseudomonas exotoxin PE38, diphtheria toxin.

[0391] Preferably, conjugation is via a cysteine ​​residue in the amino acid sequence of the specific antigen-binding molecule, which may be located anywhere in the sequence, including in optional sequences X or Y (if present).

[0392] Conjugation may be via a thiol, aminoxy or hydrazinyl moiety incorporated at the N-terminus or C-terminus of the amino acid sequence of the specific antigen-binding molecule.

[0393] Preferably, the second moiety is selected from the group comprising a detectable label, a dye, a toxin, a drug, a prodrug, a radionuclide or a biologically active molecule.

[0394] More preferably, the second moiety is at least one cytotoxic or cytostatic agent (also referred to herein as a "toxin").

[0395] More preferably, the second portion is Auristatins, anthracyclines, preferably PNU-derived anthracyclines, Maytansinoids, amanitin derivatives, preferably α-amanitin derivatives, calicheamicin, Tubulysin, Duocarmycin, Radioisotopes, e.g. alpha-emitting radionuclides, e.g. 227Th and 225Ac labels, Liposomes containing toxic payloads, Protein toxins, Taxanes, Pyrrole benzodiazepines and their dimers, Indolinobenzodiazepine pseudodimers, spliceosome inhibitors, CDK11 inhibitors, Nicotinamide phosphoribosyltransferase inhibitors (NAMPTi), Pyridinobenzodiazepines and their dimers, cyclopropapyrroloindole (CPI), cyclopropabenzoindole (CBI) or cyclopropathienoindole (CTI) and optionally their dimers, Irinotecan or exatecan and their derivatives and at least one toxin selected from the group comprising:

[0396] In other preferred embodiments according to this aspect, the second moiety may be an immunoglobulin, an immunoglobulin Fc region, a fragment of an immunoglobulin Fc region, an Fc heavy chain, a CH2 region, a CH3 region, an immunoglobulin Fab region, Fab', Fv, Fv-Fc, a single chain Fv (scFv), scFv-Fc, (scFv)2, a diabody, a triabody, a tetrabody, a bispecific T cell engager, an intein, a VNAR domain, a single domain antibody (sdAb), a VH domain, a scaffold protein (such as an affibody, centyrin, darpin) or a toxin, including but not limited to, from the group including Pseudomonas exotoxin PE38, diphtheria toxin.

[0397] In a particularly preferred embodiment, the second moiety is a VHH domain, which may be the same as or different from the specific antigen-binding molecule according to this aspect. Thus, dimers, trimers, or higher multimers of VHH domains linked by chemical conjugation are expressly contemplated herein. In such an embodiment, each individual VHH domain may have the same antigen specificity as the other VHH domains, or they may be different.

[0398] According to this embodiment, the specific antigen-binding molecule may comprise, for example, a biparatopic specific antigen-binding molecule described herein fused to an additional biologically active molecule (including, but not limited to, an Fc fusion), which is then further conjugated to a second moiety, including, but not limited to, a cytotoxic payload.

[0399] According to this embodiment, the specific antigen-binding molecule may be a placental alkaline phosphatase (ALPP)-specific antigen-binding molecule and / or germline alkaline phosphatase (ALPPL2)-specific antigen-binding molecule, which may be an ALPP- and / or ALPPL2-specific antigen-binding molecule disclosed herein.

[0400] The specific antigen-binding molecule of this embodiment may be for use in therapy. More specifically, the specific antigen-binding molecule of this embodiment may be for use in the treatment of cancer. Preferably, the cancer is an ALPP- and / or ALPPL2-positive cancer. More preferably, the cancer is selected from the group consisting of ovarian cancer, endometrial cancer, cervical cancer, gastric cancer, esophageal cancer, bladder cancer, lung cancer, pancreatic cancer, and testicular cancer.

[0401] Also provided herein is the use of a specific antigen-binding molecule of this aspect in the manufacture of a medicament for treating a disease in a patient in need thereof.

[0402] Pharmaceutical compositions comprising the specific antigen-binding molecules of this aspect are also provided. Pharmaceutical compositions may contain a variety of pharmaceutically acceptable carriers.

[0403] Furthermore, the present invention provides a method for treating a disease in a patient in need of such treatment, the method comprising administering to the patient a therapeutically effective dose of the specific antigen-binding molecule of this aspect or a pharmaceutical composition comprising the specific antigen-binding molecule of this aspect.

[0404] Preferably, the cancer is an ALPP and / or ALPPL2-positive cancer type. More preferably, the cancer is selected from the group consisting of ovarian cancer, endometrial cancer, cervical cancer, gastric cancer, esophageal cancer, bladder cancer, lung cancer, pancreatic cancer and testicular cancer.

[0405] Also provided herein is a method for assaying the presence of a target analyte in a sample, the method comprising adding a detectably labeled specific antigen-binding molecule of this embodiment to the sample and detecting binding of the molecule to the target analyte.

[0406] Further provided herein is a method for imaging a site of disease in a subject, the method comprising administering to the subject a specific antigen-binding molecule of this aspect that is detectably labeled.

[0407] Also provided herein is a method of diagnosing a disease or medical condition in a subject, the method comprising administering a specific antigen-binding molecule of this aspect.

[0408] According to a further aspect, the present invention provides engineered ALPP and / or ALPPL2 monomers that have been engineered so as to be capable of forming covalent bonds with further engineered ALPP and / or ALPPL2 monomers.

[0409] In one embodiment, the covalent bond is a disulfide bridge.

[0410] In one embodiment, the engineered ALPP and / or ALPPL2 monomers comprise an introduced cysteine ​​residue.

[0411] In one embodiment, the engineered ALPP and / or ALPPL2 monomer comprises a G503C substitution in SEQ ID NO: 275, or an equivalent substitution in another ALPP and / or ALPPL2 monomer.

[0412] Human ALPP (SEQ ID NO: 275) MLGPCMLLLLLLLGLRLQLSLGIIPVEEENPDFWNREAAEALGAAKKLQPAQTAAKNLIIFLGDGMGVSTVTAARILKGQKKDKLGPEIPLAMDRFPYVALSKTYNVDKHVPDSGATATAYLCGVKGNFQTIG LSAAARFNQCNTTRGNEVISVMNRAKKAGKSVGVVTTTRVQHASPAGTYAHTVNRNWYSDADVPASARQEGCQDIATQLISNMDIDVILGGGRKYMFRMGTPDPEYPDDYSQGGTRLDGKNLVQEWLAKRQGAR YVWNRTELMQASLDPSVTHLMGLFEPGDMKYEIHRDSTLDPSLMEMTEAALRLLSRNPRGFFLFVEGGRIDHGHHESRAYRALTETIMFDDAIERAGQLTSEEDTLSLVTADHSHVFSFGGYPLRGSSIFGLAP GKARDRKAYTVLLYGNGPGYVLKDGARPDVTESESGSPEYRQQSAVPLDEETHAGEDVAVFARGPQAHLVHGVQEQTFIAHVMAFAACLEPYTACDLAPPAGTTDAAHPGRSVVPALLPLLAGTLLLLETATAP

[0413] In one embodiment, the engineered ALPP monomer comprises a sequence according to SEQ ID NO:263.

[0414] According to a further aspect, the present invention provides an artificially engineered ALPP and / or ALPPL2 homodimer comprising a first and a second artificially engineered ALPP and / or ALPPL2 monomer disclosed herein.

[0415] Antibodies capable of binding to the target antigens described herein can be isolated from suitable antibody libraries, for example, using phage display, yeast display, ribosomal display, or mammalian display techniques known in the art. The engineered ALPP and / or ALPPL2 monomers disclosed herein or the engineered ALPP and / or ALPPL2 homodimers disclosed herein can be used to immunize animals to generate ALPP and / or ALPPL2-specific binding molecules.

[0416] According to a further aspect, the present invention provides a method for producing an ALPP and / or ALPPL2 specific binding molecule comprising the steps of: (a) immunizing an animal with an engineered ALPP and / or ALPPL2 homodimer of the thirteenth aspect; (b) isolating peripheral blood mononuclear cells (PBMCs) from the animal; (c) cloning RNA or cDNA sequences obtained from PBMCs into a vector; (d) transforming a prokaryotic cell with the vector described above under conditions that allow the production of the specific binding molecule; (e) selecting specific binding molecules by subjecting the specific binding molecules to antigen affinity selection; (f) recovering specific binding molecules having the desired specificity. The present invention provides a method comprising:

[0417] In one embodiment, the animal is a llama.

[0418] In one embodiment, antigen affinity selection is performed by biopanning.

[0419] Furthermore, any of the features described in relation to any of the above aspects of the invention may be combined with other aspects of the invention, mutatis mutandis.

[0420] In addition to the sequences listed, the following sequences are expressly disclosed, some of which relate to examples of molecules of the invention described herein.

[0421] [Table 2] JPEG2025542294000008.jpg255170JPEG2025542294000009.jpg255170JPEG2025542294000010.jpg255170

[0422] According to a further aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) an ALPP and / or ALPPL2-specific antigen-binding molecule, recombinant fusion protein, or recombinant fusion protein dimer disclosed herein, and (b) at least one toxin, cytotoxic agent, or cytostatic agent The present invention provides a target binding molecule-drug conjugate comprising:

[0423] The ALPP and / or specific antigen-binding molecule may be conjugated to a toxin, cytotoxic agent, or cytostatic agent via a linker.

[0424] At least one toxin Auristatins, anthracyclines, preferably PNU-derived anthracyclines, Maytansinoids, amanitin derivatives, preferably α-amanitin derivatives, calicheamicin, Tubulysin, Duocarmycin, Radioisotopes, e.g. alpha-emitting radionuclides, e.g. 227Th and 225Ac labels, Liposomes containing toxic payloads, Protein toxins, Taxanes, Pyrrole benzodiazepines and their dimers, Indolinobenzodiazepine pseudodimers, spliceosome inhibitors, CDK11 inhibitors, Nicotinamide phosphoribosyltransferase inhibitors (NAMPTi), Pyridinobenzodiazepines and their dimers, cyclopropapyrroloindole (CPI), cyclopropabenzoindole (CBI) or cyclopropathienoindole (CTI) and optionally their dimers, Irinotecan or exatecan and their derivatives may be selected from the group comprising:

[0425] The toxin can be an auristatin. The auristatin can be auristatin E (AE) or monomethylauristatin E (MMAE). The auristatin can be an MMAE derivative. Any of the spacer ([X]) and / or linker ([L1] and / or [L2]) groups described herein in connection with anthracycline toxins are expressly contemplated in connection with auristatins such as MMAE. In a preferred embodiment, the target binding molecule-drug conjugate can include Val-Cit-PAB-MMAE (vcPAB-MMAE).

[0426] In one embodiment, (b) is an MMAE derivative, The target binding molecule-drug conjugate has the formula (VI): [ka] [X] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof; [L1] and [L2] are optional linkers selected from the group consisting of valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), peptide, dipeptide, tripeptide, -(CH2)n-, -(CH2CHO)n-, p-aminobenzyloxycarbonyl (PAB), Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, Val-Ala, Asn-Ala, any amino acid, a D-amino acid, Phe-Lys-PAB, and combinations thereof; and Y comprises an ALPP and / or ALPPL2-specific antigen-binding molecule or recombinant fusion protein disclosed herein. It has the following structure.

[0427] Preferably, the target binding molecule-drug conjugate has the formula (VII): [ka] It has the following structure.

[0428] The toxin can be exatecan or an exatecan derivative.

[0429] Exatecan derivatives have the formula (X): [ka] where the point of attachment to the linker is represented by a wavy line. It may have the structure:

[0430] A highly attractive class of DNA-intercalating toxins for use as payloads for drug conjugates are the anthracyclines because of their proven clinical validation as chemotherapeutic agents in cancer treatment.

[0431] The stability of chemically conjugated protein-drug conjugates is an important consideration because the unintended release of highly potent anthracycline toxins, such as PNU-159682, in the patient's circulation before targeting of tumor cells can result in off-target effects and unwanted side effects. Some example molecules released from PNU conjugates include the release of PNU159682 derivatives from different Val-Cit-PAB-containing drug linkers.

[0432] Therefore, a potent toxin that can be bound to a target protein with high stability is required to avoid or at least reduce undesirable side effects. Alternatively, the linker payload is designed so that extracellular cleavage releases a derivative of the payload with reduced potency. However, sufficient potency must be maintained to avoid any reduction in side effects being offset by the need to administer higher doses to achieve efficacy.

[0433] Ease of conjugation is an important factor in producing an easily manufacturable product. The payloads of the present disclosure may use maleimide groups that can react with any available thiol group on a conjugation partner using simple and standard conditions. Furthermore, the use of maleimide / thiol chemistry for conjugation allows for site-specific conjugation to, for example, an engineered cysteine ​​residue on the side chain of the protein sequence via an engineered cysteine-containing his-myc tag (example sequences include, but are not limited to, ACAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 250)) at the C- or N-terminus of the protein.

[0434] Antibody / protein drug conjugates generated using nonselective labeling methods, for example, via reaction with amino functional groups in proteins, deliver products containing multiple different species with different drug-antibody ratios. This affects the properties of the conjugate, such as potency and PK characteristics, which affect in vivo efficacy and toxicity. Therefore, thiol-reactive payloads are of great importance because they can be reacted with naturally occurring cysteine ​​residues in proteins or engineered into specific sites at any point within the protein sequence using molecular biology / recombinant protein expression or chemical synthesis, or through chemical modification of expressed, synthetic, or native proteins, in a single process with high yield. In some cases described herein, cysteines are engineered into the Fc region of Fc fusion proteins.

[0435] The present disclosure provides anthracycline (PNU) derivatives suitable for use in drug conjugates. Specifically, a derivative of PNU159682 is provided that lacks the C14 carbon and an attached carboxyl functional group and is functionalized with an ethylenediamino (EDA) group at the C13 carbonyl of PNU159682. This EDA-PNU159682 can then be functionalized with a maleimide-containing linker via the amino group of the EDA moiety. The maleimide group is present in anthracycline (PNU) derivatives of formula (V) and may also be present in anthracycline (PNU) derivatives of formula (VI). Such payloads can react with free thiol groups on another molecule. If a free thiol is present on a protein, a protein-drug conjugate (PDC) can be formed.

[0436] Surprisingly, derivatives of PNU159682 functionalized with ethylenediamino (EDA) groups and linked to thiol groups via maleimide groups exhibit increased stability with slightly lower potency compared to non-EDA payload or free payload derivatives. A more stable payload may be advantageous due to reduced off-target effects, which in turn may lead to reduced side effects and increased patient compliance.

[0437] PCT / EP 2020 / 067210 discloses a compound of formula (V): [ka] wherein [X] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof; [L1] and [L2] are valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), peptide, -(CH2) n -, -(CH2CH2O) n -, p-aminobenzyloxycarbonyl (PAB), Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, Val-Ala, Asn-Ala, any amino acid except glycine, and combinations thereof. describes anthracycline (PNU) derivatives of the formula:

[0438] The anthracycline (PNU) derivative of formula (V) may contain [L1], [L2] or [L1] and [L2].

[0439] Preferably, when [L1] and / or [L2] are peptides, said peptides do not contain glycine.

[0440] It will be clear to one skilled in the art that in the absence of the optional spacer and / or optional linker, a bond will remain in its place.

[0441] Preferably, [X] is polyethylene glycol, [ka] and selected from the group comprising: [ka] represents the point of attachment to the rest of the molecule, and [R] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof.

[0442] Most preferably, [X] is polyethylene glycol. The polyethylene glycol may be PEG4.

[0443] Preferably, [L2] is p-aminobenzyloxycarbonyl (PAB) or alanine.

[0444] Preferably, the anthracycline (PNU) derivative comprises [L1] and / or [L2], and [X] is optional. Thus, [L1] and / or [L2] may be selected from the group consisting of valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), peptide, -(CH2) n -, -(CH2CH2O) n The linker may be selected from the group consisting of -, p-aminobenzyloxycarbonyl (PAB), Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, Val-Ala, Asn-Ala, any amino acid except glycine, and combinations thereof. The anthracycline (PNU) derivative of formula (V) may contain [L1], [L2], or [L1] and [L2]. The anthracycline (PNU) derivative of formula (V) may contain [L1] and / or [L2].

[0445] PCT / EP 2020 / 067210 discloses a compound of formula (V): [ka] wherein [X] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof; [L1] and / or [L2] are valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), peptide, -(CH2) n -, -(CH2CH2O) n -, p-aminobenzyloxycarbonyl (PAB), Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, Val-Ala, Asn-Ala, any amino acid except glycine, and combinations thereof) The present invention describes an anthracycline (PNU) derivative of formula (V) which comprises [L1], [L2] or [L1] and [L2].

[0446] Preferably, [X] is polyethylene glycol, [ka] and selected from the group comprising: [ka] represents the point of attachment to the rest of the molecule, and [R] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof.

[0447] Most preferably, [X] is polyethylene glycol. The polyethylene glycol may be PEG4.

[0448] Preferably, [L2] is p-aminobenzyloxycarbonyl (PAB) or alanine.

[0449] Preferably, the PNU derivative is [ka] [ka] [ka] [ka] The structure is selected from:

[0450] PCT / EP 2020 / 067210 discloses a compound of formula (VI): [ka] wherein [X] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof; [Z] is a reactive group. Also described are anthracycline (PNU) derivatives of the formula: The reactive group can be any reactive group suitable for use in conjugation reactions, particularly conjugation reactions to target binding molecules.

