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WO2025048716A1 - Anticorps anti-palp/palpl2, conjugués anticorps-médicaments et leurs utilisations - Google Patents

Anticorps anti-palp/palpl2, conjugués anticorps-médicaments et leurs utilisations Download PDF

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Publication number
WO2025048716A1
WO2025048716A1 PCT/SG2024/050541 SG2024050541W WO2025048716A1 WO 2025048716 A1 WO2025048716 A1 WO 2025048716A1 SG 2024050541 W SG2024050541 W SG 2024050541W WO 2025048716 A1 WO2025048716 A1 WO 2025048716A1
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seq
amino acid
antibody
variable region
chain variable
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PCT/SG2024/050541
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English (en)
Inventor
Bin Zou
Chen ZHONG
Xin Yu KOH
Bryan K.S. Yeung
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Axcynsis Therapeutics Pte. Ltd.
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Publication of WO2025048716A1 publication Critical patent/WO2025048716A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6871—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting an enzyme
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present technology relates to therapeutic antibodies, antigen-binding fragments thereof, and antibody-drug conjugates (ADCs) that target alkaline phosphatase, placental (ALPP) and/or alkaline phosphatase, placental-like 2 (ALPPL2) and uses thereof for cancer therapy.
  • ADCs antibody-drug conjugates
  • Sequence Listing associated with this application is provided electronically in XML file format and is hereby incorporated by reference into the specification in its entirety.
  • the name of the XML file containing the Sequence Listing is AXCY_001_02WO_SeqList_ST26.xml.
  • the XML file is 42,561 bytes in size and was created on August 20, 2024.
  • ALPPL2 and ALPP are highly tumorspecific and thus attractive targets for cancer therapy.
  • ALPP and ALPPL2 are membranebound proteins involved in ATP recycling from the extracellular space. Both proteins are expressed in ovarian, endometrial, gastric, and testicular cancers with limited expression in normal tissues.
  • the present technology provides optimized anti-ALPP/ALPPL2 antibodies or antigen-binding fragments thereof, as well as ADCs comprising the antibodies or antigen-binding fragments thereof and further comprising a cytotoxic payload, as novel therapeutics for cancer treatment.
  • the present technology relates to novel anti-ALPP/ALPPL2 antibodies, antigenbinding fragments thereof, and ADCs that can be employed as novel therapeutics for the treatment of cancer including, for example, mesothelioma, ovarian, pancreatic, endometrial, non-small cell lung, stomach, gastroesophageal junction, cervical, colorectal, and testicular cancers.
  • a recombinant antibody or an antigen-binding fragment thereof that specifically binds to ALPP and/or ALPPL2.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising at least one, two, or three complementarity-determining regions (CDRs) selected from SEQ ID NOs: 10, 3, 11 ; and/or a light chain variable region comprising at least one, two, or three CDRs selected from SEQ ID NOs: 6-8.
  • CDRs complementarity-determining regions
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 9, 12, or 13; and/or a light chain variable region comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 14 or 15.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising at least one, two, or three CDRs selected from GFSLTSYG (SEQ ID NO: 10), IWEX1X2ST (SEQ ID NO: 38), and AKPHYGSSYVGAMEY (SEQ ID NO: 11), wherein:
  • Xi is E, H, Q, or S
  • X2 is A, E, L, or Q; and/or a light chain variable region comprising at least one, two, or three CDRs selected from SEQ ID NOs: 6-8.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises:
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises:
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof further comprises a light chain constant region comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 33 and/or a heavy chain constant region, wherein the heavy chain constant region comprises:
  • amino acid substitutions relative to SEQ ID NO: 34 selected from the group consisting of: A121 C, S242C, L237A, L238A, D268C, K150C, V205C, C223S, A330C, and S443C; or
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof is humanized, is hypoimmune, does not bind alkaline phosphatase, intestinal (ALPI) and/or alkaline phosphatase, biomineralization associated (ALPL), and/or does not have cross reactivity to a non-human species.
  • composition comprising the anti- ALPP/ALPPL2 antibody or antigen-binding fragment thereof according to various embodiments disclosed herein.
  • nucleic acid comprising a nucleotide sequence encoding the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof according to various embodiments disclosed herein.
  • nucleotide sequence is at least 80% identical to SEQ ID NO: 35 or 36.
  • nucleic acid is in the form of a vector or virus.
  • a host cell containing the nucleic acid or the vector or virus according to various embodiments disclosed herein.
  • composition comprising the vector or virus or the host cell according to various embodiments disclosed herein.
  • an ADC comprising (1) an anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof according to various embodiments disclosed herein; and (2) one or more payloads, wherein the anti-ALPP/ALPPL2 antibody or antigenbinding fragment thereof is connected to each of the one or more payloads by a linker.
  • the ADC of has a formula of: wherein Ab is the antibody or antigen-binding fragment thereof, L is a linker, D is a payload, and z is an integer between 1 and 20.
  • the linker (L) has a structure of:
  • the linker (L) has a structure of any one of L9-L21 provided in Table 3.
  • the payload comprises monomethyl auristatin E (MMAE) or a derivative thereof, DXd/exatecan or a derivative thereof, trabectedin or a derivative thereof, lurbinectedin or a derivative thereof, and/or a pharmaceutically acceptable salt, ester, solvate, tautomer, or stereoisomer of any of the foregoing.
  • MMAE monomethyl auristatin E
  • the payload comprises MMAE or a derivative thereof having a structure of:
  • R is a saccharide, optionally a monosaccharide.
  • the payload comprises Dxd/exatecan or a derivative thereof.
  • the payload comprises lurbinectedin or a derivative thereof having a structure of: wherein:
  • R9 and R10 are independently H or a Ci 4 alkyl
  • R4 is -H or a C1-4 alkyl, optionally wherein formula (III) does not include:
  • the payload comprises lurbinectedin or a derivative thereof having a structure of: wherein:
  • R4 is -H or -CH2NHR11, wherein Rn is H or a C1-4 alkyl.
  • R5 is -H or a C1-4 alkyl, optionally wherein formula (V) does not include:
  • R2 is -OCH3
  • Rs is -OH
  • R4 is -H
  • R5 is -H.
  • the payload (D) has a structure of any one of P1 -P12 provided in Table 4.
  • the linker payload (L-D) has a structure of any one of PL1 -PL38 provided in Table 8.
  • composition comprising the ADC according to various embodiments disclosed herein.
  • a method of in vitro inhibition of the growth of a cancer cell that expresses ALPP and/or ALPPL2 comprising contacting the cancer cell with an effective amount of an anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof, an ADC, or a pharmaceutical composition containing the same, according to various embodiments disclosed herein.
  • a method of treating a cancer that expresses ALPP and/or ALPPL2 in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an anti-ALPP/ALPPL2 antibody or antigenbinding fragment thereof, an ADC, or a pharmaceutical composition containing the same, according to various embodiments disclosed herein.
  • the method further comprises administering to the subject one or more additional anti-cancer therapies.
  • the one or more additional anticancer therapies is a therapeutic antibody, an ADC, chemotherapy, radiotherapy, endocrine therapy, a targeted molecular agent, immunotherapy, or any combination thereof.
  • the cancer is a solid cancer, including, for example, mesothelioma, ovarian, pancreatic, endometrial, non-small cell lung, stomach, gastroesophageal junction, cervical, colorectal, and testicular cancers.
  • FIG. 1 shows binding of the original mouse antibody H17E2 (AT04-5A) to ALPPL2, ALPP, and similar enzymes like alkaline phosphatase, intestinal (ALPI) and alkaline phosphatase, biomineralization associated (ALPL).
  • FIG. 3 shows binding of H17E2 (AT04-5A) to various cancer cell lines.
  • FIG. 4 shows hydrophobic interaction chromatography analysis of H17E2 (AT04-5A).
  • FIG. 5A shows efforts of humanizing H17E2 (AT04-5A) to generate six new constructs (AT04-10A to AT04-15A) by combining one of two humanized light chain designs (VL1 (SEQ ID NO: 14) and VL2 (SEQ ID NO: 15)) and one of three humanized heavy chain designs (VH4 (SEQ ID NO: 9), VH5 (SEQ ID NO: 12), and VH6 (SEQ ID NO: 13)) carrying different backbone mutations.
  • FIG. 5B shows binding of the humanized antibodies to ALPPL2.
  • FIG. 6 shows hydrophobic interaction chromatography analysis of the humanized antibodies
  • FIG. 7A shows optimization of antibody AT04-13A by performing post- translational modification (PTM) removal to improve its developability and by making certain mutations at the D54 or G55 residue of the heavy chain to avoid isomerization of the antibody.
  • PTM post- translational modification
  • An alignment of the heavy chain variable region sequences of AT04-13A (SEQ ID NO: 9) and of eight optimized antibodies, AT04-16A to AT04-23A (SEQ ID NOs: 16, 18, 20, 22, 24, 26, 28, 30; see Table 6) is shown.
  • FIG. 7B shows binding of the optimized antibodies to ALPPL2.
  • FIG. 8 shows hydrophobic interaction chromatography analysis of the optimized antibodies.
  • FIG. 9 shows surface plasmon resonance (SPR) analysis of the parental and select humanized/optimized antibodies, AT04-5A, AT04-13A, AT04-23A, and AT04-26A.
  • FIG. 10 shows specificity of the parental and select humanized/optimized antibodies, AT04-5A, AT04-13A, AT04-23A, and AT04-26A, to ALPPL2, ALPP, and similar enzymes like ALPI and ALPL.