[0451] Thus, [Z] can be a moiety that contains a functional group for use in a bioconjugation reaction. Functional groups for use in a bioconjugation reaction include, but are not limited to: Maleimides or alkyl halides for reaction with thiol or selenol groups on proteins via thioether and seronoether reactions, maleimides, alkyl halides or sulfhydryl groups for reaction with thiol-functionalized molecules, including the thiol groups of protein cysteine ​​residues; activated disulfides, such as pyridyldithiol (Npys thiol) or TNB thiol (5-thiol-2-nitrobenzoic acid), for reaction with thiol groups to form disulfide bonds via thiol-disulfide exchange; Amino groups for attachment to carboxyl groups on proteins and biomolecules via amide bond formation reactions; Alkyne groups, particularly ring-constrained alkynes such as dibenzocyclooctyne (DBCO) or bicyclo[6.1.0]nonyne (BCN), for reaction with azide-functionalized biomolecules via strain-promoted alkyne-azide cycloaddition copper-free chemistry. Azide functionality can be introduced into proteins, for example, via incorporation of the unnatural amino acid para-azidomethyl-L-phenylalanine, or into protein glycans using enzyme-mediated glycoengineering to attach azide-containing sugar analogs. Azide groups for reaction with alkyne-functionalized target binding molecules via strain-promoted alkyne-azide cycloaddition copper-free chemistry; Aminoxy groups for reaction with aldehyde and ketone groups on biomolecules via oxime-forming ligation. Ketones can be introduced into proteins using amber stop codon technology, for example, through the incorporation of the unnatural amino acid para-acetylphenylalanine. Aldehydes can be found on biomolecules through the presence of reducing sugars and can be introduced into proteins through periodate oxidation of N-terminal serine residues or cis-glycol groups of carbohydrates. Aldehyde groups can also be incorporated into proteins through the conversion of protein cysteines within a defined sequence to formylglycine by formylglycine-generating enzymes. Furthermore, formylglycine-containing proteins have been conjugated to payloads via hydrazino-Picteth-Spengler (HIPS) ligation. Aldehyde or ketone groups for reaction with aminoxy-, hydrazide-, or hydrazinyl-functionalized biomolecules via oxime- or hydrazine-bond-forming ligation reactions. Aminoxy- and hydrazide-functionalized proteins can be generated via cleavage of intein fusion proteins.

[0452] Thus, [Z] may be selected from the group consisting of maleimide, alkyl halide, sulfhydryl group, activated disulfide (e.g., pyridyldithiol (Npys thiol) or TNB thiol (5-thiol-2-nitrobenzoic acid)), amino group, alkyne group (e.g., ring-constrained alkynes such as dibenzocyclooctyne (DBCO) or bicyclo[6.1.0]nonyne (BCN)), azide group, aminoxy group, aldehyde group, and ketone group.

[0453] [Z] can be a moiety for enzyme-mediated bioconjugation reactions. Moieties for use in enzyme-mediated bioconjugation reactions include, but are not limited to, polyGly [(Gly) ] for use in sortase enzyme-mediated antibody conjugation. n ] or Lys-Lys-Gln-Gly and Lys-Pro-Glu-Thr-Gly and contain a suitable primary amine for bacterial transglutaminase-mediated conjugation to the glutamine γ-carboxyamide group.

[0454] Thus, [Z] may be selected from the group consisting of poly-Gly and primary amines.

[0455] Thus, the PNU derivative according to formula (IV) may correspond to a PNU derivative of formula (V) in which L1 is Val-Cit-PAB, L2 is absent, and the maleimide group may be replaced with another reactive group as defined above.

[0456] Preferably, [X] is polyethylene glycol, [ka] and selected from the group comprising: [ka] represents the point of attachment to the rest of the molecule, and [R] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof.

[0457] Most preferably, [X] is polyethylene glycol. The polyethylene glycol may be PEG4.

[0458] The PNU derivative according to formula (V) or formula (VI) may be conjugated to the ALPP- and / or ALPPL2-specific antigen-binding molecule according to the present invention or the recombinant fusion protein or recombinant fusion protein dimer of the present invention.

[0459] According to a further aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) an ALPP and / or ALPPL2-specific antigen-binding molecule disclosed herein, or a recombinant fusion protein disclosed herein, or a recombinant fusion protein dimer disclosed herein; (b) anthracycline (PNU) derivatives and 1. A target binding molecule-drug conjugate comprising the formula (III): [ka] wherein [X] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof; [L1] and [L2] are valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), peptide, -(CH2) n -, -(CH2CH2O) nan optional linker selected from the group consisting of -, p-aminobenzyloxycarbonyl (PAB), Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, Val-Ala, Asn-Ala, any amino acid except glycine, and combinations thereof; and Y comprises an ALPP and / or ALPPL2-specific antigen-binding molecule, recombinant fusion protein, or recombinant fusion protein dimer disclosed herein. The present invention provides a target-binding molecule-drug conjugate having the structure:

[0460] The target-binding molecule-drug conjugate of formula (III) may comprise [L1], [L2], or [L1] and [L2].

[0461] Preferably, in the target-binding molecule-drug conjugate in which [L1] and / or [L2] is a peptide, the peptide does not contain glycine.

[0462] It will be clear to one skilled in the art that in the absence of the optional spacer and / or optional linker, a bond will remain in its place.

[0463] Preferably, the target binding molecule-drug conjugate comprises: [ka] The structure is selected from:

[0464] According to a further aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) an ALPP and / or ALPPL2-specific antigen-binding molecule disclosed herein, or a recombinant fusion protein or recombinant fusion protein dimer disclosed herein; (b) anthracycline (PNU) derivatives and 1. A target binding molecule-drug conjugate comprising formula (IV): [ka] wherein [X] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof; [Z] is a linker derived from a reactive group used to conjugate the anthracycline (PNU) derivative and the target binding molecule, and Y comprises an ALPP-specific antigen-binding molecule of the first, second or ninth aspect, or a recombinant fusion protein or recombinant fusion protein dimer according to the third, fourth or fifth aspect. The present invention provides a target-binding molecule-drug conjugate having the structure:

[0465] [Z] is typically a moiety derived from a reactive group used to conjugate an anthracycline (PNU) derivative and a target-binding molecule. [Z] may be a moiety derived from a reactive group selected from the group consisting of maleimide, alkyl halide, sulfhydryl group, activated disulfide, amino group, alkyne group, azide group, aminoxy group, aldehyde group, and ketone group.

[0466] Thus, [Z] may be selected from the group consisting of a disulfide bond, an amide bond, an oxime bond, a hydrazone bond, a thioether bond, a 1,2,3 triazole, and a polyGly.

[0467] Preferably, [X] is polyethylene glycol, [ka] and selected from the group comprising: [ka] represents the point of attachment to the rest of the moiety, and [R] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof.

[0468] Most preferably, [X] is polyethylene glycol. The polyethylene glycol may be PEG4.

[0469] In one embodiment of the target-binding molecule-drug conjugate of the present invention, Y comprises an ALPP- and / or ALPPL2-specific antigen-binding molecule disclosed herein conjugated to a PNU derivative via a human immunoglobulin Fc region or an Fc fragment thereof.

[0470] In one embodiment, the fragment of a human immunoglobulin Fc region may be selected from the group consisting of an Fc heavy chain, a CH2 region, and a CH3 region.

[0471] In a further aspect, the present invention provides biparatopic placental alkaline phosphatase (ALPP) and / or germline alkaline phosphatase (ALPPL2)-specific antigen-binding molecules.

[0472] According to a further aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) a biparatopic placental alkaline phosphatase (ALPP) and / or germline alkaline phosphatase (ALPPL2)-specific antigen-binding molecule; (b) at least one toxin, cytotoxic agent, or cytostatic agent; The present invention provides a biparatopic target binding molecule-drug conjugate comprising:

[0473] In one embodiment of the biparatopic target-binding molecule-drug conjugate, the biparatopic ALPP- and / or ALPPL2-specific antigen-binding molecule is conjugated to a cytotoxic or cytostatic agent via a linker.

[0474] In one embodiment of the biparatopic target binding molecule-drug conjugate, (b) is Auristatins, anthracyclines, preferably PNU-derived anthracyclines, Maytansinoids, amanitin derivatives, preferably α-amanitin derivatives, calicheamicin, Tubulysin, Duocarmycin, Radioisotopes, e.g. alpha-emitting radionuclides, e.g. 227Th and 225Ac labels, Liposomes containing toxic payloads, Protein toxins, Taxanes, Pyrrole benzodiazepines and their dimers, Indolinobenzodiazepine pseudodimers, spliceosome inhibitors, CDK11 inhibitors, Nicotinamide phosphoribosyltransferase inhibitors (NAMPTi), Pyridinobenzodiazepines and their dimers, cyclopropapyrroloindole (CPI), cyclopropabenzoindole (CBI) or cyclopropathienoindole (CTI) and optionally their dimers, Irinotecan or exatecan and their derivatives The toxin is selected from the group consisting of:

[0475] In one embodiment of the biparatopic target binding molecule-drug conjugate, the toxin is an auristatin.

[0476] In one embodiment of the biparatopic target binding molecule-drug conjugate, the toxin is monomethyl auristatin.

[0477] In one embodiment of the biparatopic target binding molecule-drug conjugate, the toxin is monomethyl auristatin E.

[0478] In one embodiment of the biparatopic target binding molecule-drug conjugate, (b) is an MMAE derivative; The target binding molecule-drug conjugate has the formula (VI): [ka] [X] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof; [L1] and [L2] are optional linkers selected from the group consisting of valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), peptide, dipeptide, tripeptide, -(CH2)n-, -(CH2CHO)n-, p-aminobenzyloxycarbonyl (PAB), Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, Val-Ala, Asn-Ala, any amino acid, a D-amino acid, Phe-Lys-PAB, and combinations thereof; and Y comprises a biparatopic ALPP and / or ALPPL2-specific antigen-binding molecule disclosed herein. It has the following structure.

[0479] In one embodiment of the biparatopic target binding molecule-drug conjugate, (b) is an MMAE derivative; The target binding molecule-drug conjugate has the formula (VII): [ka] It has the following structure.

[0480] In one embodiment of the biparatopic target binding molecule-drug conjugate, (b) is an anthracycline (PNU) derivative; The target binding molecule-drug conjugate has the formula (III): [ka] wherein [X] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof; [L1] and [L2] are valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), peptide, -(CH2) n -, -(CH2CH2O) n an optional linker selected from the group consisting of -, p-aminobenzyloxycarbonyl (PAB), Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, Val-Ala, Asn-Ala, any amino acid except glycine, and combinations thereof; and Y comprises a biparatopic ALPP and / or ALPPL2-specific antigen-binding molecule disclosed herein. It has the following structure.

[0481] In one embodiment of a biparatopic target binding molecule-drug conjugate, the target binding molecule-drug conjugate comprises: [ka] The structure is selected from:

[0482] Also provided herein is a target binding molecule-drug conjugate according to the above aspects for use in therapy.

[0483] Also provided herein is a target binding molecule-drug conjugate according to the above embodiment for use in the treatment of cancer.

[0484] Also provided herein is the use of a target binding molecule-drug conjugate according to the above aspects in the manufacture of a medicament for treating a disease in a patient in need thereof.

[0485] Also provided herein is a method of treating a disease in a patient in need thereof, comprising administering to the patient a therapeutically effective dose of a target binding molecule-drug conjugate according to the above embodiment. The disease may be cancer.

[0486] Preferably, the cancer is an ALPP and / or ALPPL2-positive cancer type. More preferably, the cancer is selected from the group consisting of ovarian cancer, endometrial cancer, cervical cancer, gastric cancer, esophageal cancer, bladder cancer, lung cancer, pancreatic cancer and testicular cancer.

[0487] Also provided herein is a pharmaceutical composition comprising a target binding molecule-drug conjugate according to any of the above aspects and at least one other pharmaceutically acceptable ingredient.

[0488] definition Antigen-specific binding molecules of the invention comprise amino acid sequences derived from a synthetic library of VHH molecules or a library derived from immunization of camelids. The terms VHH and single domain antibody (sdAb) may also be used interchangeably.

[0489] Amino acids are represented herein as one-letter codes or three-letter codes or both.

[0490] The term "affinity purification" refers to the purification of a molecule based on specifically attracting or binding a molecule to a chemical or binding partner to form a combination or complex that allows the molecule to bind to or be attracted to the partner moiety while being separated from impurities.

[0491] The term "complementarity determining region" or CDR (i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of a VHH domain whose presence is typically involved in antigen binding. Each VHH typically has three CDR regions, identified as CDR1, CDR2, and CDR3.

[0492] "Framework regions" (FW) are those VHH residues other than the CDR residues. Each VHH typically has four framework regions identified as FW1, FW2, FW3 and FW4.

[0493] The boundaries between the FW and CDR regions in VHHs are not intended to be fixed, and therefore some variation in the length and composition of these regions should be expected. This will be understood by those skilled in the art, particularly with reference to studies that have been conducted on the analysis of these regions (Wesolowski et al., Med Microbiol Immunol (2009) 198, 157-174; Vattekatte et al., PeerJ (2020) e8408). The molecules of the present invention are defined herein by reference to the FW and CDR regions, but are not limited by their precise definition. Thus, variations consistent with art-recognized structure of VHH domains are expressly contemplated herein.

[0494] A "codon set" refers to a set of different nucleotide triplet sequences used to encode desired variant amino acids. A set of oligonucleotides, representing all possible combinations of nucleotide triplets provided by the codon set, contains sequences encoding the desired group of amino acids and can be synthesized, for example, by solid-phase synthesis. A standard format for codon designation is that of the IUB code, known in the art and described herein.

[0495] Codon sets are typically represented by three capital letters in italics, such as NNK, NNS, XYZ, DVK, etc. Thus, a "non-random codon set" refers to a codon set that encodes selected amino acids that partially, preferably completely, satisfy the amino acid selection criteria described herein. The synthesis of oligonucleotides with selected nucleotide "degeneracy" at certain positions is well known in the art, such as the TRIM approach (Knappek et al.; J. Mol. Biol. (1999), 296, 57-86; Garrard & Henner, Gene (1993), 128, 103). Such a set of oligonucleotides with a certain codon set can be synthesized using commercially available nucleic acid synthesizers (e.g., available from Applied Biosystems, Foster City, CA) or can be obtained commercially (e.g., from Life Technologies, Rockville, MD). A set of synthetic oligonucleotides with a specific codon set typically includes multiple oligonucleotides with different sequences, the differences being established by the codon sets within the overall sequence. Oligonucleotides used according to the present invention have a sequence that allows hybridization to a VHH nucleic acid template and may also contain restriction enzyme sites, as appropriate.

[0496] "Cell," "cell line," and "cell culture" are used interchangeably (unless the context indicates otherwise), and such designations include all progeny of the cell or cell line. Thus, for example, terms such as "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.

[0497] "Control sequences," when referring to expression, refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences that are suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, a ribosome binding site, etc. Eukaryotic cells use control sequences such as promoters, polyadenylation signals, and enhancers.

[0498] The term "coat protein" refers to a protein, at least a portion of which is present on the surface of a virus particle. From a functional standpoint, a coat protein is any protein that associates with a virus particle during the virus assembly process in a host cell and remains associated with the assembled virus until it infects another host cell.

[0499] The "detection limit" for a chemical entity in a particular assay is the lowest concentration of that entity that can be detected above background levels for that assay. For example, in a phage ELISA, the "detection limit" for a particular phage displaying a particular antigen-binding fragment is the phage concentration at which the particular phage produces an ELISA signal that exceeds the signal produced by a control phage that does not display the antigen-binding fragment.

[0500] "Fusion protein" and "fusion polypeptide" refer to a polypeptide having two portions covalently linked to each other, each of which has a different property. The property can be a biological property, such as in vitro or in vivo activity. The property can be a simple chemical or physical property, such as binding to a target antigen, catalysis of a reaction, etc. The two portions can be linked directly by a single peptide bond or through a peptide linker containing one or more amino acid residues. Generally, the two portions and the linker are in reading frame with each other. Preferably, the two portions of the polypeptide are derived from heterologous or different polypeptides.

[0501] As used herein, the term "fusion protein" generally refers to one or more proteins linked together by chemical means, such as hydrogen bonds or salt bridges, or by peptide bonds via protein synthesis, or both. Typically, fusion proteins are prepared by recombinant DNA techniques and may be referred to herein as recombinant fusion proteins.

[0502] "Heterologous DNA" is any DNA introduced into a host cell. The DNA can be from a variety of sources, such as genomic DNA, cDNA, synthetic DNA, and fusions, or combinations thereof. The DNA can include DNA from the same cell or cell type as the host or recipient cell, or DNA from a different cell type, e.g., an allogenic or xenogenic source. The DNA can optionally include a marker or selection gene, e.g., an antibiotic resistance gene, a temperature resistance gene, etc.

[0503] "Highly diverse positions" refer to amino acid positions located in the variable regions of the light and heavy chains that have a large number of different amino acids represented at that position when known amino acid sequences and / or naturally occurring antibodies or antigen-binding fragments are compared. Highly diverse positions are typically found in the CDR or HV regions.

[0504] "Identity" describes a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. Identity, in some cases, also refers to the degree of sequence relatedness (homology) between polypeptide or polynucleotide sequences, as determined by the match between strings of polypeptide or polynucleotide sequences. While numerous methods exist for measuring identity between two polypeptide or two polynucleotide sequences, commonly used methods for determining identity are codified in computer programs. Preferred computer programs for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux, et al., Nucleic Acids Research, 12, 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec. Biol. (1990) 215, 403).

[0505] Preferably, the amino acid sequence of the protein has at least 45% identity at the amino acid level with the amino acid sequence disclosed herein using the default parameters of the BLAST computer program (Atschul et al., J. Mol. Biol. (1990) 215, 403-410) provided by the HGMP (Human Genome Mapping Project).

[0506] More preferably, the protein sequences may have at least 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%, and even more preferably 95% (and even more preferably at least 96%, 97%, 98% or 99%) identity at the nucleic acid or amino acid level to the amino acid sequences set out herein.

[0507] Proteins may also include sequences having at least 45%, 46%, 47%, 48%, 49%, 50%, 52%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a sequence disclosed herein using the default parameters of the BLAST computer program provided by HGMP.

[0508] A "library" refers to a plurality of VHH or VHH fragment sequences (e.g., polypeptides of the invention) or nucleic acids encoding those sequences, which differ in the combinations of variant amino acids introduced into them by the methods of the invention.

[0509] "Ligation" is the process of forming phosphodiester bonds between two nucleic acid fragments. For two fragments to ligate, their ends must be compatible with each other. In some cases, the ends become directly compatible after endonuclease digestion. However, it may first be necessary to convert the protruding ends typically produced after endonuclease digestion to blunt ends to make them compatible for ligation. To blunt the ends, DNA is treated with approximately 10 units of the Klenow fragment of DNA polymerase I or T4 DNA polymerase in a suitable buffer in the presence of four deoxyribonucleotide triphosphates at 15°C for at least 15 minutes. The DNA is then purified by phenol-chloroform extraction and ethanol precipitation or silica purification. The DNA fragments to be ligated together are placed in a solution in approximately equimolar amounts. The solution also contains ATP, ligase buffer, and approximately 10 units of ligase, such as T4 DNA ligase, per 0.5 μg of DNA. If the DNA is to be ligated into a vector, the vector is first linearized by digestion with the appropriate restriction endonuclease. The linearized fragment is then treated with bacterial alkaline phosphatase or calf intestinal phosphatase to prevent self-ligation during the ligation step.