  • ITC isotype control antibody lacking specificity to the target, was used as negative control.
  • FIG. 11 shows cross reactivity of the parental and select humanized/optimized antibodies, AT04-5A, AT04-13A, AT04-23A, and AT04-26A, to mouse and monkey ALPPL2.
  • ITC isotype control.
  • FIG. 12 shows internalization data of AT04-23A on ALPPL2-expressing cell lines.
  • FIG. 13 shows exemplary structures of monomethyl auristatin E (MMAE)-based antibody-drug conjugates (ADCs).
  • MMAE monomethyl auristatin E
  • FIGS. 14A-14E show cytotoxicity data of ADCs made from various humanized and/or optimized antibodies on cancer cells.
  • FIG. 15 shows strong antitumor activity of ADC-14 in tumor cell derived xenografts in mice.
  • FIG. 16 shows potent inhibition of tumor growth by ADC-14 in a patient-derived xenograft tumor model in mice.
  • FIG. 17 shows potent inhibition of tumor growth by ADC-14 in a patient-derived xenograft tumor model in mice.
  • the present technology provides novel anti- ALPP/ALPPL2 antibodies, antigen-binding fragments thereof, and ADCs that can be employed as novel therapeutics for the treatment of cancer including, for example, mesothelioma, ovarian, pancreatic, endometrial, non-small cell lung, stomach, gastroesophageal junction, cervical, colorectal, and testicular cancers.
  • cancer including, for example, mesothelioma, ovarian, pancreatic, endometrial, non-small cell lung, stomach, gastroesophageal junction, cervical, colorectal, and testicular cancers.
  • H17E2 the first anti-ALPP/ALPPL2 murine monoclonal antibodies
  • H17E2 the first anti-ALPP/ALPPL2 murine monoclonal antibodies
  • the original mouse H17E2 clone has been “reshaped” and humanized, but the humanized antibody has limited developability to generate ADCs due to its poor humanness score, yield, and pharmacokinetic properties (high hydrophobicity and high propensity for aggregation), causing it to be unsuitable for use for conjugation to a payload.
  • the present technology provides optimized anti-ALPP/ALPPL2 antibodies or antigen-binding fragments thereof with improved binding affinity (around 10-fold), improved hydrophilicity, and increased yield (around 5 to 15-fold). Moreover, with improved hydrophilicity and reduced propensity for aggregation, the anti-ALPP/ALPPL2 antibodies or antigen-binding fragments thereof of the present technology are suitable for the development of ADCs, for example, by conjugation with a cytotoxic payload. Therefore, the present technology presents promising therapeutic agents for treatment of various cancers, including, for example, ovarian, endometrial, gastric, and testicular cancers, that bear the ALPP and/or ALPPL2 antigens.
  • a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios, such as about 2, about 3, and about 4, and sub-ranges, such as about 10 to about 50, about 20 to about 100, and so forth. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified.
  • Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid. Such analogs have modified R groups or modified peptide backbones but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.
  • antibody is used herein in the broadest sense and includes polyclonal and monoclonal antibodies, such as intact antibodies and functional (antigenbinding) fragments thereof.
  • the term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, synthetic antibodies, and heteroconjugate antibodies, multi-specific (e.g., bispecific) antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, and tandem tri-scFv.
  • the term encompasses intact or full-length antibodies, including antibodies of any class or subclass (e.g., IgG and sub-classes thereof such as IgGI, lgG2, lgG3, and lgG4; IgM; IgE; IgA; and IgD), as well as antibody fragments.
  • IgG and sub-classes thereof such as IgGI, lgG2, lgG3, and lgG4; IgM; IgE; IgA; and IgD
  • an antibody comprises an antigen-binding fragment of an immunoglobulin molecule
  • the antibody may include, but is not limited to, a single chain variable fragment antibody (scFv), a disulfide linked Fv, a single domain antibody (sdAb), a VHH antibody, an antigen-binding fragment F(ab), a F(ab’) fragment, a F(ab’)2fragment, or a diabody.
  • scFv antibody is derived from a natural antibody by linking the variable regions of the heavy (VH) and light (VL) chains of the immunoglobulin with a short linker peptide.
  • a disulfide linked Fv can be generated by linking the Vn and VL using an interdomain disulfide bond.
  • sdAbs consist of only the variable region from either the heavy or light chain and usually are the smallest antigen-binding fragments of antibodies.
  • a VHH antibody is the antigen binding fragment of heavy chain only.
  • a diabody is a dimer of scFv fragment that consists of the VH and VL regions noncovalent connected by a small peptide linker or covalently linked to each other.
  • the term “antigen-binding fragment” may refer to an immunogenically active fragment of an antibody as described that possesses the ability to specifically recognize, associate, unite, or combine with an antigen or target molecule.
  • An antigen-binding fragment includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced. Both the antibodies and antigen-binding fragments thereof disclosed herein retain the ability to bind a specific antigen. In some embodiments, an antigen-binding fragment of a full-length antibody may be used in making an antibody-drug conjugate of the present invention.
  • An antibody may include a heavy chain (or a polypeptide sequence derived therefrom) and a light chain (or a polypeptide sequence derived therefrom).
  • the term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in the antibody's binding to an antigen.
  • the variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions and three complementarity-determining regions. A single VH or VL domain may sometimes be sufficient to confer all or a majority of the antigen-binding specificity of an antibody.
  • antibodies that bind a particular antigen may be isolated by using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991 ).
  • antigen refers to a molecule capable of provoking an immune response.
  • Antigens include but are not limited to cells, cell extracts, proteins, polypeptides, peptides, polysaccharides, polysaccharide conjugates, peptide and nonpeptide mimics of polysaccharides and other molecules, small molecules, lipids, glycolipids, carbohydrates, viruses and viral extracts and multicellular organisms such as parasites and allergens.
  • antigen broadly includes any type of molecule which is recognized by a host immune system as being foreign.
  • the term “neoantigen” can be used to refer to a cancer-specific antigen (i.e. , an antigen found on cancer cells but not on non-cancer cells) that is specifically recognized by a cognate binding molecule as described.
  • the term “specific binding,” “specifically binds,” or “specifically recognize” refers to an antibody or antigen binding fragment thereof which binds to a predetermined antigen/target molecule.
  • Specific binding of an antibody or antigen binding fragment thereof typically describes an antibody or antigen binding fragment thereof having an affinity of at least 10- 7 M (as Kd value; i.e. preferably those with Kd values smaller than 10 -7 M), with the antibody or antigen binding fragment thereof having an at least two times higher affinity for the predetermined antigen/target molecule than for a non-specific antigen/target molecule (e.g. bovine serum albumin, or casein) which is not the predetermined antigen/target molecule or a closely related antigen/target molecule.
  • a non-specific antigen/target molecule e.g. bovine serum albumin, or casein
  • Specific binding of an antibody or antigen binding fragment thereof does not exclude the antibody or binder binding to a plurality of antigens/target molecules.
  • the antibodies preferably have an affinity of at least 10 ⁇ 7 M, at least 10 ⁇ 8 M, or at least 10 ⁇ 9 M to 10 ⁇ 11 M.
  • the Kd values may be determined, for example, by means of surface plasmon resonance spectroscopy.
  • a “clinically effective amount,” “clinically effective concentration,” or “clinically effective dose” refers to a concentration or dose of a peptide, composition, or pharmaceutical composition that is shown to be effective in clinical trials or is predicted to be effective based on early phase or pre-clinical trials.
  • a “clinically effective amount” is the same as a “therapeutically effective amount.”
  • a “clinically effective amount” is higher or lower than a “therapeutically effective amount.”
  • the effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the subject’s response to treatment.
  • the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition may require an increase or decrease in the actual effective amount administered.
  • codon-optimized or “codon optimization” when referring to a nucleotide sequence is based on the discovery that the frequency of occurrence of synonymous codons (i.e., codons that code for the same amino acid) in coding nucleotide is biased in different species. Such codon degeneracy allows an identical polypeptide to be encoded by a variety of nucleotide sequences. Codon optimization refers to the process of substituting certain codons in a coding nucleotide sequence with synonymous codons based on the host cell’s preference without changing the resulting polypeptide sequence. A variety of codon optimization methods are known in the art, and include, for example, methods disclosed in at least U.S. Pat.
  • CDRs complementarity determining regions
  • HVR hypervariable region
  • FRs framework regions
  • Variable domain sequences can be aligned to a numbering scheme (e.g., Kabat, EU, international ImMunoGeneTics information system® (IMGT®), and Aho), which can allow equivalent residue positions to be annotated and for different molecules to be compared using the Antibody Numbering and Antigen Receptor Classification (ANARCI) software tool (2016, Bioinformatics 15:298-300).
  • a numbering scheme e.g., Kabat, EU, international ImMunoGeneTics information system® (IMGT®), and Aho
  • ANARCI Antibody Numbering and Antigen Receptor Classification
  • IMGT IMGT numbering scheme
  • Hithia based on structural information
  • Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a.” The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering.
  • the contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. Unless indicated otherwise, the CDRs of the antibodies referred to herein may be identified according to any of the Kabat, Chothia, IMGT, and contact methods.
  • conservative substitution when referring to amino acid sequences, is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties.
  • a variety of criteria known to persons skilled in the art indicate whether an amino acid that is substituted at a particular position in a peptide or polypeptide is conservative (or similar).