[0510] A "mutation" is a deletion, insertion or substitution of a nucleotide relative to a reference nucleotide sequence, e.g., a wild-type sequence.

[0511] "Natural" or "naturally occurring" VHHs refer to VHHs identified from non-synthetic sources, such as ex vivo tissue sources or serum from Camelidae animals. These VHHs can include VHHs produced in any type of immune response, natural or otherwise induced. Natural VHHs include the amino acid sequences and nucleotide sequences that constitute or encode those antibodies. As used herein, natural VHHs differ from "synthetic VHHs," which refer to VHH sequences that have been altered from a source or template sequence, for example, by substituting, deleting, or adding one or more amino acids at a certain position with different amino acids to provide an antibody sequence that differs from the source antibody sequence.

[0512] The term "nucleic acid construct" generally refers to a nucleic acid of any length, which may be DNA, cDNA, or RNA, such as mRNA, obtained by cloning or produced by chemical synthesis. DNA may be single-stranded or double-stranded. Single-stranded DNA may be the coding sense strand, or it may be the non-coding or antisense strand. For therapeutic use, the nucleic acid construct is preferably in a form that can be expressed in the subject to be treated.

[0513] "Operably linked," when referring to a nucleic acid, means that the nucleic acid is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, concatenated and in reading frame. However, enhancers need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

[0514] The term "protein" generally refers to multiple amino acid residues linked together by peptide bonds. It is used interchangeably with peptide, oligopeptide, oligomer, or polypeptide to mean the same thing, including glycoproteins and derivatives thereof. The term "protein" is also intended to include fragments, analogs, variants, and derivatives of proteins, which fragments, analogs, variants, or derivatives retain essentially the same biological activity or function as the reference protein. Examples of protein analogs and derivatives include peptide nucleic acids and DARPins (designed ankyrin repeat proteins).

[0515] Protein fragments, analogs, variants or derivatives may be at least 25, preferably 30 or 40, or up to 50, 100 or 60-120 amino acids in length, depending on the length of the original protein sequence from which they are derived, although lengths of 90-120, 100-110 amino acids may be convenient in some cases.

[0516] Fragments, derivatives, variants, or analogs of proteins include those in which (i) one or more of the amino acid residues are substituted with a conservative or non-conservative amino acid residue (preferably a conservative amino acid residue), which may or may not be an amino acid residue encoded by the genetic code, or (ii) one or more of the amino acid residues includes a substituent group, or (iii) additional amino acids are fused to the mature polypeptide, for example, to a leader or accessory sequence used in purification of the polypeptide. Such fragments, derivatives, variants, and analogs are deemed to be within the skill of one of ordinary skill in the art from the teachings herein.

[0517] As used herein, the term "VHH" refers to the variable domain of the heavy chain of a heavy-chain antibody that naturally lacks a light chain. The amino acid sequence and structure of a VHH can be considered to be composed of four framework regions or "FWs" referred to in the art and hereinafter as "framework region 1" or "FW1," "framework region 2" or "FW2," "framework region 3" or "FW3," and "framework region 4" or "FW4," respectively, separated by three complementarity-determining regions or "CDRs" referred to in the art as "complementarity-determining region 1" or "CDR1," "complementarity-determining region 2" or "CDR2," and "complementarity-determining region 3" or "CDR3," respectively, but are not limited thereto. As further described below, the total number of amino acid residues contained in a VHH can be approximately 110 to 130, preferably 112 to 115, and most preferably 113. However, it should be noted that portions, fragments, or analogs of antibody VHH domains are not particularly limited in length and / or size, so long as such portions, fragments, or analogs retain (at least a portion of) their functional activity, such as cell-killing activity (as defined herein), and / or retain (at least a portion of) the binding specificity of the original VHH from which they are derived. Portions, fragments, or analogs that retain (at least a portion of) their functional activity, such as cell-killing activity (as defined herein) and / or retain (at least a portion of) the binding specificity of the VHH from which they are derived are also referred to herein as "functional fragments" or "functional variants" of VHH. The numbering method for amino acid residues in VHH domains is the method described by Chothia et al. (Nature 342, 877-883 (1989)), the so-called "AbM definition" and the so-called "contact definition." This is the numbering system adopted herein.Alternatively, the amino acid residues of the variable domains of antibody heavy chain variable domains (including VHHs) may be numbered according to the general numbering for heavy chain variable domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, Md., Publication No. 91), as applied to VHH domains from camelids in Riechmann and Muyldermans (J Immunol Methods. 1999 Dec 10;231(1-2):25-38).

[0518] An "oligonucleotide" is a short, single- or double-stranded polydeoxynucleotide that is chemically synthesized by known methods (e.g., phosphotriester, phosphorous, or phosphoramidite chemistry using solid-phase techniques). Additional methods include polymerase chain reaction (PCR), which is used when the entire nucleic acid sequence of a gene is known or when the sequence of a nucleic acid complementary to the coding strand is available. Alternatively, if the target amino acid sequence is known, known preferred coding residues for each amino acid residue can be used to deduce potential nucleic acid sequences. Oligonucleotides can be purified on polyacrylamide gels or molecular sizing columns, or by precipitation. DNA is "purified" when it is separated from non-nucleic acid impurities (which may be polar, non-polar, ionic, etc.).

[0519] As used herein, a "source" or "template" VHH refers to a VHH or VHH antigen-binding fragment whose antigen-binding sequence serves as a template sequence when diversification is performed according to the criteria described herein. The antigen-binding sequence generally preferably comprises at least one CDR, and preferably framework regions, within the VHH.

[0520] A "transcriptional regulatory element" contains one or more of the following components: an enhancer element, a promoter, an operator sequence, a repressor gene, and a transcription termination sequence.

[0521] "Transformation" refers to the process by which a cell takes up DNA and becomes a "transformant." DNA uptake can be permanent or transient. A "transformant" is a cell that has taken up and maintained DNA, as evidenced by the expression of a phenotype associated with the DNA (e.g., antibiotic resistance conferred by a protein encoded by the DNA).

[0522] A "variant" or "mutant" of a starting or reference polypeptide (e.g., a resource VHH or its CDR), such as a fusion protein (polypeptide) or heterologous polypeptide (heterologous to a phage), is a polypeptide that (1) has an amino acid sequence that differs from that of the starting or reference polypeptide and (2) is derived from the starting or reference polypeptide through either natural or artificial mutagenesis. Such variants include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the polypeptide of interest. For example, a fusion polypeptide of the invention generated using an oligonucleotide comprising a non-random codon set that encodes a sequence having a variant amino acid (with respect to the amino acid found at the corresponding position in the resource VHH or antigen-binding fragment) is a variant polypeptide with respect to the resource VHH or antigen-binding fragment. Thus, a variant CDR refers to a CDR that comprises a variant sequence with respect to the starting or reference polypeptide sequence (e.g., of a resource VHH or antigen-binding fragment). In this context, a variant amino acid refers to an amino acid that differs from the amino acid at the corresponding position in the starting or reference polypeptide sequence (e.g., of a resource VHH or antigen-binding fragment). Any combination of deletion, insertion, and substitution can be made to arrive at the final variant or mutant construct, provided that the final construct possesses the desired functional characteristics. Amino acid changes can also alter post-translational processing of the polypeptide, for example, changing the number or position of glycosylation sites.

[0523] A "wild-type" or "reference" sequence or the sequence of a "wild-type" or "reference" protein / polypeptide, e.g., a coat protein or a CDR of a source VHH, can be a reference sequence from which a variant polypeptide is derived through the introduction of mutations. Generally, the "wild-type" sequence for a given protein is the most common sequence found in nature. Similarly, a "wild-type" gene sequence is the sequence for that gene that is most commonly found in nature. Mutations can be introduced into a "wild-type" gene (and thus the protein it encodes) either through natural processes or by artificial means. The product of such a process is a "variant" or "mutant" form of the original "wild-type" protein or gene.

[0524] A "humanized" antigen-specific antigen-binding molecule can be modified at one or more amino acid sequence positions to reduce the potential for immunogenicity in vivo while retaining functional binding activity for a specific epitope on a specific antigen.

[0525] Humanization of antibody variable domains is a technique well known in the art for modifying antibodies raised in non-human species against therapeutically useful targets so that the humanized forms can avoid undesired immunological responses when administered to human subjects. Methods related to humanization are summarized in Almagro JC and William Strohl W. Antibody Engineering: Humanization, Affinity Maturation, and Selection Techniques in Therapeutic Monoclonal Antibodies: From Bench to Clinic. Edited by An J. 2009 John Wiley & Sons, Inc. and Strohl WR and Strohl LM, Therapeutic Antibody Engineering, Woodhead Publishing 2012.

[0526] Humanization of single domain antibodies is a well-known technique in the art, and humanized forms can avoid unwanted immunological reactions when administered to human subjects. Methods involved in humanization are summarized in Vincke et al., Prot Structure and Folding (2009) 284 (5) 3273-3284; Rossotti et al., FEBS J (2022) 289 (14) 3917-4328; Sulea T, Methods Mol Biol (2022) 2446.

[0527] The term "chimeric antigen receptor (CAR)" as used herein can refer to, for example, an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immune receptor, and encompasses artificially engineered receptors that graft artificial specificities onto specific immune effector cells. CARs can be used to confer the specificity of an antigen-specific binding protein, such as a monoclonal antibody or a VHH domain, onto T cells, thereby enabling the generation of large numbers of specific T cells for use in, for example, adoptive cell therapy. CARs can, for example, direct the specificity of cells to a tumor-associated antigen. CARs can comprise an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising a tumor-associated antigen-binding region. In certain embodiments, a CAR comprises a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to a CD3-zeta transmembrane and endodomain. In other specific embodiments, a CAR comprises a fusion of a VHH domain described herein with a CD3-zeta transmembrane and endodomain. The specificity of other CAR designs can be derived from the receptor's ligand (e.g., a peptide) or a pattern recognition receptor, such as dectin. In certain embodiments, malignant B cells can be targeted by redirecting T cell specificity using a CAR specific for the B-lineage molecule CD19. In some cases, the spacing of the antigen recognition domain can be modified to reduce activation-induced cell death. In some cases, the CAR contains additional domains for costimulatory signaling, such as CD3-zeta, FcR, CD27, CD28, CD137, DAP10, and / or OX40. In some cases, molecules such as costimulatory molecules, reporter genes for imaging (e.g., positron emission tomography), gene products that conditionally eliminate T cells upon addition of a prodrug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors can be co-expressed with the CAR.

[0528] As used herein, the term "conjugation" may refer to any method of chemically linking two or more chemical moieties. Typically, conjugation is via a covalent bond. In the context of the present invention, at least one of the chemical moieties is a polypeptide, and in some cases, the conjugation involves two or more polypeptides, one or more of which may be produced by recombinant DNA technology. Numerous systems for conjugating polypeptides are known in the art. For example, conjugation can be achieved through lysine residues present in the polypeptide molecule using N-hydroxysuccinimide, or through cysteine ​​residues present in the polypeptide molecule using maleimidobenzoyl sulfosuccinimide ester. In some embodiments, conjugation occurs via a short-acting degradable bond, including, but not limited to, physiologically cleavable bonds, such as esters, carbonates, carbamates, sulfates, phosphates, acyloxyalkyl ethers, acetals and ketals, hydrazones, oximes, and disulfides. In some embodiments, a linker is incorporated that is cleavable by an intracellular or extracellular enzyme, such as a cathepsin family member or a glucuronidase family member, e.g., beta-glucuronidase, and is cleavable under reducing conditions or acidic pH, to allow release of the conjugated moiety from the polypeptide or protein to which it is conjugated.

[0529] A particularly preferred method of conjugation is via engineered cysteine ​​residues at specific positions within the Fc sequence, or alternatively, to naturally occurring cysteine ​​residues within the Fc sequence, including but not limited to cysteine ​​residues involved in interchain disulfide bonds.

[0530] A particularly preferred method of conjugation is the use of intein-based technology (US Patent Application Publication No. 2006247417). Briefly, a protein of interest is expressed as an N-terminal fusion of an engineered intein domain (Muir 2006 Nature 442, 517-518). Subsequent N-to-S acyl shift in the protein-intein conjugate leads to a thioester bond intermediate, which can be chemically cleaved with bis-aminoxyl reagents or amino-thiols to yield the desired protein C-terminal aminoxy or thiol derivatives, respectively. These C-terminal aminoxy and thiol derivatives can be chemoselectively reacted with aldehyde / ketone and maleimide-functionalized moieties, respectively, to yield site-specific C-terminally modified proteins.

[0531] In another preferred method of conjugation, VHHs are directly expressed with an additional cysteine ​​incorporated at or near the C-terminal region of the VHH or within a short C-terminal tag sequence, allowing conjugation with a thiol-reactive payload, such as a maleimide-functionalized moiety.

[0532] Conjugation, as referred to herein, is also intended to encompass the use of linker moieties, which can impart a number of useful properties. Linker moieties include, but are not limited to, peptide sequences such as poly-glycine, gly-ser, val-cit, or val-ala. In some cases, the linker moiety can be selected to be cleavable under certain conditions, such as through the use of enzymes, nucleophilic / basic reagents, reducing agents, light irradiation, electrophilic / acidic reagents, organometallic and metallic reagents, or oxidizing reagents, or the linker can be specifically selected to resist cleavage under such conditions.

[0533] In some cases, the linker may include a PAB (used interchangeably with PABC) moiety, shown below. [ka]

[0534] In some cases, the linker may include the moiety val-cit-PAB shown below. [ka]

[0535] Polypeptides can be conjugated to a variety of functional moieties to achieve a number of goals. Examples of functional moieties to reduce immunogenicity and antigenicity or improve solubility include, but are not limited to, polymers such as polyethylene glycol. Further non-limiting examples include conjugation of polypeptides to therapeutic or cytotoxic agents.

[0536] The term "detectable label" is used herein to define that an entity can be visualized or otherwise detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical, or other means. A detectable label can be selected so that it generates a signal that can be measured and whose intensity is proportional to the amount of bound entity. A wide variety of systems for labeling and / or detecting proteins and peptides are known in the art. A label can be directly detectable (i.e., does not require any further reaction or manipulation to become detectable; e.g., fluorophores are directly detectable), or it can be indirectly detectable (i.e., becomes detectable through reaction or binding with another entity that is detectable; e.g., haptens are detectable by immunostaining after reaction with an appropriate antibody containing a reporter, e.g., a fluorophore). Suitable detectable agents include, but are not limited to, radionuclides, fluorophores, chemiluminescent agents, microparticles, enzymes, colorimetric labels, magnetic labels, haptens, molecular beacons, and aptamer beacons.

[0537] Contemplated herein are methods of killing or inhibiting the growth of cells expressing ALPP and / or ALPPL2 in vitro or in patients. Generally, the term "killing," as used herein in reference to cells, means causing cell death. This can be achieved by a number of mechanisms, for example, by inducing necrosis or other cell damage or apoptosis. As used herein, the phrases "inhibiting growth" or "inhibiting proliferation" are intended to encompass prevention of cell development, more specifically, prevention of cell division.

[0538] As used herein, an alkyl group is a straight-chain or branched-chain, substituted or unsubstituted (preferably unsubstituted) group containing 1 to 40 carbon atoms. The alkyl group can be optionally substituted at any position. As used herein, the term "alkenyl" refers to a group derived by removal of a single hydrogen atom from a straight-chain or branched-chain aliphatic moiety having at least one carbon-carbon double bond. As used herein, the term "alkynyl" refers to a group derived by removal of a single hydrogen atom from a straight-chain or branched-chain aliphatic moiety having at least one carbon-carbon triple bond.

[0539] Terms such as "alkyl," "aryl," and "heteroaryl" also include polyvalent species such as alkylene, arylene, and "heteroarylene." Examples of alkylene groups include ethylene (-CH-CH-) and propylene (-CH-CH-CH-). An exemplary arylene group is phenylene (-CH-), and an exemplary heteroarylene group is pyridinylene (-CHN-).

[0540] An aromatic ring is a cyclic aromatic group that may have 0, 1, 2 or more, preferably 0, 1 or 2, ring heteroatoms. The aromatic ring may be optionally substituted and / or fused to one or more aromatic or non-aromatic rings (preferably aromatic), which may contain 0, 1, 2 or more ring heteroatoms to form a polycyclic ring system.

[0541] Aromatic rings include both aryl and heteroaryl groups. Aryl and heteroaryl groups can be mononuclear, i.e., have only one aromatic ring (e.g., phenyl or phenylene), or polynuclear, i.e., have two or more aromatic rings that may be fused (e.g., naphthyl or naphthylene), individually covalently bonded (e.g., biphenyl), and / or have a combination of both fused and individually bonded aromatic rings. Preferably, the aryl or heteroaryl group is substantially conjugated aromatic throughout the group. Aryl groups can contain 5 to 40 ring carbon atoms, 5 to 25 carbon atoms, 5 to 20 carbon atoms, or 5 to 12 carbon atoms. Heteroaryl groups can be 5 to 40-, 5 to 25-, 5 to 20-, or 5 to 12-membered rings containing one or more ring heteroatoms selected from N, O, S, and P. An aryl or heteroaryl may be fused to one or more aromatic or non-aromatic rings (preferably aromatic rings) to form a polycyclic ring system.

[0542] Aryl and heteroaryl preferably denote mono-, bi- or tricyclic aromatic or heteroaromatic groups having up to 25 ring atoms, which may also include fused rings and which are optionally substituted.

[0543] Preferred aryl groups include, but are not limited to, benzene, biphenylene, triphenylene, [1,1':3',1'']terphenyl-2'-ylene, naphthalene, anthracene, binaphthylene, phenanthrene, pyrene, dihydropyrene, chrysene, perylene, tetracene, pentacene, benzopyrene, fluorene, indene, indenofluorene, spirobifluorene, and the like.