  • a similar amino acid or a conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • amino acids with acidic side chains e.g., aspartic acid, glutamic acid
  • amino acids with uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, histidine
  • amino acids with nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • amino acids with beta-branched side chains e.g., threonine, valine, isoleucine
  • amino acids with aromatic side chains e.g., tyrosine, phenylalanine, tryptophan
  • Proline which is considered more difficult to classify, shares properties with amino acids that have aliphatic side chains (e.g., leucine, valine, isoleucine, and alanine).
  • substitution of glutamine for glutamic acid or asparagine for aspartic acid may be considered a similar substitution in that glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively.
  • epitope includes any molecule, structure, amino acid sequence or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an antibody or a T cell receptor, or other binding molecule, domain, or protein.
  • the term “expression” refers to the process by which a polypeptide is produced based on the encoding sequence of a nucleic acid molecule, such as a gene.
  • the process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof.
  • An expressed nucleic acid molecule is typically operably linked to an expression control sequence (e.g., a promoter).
  • host cell refers to a cell or microorganism targeted for genetic modification by introduction of a construct or vector carrying a nucleotide sequence for expression of a protein or polypeptide of interest.
  • nucleic acid refers to a polymeric compound including covalently linked nucleotides comprising natural subunits (e.g., purine or pyrimidine bases).
  • Purine bases include adenine and guanine
  • pyrimidine bases include uracil, thymine, and cytosine.
  • Nucleic acid molecules include polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), which includes cDNA, genomic DNA, and synthetic DNA, either of which may be single- or double-stranded.
  • a nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence.
  • operably linked refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other.
  • polypeptide refers to a polymer of amino acid residues, and are not limited to a minimum length, though a number of amino acid residues may be specified.
  • Polypeptides may include amino acid residues including natural and/or non-natural amino acid residues.
  • the terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like.
  • the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site- directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
  • subject refers to a mammalian subject, preferably a human.
  • a “subject in need thereof” refers to a subject who has been diagnosed with cancer or is at an elevated risk of developing cancer.
  • subject and patient are used interchangeably herein.
  • treat refers to alleviating the cancer partially or entirely, inhibiting cancer cell growth, reducing the number of cancer cells, preventing the cancer, decreasing the likelihood of occurrence or recurrence of the cancer, slowing the progression or development of the cancer, or eliminating, reducing, or slowing the development of one or more symptoms associated with the cancer.
  • treating may refer to preventing or slowing the existing tumor from growing larger, preventing or slowing the formation or metastasis of cancer, and/or slowing the development of certain symptoms of the cancer.
  • the term “treat,” “treating,” or “treatment” means that the subject has a reduced number or size of tumor compared to a subject not being administered the treatment. In some embodiments, the term “treat,” “treating,” or “treatment” means that one or more symptoms of the cancer are alleviated in a subject receiving the pharmaceutical compositions as disclosed and described herein, compared to a subject who does not receive such treatment.
  • a therapeutically effective amount for a particular composition will vary based on a variety of factors, including, but not limited to, the characteristics of the therapeutic composition (e.g., activity, pharmacokinetics, pharmacodynamics, and bioavailability); the physiological condition of the subject (e.g., age, body weight, sex, disease type and stage, medical history, general physical condition, responsiveness to a given dosage, and other present medications); the nature of any pharmaceutically acceptable carriers, excipients, and preservatives in the composition; and the route of administration.
  • One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, namely, by monitoring a subject’s response to administration of the therapeutic composition and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy, 21st Edition, Univ, of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005.
  • variable region refers to a fragment of an antibody heavy or light chain that is involved in antigen binding.
  • Variable domains of antibody heavy (VH) and light (VL) chains each generally comprise four generally conserved framework regions (FRs) and three complementarity determining regions (CDRs). Framework regions separate CDRs, such that CDRs are situated between framework regions.
  • a “vector” refers to a DNA construct containing a nucleic acid molecule that is operably linked to a suitable control sequence capable of effecting the expression of the nucleic acid molecule in a suitable host.
  • control sequences may include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation.
  • the vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region derived from the murine monoclonal antibody H17E2.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises one or more (e.g., one, two, three, four, five, or six) complementarity-determining regions (CDRs) having amino acid sequences selected from SEQ ID NOs: 2-4 and 6-8.
  • CDRs complementarity-determining regions
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from SEQ ID NOs: 2-4; and/or a light chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from SEQ ID NOs: 6-8.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 1 , or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 1 ; and/or a light chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 5, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 5.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region modified from the murine monoclonal antibody H17E2 to have improved binding affinity, improved hydrophilicity, improved yield, improved hydrophilicity, and/or reduced propensity for aggregation compared to the original H17E2 antibody.
  • the modification can comprise one or more amino acid mutations, substitutions, additions, or deletions in one or more of the CDRs and/or the framework regions (FRs) of the heavy and/or light chains.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from GFSLTSYG (SEQ ID NO: 10), IWEDGST (SEQ ID NO: 3), and AKPHYGSSYVGAMEY (SEQ ID NO: 11 ); and/or a light chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from SEQ ID NOs: 6-8.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 9, 12, or 13, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 9, 12, or 13; and/or a light chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 14 or 15, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 14 or 15.