[0544] Preferred heteroaryl groups include, but are not limited to, five-membered rings such as pyrrole, pyrazole, silole, imidazole, 1,2,3-triazole, 1,2,4-triazole, tetrazole, furan, thiophene, selenophene, oxazole, isoxazole, 1,2-thiazole, 1,3-thiazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, 1,2,3-thiadiazole, and 1,2,4-thiadiazole. 1,2,5-thiadiazole, 1,3,4-thiadiazole, 6-membered rings such as pyridine, pyridazine, pyrimidine, pyrazine, 1,3,5-triazine, 1,2,4-triazine, 1,2,3-triazine, 1,2,4,5-tetrazine, 1,2,3,4-tetrazine, 1,2,3,5-tetrazine and condensed systems such as carbazole, indole, isoindole, indolizine, indazole, benzimidazole, benzotriazole, purine, naphthimidazole, phenanthrimidazole, pyridoimidine dazole, pyrazineimidazole, quinoxalineimidazole, benzoxazole, naphthoxazole, anthroxazole, phenanthroxazole, isoxazole, benzothiazole, benzofuran, isobenzofuran, dibenzofuran, quinoline, isoquinoline, pteridine, benzo-5,6-quinoline, benzo-6,7-quinoline, benzo-7,8-quinoline, benzisoquinoline, acridine, phenothiazine, phenoxazine, benzopyridazine, benzopyrimidine, quinoxaline, phenanthroxazole, azine, naphthyridine, azacarbazole, benzocarboline, phenanthridine, phenanthroline, thieno[2,3b]thiophene, thieno[3,2b]thiophene, dithienothiophene, dithienopyridine, isobenzothiophene, dibenzothiophene, benzothiadiazothiophene, 2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione (diketopyrrolopyrrole, DPP), 2-oxo-1H-indol-3-ylidene, [3,3'-bipyrrolo[2,3-b]pyridinylidene]-2,2'(1H,1'H)-dione (pyridine isoindigo) and (3E)-3-(2-oxo-1H-indol-3-ylidene)-1H-indol-2-one (isoindigo), or a combination thereof. The heteroaryl group may be substituted with alkyl, alkoxy, thioalkyl, fluoro, fluoroalkyl, or further aryl or heteroaryl substituents. Preferably, the heteroaryl group is thiophene.

[0545] Particularly preferred heteroatoms are selected from O, S, N, P, and Si. Typically, hydrogen completes the valence of heteroatoms contained in the molecules of the invention, for example, -NH- or -NH2 may be present, involving one or two other groups for N.

[0546] As used herein, the term "optionally substituted" means that one or more of the hydrogen atoms in the optionally substituted moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and if more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituents may be the same or different at all positions. The substituent combinations envisioned by the present invention are preferably those that result in the production of stable compounds. As used herein, the term "stable" refers to compounds that are chemically feasible and can exist at room temperature (i.e., 16-25°C) long enough to allow for their detection, isolation, and / or use in chemical synthesis.

[0547] Any of the above groups (e.g., those referred to herein as "optionally substituted," e.g., alkyl, aryl, and heteroaryl groups) may optionally include one or more substituents, which are preferably silyl, sulfo, sulfonyl, formyl, amino, imino, nitrilo, mercapto, cyano, nitro, halogen, -NCO, -NCS, -OCN, -SCN, -C(=O)NR 0 R 00 , -C(=O)X 0, C(=O)R 0 , -NR 0 R 00 , C 1~12 Alkyl, C 1~12 Alkenyl, C 1~12 Alkynyl, C 6~12 Aryl, C 3~12 Cycloalkyl, heterocycloalkyl having 4 to 12 ring atoms, heteroaryl having 5 to 12 ring atoms, C 1~12 Alkoxy, hydroxy, C 1~12 Alkyl carbonyl, C 1~12 Alkoxy-carbonyl, C 1~12 Alkylcarbonyloxy or C 1~12 alkoxycarbonyloxy, wherein one or more H atoms are optionally replaced with F or Cl and / or combinations thereof; 0 is a halogen and R 0 and R 00 are independently H or optionally substituted C 1~12 It is alkyl. Optional substituents may include all chemically possible combinations of the same and / or multiple of the above groups (e.g., amino and sulfonyl, when directly attached to each other, represent a sulfamoyl group). In one embodiment, the substituent is not acyl. As used herein, acyl refers to an acyl group, which is a moiety derived from an oxoacid, such as a carboxylic acid, by removal of one or more hydroxyl groups. It contains a double-bonded oxygen atom and an alkyl group.

[0548] In some embodiments, the group can be unsubstituted. For example, an anthracycline (PNU) derivative can be represented by formula (V): [ka] wherein [X] is an optional spacer selected from the group comprising an unsubstituted alkyl group, an unsubstituted heteroalkyl group, an unsubstituted aryl group, an unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof; [L1] and [L2] are valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), peptide, -(CH2) n -, -(CH2CH2O) n -, p-aminobenzyloxycarbonyl (PAB), Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, Val-Ala, Asn-Ala, any amino acid except glycine, and combinations thereof. It can be of the following type.

[0549] In embodiments where the group is unsubstituted, [X] is preferably polyethylene glycol, and [ka] and selected from the group comprising: [ka] represents the point of attachment to the rest of the molecule, and [R] is an optional spacer selected from the group comprising an unsubstituted alkyl group, an unsubstituted heteroalkyl group, an unsubstituted aryl group, an unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof.

[0550] Generally, the term PAB is intended to mean p-aminobenzyloxycarbonyl. Occasionally, in the literature, the term PAB may be used to refer to p-aminobenzyl. Herein, PAB is intended to refer to p-aminobenzyloxycarbonyl. The terms "vc-PAB-MMAE," "vcPAB-MMAE," "vcMMAE," and "MMAE" are used interchangeably herein to describe conjugates obtained from conjugation with MC-vc-PAB-MMAE.

[0551] The term "target-binding molecule" refers to any molecule that binds to a given target. In this context, "target" and "antigen" may be used interchangeably. Examples of target-binding molecules include natural or recombinant proteins, such as immunoglobulins or antibodies, immunoglobulin Fc regions, immunoglobulin Fab regions, Fab, Fab', Fv, Fv-Fc, single-chain Fv (scFv), scFv-Fc, (scFv)2, diabodies, triabodies, tetrabodies, bispecific T cell engagers, inteins, intein fusions, VNAR domains, single-domain antibodies (sdAbs), VH domains, scaffold proteins (affibodies, centyrins, darpins, etc.), and nucleic acids, such as aptamers or small molecules or natural products that are developed to bind to or that naturally bind to a target.

[0552] Chemical modification of proteins and biomolecules to introduce thiols is well established. Methods include reaction of amine groups with 2-iminothiolane (Traut's reagent), modification of amine groups with NHS-ester-containing heterobifunctional agents such as N-succinimidyl S-acetylthiolate (SATA) or N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) followed by treatment with hydroxylamine and reducing agents, respectively, and cleavage of engineered intein fusion proteins with cysteamine to generate C-terminal thiol proteins and peptides.

[0553] The phrase "selected from the group comprising" whenever it appears in this specification may be replaced with the phrase "selected from the group consisting of" and vice versa.

[0554] The PNU derivatives described herein can be prepared according to standard synthetic methods. Mass spectrometry can be used to confirm that the correct molecule has been produced (Table 4).

[0555] [Table 3]

[0556] The present invention will be further understood with reference to the following examples. [Example]

[0557] Example 1 – Generation of anti-ALPP and ALPPL2 binders VHH production VHHs targeting ALPP and ALPPL2 were generated through immunization of llamas with an engineered ALPP homodimer (ALPP CysHis, SEQ ID NO: 263). To ensure that the dimeric form of the protein was maintained in solution, the engineered ALPP homodimer was used for immunization. Briefly, two llamas were immunized five times with ALPP CysHis and periodically checked for antibody titers over a 98-day period, after which they were bled and PBMCs were isolated. PBMCs from both animals were combined to generate a single-domain antibody library from RNA / cDNA. Three rounds of biopanning were performed against ALPP CysHis, with negative selection against the ALPI His homodimer (SEQ ID NO: 264 at each stage).

[0558] After panning, 94 individual clones expressed as monoclonal phages were screened for specific binding to ALPP. These were analyzed by ELISA for specific binding to ALPP and any off-target binding to ALPI. Clones with high specificity for ALPP and little binding to ALPI were selected for off-phage testing.

[0559] Twenty-five hits identified from the phage library screen were expressed in Escherichia coli (E. coli) periplasmically, and C-terminal His-tagged proteins were extracted by osmotic shock and purified by IMAC chromatography using cOmplete™ His-tag purification resin (Roche). Eluted samples were dialyzed into PBS pH 7.4. The concentration of each sample was calculated from OD280 readings using a predicted extinction coefficient based on the nanobody sequence. All proteins were then characterized by SDS-PAGE under reducing and non-reducing conditions, size exclusion chromatography (SEC), and electrospray mass spectrometry to determine expression levels, purity, monomericity, and confirm protein identity. Binding to ALPP and ALPI was assessed by ELISA, thermostability by thermal shift assay, and ALPP and ALPI binding kinetics (K ). D A subset of monomers was also tested by BLI against ALPPL2 to determine cross-reactivity between homologs.

[0560] [Table 4] JPEG2025542294000043.jpg255170

[0561] Binding to ALPP and ALPI by ELISA ELISA was used to test the binding and specificity of VHHs. ALPP CysHis (SEQ ID NO: 263), ALPI His homodimer (SEQ ID NO: 264), or BSA (Thermo Fisher) was coated onto plates, and 500 ng of each purified VHH was added. Anti-llama-HRP antibody was used for detection.

[0562] Size exclusion chromatography The monomericity and biophysical properties of the VHH variants were assessed by size exclusion chromatography (SEC) using an analytical SEC column (Superdex75 10 / 300GL). Chromatography was performed in PBS pH 7.4.

[0563] Binding to human ALPP / ALPPL2, ALPI, and ALPL by BLI Binding kinetics were determined using a BioLayer Interferometry (BLI) Octet K2 system (Sartorius). Human ALPP, ALPPL2, ALPL (each fused to a cysteine-stabilized leucine zipper), and ALPI (SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267, and SEQ ID NO: 264, respectively) were immobilized to an AR2G sensor using amine coupling in sodium acetate pH 5 buffer. All assays were performed in PBS at a plate temperature of 25°C. Ka (M) was calculated using Octet Data Analysis High Throughput software for BioLayer Interferometry (ForteBio). -1 s -1 ) value, Kd(s -1 ) value and K D (nM) values ​​were determined.

[0564] Thermal stability experiments The Applied Biosystems StepOne Real Time PCR system was used for the thermostability assay using the Protein Thermal Shift™ dye kit (Thermo Fisher). The assay mix was set up to a final protein concentration of 20 μM in 20 μL. 5 μL of Thermal Shift™ buffer was added along with 2.5 μL of 8× Thermal Shift™ Dye. The assay was run using StepOne software, and data was analyzed using Protein Thermal Shift™ software. All data are from first derivative analysis.

[0565] [Table 5]

[0566] High affinity ALPP and ALPPL2 specific binders with good thermal stability were generated, as shown in Table 5. Neither binding to ALPI nor to ALPL was detectable by BLI.

[0567] Epitope binning experiments To determine epitope cross-reactivity, a BLI competition assay was performed. ALPP was immobilized as described above using the AR2G sensor. The ALPP sensor was then immersed in a well containing a saturating concentration of the initial binder for 10 minutes to obtain an association curve. The sensor was then immersed in a well containing the same concentration of the initial binder plus 20 nM of the secondary binder. An increase in the association curve suggested that the second VHH bound to a different epitope than the initial binder. Monomers with identical CDR3s were grouped as likely to be in the same epitope bin, and only one from each group was used for the epitope binning experiment.

[0568] [Table 6]

[0569] The epitope binning experiment described above (Table 6) suggests that the 25 different binding sequences can be grouped into five different epitope bins. A05, B09, B11, E06, E10, F05, F08 and G03 appear to cross-compete and are therefore in epitope bin 1. C07 and D04 constitute epitope bin 4, while A06, B01 and D10 do not cross-react with any other VHH and therefore constitute epitope bins 2, 3 and 5, respectively.

[0570] Binding of VHH proteins to cell surface ALPP, ALPPL2, ALPI, and ALPL by flow cytometry C-terminal His-tagged VHH proteins were evaluated for cell surface binding of ALPP, ALPPL2, ALPI, and ALPL by flow cytometry. Binders containing different epitope bins were screened on CHO cells stably expressing ALPP, ALPPL2, ALPI, or ALPL. High levels of cell surface protein expression were confirmed for each cell line by flow cytometry.

[0571] The cell surface binding of test agents to hALPP isoforms was characterized in four CHO-ALPP isoform stably transfected cell lines (CHO-ALPP, CHO-ALPPL2, CHO-ALPI, and CHO-ALPL), and the resulting K DappThe values ​​were determined. Adherent cells were detached from tissue culture flasks by incubation with 0.1% EDTA / PBS solution at 37°C for approximately 10 minutes or until cells readily detached. Cells were resuspended in ice-cold PBS / 2% FCS in 15 ml tubes and centrifuged at 1700 rpm for 5 minutes at 4°C. The supernatant was removed, and the cell pellet was resuspended in PBS / 2% FCS. Cell counts were performed using a Chemometec Nucleocounter NC-202, and 2.5 x 10^5 cells per test sample were aliquoted into a 96-well plate. Cells were incubated on ice for 1 hour with 100 μl of test agent at a concentration series and controls. The sample plate was centrifuged at 1700 rpm at 4°C for 5 minutes. Washing was performed by removing the supernatant and resuspending the cell pellet in 0.25 mL of ice-cold PBS / 2% FCS using a multichannel pipette. The samples were again centrifuged at 1700 rpm at 4°C for 5 minutes. The supernatant was removed, and two additional washes were performed as described. After the final wash and centrifugation step, excess liquid was removed by blotting the plate on tissue paper. Where appropriate, binding of the His6-tagged VHH drug was determined by adding 100 μl of anti-x6His tag Ab (Abcam) per cell pellet sample and incubating on ice for 30 min. Washing steps were performed as described previously. Binding of the His6-tagged VHH drug was detected using Fitc-anti-mouse antibody (ThermoFisher) by incubating with the appropriate sample on ice in the dark for 30 min. Washing steps were performed as described previously. Finally, all cell pellets were resuspended in 0.28 ml of ice-cold PBS / 2% FCS and left on ice in the dark before analysis on a Merck-Millipore Guava EasyCyte HT or Thermo Fisher Attune NxT flow cytometer.

[0572] The binding of the different domains to the different cell surface ALPP forms is summarized in Table 7 and FIG.

[0573] [Table 7]

[0574] For 350 nM D04-His, the MFI=629 for binding to CHO-ALPI compared to an MFI of 6652 for binding to CHO ALPP (ALPI MFI is 9.5% of the ALPP MFI).

[0575] As shown in Table 7 and Figure 1, highly specific VHH protein binders were generated for ALPP and ALPPL2 that showed no binding to either ALPI or ALPL, even at very high concentrations.

[0576] D10 shows preferential binding to ALPP over ALPPL2, D04 exhibits weak binding to ALPI, and F08 exhibits minimal binding, with an MFI at saturation (350 nM) that is 75-fold lower for ALPI binding compared to ALPP.

[0577] Example 2 - VHH reformatting as an Fc fusion protein Generation of VHH-Fc fusion proteins Fusion of proteins to the Fc domain can improve protein solubility and stability, significantly increase plasma half-life, and improve overall therapeutic efficacy. The human IgG1 Fc sequence and further examples are shown below.

[0578] Human IgG1 Fc (hFc) EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 209)

[0579] hFc S293C EPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 268)

[0580] hFc S442C EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 269)

[0581] hFc S293C+S442C EPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 270)

[0582] VHH variants were genetically fused to engineered hIgG1 Fc domains (see sequence listing) containing various cysteine ​​substitutions in the hIgG1 Fc sequence, including S239C, S442C, and S239C+S442C (EU numbering), via either a long [G4S]3 linker or a shorter [G4S]1 linker. VHH-hFc fusion proteins were transiently expressed as secreted proteins in CHO K1 cells and purified from the culture medium using MabSelect™ SuRe™ (Evitria, Switzerland). Purified proteins were exchanged into PBS pH 7.4 or PBS + 100 mM Arg pH 7.4 and analyzed by SEC (AdvanceBio, Agilent, running buffer DPBS pH 7.4 or Superdex200 Increase 10 / 300, Cytivia, running buffer PBS pH 7), SDS-PAGE under reducing and native conditions, and mass spectrometry to confirm sequence and protein integrity. For MS analysis, proteins were deglycosylated with PNGaseF, reduced with 50 mM DTT (reduced mass) or analyzed without reduction (intact mass), and run on a Sciex X500B QTOF with a MassPREP desalting column (Waters). Characterization data for VHH-hFc proteins are shown in Table 8.

[0583] [Table 8]

[0584] The purified target protein was produced in good yield and exhibited a high percentage of monomericity as determined by SEC.

[0585] Binding of VHH-hFc proteins to ALPP homologs by BLI Binding kinetics were determined using a BioLayer Interferometry (BLI) Octet K2 or R4 system (Sartorius). Human ALPP, ALPPL2, and ALPL (SEQ ID NO: 265, SEQ ID NO: 266, and SEQ ID NO: 267), all fused with a disulfide-stabilized leucine zipper, and ALPI His (SEQ ID NO: 264) were used in BLI experiments. D For apparent measurements, ALPP proteins were immobilized on AR2G sensors in sodium acetate pH 5 buffer using amine coupling. To measure 1:1 interactions, Fc fusion proteins were immobilized using AHC biosensors. All assays were performed in HBS-EP at a plate temperature of 25°C. Ka (M -1 s -1 ), Kd(s -1 ) and K D (nM) values ​​were determined.

[0586] [Table 9]

[0587] BLI data (Table 9) confirm the high affinity binding of VHH-hFc fusion proteins to ALPP and ALPPL2. B01 and F08 have K values ​​of 148 nM and 10.8 nM, respectively. D Although both of these proteins showed very low levels of binding to ALPI in the ALPP / ALPPL2 response, both of these proteins gave very low response values ​​compared to those observed for ALPP / ALPPL2. All other fusions showed no binding to ALPI or ALPL.

[0588] D04-hFc and D10-hFc show a slightly reduced binding affinity for ALPPL2 compared to ALPP, and lower response values ​​are also observed.

[0589] ALPP species cross-reactivity of VHH-hFc proteins To assess the species cross-reactivity of VHH-hFc fusions targeting ALPP / ALPPL2, BLI was performed using mouse ALPPL2 (NCBI# NP_031459), rhesus ALPP (NCBI# XP_028687061), and cynomolgus ALPP (NCBI# XP_045223825). Each of these was expressed as a cysteine-stabilized leucine zipper fusion (SEQ ID NO: 271, SEQ ID NO: 272, and SEQ ID NO: 273). BLI was performed as described above. The data are shown in Table 10.

[0590] [Table 10]

[0591] A05, A06, E10, and F05-hFc showed binding to mouse ALPPL2, although the response values ​​were significantly lower than those observed for binding to human ALPP and ALPPL2. All hFc fusions bound with high affinity to rhesus and cynomolgus monkey ALPP proteins.

[0592] Binding of VHH-hFc proteins to cell surface ALPP, ALPPL2, ALPI, and ALPL by flow cytometry VHH-hFc proteins were evaluated for human ALPP, ALPPL2, ALPI, and ALPL cell surface binding by flow cytometry. Binders were screened on CHO cells stably expressing human ALPP, ALPPL2, ALPI, or ALPL.

[0593] Cell surface binding of test agents to hALPP isoforms was characterized in four CHO-ALPP isoform transfectant cell lines (CHO-ALPP, CHO-ALPPL2, CHO-ALPI, and CHO-ALPL), and the resulting K DappThe values ​​were determined. Adherent cells were detached from tissue culture flasks by incubation with 0.1% EDTA / PBS solution at 37°C for approximately 10 minutes or until the cells readily detached. Cells were resuspended in ice-cold PBS / 2% FCS in 15 ml tubes and centrifuged at 1700 rpm at 4°C for 5 minutes. The supernatant was removed, and the cell pellet was resuspended in PBS / 2% FCS. Cell counts were performed using a Chemometec Nucleocounter NC-202, and 2.5 x 10^5 cells per test sample were aliquoted into a 96-well plate. Cells were incubated on ice for 1 hour with 100 μl of test agent at a concentration series and controls. The sample plate was centrifuged at 1700 rpm at 4°C for 5 minutes. Washing was performed by removing the supernatant and resuspending the cell pellet in 0.25 mL of ice-cold PBS / 2% FCS using a multichannel pipette. The samples were again centrifuged at 1700 rpm at 4°C for 5 minutes. The supernatant was removed, and two further washes were performed as described. After the final wash and centrifugation step, excess liquid was removed by blotting the plates on tissue paper. Binding of VHH-hFc agents was determined by adding 100 μl of Fitc-anti-human Ab (ThermoFisher) and incubating on ice in the dark for 30 minutes. Washing steps were performed as previously described. Finally, all cell pellets were resuspended in 0.28 ml of ice-cold PBS / 2% FCS and left on ice in the dark before analysis on a Merck-Millipore Guava EasyCyte HT or ThermoFisher Attune NxT flow cytometer.

[0594] The binding of the different domains to the different cell surface ALPP forms is summarized in Table 11 and FIG.

[0595] [Table 11]

[0596] Figure 3 and Table 11 show that VHH-hFc fusions specifically bind to ALPP / ALPPL2 on the cell surface without binding to ALPI or ALPL. B01-hFc showed very weak binding to CHO-ALPI cells at the highest concentration of protein used (350 nM).

[0597] Example 3 Biparatopic ALPP / ALPPL2 Binding Proteins Biparatopic VHH-Fc fusion protein ALPP / ALPPL2-binding VHH proteins were genetically fused to engineered human IgG1 hFc for heterodimerization via long [G4S]3 or short [G4S]1 linkers (Ridgway 1996 Protein Engineering 9(7):617-21). The knob variant has a tyrosine substitution at position 366 (T366Y), and the hole variant has a threonine substitution at position 407 (Y407T) (EU numbering). This approach was used to generate biparatopic ALPP / ALPPL2 binders, in which one arm contains the ALPP / ALPPL2 VHH protein and the other arm contains a second, different ALPP / ALPPL2-binding VHH domain.

[0598] Additionally, cysteine ​​substitutions were incorporated into the hIgG1 Fc sequence of both the knob and hole variants to facilitate bioconjugation with different payloads.

[0599] The human IgG1 sequences contained one or two site-specific Cys substitutions (S239C±S442C) for site-specific conjugation, and either a T366Y or a Y407T (EU numbering) knob-into-hole (KIH) mutation to facilitate biparatopic chain pairing via Fc chain heterodimerization.

[0600] Biparatopic VHH-hFc fusion proteins were transiently coexpressed as secreted proteins in CHO K1 cells and purified from the culture medium using MabSelect™ SuRe™ (Evitria, Switzerland). The purified proteins were exchanged into PBS pH 7.4 and analyzed by SEC (AdvanceBio, Agilent, running buffer DPBS), SDS-PAGE, and mass spectrometry to confirm sequence and protein integrity and correct bispecific pairing. For MS analysis, the proteins were deglycosylated with PNGaseF, reduced (if necessary) with 50 mM DTT, and run on a Sciex X500B QTOF with a MassPREP desalting column (Waters). Characterization data for the biparatopic VHH-hFc proteins are shown in Table 12.

[0601] [Table 12] JPEG2025542294000052.jpg255170JPEG2025542294000053.jpg255170

[0602] The purified target biparatopic protein was produced in good yield and showed a high percentage of monomericity as determined by SEC, i.e., the desired heterodimeric product was obtained along with low amounts of high molecular weight aggregates.

[0603] Binding of biparatopic VHH-hFc proteins to ALPP homologs by BLI Binding kinetics were determined using a BioLayer Interferometry (BLI) Octet K2 or R4 system (Sartorius). Human ALPP, ALPPL2, and ALPL (SEQ ID NO: 265, SEQ ID NO: 266, and SEQ ID NO: 267), all fused with a disulfide-stabilized leucine zipper, and ALPI His (SEQ ID NO: 264) were used in BLI experiments. DFor apparent measurements, ALPP proteins were immobilized on AR2G sensors in sodium acetate pH 5 buffer using amine coupling. To measure 1:1 interactions, Fc fusion proteins were immobilized using AHC biosensors. All assays were performed in HBS-EP at a plate temperature of 25°C. Ka (M -1 s -1 ), Kd(s -1 ) and K D The (nM) values ​​were determined and the binding kinetics are shown in Tables 13 and 14.

[0604] [Table 13]

[0605] [Table 14]

[0606] BLI data confirm high affinity binding of biparatopic VHH-hFc fusion proteins to ALPP and ALPPL2. No binding to ALPI or ALPL was observed.

[0607] Example 4 – ALPP / ALPPL2 protein drug conjugates VHH-hFc drug conjugates An approach to generating ADCs is to engineer cysteine ​​substitutions or additions at positions on the light and heavy chains of antibodies, where these cysteines provide reactive thiol groups for site-specific labeling (Junutula 2008 Nature Biotechnology 26,925-932, Jeffrey 2013, Sutherland 2016).

[0608] As previously described, anti-ALPP / ALPPL2 VHH-hFc fusions were generated containing an additional engineered cysteine ​​residue in the Fc region, allowing site-specific labeling of payloads and cytotoxic drugs with maleimide derivatives (MC-vc-PAB-MMAE, MA-PEG4-vc-PAB-EDA-PNU159682, and MA-PEG4-va-PAB-EDA-PNU159682) (Figure 4).

[0609] Generation of VHH-hFc-MMAE conjugates These proteins were site-specifically labeled with maleimide MMAE derivatives using a partial reduction, refolding, and labeling method adapted from the literature [Junutula et al., 2008 Nat Biotech; Jeffrey et al., 2013 Bioconj Chem]. Briefly, 1–6 mg / ml VHH hFc solutions were prepared in PBS pH 7.4 with 1 mM EDTA, and 20–40 molar equivalents of TCEP were added and incubated at 4°C for 16 hours. 30–50 molar equivalents of DHAA were then added, the pH adjusted to 6.5, and incubated at room temperature for 1 hour. The refolded VHH Fc was either extensively dialyzed or buffer exchanged into PBS, quantified by UV, and then reacted with 4–8 molar equivalents of maleimide MMAE solution at room temperature for 2 hours. The conjugates were purified by SEC and analyzed by reducing and non-reducing SDS-PAGE and LC-MS. Table 15 also lists the prepared conjugates.

[0610] [Table 15]

[0611] SDS-PAGE and mass spectrometry of the final conjugates determined that labeling proceeded quantitatively to give highly pure, homogenous protein-drug conjugates with the desired drug-to-antibody ratios (DAR of 2 or 4).

[0612] Generation of VHH-hFc-PNU conjugates Similar partial reduction, refolding, and labeling procedures were used for PNU conjugation as described above, with some modifications. Briefly, 1-6 mg / ml VHH hFc solutions were prepared in PBS + 100 mM L-Arg, pH 7.4, with 1 mM EDTA. 20 molar equivalents of TCEP were added and incubated at 4°C for 16 hours. 30 molar equivalents of DHAA were then added, the pH adjusted to 6.5, and incubated at room temperature for 3 hours. The refolded VHH Fc was buffer exchanged into PBS using a NAP-25 column and quantified by UV. Propylene glycol was then added to a final concentration of 20%, followed by the addition of 4 molar equivalents of maleimide-PNU solution. This was incubated at room temperature for 2 hours. The conjugates were purified using activated charcoal and a NAP-25 desalting column and subsequently analyzed by reducing and non-reducing SDS-PAGE, analytical HIC, and LC-MS. Table 16 summarizes the conjugates prepared.

[0613] [Table 16]

[0614] SDS-PAGE, SEC, and mass spectrometry of the final conjugate determined that labeling proceeded quantitatively to give a highly pure, homogenous protein-drug conjugate with the desired drug-to-antibody ratio (DAR of 2) and low levels (<4%) of high molecular weight aggregates.

[0615] Characterization of cancer cell lines for ALPP / ALPPL2 expression The following cell lines were characterized for cell surface ALPP isoform expression analysis and most were also used in cell viability assays.

[0616] CHO-ALPP isoform stable transfectants were generated using CHO-K1 cells (CCL-61) provided by the American Type Culture Collection (ATCC) and grown in ATCC-standard F-12K medium plus 10% FCS.

[0617] MKN1 cells (RCB1003, RIKEN BRC). Ref: Cancer Sci 2006;97:387-394, provided by RIKEN BRC through the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology of Japan, were grown in RPMI1640 + 10% FCS.

[0618] NCI-H1651 cells, CRL-5884, were provided by the American Type Culture Collection (ATCC) and grown in ATCC DMEM:F12 medium + 10% FCS + supplements required to make ACL-4 medium as recommended by ATCC.

[0619] NCI-N87 cells, CRL-5822, provided by the American Type Culture Collection (ATCC) were grown in RPMI 1640 + 10% FCS.

[0620] HPAC cells, CRL-2119, were provided by the American Type Culture Collection (ATCC) and grown in 1:1 DMEM:Ham's F12 + 5% FCS + supplements as recommended by ATCC.

[0621] Caov3 cells, HTB-75, provided by the American Type Culture Collection (ATCC), were grown in DMEM + 10% FCS.

[0622] RMUG-S cells, IFO50320, provided by the Japanese Collection of Research Bioresources (JCRB) through Tebu-bio. Ref: M. Sakayori et.al. Hum Cell 1990 Mar;3(1):52-6. Grown in Ham's F12 medium + 10% FCS.

[0623] HeLa cells, CCL-2, provided by the American Type Culture Collection (ATCC), were grown in EMEM + 10% FCS.

[0624] MDA-MB-468 cells, HTB-132, provided by the American Type Culture Collection (ATCC), were grown in DMEM + 10% FCS.

[0625] The expression levels of ALPP / ALPPL2 protein on the surface of different cancer cell lines were determined by flow cytometry. ALPP / ALPPL2 was assessed using anti-PLAP mAb (PE-8B6 from Santa Cruz).

[0626] Adherent cells were detached from tissue culture flasks by incubation with 0.1% EDTA / PBS solution at 37°C for approximately 10 minutes or until cells readily detached. Cells were resuspended in ice-cold PBS / 2% FCS in 15 ml tubes and centrifuged at 1700 rpm for 5 minutes at 4°C. The supernatant was removed, and the cell pellet was resuspended in PBS / 2% FCS. Cell counts were performed using a Chemometec Nucleocounter NC-202, and 2.5 x 10^5 cells per test sample were aliquoted into 96-well plates. Cells were incubated with 100 μl of PE-anti-PLAP mAb or control on ice for 1 hour. The sample plate was centrifuged at 1700 rpm at 4°C for 5 minutes. Washing was performed by removing the supernatant and resuspending the cell pellet in 0.25 mL of ice-cold PBS / 2% FCS using a multichannel pipette. The samples were again centrifuged at 1700 rpm for 5 minutes at 4°C. The supernatant was removed, and two additional washes were performed as described. After the final wash and centrifugation step, excess liquid was removed by blotting the plate on tissue paper. The cell pellet was resuspended in 0.28 ml of ice-cold PBS / 2% FCS and kept on ice in the dark before analysis on a Merck-Millipore Guava EasyCyte HT or Thermo Fisher Attune NxT flow cytometer.

[0627] Protein expression levels and mRNA expression values ​​are shown in Table 17 below.

[0628] ALPP / ALPPL2 mRNA expression levels were obtained from the Cancer Cell Line Encyclopedia (CCLE).

[0629] [Table 17]

[0630] In vitro cell viability assay for cancer cells treated with anti-ALPP / ALPPL2 drug conjugates As described above, cells were seeded into white, clear-bottom 96-well plates (Costar) in the appropriate complete growth medium and incubated at 37°C and 5% CO2 for 24 hours. The following day, a dilution series was set up with a ×10 working stock for each test agent. Dose-response ×10 stocks were, for example, 10,000, 5,000, 1,000, 500, 100, 50, 10, 5, 1, 0.5 nM, etc. Ten microliters of the ×10 stock solution were added to the cell plate using a multichannel pipette (90 μl per well). This resulted in a 1:10 dilution into the wells and a dose response ranging from 1,000 nM (column 1) to 0.05 nM (column 10), or down to 0.5 fM as appropriate for the most sensitive cell line. Ten microliters of vehicle control (PBS) was added to control wells (columns 11 and 12). Plates were incubated at 37°C and 5% CO2 for 96 hours. Cell viability was assessed using Promega Cell Titre Glo reagent according to the manufacturer's instructions. Briefly, assay plates were removed from the incubator and allowed to equilibrate to room temperature before adding 100 μl of room-temperature Cell Titre Glo reagent to each 100 μl assay well. The plate was placed on a plate shaker at 600 rpm for 2 minutes. The plate was allowed to sit at room temperature for an additional 10 minutes before measuring the luminescence readout using a Clariostar plate reader (BMG). Data were analyzed by calculating the mean for untreated (vehicle only) control wells and determining the % of control for each treatment well. The % of control data was then plotted against the Log [treatment] concentration, and IC50 values ​​were derived using nonlinear regression fitting in GraphPad Prism software.

[0631] Table 18 shows the IC values ​​for cell killing of CHO-ALPP isoform transfectant cell lines stably expressing either ALPP, ALPPL2, ALPI, or ALPL by monoparatopic VHH-hFc drug conjugates [-vc-PAB-MMAE (DAR4) or -PEG4-vc-PAB-EDA-PNU159682 (DAR2)]. 50 Values ​​are shown. αGFP-hFc(S239C+S442C)-vc-PAB-MMAE (DAR4) and αGFP-hFc(S239C)-vc-PAB-EDA-PNU159682 (DAR2) drugs were included as non-binding negative controls. αGFP-hFc is a control non-binding protein consisting of a GFP-targeting VHH domain fused to human IgG1 Fc in a manner similar to the ALPP / ALPPL2-targeting protein. Figure 5 shows representative dose-response curves for cell killing of CHO-ALPP, CHO-ALPPL2, CHO-ALPI, and CHO-ALPL cells by VHH-hFc-PNU drug conjugates.

[0632] [Table 18]

[0633] As shown in Table 18 and Figure 5, VHH-hFc-MMAE and PNU conjugates were generated that exhibited potent selective killing of ALPP- and ALPPL2-expressing CHO cells with much lower potency for killing ALPI- and ALPL-expressing CHO cells. This high selectivity for killing ALPP- and ALPPL2-expressing cells is observed with both VHH-Fc proteins conjugated with an MMAE payload and VHH-Fc proteins conjugated with a PNU payload. The D10-hFc drug conjugate exhibits increased potency against ALPP-expressing CHO cells over ALPPL2-expressing CHO cells.

[0634] Table 19 and Figure 6 show the corresponding IC for cell killing of ALPP / ALPPL2-positive cancer cells by monoparatopic VHH-hFc drug conjugates [-vc-PAB-MMAE (DAR4) or -PEG4-vc-PAB-EDA-PNU159682 (DAR2)]. 50 Dose-response curves are shown with values. The αGFP-hFc(S239C+S442C)-vc-PAB-MMAE (DAR4) and αGFP-hFc(S239C)-vc-PAB-EDA-PNU159682 (DAR2) agents were included as non-binding negative controls.

[0635] [Table 19]

[0636] As shown in Table 19 and Figure 6, ALPP / ALPPL2 VHH-hFc-MMAE and PNU conjugates were generated that exhibited potent killing of ALPP / ALPPL2-expressing cancer cells. A large window of cell killing is observed between the ALPP / ALPPL2-targeted VHH-hFc-MMAE and ALPP / ALPPL2-targeted VHH-hFc-PNU conjugates compared to the respective non-binding αGFP-hFc conjugate controls. Thus, highly selective and potent cell killing of ALPP- and ALPPL2-expressing cancer cells is observed for both VHHFc proteins conjugated with an MMAE payload and VHHFc proteins conjugated with a PNU payload.

[0637] Example 5 – Biparatopic ALPP / ALPPL2 drug conjugates Biparatopic VHH-hFc drug conjugates As previously described, biparatopic anti-ALPP / ALPPL2 VHH-hFc fusions were generated with an additional engineered cysteine ​​residue in the Fc region, allowing site-specific labeling of payload and cytotoxic drugs with maleimide derivatives (MC-vc-PAB-MMAE and MA-PEG4-vc-PAB-EDA PNU159682).

[0638] Biparatopic anti-ALPP / ALPPL2 VHH-hFc proteins were conjugated with either MMAE or PNU derivatives and analyzed as described in Example 4. Tables 20 and 21 summarize the conjugates that were prepared.

[0639] [Table 20] JPEG2025542294000062.jpg255170

[0640] SEC and SDS-PAGE and mass spectrometry of the final conjugates determined that labeling proceeded quantitatively to give highly pure, homogenous protein-drug conjugates with the desired drug-to-antibody ratio (either DAR2 or DAR4) and low levels of observed high molecular weight aggregates.

[0641] [Table 21]

[0642] SDS-PAGE, SEC, and mass spectrometry of the final conjugate determined that labeling proceeded quantitatively with good overall yield to give highly pure, homogenous protein-drug conjugates with the desired drug-to-antibody ratio (DAR of 2) and low levels (<4%) of high molecular weight aggregates.