  • a heavy chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 9, 12, or 13, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region further modified from the murine monoclonal antibody H17E2, e.g., by post-translational modification (PTM) removal, to have improved binding affinity, improved hydrophilicity, improved yield, improved hydrophilicity, and/or reduced propensity for aggregation compared to the original H17E2 antibody.
  • the modification can comprise one or more amino acid mutations, substitutions, additions, or deletions in one or more of the CDRs of the heavy and/or light chains.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from GFSLTSYG (SEQ ID NO: 10), IWEX1X2ST (SEQ ID NO: 38), and AKPHYGSSYVGAMEY (SEQ ID NO: 11 ), wherein:
  • Xi is E, H, Q, or S
  • X2 is A, E, L, or Q.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a light chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from SEQ ID NOs: 6-8.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from GFSLTSYG (SEQ ID NO: 10), IWEEGST (SEQ ID NO: 17; comprising a D54E mutation compared to the original H17E2 antibody heavy chain variable region sequence (SEQ ID NO: 1 )), and AKPHYGSSYVGAMEY (SEQ ID NO: 11 ); and/or a light chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from SEQ ID NOs: 6-8.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 16; and/or a light chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 14 or 15, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 14 or 15.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from GFSLTSYG (SEQ ID NO: 10), IWEHGST (SEQ ID NO: 19; comprising a D54H mutation compared to the original H17E2 antibody heavy chain variable region sequence (SEQ ID NO: 1 )), and AKPHYGSSYVGAMEY (SEQ ID NO: 11 ); and/or a light chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from SEQ ID NOs: 6-8.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 18, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 18; and/or a light chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 14 or 15, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 14 or 15.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 20, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 20; and/or a light chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 14 or 15, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 14 or 15.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from GFSLTSYG (SEQ ID NO: 10), IWESGST (SEQ ID NO: 23; comprising a D54S mutation compared to the original H17E2 antibody heavy chain variable region sequence (SEQ ID NO: 1 )), and AKPHYGSSYVGAMEY (SEQ ID NO: 11 ); and/or a light chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from SEQ ID NOs: 6-8.
  • GFSLTSYG SEQ ID NO: 10
  • IWESGST SEQ ID NO: 23
  • AKPHYGSSYVGAMEY SEQ ID NO: 11
  • a light chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 22, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 22; and/or a light chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 14 or 15, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 14 or 15.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from GFSLTSYG (SEQ ID NO: 10), IWEDAST (SEQ ID NO: 25; comprising a G55A mutation compared to the original H17E2 antibody heavy chain variable region sequence (SEQ ID NO: 1 )), and AKPHYGSSYVGAMEY (SEQ ID NO: 11 ); and/or a light chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from SEQ ID NOs: 6-8.
  • GFSLTSYG SEQ ID NO: 10
  • IWEDAST SEQ ID NO: 25
  • AKPHYGSSYVGAMEY SEQ ID NO: 11
  • a light chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from GFSLTSYG (SEQ ID NO: 10), IWEDEST (SEQ ID NO: 27; comprising a G55E mutation compared to the original H17E2 antibody heavy chain variable region sequence (SEQ ID NO: 1 )), and AKPHYGSSYVGAMEY (SEQ ID NO: 11 ); and/or a light chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from SEQ ID NOs: 6-8.
  • GFSLTSYG SEQ ID NO: 10
  • IWEDEST SEQ ID NO: 27
  • AKPHYGSSYVGAMEY SEQ ID NO: 11
  • a light chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from GFSLTSYG (SEQ ID NO: 10), IWEDLST (SEQ ID NO: 29; comprising a G55L mutation compared to the original H17E2 antibody heavy chain variable region sequence (SEQ ID NO: 1 )), and AKPHYGSSYVGAMEY (SEQ ID NO: 11 ); and/or a light chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from SEQ ID NOs: 6-8.