[0643] Binding of biparatopic VHH-hFc drug conjugates to cell surface ALPP, ALPPL2, ALPI, and ALPL by flow cytometry Biparatopic VHH-hFc drug proteins were evaluated for cell surface binding to human ALPP, ALPPL2, ALPI, and ALPL by flow cytometry. Binders were screened on CHO cells stably expressing human ALPP, ALPPL2, ALPI, or ALPL.

[0644] The cell surface binding of test agents to hALPP isoforms was characterized in four CHO-ALPP isoform transfectant cell lines (CHO-ALPP, CHO-ALPPL2, CHO-ALPI, and CHO-ALPL), and the resulting K Dapp Values ​​were determined. Adherent cells were detached from tissue culture flasks by incubation with 0.1% EDTA / PBS solution at 37°C for approximately 10 minutes or until cells readily detached. Cells were resuspended in ice-cold PBS / 2% FCS in 15 ml tubes and centrifuged at 1700 rpm for 5 minutes at 4°C. The supernatant was removed, and the cell pellet was resuspended in PBS / 2% FCS. Cell counts were performed using a Chemometec Nucleocounter NC-202, and 2.5 x 10^5 cells per test sample were aliquoted into a 96-well plate. Cells were incubated on ice for 1 hour with 100 μl of test agent and control concentrations. The sample plate was centrifuged at 1700 rpm at 4°C for 5 minutes. Washing was performed by removing the supernatant and resuspending the cell pellet in 0.25 mL of ice-cold PBS / 2% FCS using a multichannel pipette. The samples were again centrifuged at 1700 rpm for 5 minutes at 4°C. The supernatant was removed, and two further washes were performed as described. After the final wash and centrifugation step, excess liquid was removed by blotting the plates on tissue paper. Binding of VHH-hFc agents was determined by adding 100 μl of Fitc-anti-human Ab (ThermoFisher) and incubating on ice in the dark for 30 minutes. Washing steps were performed as previously described. Finally, all cell pellets were resuspended in 0.28 ml of ice-cold PBS / 2% FCS and left on ice in the dark before analysis on a Merck-Millipore Guava EasyCyte HT or ThermoFisher Attune NxT flow cytometer.

[0645] The binding of the different domains to the different cell surface ALPP forms is summarized in Table 22 and FIG.

[0646] [Table 22]

[0647] Figure 7 and Table 22 show that biparatopic VHH-hFc drug conjugates specifically bind to ALPP / ALPPL2 on the surface of cells.

[0648] In vitro cell viability assay for cancer cells treated with anti-ALPP / ALPPL2 biparatopic drug conjugates The Cell Titre Glo assay was performed as described in Examples 4 and 5. Cells were incubated with biparatopic VHH-hFc-drug conjugates for 96 hours at 37°C, 5% CO2, and the % cell survival was determined as a dose response. The % control data was plotted against the Log [treatment] concentration, and IC was calculated using nonlinear regression fitting in GraphPad Prism software. 50 The value was derived.

[0649] MMAE conjugates Table 23 shows the IC for cell killing of CHO-ALPP isoform transfectant cell lines stably expressing either ALPP, ALPPL2, ALPI, or ALPL by biparatopic VHH-hFc-vc-PAB-MMAE(DAR4) drug conjugates. 50 Values ​​are shown. The corresponding αGFP-hFc(S239C+S442C)-vc-PAB-MMAE(DAR4) drug was included as a non-binding negative control. Figure 8 shows representative dose-response curves for cell killing of CHO-ALPP, CHO-ALPPL2, CHO-ALPI, and CHO-ALPL cells by biparatopic VHH-hFc MMAE drug conjugates.

[0650] [Table 23]

[0651] As shown in Table 23 and Figure 8, biparatopic VHH-hFc-MMAE conjugates were generated that exhibited potent and selective killing of ALPP- and ALPPL2-expressing CHO cells, with a significantly reduced efficacy for killing of ALPI- and ALPL-expressing CHO cells.

[0652] Table 24 shows the IC for cell killing of ALPP / ALPPL2-positive cancer cells (MKN1, NCI-H1651, and NCI-N87) and ALPP / ALPPL2-negative HeLa cancer cell lines by biparatopic VHH-hFc-vc-PAB-MMAE(DAR4) drug conjugates. 50 Values ​​are shown. The corresponding αGFP-hFc(S239C+S442C)-vc-PAB-MMAE(DAR4) drug was included as a non-binding negative control.

[0653] [Table 24]

[0654] As shown in Table 24 and Figure 9, biparatopic VHH-hFc-MMAE conjugates were generated that exhibited potent and selective killing of ALPP / ALPPL2-expressing cancer cells, but little efficacy against HeLa cancer cells, which express little to no ALPP / ALPPL2.

[0655] Compared to the control non-binding protein-drug conjugate, a larger window was observed for αGFP-hFc(S239C+S442C)-vc PAB MMAE(DAR4).

[0656] Treatment of cancer cell lines with free MMAE payloads was also performed IC 50 Dose response is shown with values. The % remaining cell viability at the highest concentration of MMAE is consistent with the % remaining cell viability observed for VHH-FcMMAE drug conjugate binders in ALPP / ALPPL2-positive cancer cell lines.

[0657] PNU conjugates Table 25 shows the IC for cell killing of CHO-ALPP isoform transfectant cell lines stably expressing either ALPP, ALPPL2, ALPI, or ALPL by biparatopic VHH-hFc-PEG4-vc-PAB-EDA-PNU159682 (DAR2) drug conjugates. 50 Values ​​are shown. The corresponding αGFP-hFc(S239C)-PEG4-vc-PAB-EDA-PNU159682 (DAR2) drug was included as a non-binding negative control. Figure 10 shows representative dose-response curves for cell killing of CHO-ALPP, CHO-ALPPL2, CHO-ALPI, and CHO-ALPL cells by biparatopic VHH-hFc-PNU drug conjugates.

[0658] [Table 25]

[0659] As shown in Table 25 and Figure 10, biparatopic VHH-hFc-PNU conjugates were generated that exhibited potent and selective killing of ALPP- and ALPPL2-expressing CHO cells, with much lower efficacy in killing ALPI- and ALPL-expressing CHO cells.

[0660] For CHO-ALPP and CHO-ALPPL2 killing, IC for ALPP / ALPPL2-targeted VHH-hFc-PNU conjugates versus control non-binding αGFP-hFc-PNU control. 50 A large window of cell killing was observed, and this window of cell killing between drug and control was abolished for CHO-ALPI and CHO-ALPL, indicating the highly selective nature of ALPP / ALPPL2-mediated cell killing.

[0661] Table 26 and Figure 11 show the IC for cell killing of ALPP / ALPPL2-positive cancer cells (MKN1, NCI-N87, NCI-H1651, Caov-3, and HPAC) by biparatopic VHH-hFc-PEG4-vc-PAB-EDA-PNU(DAR2) drug conjugates. 50 Values ​​and dose-response curves are shown. The corresponding αGFP-hFc(S239C)-PEG4-vc-PAB-EDA-PNU(DAR4) drug was included as a non-binding negative control.

[0662] [Table 26]

[0663] As shown in Table 26 and Figure 11, biparatopic VHH-hFc-PNU conjugates were generated that showed potent and selective killing of a range of different ALPP / ALPPL2-expressing cancer cell lines.

[0664] A large window between ALPP / ALPPL2-targeted PDC and the non-binding αGFP-hFc(S239C)-PEG4-vc-PAB-EDA-PNU(DAR2) control was observed across all cancer cell lines.

[0665] For the same cancer cell lines, the remaining cell viability (%) at the highest concentration for the VHH-Fc-PNU drug conjugate was observed to be lower than the remaining cell viability (%) for the corresponding VHH-hFc-MMAE drug conjugate (see data above).

[0666] Example 6 – In vivo efficacy of ALPP / ALPPL2 protein-drug conjugates Procedures and protocols involving animals were conducted after review and approval by the institutional Institutional Animal Care and Use Committee (IACUC), and animal care and use was in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

[0667] NCI-N87 human gastric cancer CDX and HPAC human pancreatic ductal adenocarcinoma CDX - biparatopic VHH-hFc-vcMMAE drug conjugates Efficacy studies were conducted at Crown Bioscience (Beijing) in the ALPP / ALPPL2+NCI-N87 gastric cancer xenograft model and the HPAC human pancreatic ductal adenocarcinoma xenograft model, which were shown to express ALPP / ALPPL2 by IHC (Figure 12).

[0668] ALPP / ALPPL2 expression was confirmed in both NCI-N87 and HPAC cell line-derived xenograft FFPE samples by immunohistochemical (IHC) staining using an anti-ALPP / ALPPL2 antibody (MS Validated Antibodies) and a rabbit isotype negative control.

[0669] For the NCI-N87 study, female BALB / c nude mice were inoculated with NCI-N87 human gastric cancer cells (ATCC, Crown ID CL-00455). NCI-N87 tumor cells (1 × 10) in 0.1 mL of a 1:1 PBS / Matrigel suspension were used. 7 ) was inoculated subcutaneously into the right flank of each mouse.

[0670] For the HPAC study, female NOD / SCID mice were inoculated with HPAC human pancreatic ductal adenocarcinoma cells (ATCC, Crown ID CL-00891). HPAC tumor cells (1 × 10) in 0.1 mL of a 1:1 PBS / Matrigel suspension were inoculated. 7 ) was inoculated subcutaneously into the right anterior flank of each mouse.

[0671] Tumor volumes of approximately 200 mm were obtained in a sufficient number of animals. 3 Mice were monitored until they reached the inclusion criteria of 100 mg / kg / day. The mean tumor volume for each group was approximately 200 mm. 3Mice were randomized into treatment groups (n=5) so that tumor volumes were consistent across groups and that there were no statistical differences between tumor volumes across groups. The day of randomization was designated day 0, and mice were treated with vehicle or protein-drug conjugate (see Table 27 below) on day 1. Protein-drug conjugates were administered intravenously at either 1 mg / kg or 3 mg / kg once every four days (Q4D x 4). All mice were pre-primed with mouse IgG 20 h before the first PDC administration.

[0672] [Table 27]

[0673] Tumor volume was assessed by measuring the perpendicular tumor diameter three times a week using a caliper during the experimental period. The formula is TV (mm 3 Absolute tumor volume (ATV) was calculated using the formula: ) = [length (mm) x width (mm) x width (mm)] x 0.5, where length (L) is the longest tumor dimension and width (W) is the longest tumor dimension perpendicular to L. All animals were weighed simultaneously with tumor size measurements and also on the day of dosing. Mice were observed daily and reported for changes in physical appearance, behavior, adverse clinical signs, and general welfare in accordance with local welfare and best veterinary practice guidelines.

[0674] Figure 13 shows the effect of protein-drug conjugates on tumor growth compared to vehicle controls for the NCI-N87 CDX study. All protein-drug conjugates were well tolerated and demonstrated highly statistically significant in vivo efficacy compared to vehicle-treated groups at both the 1 mg / kg and 3 mg / kg dose levels. PDC8, with a DAR of 2, demonstrated slightly lower tumor growth inhibition compared to the corresponding DAR4 version at equivalent doses. Vehicle data are plotted up to the last day, when tumor volumes for all animals in a group were within the ethical tumor size limit.

[0675] Figure 14 shows the effect of protein-drug conjugates on tumor growth compared to vehicle controls for the HPAC CDX study. All protein-drug conjugates were well tolerated and demonstrated highly statistically significant in vivo efficacy compared to vehicle-treated groups at both the 1 mg / kg and 3 mg / kg dose levels. PDC8, with a DAR of 2, demonstrated slightly lower tumor growth inhibition compared to the corresponding DAR4 version at equivalent doses. Vehicle data are plotted up to the last day when tumor volumes for all animals within a group are within the ethical tumor size limit.

[0676] Conjugates of the present invention were produced as either DAR2 or DAR4 molecules. The DAR, or drug-to-antibody ratio, represents the average number of drug molecules conjugated to an antigen-binding domain of the present invention. The exact DAR values ​​for each batch of PDC molecules are shown in Table 28 below.

[0677] [Table 28]

[0678] Example 7 – ALPP / ALPPL2 bispecific T cell engaging protein ALPP / ALPPL2xCD3 bispecific sequences combining the N-terminal ALPP / ALPPL2 VHH and the C-terminal anti-CD3 scFv (clone OKT3) via two different lengths of G4S linkers were expressed in CHO cells (Evitria) and purified by IMAC (HisTrap Excel, GE Healthcare) followed by SEC (Superdex 200 26 / 60, GE Healthcare). Similarly, biparatopic ALPP / ALPPL2xCD3 bispecific sequences combining the N-terminal biparatopic ALPP / ALPPL2 VHH and the C-terminal anti-CD3 scFv were also expressed in CHO (Evitria).

[0679] CD3 BiTE-like approach; Example and orientation of CD3 binding sequences for use as ALPP / ALPPL2 VHH bispecific anti-CD3 scFv clone OKT3 (WO 2014028776 Zyngenia) and their humanized derivatization

[0680] VH-[G4S]3-VL DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKS (SEQ ID NO: 149)

[0681] Humanized anti-CD3 scFv UCHT1 (Arnett et al. PNAS 2004 101(46)16268-16273) and its derivatives VL-[G4S]3-VH MDIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKGGGGSGGGGSGGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 150)

[0682] Example 8 – ALPP and ALPPL2 CAR-T Approach Chimeric antigen receptors (CARs) based on the ALPP- and ALPPL2-specific antigen binding molecules described in this application can be generated. Furthermore, engineered T cells expressing such CARs can also be generated, which can then be used, for example, in adoptive cell therapy.

[0683] Briefly, a nucleic acid construct encoding an ALPP or ALPPL2-specific CAR is produced. The ALPP / ALPPL2-specific CAR may comprise an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising an ALPP / ALPPL2-specific antigen-binding molecule described herein. The nucleic acid construct is then incorporated into a viral vector, such as a retroviral vector (e.g., a lentiviral vector).

[0684] T cells can be isolated from a patient in need of treatment and then engineered to express a nucleic acid construct encoding a CAR, for example, by retroviral transfection or gene editing using approaches such as CRISPR-CAS-9.

[0685] The engineered T cells are then re-infused into the patient to treat the condition, for example to treat cancer.

[0686] Example 9 – Internalization of ALPP / ALPPL2 binding proteins Internalization of ALPP / ALPPL2-targeting VHH-hFc binders, a monoclonal antibody (h12F3) targeting ALPP / ALPPL2, and a non-binding control VHH-hFc fusion of αGFP was assessed in Hep2 cells using an IncuCyte S3 live cell analyzer (Sartorius). Cells were seeded at a density of 3000 cells / well in black clear-bottom 96-well plates (Corning #3340) and allowed to adhere for 24 hours at 37°C and 5% CO2. Test agents were mixed with FabFluor-pH Red Antibody Labeling Reagent (Sartorius, #4722) at a 1:2 molar ratio in culture medium at ×2 the final assay concentration and incubated for 15 minutes at 37°C to allow conjugation. 50 μl of the resulting mixture was added to the appropriate wells containing cells (50 μl) to give a final concentration of 25 nM for each test agent. Images were captured every hour for 30 hours, with three regions of interest imaged from each well. Individual cell-level analysis was performed using the Incucyte Integrate module. Data are presented as the mean red intensity (Red Calibration Units, RCU) over time. Assays were performed in triplicate.

[0687] As shown in Figure 15, VHH-hFc fusions targeting ALPP / ALPPL2 that bind in a monoparatopic manner were internalized in Hep2 cells, whereas the non-binding control αGFP VHH-hFc protein showed no internalization.

[0688] The internalization of biparatopic VHH-hFc fusion proteins targeting ALPP / ALPPL2 was similarly evaluated. As shown in Figure 16, the biparatopic VHH-hFc fusion proteins exhibited enhanced internalization into Hep2 cells. These biparatopic proteins exhibited increased internalization compared with the monoclonal antibody h12F3 HGLF targeting ALPP / ALPPL2. This humanized monoclonal antibody h12F3 HGLF (described as SEQ ID NO: 40 and SEQ ID NO: 50 in WO 2022197890) was transiently expressed as a secreted protein in CHO K1 cells and purified from the culture medium using MabSelect™ SuRe™ (Evitria, Switzerland). The purified protein was exchanged into PBS pH 7.4 and analyzed by SEC (AdvanceBio, Agilent, running buffer DPBS pH 7.4 or Superdex200 Increase 10 / 300, Cytivia, running buffer PBS pH 7), SDS PAGE under reducing and native conditions, and mass spectrometry to confirm the sequence and protein integrity.

[0689] Example 10 – Efficacy of biparatopic and monoparatopic PDCs and ADCs (DAR2 MMAE format) targeting ALPP / ALPPL2 Generation of DAR2 MMAE biparatopic VHH-hFc (F405L / K409R) drug conjugate and h12F3 HGLF drug conjugate A series of biparatopic VHH-hFc proteins was generated using the FAB arm exchange method by expressing homozygous ALPP / ALPPL2 VHH-hFc proteins with a [G4S]3 linker, each containing either an F405L or K409R substitution and also an S239C substitution for downstream conjugation.

[0690] The VHH-hFc fusion protein was transiently expressed as a secreted protein in CHO K1 cells and purified from the culture medium using MabSelect™ SuRe™ (Evitria, Switzerland). The purified protein was exchanged into PBS pH 7.4 and analyzed by SEC (AdvanceBio, Agilent, running buffer DPBS), SDS-PAGE, and mass spectrometry to confirm the sequence and protein integrity.

[0691] To form the biparatopic complex, equal ratios of two homodimeric proteins, one carrying F405L and the other K409R, were mixed, 200 mM cysteamine was added, and the mixture was then incubated for 5 hours at 30° C., followed by 16 hours at 4° C. The resulting heterodimeric biparatopic VHH-hFc protein was then purified by SEC, and mass spectrometry was used to confirm the production of the desired biparatopic VHH-hFc protein.

[0692] Using the methods and analyses described in Example 4, biparatopic anti-ALPP / ALPPL2 VHH-hFc proteins generated via FAB arm exchange were conjugated with MMAE (MC-vc-PAB-MMAE) to give the corresponding DAR2 vc-PAB-MMAE PDC.