  • GFSLTSYG SEQ ID NO: 10
  • IWEDLST SEQ ID NO: 29
  • AKPHYGSSYVGAMEY SEQ ID NO: 11
  • a light chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 28; and/or a light chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 14 or 15, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 14 or 15.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from GFSLTSYG (SEQ ID NO: 10), IWEDQST (SEQ ID NO: 31 ; comprising a G55Q mutation compared to the original H17E2 antibody heavy chain variable region sequence (SEQ ID NO: 1 )), and AKPHYGSSYVGAMEY (SEQ ID NO: 11 ); and/or a light chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from SEQ ID NOs: 6-8.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 30, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 30; and/or a light chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 14 or 15, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 14 or 15.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises one or more (e.g., one, two, three, four, five, or six) CDRs having amino acid sequences selected from SEQ ID NOs: 10, 19, 11 , and 6-8.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from SEQ ID NOs: 10, 19, and 11 ; and/or a light chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from SEQ ID NOs: 6-8.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 18, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 18; and/or a light chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 15, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 15.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from SEQ ID NOs: 10, 21 , and 11 ; and/or a light chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from SEQ ID NOs: 6-8.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 20, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 20; and/or a light chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 15, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 15.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises one or more (e.g., one, two, three, four, five, or six) CDRs having amino acid sequences selected from SEQ ID NOs: 10, 23, 11 , and 6-8.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from SEQ ID NOs: 10, 23, and 11 ; and/or a light chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from SEQ ID NOs: 6-8.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 22, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 22; and/or a light chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 15, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 15.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises one or more (e.g., one, two, three, four, five, or six) CDRs having amino acid sequences selected from SEQ ID NOs: 10, 25, 11 , and 6-8.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from SEQ ID NOs: 10, 27, and 11 ; and/or a light chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from SEQ ID NOs: 6-8.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 26, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 26; and/or a light chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 15, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 15.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises one or more (e.g., one, two, three, four, five, or six) CDRs having amino acid sequences selected from SEQ ID NOs: 10, 29, 11 , and 6-8.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from SEQ ID NOs: 10, 29, and 11 ; and/or a light chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from SEQ ID NOs: 6-8.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises one or more (e.g., one, two, three, four, five, or six) CDRs having amino acid sequences selected from SEQ ID NOs: 10, 31 , 11 , and 6-8.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from SEQ ID NOs: 10, 31 , and 11 ; and/or a light chain variable region comprising one or more (e.g., one, two, or three) CDRs having amino acid sequences selected from SEQ ID NOs: 6-8.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 30, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 30; and/or a light chain variable region that comprises or consists of an amino acid sequence of SEQ ID NO: 15, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 15.
  • the anti-ALPP/ALPPL2 antibodies or antigen-binding fragments thereof may comprise one or more amino acid substitutions (e.g., conservative substitutions), insertions, and/or deletions in any of the exemplary amino acid sequences, including, for example, one or more (e.g., one, two, or three) substitutions, insertions, and/or deletions in any of the exemplary CDR sequences.
  • the present technology especially contemplates anti-ALPP/ALPPL2 antibodies or antigen-binding fragments thereof having one or more (e.g., one, two, or three) substitutions, insertions, and/or deletions compared to a reference CDR sequence as described herein that still maintain a certain level of binding affinity to ALPP and/or ALPPL2, e.g., human ALPP/ALPPL2.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof having one or more (e.g., one, two, or three) substitutions, insertions, and/or deletions compared to a reference CDR sequence as described herein would maintain at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) of the binding affinity exhibited by the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof having the reference CDR sequence, when tested under same or similar conditions.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof having one or more (e.g., one, two, or three) substitutions, insertions, and/or deletions compared to a reference CDR sequence as described herein would bind to ALPP/ALPPL2 (e.g., human ALPP/ALPPL2) with an affinity (KD) of about 1 x10- 11 -1 x10 7 M, e.g., about 1x10 11 -1 x10 8 M, about 1 x10 11 -1 x10 9 M, about 1 x10 11 -1x10 10 M, about 1 x10 10 -1 x10 7 M, about 1x10 10 -1 x10 8 M, about 1x10 10 -1 x10 9 M, about 1 x10 11 -1 x10 7 M, about 1x10 -9 -1 x10 -7 M, about 1 x10 9 -1 x10- 8 M, or about 1 x10- 81 -1 x
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof according to various embodiments disclosed herein is humanized.
  • a “humanized” form of a non-human (e.g., rodent) antibody is a chimeric antibody including a sequence derived from a non-human antibody.
  • the antibody or antigenbinding fragment thereof may be humanized to reduce immunogenicity in a human subject.
  • the non-human antibody can be humanized through CDR grafting, in which the CDRs from the non-human antibody are placed into the respective positions in a framework of a compatible human antibody.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain constant region that comprises or consists of an amino acid sequence of SEQ ID NO: 32, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 32; and/or a light chain constant region that comprises or consists of an amino acid sequence of SEQ ID NO: 33, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 33.
  • the constant region of the heavy and/or light chains of the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof is further modified to include strategically placed reactive cysteine residues, for example, for subsequent conjugation with a drug payload to generate ADCs.
  • the constant region of the modified anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises one or more of the following mutations compared to the original H17E2 antibody heavy chain sequence (SEQ ID NO: 34): A121 C, K150C, V205C, C223S, S242C, D268C, A330C, and S443C.