[0693] The monoclonal antibody h12F3 HGLF targeting ALPP / ALPPL2, described in Example 9, was similarly expressed with the S239C substitution. This protein was then conjugated with MMAE (MC-vc-PAB-MMAE) using the methods and assays described in Example 4 to give the corresponding DAR2 vc-PAB-MMAE ADC. Table 29 summarizes the DAR2 MMAE conjugates that were prepared.

[0694] [Table 29]

[0695] FF in vitro cell viability assay for cancer cells treated with anti-ALPP / ALPPL2 DAR2 MMAE biparatopic VHH-hFc (F405L / K409R) drug conjugate and h12F3 HGLF drug conjugate The Cell Titre Glo assay was performed as described in Example 5. Cells were incubated with VHH-hFc-drug conjugates and h12F3-drug conjugates for 96 hours at 37°C and 5% CO2, and the % cell survival was determined as a dose response. The % control data was plotted against the Log [treatment] concentration, and IC was calculated using nonlinear regression fitting in GraphPad Prism software. 50 The value was derived.

[0696] Table 30 and Figure 17 show the IC for cell killing of ALPP / ALPL2-positive cancer cells (NCI-H1651) by mono- and biparatopic VHH-hFc-vc-PAB-MMAE (DAR2) drug conjugates. 50 Values ​​and corresponding dose-response plots are shown.

[0697] [Table 30]

[0698] As shown in Table 30 and Figure 17, biparatopic VHH-hFc-MMAE(DAR2) conjugates are more potent in killing ALPP / ALPPL2-expressing cancer cells than the corresponding monoparatopic VHH-hFc-MMAE(DAR2) conjugates.

[0699] Table 31 and Figure 18 show the IC values ​​for cell killing of CHO-ALPP isoform transfectant cell lines stably expressing either ALPP or ALPPL2 by biparatopic VHH-hFc-vc-PAB-MMAE(DAR2) drug conjugates and monoclonal antibody h12F3 HGLF-vc-PAB-MMAE(DAR2) drug conjugates. 50 Values ​​and dose-response plots are shown.

[0700] [Table 31]

[0701] As shown in Table 31 and Figure 18, the biparatopic VHH-hFc-MMAE(DAR2) conjugate is more potent in killing ALPP- and ALPPL2-expressing CHO cells than the monoparatopic h12F3 HGLF-vc-PAB-MMAE(DAR2) antibody-drug conjugate.

[0702] Example 11 In vitro cell viability assay for cancer cells treated with anti-ALPP / ALPPL2 DAR4 MMAE biparatopic VHH-hFc drug conjugate and h12F3 HGLF MMAE (DAR4) antibody drug conjugate Biparatopic VHH-hFc-MMAE(DAR4) drug conjugates are as described in Examples 5 and 6.

[0703] The ALPP / ALPPL2-targeting monoclonal antibody h12F3 HGLF described in Example 9 was stochastically labeled with MMAE (MC-vc-PAB-MMAE) using endogenous cysteine ​​residues within the antibody sequence to yield the corresponding vc-PAB-MMAE antibody-drug conjugate with an average DAR of 4. Briefly, the h12F3 HGLF antibody solution was pH-adjusted using 2% v / v 0.5 M Tris, 0.0025 M EDTA, pH 8.5. 2.2 molar equivalents of TCEP were then added and allowed to reduce for 1 hour at 20°C. Next, 7 molar equivalents of MC-vc-PAB-MMAE were added to the reduced protein along with 10% v / v DMA. The reaction was allowed to proceed for 1 hour at 20°C before being quenched by the addition of N-acetylcysteine ​​in an amount equal to the molar equivalent of the payload. Free payload was removed by desalting into PBS pH 7.4 using Desalt Hiprep 26 / 10, followed by the addition of 1 mg of carbon per mg of ADC. After 2 hours, the carbon was removed by centrifugation.

[0704] The Cell Titre Glo assay was performed as described in Example 5. Cells were incubated with biparatopic VHH-hFc-drug conjugates and h12F3 drug conjugates for 96 hours at 37°C and 5% CO2, and the % cell survival was determined as a dose response. % control data was plotted against Log [treatment] concentration, and IC was calculated using nonlinear regression fitting in GraphPad Prism software. 50 The value was derived.

[0705] Table 32 shows the IC values ​​for cell killing of ALPP / ALPPL2-positive cancer cells (MKN-1, NCI-H1651, NCI-N87, MDA-MB-468) by biparatopic VHH-hFc-vc-PAB-MMAE(DAR4) drug conjugates and h12F3-vc-PAB-MMAE(DAR4) ADC. 50 Indicates the value.

[0706] [Table 32]

[0707] As shown in Table 32, the biparatopic VHH-hFc-vc-PAB-MMAE(DAR4) conjugate is consistently more potent in killing ALPP- and ALPPL2-expressing cancer cells than the antibody-drug conjugate h12F3 HGLF-vc-PAB-MMAE(DAR4).

[0708] αGFP-hFc-vc-MMAE is a control non-binding VHH-hFc-vc-PAB-MMAE (DAR4) conjugate generated in the same manner as the biparatopic VHH-Fc-MMAE conjugate (DAR4) targeting ALPP / ALPPL2, but the VHH domain recognizes the GFP protein.

[0709] Example 12 In vivo efficacy of ALPP / ALPPL2 biparatopic VHH-hFc MMAE drug conjugate and h12F3 HGLF MMAE (DAR4) antibody drug conjugate NCI-N87 Human Gastric Cancer CDX - Biparatopic VHH-hFc-vcMMAE Drug Conjugate An efficacy study was conducted in the ALPP / ALPPL2+NCI-N87 gastric cancer xenograft model at Crown Bioscience (Taicang) as described in Example 6, but using a single intravenous dose of drug (drugs used in this study are listed in Table 33).

[0710] Female BALB / c nude mice were inoculated with NCI-N87 human gastric cancer cells (ATCC, Crown ID CL-00455). NCI-N87 tumor cells (1 × 10) were inoculated in 0.1 mL of a 1:1 PBS / Matrigel suspension. 7 ) was inoculated subcutaneously into the right flank of each mouse.

[0711] Tumor volumes of approximately 200 mm were obtained in a sufficient number of animals. 3 Mice were monitored until they reached the inclusion criteria of 100 mg / kg / day. The mean tumor volume for each group was approximately 200 mm. 3 Mice were randomized into treatment groups (n=5) so that tumor volumes were consistent across groups and that there were no statistical differences between tumor volumes across groups. The day of randomization was designated day 0, and mice were treated with vehicle, biparatopic protein-drug conjugates, or ADC h12F3-MMAE(DAR4) (see Table 33 below) on day 1.

[0712] The biparatopic VHH-hFc-MMAE(DAR4) protein drug conjugate was administered intravenously as a single dose at either 1.25 mg / kg or 2.5 mg / kg.

[0713] The biparatopic VHH-hFc-MMAE(DAR2) protein drug conjugate was administered intravenously as a single dose at either 1.25 mg / kg or 2.5 mg / kg.

[0714] The h12F3-MMAE(DAR4) antibody drug conjugate was administered intravenously as a single dose at either 1.25 mg / kg or 2.5 mg / kg.

[0715] All mice were pre-primed with mouse IgG 20 h before the first PDC administration.

[0716] [Table 33]

[0717] Tumor volume was assessed by measuring the perpendicular tumor diameter three times a week using a caliper during the experimental period. The formula is TV (mm 3 Absolute tumor volume (ATV) was calculated using the formula: ) = [length (mm) x width (mm) x width (mm)] x 0.5, where length (L) is the longest tumor dimension and width (W) is the longest tumor dimension perpendicular to L. All animals were weighed simultaneously with tumor size measurements and also on the day of dosing. Mice were observed daily and reported for changes in physical appearance, behavior, adverse clinical signs, and general welfare in accordance with best veterinary practice and welfare guidelines.

[0718] Figures 19a and 19b show the effects of ALPP / ALPPL2-targeting ADCs and biparatopic protein-drug conjugates on tumor growth compared to vehicle controls for the NCI-N87 CDX study. Highly statistically significant in vivo efficacy was observed at the 2.5 mg / kg dose level for all biparatopic protein-drug conjugates (PDC1-5) compared to the vehicle-treated group, but not for ADC h12F3-MMAE (PDC6). Statistically significant antitumor efficacy was also observed at the 1.25 mg / kg dose level for all DAR4 biparatopic protein-drug conjugates (PDC1-4), but not for ADC h12F3-MMAE (PDC6). Complete regressions were observed at the 1.25 mg / kg dose level in all biparatopic PDC-treated groups (PDC1-5), but no complete regressions (0 / 5) were observed for ADC h12F3-MMAE (PDC6). Complete regression was defined as TV = 0 mm at the end of the study (day 61). 3 Mice were defined as those with

[0719] Figure 19c shows the same antitumor efficacy data plotted on an MMAE payload μmol / kg basis.

[0720] As shown in Figure 19c, the biparatopic VHH-hFc-MMAE(DAR4 and DAR2) conjugates of the invention are more efficacious than ADC h12F3-MMAE(DAR4) on an MMAE payload equivalent basis (i.e., based on the total moles of MMAE payload administered to the mice). The biparatopic VHH-hFc-MMAE(DAR4 and DAR2) conjugates induce stronger antitumor responses than ADC h12F3-MMAE(DAR4) while using less MMAE payload.

[0721] HPAC Human Pancreatic Ductal Adenocarcinoma CDX - Biparatopic VHH-hFc-vcMMAE Drug Conjugate As described in Example 6, an efficacy study was conducted at Crown Bioscience (Taicang) in the ALPP / ALPPL2+HPAC human pancreatic ductal adenocarcinoma xenograft model using a single intravenous dose of drug (the drugs used in this study are listed in Table 34).

[0722] Female NOD / SCID mice were inoculated with HPAC human pancreatic ductal adenocarcinoma cells (ATCC, Crown ID CL-00891). HPAC tumor cells (1 × 10) were inoculated in 0.1 mL of a 1:1 PBS / Matrigel suspension. 7 ) was inoculated subcutaneously into the right anterior flank of each mouse.

[0723] Tumor volumes of approximately 200 mm were obtained in a sufficient number of animals. 3 Mice were monitored until they reached the inclusion criteria of 100 mg / kg / day. The mean tumor volume for each group was approximately 200 mm. 3Mice were randomized into treatment groups (n=5) so that tumor volumes were consistent across groups and that there were no statistical differences between tumor volumes across groups. The day of randomization was designated day 0, and mice were treated with vehicle, biparatopic protein-drug conjugates, or ADC h12F3-vc-PAB-MMAE(DAR4) (see Table 34) on day 1.

[0724] Biparatopic VHH-hFc-MMAE(DAR4) protein-drug conjugates were administered intravenously as a single dose at either 5 mg / kg or 10 mg / kg.

[0725] The biparatopic VHH-hFc-MMAE(DAR2) protein-drug conjugate was administered intravenously as a single dose at either 5 mg / kg or 10 mg / kg.

[0726] The h12F3-MMAE(DAR4) antibody-drug conjugate was administered intravenously as a single dose at either 5 mg / kg or 10 mg / kg.

[0727] All mice were pre-primed with mouse IgG 20 h before the first PDC administration.

[0728] [Table 34]

[0729] Tumor volume was assessed by measuring the perpendicular tumor diameter three times a week using a caliper during the experimental period. The formula is TV (mm 3 Absolute tumor volume (ATV) was calculated using the formula: ) = [length (mm) x width (mm) x width (mm)] x 0.5, where length (L) is the longest tumor dimension and width (W) is the longest tumor dimension perpendicular to L. All animals were weighed simultaneously with tumor size measurements and also on the day of dosing. Mice were observed daily and reported for changes in physical appearance, behavior, adverse clinical signs, and general well-being in accordance with best veterinary practice and welfare guidelines.

[0730] Figures 20a and 20b show the effect of VHH-hFc-MMAE (DAR4 and DAR2) conjugates and ADC h12F3-MMAE (DAR4) agents on tumor growth compared to vehicle controls in a HPAC CDX study. All molecules were well tolerated and demonstrated highly statistically significant in vivo efficacy compared to vehicle-treated groups at both the 5 mg / kg and 10 mg / kg dose levels. Complete regressions were observed in groups treated with biparatopic PDC#1-5 (complete regression is defined as TV = 0 mm for three consecutive measurements). 3 (Definition: mice with pulmonary fibrosis were defined as those with pulmonary fibrosis). Complete regression was not observed at any time point in the ADC h12F3-vc-PAB-MMAE (PDC6) treatment group.

[0731] Figure 20c shows the same antitumor efficacy data plotted on an MMAE payload μmol / kg basis.

[0732] As shown in Figure 20c, the biparatopic VHH-hFc-MMAE(DAR4 and DAR2) conjugates of the invention are more efficacious than ADC h12F3-MMAE(DAR4) on an MMAE payload equivalent basis (i.e., based on the total moles of MMAE payload administered to the mice). The biparatopic VHH-hFc-MMAE(DAR4 and DAR2) conjugates of the invention induce stronger antitumor responses with less MMAE payload than ADC h12F3-MMAE(DAR4).

[0733] Caov-3 high-grade ovarian serous adenocarcinoma CDX - biparatopic VHH-hFc-vcMMAE drug conjugate At Crown Bioscience (Beijing), efficacy studies were performed in the ALPP / ALPPL2+Caov-3 ovarian cancer xenograft model, which was shown to express ALPP / ALPPL2 by IHC, although the staining was weaker than that of the NCI-N87 and HPAC CDX models (Figure 12).

[0734] ALPP / ALPPL2 expression was confirmed in FFPE xenograft samples derived from the Caov-3 cell line by immunohistochemical (IHC) staining using an anti-ALPP / ALPPL2 antibody (MS Validated Antibodies) and a rabbit isotype negative control.

[0735] For the Caov-3 study, female NCG mice were inoculated with Caov-3 ovarian adenocarcinoma cells (Peking Union Medical College, Crown ID CL-00780). Caov-3 tumor cells (1 × 10) in 0.2 mL of a 1:1 PBS / Matrigel suspension were used. 7 ) was inoculated into the mammary fat pad of each mouse.

[0736] Tumor volumes of approximately 200 mm were obtained in a sufficient number of animals. 3 Mice (n = 3 / group) were monitored until they reached the inclusion criteria of 100 mg / kg / day. The mean tumor volume for each group was approximately 200 mm. 3 Mice were randomized into treatment groups so that tumor volumes were consistent across groups and so that there were no statistical differences between tumor volumes across groups. The day of randomization was designated day 0, and mice were treated with vehicle or biparatopic protein-drug conjugates or the ADC h12F3-MMAE(DAR4) (see Table 35 below) on day 1. Protein-drug conjugates and h12F3 ADC were administered intravenously at either 1 mg / kg or 3 mg / kg once every four days (Q4D x 4). All mice were pre-primed with mouse IgG 20 h before the first PDC administration.

[0737] [Table 35]

[0738] Tumor volume was assessed by measuring the perpendicular tumor diameter twice weekly using calipers throughout the experimental period. Absolute tumor volume (ATV) was calculated using the formula TV (mm3) = [length (mm) × width (mm) × width (mm)] × 0.5, where length (L) is the longest tumor dimension and width (W) is the longest tumor dimension perpendicular to L. All animals were weighed simultaneously with tumor size measurements and on the day of dosing. Mice were observed daily and reported for changes in physical appearance, behavior, adverse clinical signs, and general welfare in accordance with best veterinary practice and welfare guidelines.

[0739] Figure 21a shows the effect of biparatopic VHH-hFc-MMAE (DAR4 and DAR2) conjugates and ADC hF12-MMAE (DAR4) on tumor growth compared to vehicle control in the Caov-3 ovarian CDX model. Treatment with all biparatopic VHH-hFc-MMAE agents (PDC1-5) produced statistically significant antitumor efficacy. ADC h12F3-MMAE (PDC6) did not produce statistically significant antitumor efficacy at any dose tested. One animal from the ADC h12F3-MMAE (PDC6) 3 mg / kg Q4Dx4 group died on day 49, and an additional animal was humanely euthanized on day 55. At the end of the study period (day 61), mice with complete regression (CR) were observed in the 3 mg / kg Q4D × 4 groups of PDC1–4, but not in the ADC h12F3-MMAE (PDC6).

[0740] Figure 21b shows the same antitumor efficacy data for biparatopic PDC1-4 at 1 mg / kg and ADC h12F3-MMAE (PDC6) at 3 mg / kg replotted on an MMAE payload μmol / kg basis.

[0741] As shown in Figure 21b, biparatopic VHH-hFc-MMAE PDCs provided similar or better antitumor efficacy using significantly less MMAE payload than ADC h12F3-MMAE (PDC6), as also demonstrated in Figure 21c, where DAR2 biparatopic VHH-hFc-MMAE (PDC5) induced a stronger antitumor response with less MMAE payload than the DAR4 ADC h12F3-MMAE conjugate.

Claims

1. Formula (I): FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4 (I) (In the formula, FW1 is the framework region, CDR1 is a CDR sequence, FW2 is the framework region, CDR2 is a CDR sequence, FW3 is the framework region, CDR3 is a CDR sequence, FW4 is the framework region) An antigen-binding molecule specific for placental alkaline phosphatase (ALPP) and / or germline alkaline phosphatase (ALPPL2), comprising an amino acid sequence represented by:

2. CDR1 is a CDR sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-15; FW2 is the framework region, CDR2 is a CDR sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-10; FW3 is the framework region, CDR3 is a CDR sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5; The ALPP- and / or ALPPL2-specific antigen-binding molecule of claim 1 , wherein FW4 is a framework region.

3. CDR1 is a CDR sequence having the amino acid sequence of SEQ ID NO: 11, CDR2 is a CDR sequence having the amino acid sequence of SEQ ID NO: 6, and CDR3 is a CDR sequence having the amino acid sequence of SEQ ID NO: 1; or CDR1 is a CDR sequence having the amino acid sequence of SEQ ID NO: 12, CDR2 is a CDR sequence having the amino acid sequence of SEQ ID NO: 7, and CDR3 is a CDR sequence having the amino acid sequence of SEQ ID NO: 2; or CDR1 is a CDR sequence having the amino acid sequence of SEQ ID NO: 13, CDR2 is a CDR sequence having the amino acid sequence of SEQ ID NO: 8, and CDR3 is a CDR sequence having the amino acid sequence of SEQ ID NO: 3; CDR1 is a CDR sequence having the amino acid sequence of SEQ ID NO: 14, CDR2 is a CDR sequence having the amino acid sequence of SEQ ID NO: 9, and CDR3 is a CDR sequence having the amino acid sequence of SEQ ID NO: 4, and / or CDR1 is a CDR sequence having the amino acid sequence of SEQ ID NO: 15, CDR2 is a CDR sequence having the amino acid sequence of SEQ ID NO: 10, and CDR3 is a CDR sequence having the amino acid sequence of SEQ ID NO:

5. The ALPP- and / or ALPPL2-specific antigen-binding molecule according to any one of claims 1 to 2.

4. 2. An ALPP- and / or ALPPL2-specific antigen-binding molecule according to any one of the preceding claims, having the amino acid sequence of any one of SEQ ID NOs: 17, 28, 30, 31 or 34.

5. A recombinant fusion protein comprising an ALPP- and / or ALPPL2-specific antigen-binding molecule according to any one of the preceding claims.

6. The recombinant fusion protein of claim 5 , wherein the ALPP and / or ALPPL2-specific antigen-binding molecule is fused to one or more biologically active proteins via one or more linker domains.

7. The linker may be G ４ S (SEQ ID NO: 236) and [G ４ S] ３ 7. The recombinant fusion protein of claim 6, wherein the fusion protein is selected from the group consisting of: (SEQ ID NO: 237).

8. 8. The recombinant fusion protein of any one of claims 5 to 7, wherein the at least one biologically active molecule is an immunoglobulin, an immunoglobulin Fc region, a fragment of an immunoglobulin Fc region, an Fc heavy chain, a CH2 region, a CH3 region, an immunoglobulin Fab region, Fab', Fv, Fv-Fc, a single chain Fv (scFv), scFv-Fc, (scFv)2, a diabody, a triabody, a tetrabody, a bispecific T cell engager, an intein, a VNAR domain, a single domain antibody (sdAb), a VH domain, or a scaffold protein (such as an affibody, centyrin, or darpin).

9. The recombinant fusion protein of any one of claims 5 to 8, wherein the at least one biologically active protein is an immunoglobulin Fc region or a fragment thereof.

10. The recombinant fusion protein according to claims 5 to 9, having an amino acid sequence according to SEQ ID NOs: 87 to 96.

11. 11. The recombinant fusion protein of any one of claims 5 to 10, wherein the immunoglobulin Fc region comprises a mutation to Cys (C), optionally wherein the C mutation is at position S239 and / or S442.

12. The recombinant fusion protein of any one of claims 5 to 11, having an amino acid sequence of SEQ ID NO: 97 to 121.

13. 13. The recombinant fusion protein of any one of claims 5 to 12, wherein the fragment of an immunoglobulin Fc region is engineered to dimerize with a second fragment of an immunoglobulin Fc region.

14. 14. The recombinant fusion protein of any one of claims 5 to 13, wherein one or more residues of the first fragment of an immunoglobulin Fc region comprise one or more amino acid substitutions that are suitable for knobs-in-holes (KIH) dimerization with the second fragment of an immunoglobulin Fc region, and one or more residues of the second fragment of an immunoglobulin Fc region comprise one or more amino acid substitutions that are suitable for knobs-in-holes (KIH) dimerization with the first fragment of an immunoglobulin Fc region.

15. The recombinant fusion protein of any one of claims 5 to 14, wherein the fragment of an immunoglobulin Fc region comprises a T366Y substitution or a Y407T substitution.

16. The recombinant fusion protein of any one of claims 5 to 15, having an amino acid sequence of SEQ ID NO: 188 to 208.

17. The recombinant fusion protein of any one of claims 5 to 15, having an amino acid sequence of SEQ ID NO: 127 to 186.

18. (a) a first recombinant fusion protein according to any one of claims 5 to 17, and (b) a second recombinant fusion protein according to any one of claims 5 to 17. A recombinant fusion protein dimer comprising:

19. 19. The recombinant fusion protein dimer of claim 18, comprising any one or any two of SEQ ID NOs: 87-186 or 188-208.

20. (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 138 (D10-hFc(long linker)S239C+T366Y); and (b) the second recombinant fusion protein comprises a sequence according to SEQ ID NO: 129 (E06-hFc(long linker)S239C+Y407T); or (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 138 (D10-hFc(long linker)S239C+T366Y); and (b) the second recombinant fusion protein comprises a sequence according to SEQ ID NO: 130 (E10-hFc(long linker)S239C+Y407T); or (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 138 (D10-hFc(long linker)S239C+T366Y); and (b) the second recombinant fusion protein comprises a sequence according to SEQ ID NO: 131 (F05-hFc(long linker)S239C+Y407T); or (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 137 (A06-hFc S239C T366Y), and (b) the second recombinant fusion protein comprises a sequence according to SEQ ID NO: 131 (F05-hFc(long linker)S239C+Y407T); or (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 143 (D10-hFc(short linker)S239C+T366Y); and (b) the second recombinant fusion protein comprises a sequence according to SEQ ID NO: 134 (E06-hFc(short linker)S239C+Y407T); or (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 143 (D10-hFc(short linker)S239C+T366Y); and (b) the second recombinant fusion protein comprises a sequence according to SEQ ID NO: 135 (E10-hFc (short linker) S239C Y407T); or (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 143 (D10-hFc(short linker)S239C+T366Y); and (b) the second recombinant fusion protein comprises a sequence according to SEQ ID NO: 136 (F05-hFc(short linker)S239C+Y407T); or (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 178 (D10-hFc S239C and S442C T366Y); and (b) the second recombinant fusion protein comprises a sequence according to SEQ ID NO: 169 (E06-hFc S239C and S442C+Y407T); or (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 178 (D10-hFc S239C and S442C+T366Y), and (b) the second recombinant fusion protein comprises a sequence according to SEQ ID NO: 170 (E10-hFc S239C and S442C+Y407T); or (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 178 (D10-hFc S239C and S442C+T366Y), and (b) the second recombinant fusion protein comprises a sequence according to SEQ ID NO: 171 (F05-hFc S239C and S442C+Y407T); or (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 177 (A06-hFc S239C and S442C+T366Y), and (b) the second recombinant fusion protein comprises a sequence according to SEQ ID NO: 171 (F05-hFc S239C and S442C+Y407T); or (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 183 (D10-hFc (short linker) S239C and S442C + T366Y); and (b) the second recombinant fusion protein comprises a sequence according to SEQ ID NO: 174 (E06-hFc (short linker) S239C and S442C + Y407T); or (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 183 (D10-hFc (short linker) S239C and S442C + T366Y); and (b) the second recombinant fusion protein comprises a sequence according to SEQ ID NO: 175 (E10-hFc (short linker) S239C and S442C + Y407T); or (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 183 (D10-hFc (short linker) S239C and S442C + T366Y); and (b) the second recombinant fusion protein comprises a sequence according to SEQ ID NO: 176 (F05-hFc (short linker) S239C and S442C + Y407T); 20. The recombinant fusion protein dimer of claim 18 or 19.

21. (a) an ALPP- and / or ALPPL2-specific antigen-binding molecule according to any one of claims 1 to 4, a recombinant fusion protein according to any one of claims 5 to 17, or a recombinant fusion protein dimer according to any one of claims 18 to 20, and (b) at least one toxin, cytotoxic agent, or cytostatic agent; A target binding molecule-drug conjugate comprising:

22. The target-binding molecule-drug conjugate of claim 21, wherein the ALPP- and / or ALPPL2-specific antigen-binding molecule is conjugated to the cytotoxic or cytostatic agent via a linker.

23. (b) is auristatins, anthracyclines, preferably PNU-derived anthracyclines; Maytansinoids, amanitin derivatives, preferably α-amanitin derivatives, calicheamicin, - Tubulysin, - duocarmycin, radioisotopes, such as alpha-emitting radionuclides, e.g., 227Th and 225Ac labels; Liposomes containing a toxic payload, - Protein toxins, Taxanes, - pyrrole benzodiazepines and their dimers, - indolinobenzodiazepine pseudodimers, - spliceosome inhibitors, CDK11 inhibitors, - nicotinamide phosphoribosyltransferase inhibitors (NAMPTi), - pyridinobenzodiazepines and their dimers, cyclopropapyrroloindole (CPI), cyclopropabenzoindole (CBI) or cyclopropathienoindole (CTI) and optionally dimers thereof, - Irinotecan or exatecan and their derivatives 23. The target-binding molecule-drug conjugate of claim 21 or 22, wherein the toxin is selected from the group consisting of:

24. The target binding molecule-drug conjugate of any one of claims 21 to 23, wherein the toxin is an auristatin.

25. The target binding molecule-drug conjugate of any one of claims 21 to 24, wherein the toxin is monomethyl auristatin.

26. The target binding molecule-drug conjugate of any one of claims 21 to 24, wherein the toxin is monomethyl auristatin E.

27. (b) is a MMAE derivative, The target binding molecule-drug conjugate has the formula (VI): 【Chemistry 1】 [X] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof; [L1] and [L2] are optional linkers selected from the group consisting of valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), peptide, dipeptide, tripeptide, -(CH2)n-, -(CH2CHO)n-, p-aminobenzyloxycarbonyl (PAB), Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, Val-Ala, Asn-Ala, any amino acid, a D-amino acid, Phe-Lys-PAB, and combinations thereof; and Y comprises an ALPP and / or ALPPL2-specific antigen-binding molecule or recombinant fusion protein according to any one of claims 1 to 20. The target-binding molecule-drug conjugate of any one of claims 21 to 23, having the structure:

28. (b) is a MMAE derivative, The target binding molecule-drug conjugate has the formula (VII): 【Chemistry 2】 28. The target-binding molecule-drug conjugate of claim 27, having the structure:

29. (b) an anthracycline (PNU) derivative; The target binding molecule-drug conjugate has the formula (III): 【Transformation 3】 wherein [X] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof; [L1] and [L2] are valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), peptide, -(CH ２ ) ｎ -, -(CH ２ CH ２ O) ｎ an optional linker selected from the group consisting of p-aminobenzyloxycarbonyl (PAB), Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, Val-Ala, Asn-Ala, any amino acid except glycine, and combinations thereof; and Y comprises an ALPP and / or ALPPL2-specific antigen-binding molecule or recombinant fusion protein according to any one of claims 1 to 20. The target-binding molecule-drug conjugate of any one of claims 21 to 23, having the structure: 【Request Item 30】 【Chemistry 4】 30. The target-binding molecule-drug conjugate of claim 29, having a structure selected from:

31. 24. The target binding molecule-drug conjugate of claim 23, wherein the toxin is exatecan or an exatecan derivative.

32. An antibody, antibody fragment, or antigen-binding molecule that competes with the ALPP- and / or ALPPL2-specific antigen-binding molecule of claims 1 to 4 for binding to ALPP and / or ALPPL2.

33. 10. An ALPP- and / or ALPPL2-specific chimeric antigen receptor (CAR) comprising at least one ALPP- and / or ALPPL2-specific antigen-binding molecule according to claim 1 , which is fused or conjugated to at least one transmembrane region and at least one intracellular domain.

34. 34. A cell comprising the chimeric antigen receptor of claim 33, preferably an engineered T cell.

35. A nucleic acid sequence comprising a polynucleotide sequence encoding the specific antigen-binding molecule, recombinant fusion protein, recombinant fusion protein dimer, or chimeric antigen receptor according to any one of claims 1 to 33.

36. A vector comprising the nucleic acid sequence of claim 35.

37. A cell comprising the vector of claim 36.

38. A pharmaceutical composition comprising the ALPP- and / or ALPPL2-specific antigen-binding molecule according to claims 1 to 4, the recombinant fusion protein according to claims 5 to 17, the recombinant fusion protein dimer according to claims 18 to 20, the CAR according to claim 33, the cell according to claim 34 or 37, the nucleic acid sequence according to claim 35, the vector according to claim 36, or the target-binding molecule-drug conjugate according to claims 21 to 31.

39. 36. The ALPP- and / or ALPPL2-specific antigen-binding molecule of claims 1 to 4, the recombinant fusion protein of claims 5 to 17, the recombinant fusion protein dimer of claims 18 to 20, the CAR of claim 33, the cell of claim 34 or 37, the nucleic acid sequence of claim 35, the vector of claim 36, or the target-binding molecule-drug conjugate of claims 21 to 31, for use in therapy.

40. 36. The ALPP- and / or ALPPL2-specific antigen-binding molecule of claims 1 to 4, the recombinant fusion protein of claims 5 to 17, the recombinant fusion protein dimer of claims 18 to 20, the CAR of claim 33, the cell of claim 34 or 37, the nucleic acid sequence of claim 35, the vector of claim 36, or the target-binding molecule-drug conjugate of claims 21 to 31, for use in treating cancer, wherein the cancer is optionally an ALPP- and / or ALPPL2-positive cancer type.

41. The ALPP- and / or ALPPL2-specific antigen-binding molecule, recombinant fusion protein, recombinant fusion protein dimer, CAR, cell, nucleic acid sequence, vector, or target-binding molecule-drug conjugate for use according to claim 40, wherein the cancer is selected from the group consisting of ovarian cancer, endometrial cancer, cervical cancer, gastric cancer, esophageal cancer, bladder cancer, lung cancer, pancreatic cancer, and testicular cancer.

42. Use of the ALPP- and / or ALPPL2-specific antigen-binding molecule of claims 1 to 4, the recombinant fusion protein of claims 5 to 17, the recombinant fusion protein dimer of claims 18 to 20, the CAR of claim 33, the cell of claim 34 or 37, the nucleic acid sequence of claim 35, the vector of claim 36, or the target-binding molecule-drug conjugate of claims 21 to 31 in the manufacture of a pharmaceutical for treating a disease in a patient in need thereof.

43. A method for treating a disease in a patient in need thereof, comprising administering to the patient a therapeutically effective dose of the ALPP- and / or ALPPL2-specific antigen-binding molecule of claims 1 to 4, the recombinant fusion protein of claims 5 to 17, the recombinant fusion protein dimer of claims 18 to 20, the CAR of claim 33, the cell of claim 34 or 37, the nucleic acid sequence of claim 35, the vector of claim 36, or the target-binding molecule-drug conjugate of claims 21 to 31.

44. 44. The method of treatment of claim 43, wherein the disease to be treated is cancer, and optionally the cancer is an ALPP and / or ALPPL2 positive cancer type.

45. 45. The method of claim 44, wherein the cancer is selected from the group consisting of ovarian cancer, endometrial cancer, cervical cancer, gastric cancer, esophageal cancer, bladder cancer, lung cancer, pancreatic cancer and testicular cancer.

46. A method for preparing an ALPP- and / or ALPPL2-specific antigen-binding molecule according to claims 1 to 4, a recombinant fusion protein according to claims 5 to 17, a recombinant fusion protein dimer according to claims 18 to 20, a CAR according to claim 33, a cell according to claim 34 or 37, a nucleic acid sequence according to claim 35, a vector according to claim 36, or a target-binding molecule-drug conjugate according to claims 21 to 31.

47. An artificially engineered ALPP and / or ALPPL2 monomer, which has been artificially engineered so that it can form a covalent bond with a further artificially engineered ALPP and / or ALPPL2 monomer.

48. 48. The artificially engineered ALPP and / or ALPPL2 monomer of claim 47, wherein the covalent bond is a disulfide bridge and / or the artificially engineered ALPP and / or ALPPL2 monomer comprises an introduced cysteine ​​residue.

49. 49. The artificially engineered ALPP and / or ALPPL2 monomer of claim 48, the engineered ALPP and / or ALPPL2 monomer comprises a G503C substitution in SEQ ID NO: 263 or an equivalent substitution in another ALPP and / or ALPPL2 monomer; or The engineered ALPP monomer comprises a sequence according to SEQ ID NO:

263. Engineered ALPP and / or ALPPL2 monomers.

50. An artificially engineered ALPP and / or ALPPL2 homodimer comprising a first and a second artificially engineered ALPP and / or ALPPL2 monomer according to claims 47 to 49.

51. 1. A method for producing an ALPP and / or ALPPL2 specific binding molecule, comprising: (a) immunizing an animal with the engineered ALPP and / or ALPPL2 homodimer of claim 50; (b) isolating peripheral blood mononuclear cells (PBMCs) from the animal; (c) cloning the RNA or cDNA sequences obtained from the PBMCs into a vector; (d) transforming a prokaryotic cell with the vector under conditions that allow the production of the specific binding molecule; (e) selecting the specific binding molecules by subjecting the specific binding molecules to antigen affinity selection; (f) recovering said specific binding molecules having a desired specificity. A method comprising:

52. Biparatopic placental alkaline phosphatase (ALPP) and / or germline alkaline phosphatase (ALPPL2)-specific antigen-binding molecules.

53. (a) the biparatopic placental alkaline phosphatase (ALPP) and / or germline alkaline phosphatase (ALPPL2)-specific antigen-binding molecule of claim 52, and (b) at least one toxin, cytotoxic agent, or cytostatic agent; A biparatopic target binding molecule-drug conjugate comprising:

54. 54. The biparatopic target binding molecule-drug conjugate of claim 53, wherein the toxin is MMAE.

55. (b) is a MMAE derivative, The target binding molecule-drug conjugate has the formula (VI): 【Transformation 5】 [X] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof; [L1] and [L2] are optional linkers selected from the group consisting of valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), peptide, dipeptide, tripeptide, -(CH2)n-, -(CH2CHO)n-, p-aminobenzyloxycarbonyl (PAB), Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, Val-Ala, Asn-Ala, any amino acid, a D-amino acid, Phe-Lys-PAB, and combinations thereof; and Y comprises a biparatopic placental alkaline phosphatase (ALPP) and / or germline alkaline phosphatase (ALPPL2)-specific antigen-binding molecule according to claim 52.

54. The biparatopic target binding molecule-drug conjugate of claim 53, having the structure:

56. (b) an anthracycline (PNU) derivative; The target binding molecule-drug conjugate has the formula (III): 【Transformation 6】 wherein [X] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof; [L1] and [L2] are valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), peptide, -(CH ２ ) ｎ -, -(CH ２ CH ２ O) ｎ an optional linker selected from the group consisting of p-aminobenzyloxycarbonyl (PAB), Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, Val-Ala, Asn-Ala, any amino acid except glycine, and combinations thereof; and Y comprises a biparatopic placental alkaline phosphatase (ALPP) and / or germline alkaline phosphatase (ALPPL2)-specific antigen-binding molecule according to claim 52.

54. The biparatopic target binding molecule-drug conjugate of claim 53, having the structure:

57. 54. The biparatopic target binding molecule-drug conjugate of claim 53, wherein the toxin is exatecan or an exatecan derivative.

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