  • the anti-ALPP/ALPPL2 antibody or antigenbinding fragment thereof comprises a heavy chain constant region that comprises or consists of an amino acid sequence of SEQ ID NO: 35 (comprising A121 C and S242C mutations compared to the original H17E2 antibody heavy chain sequence (SEQ ID NO: 34)), or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 35; and/or a light chain constant region that comprises or consists of an amino acid sequence of SEQ ID NO: 33, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 33.
  • SEQ ID NO: 35 comprising A121 C and S242C mutations compared to the original H17E2 antibody heavy chain sequence (SEQ ID NO: 34)
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain constant region that comprises or consists of an amino acid sequence of SEQ ID NO: 36 (comprising A121 C, L237A, L238A, and D268C mutations compared to the original H17E2 antibody heavy chain sequence (SEQ ID NO: 34)), or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 36; and/or a light chain constant region that comprises or consists of an amino acid sequence of SEQ ID NO: 33, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 33.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof comprises a heavy chain constant region that comprises or consists of an amino acid sequence of SEQ ID NO: 37 (comprising A121C, L237A, L238A, and S242C mutations compared to the original H17E2 antibody heavy chain sequence (SEQ ID NO: 34)), or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 37; and/or a light chain constant region that comprises or consists of an amino acid sequence of SEQ ID NO: 33, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 33.
  • SEQ ID NO: 37 comprising A121C, L237A, L238A, and S242C mutation
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof according to various embodiments disclosed herein is hypoimmune, e.g., less prone to elicit an immune response or an immune rejection by a subject into which the antibody is transplanted.
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof according to various embodiments disclosed herein is specific to ALPP/ALPPL2 and does not bind a protein that shares a degree of sequence homology to ALPP/ALPPL2, including, for example, alkaline phosphatase, intestinal (ALPI) and/or alkaline phosphatase, biomineralization associated (ALPL).
  • ALPI alkaline phosphatase
  • APL biomineralization associated
  • the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof according to various embodiments disclosed herein is specific to human ALPP/ALPPL2, i.e., does not have cross reactivity to ALPP/ALPPL2 from a non-human species.
  • compositions comprising a recombinant anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof according to various embodiments disclosed and described herein.
  • the pharmaceutical compositions may further comprise one or more pharmaceutically acceptable carriers, excipients, preservatives, or a combination thereof.
  • a “pharmaceutically acceptable carrier or excipient” refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or fragment of the body to another tissue, organ, or fragment of the body.
  • the carrier or excipient may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or some combination thereof.
  • Each component of the carrier or excipient must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It also must be suitable for contact with any tissue, organ, or fragment of the body that it may encounter, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.
  • Non-limiting examples of such carriers or excipients include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.
  • the pharmaceutical composition can be co-formulated in the same dosage unit or can be individually formulated in separate dosage units.
  • dosage unit herein refers to a fragment of a pharmaceutical composition that contains an amount of a therapeutic agent suitable for a single administration to provide a therapeutic effect. Such dosage units may be administered one to a plurality (e.g., 1 to about 10, 1 to 8, 1 to 6, 1 to 4, or 1 to 2) of times per day, or as many times as needed to elicit a therapeutic response.
  • nucleic acids comprising a nucleotide sequence encoding a recombinant anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof according to various embodiments according to various embodiments disclosed and described herein.
  • the nucleic acids may be used (for example, in the form of a vector) to transfect or transduce a host cell so that the host cell would express the encoded recombinant anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof.
  • Exemplary nucleotide sequences to be used in the present technology are provided in Table 2.
  • the nucleic acid comprises a nucleotide sequence encoding an anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof.
  • the nucleic acid comprises a nucleotide sequence of SEQ ID NO: 39, or a nucleotide sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 39.
  • the nucleic acid comprises a nucleotide sequence of SEQ ID NO: 40, or a nucleotide sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 40.
  • the nucleic acid comprises a nucleotide sequence that is codon-optimized for a host cell (for example, a human cell) according to techniques known to one of ordinary skill in the art. Codon-optimized sequences include sequences that are partially or fully codon-optimized.
  • the nucleic acid comprising a nucleotide sequence encoding an anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof may be present in the form of a vector (e.g., a plasmid or a viral vector) or packaged into a virus for introduction into a host cell.
  • the vector can be any type of vector suitable for introduction of nucleotide sequences into a host cell, including, for example, plasmids, adenoviral vectors, adeno-associated viral (AAV) vectors, retroviral vectors, lentiviral vectors, phages, and homology-directed repair (HDR)-based donor vectors.
  • the virus can be any type of virus suitable for transducing a host cell and introducing nucleotide sequences into the host cell, including, for example, adenoviruses, adeno-associated viruses (AAVs), retroviruses, lentiviruses, and phages.
  • the nucleotide sequence is in a vector (e.g., a viral vector) or a virus which facilitates integration of the nucleotide sequence into a host cell’s genome upon introduction into the host cell and thereby replication along with the host genome.
  • such nucleotide sequence may be present inside a host cell, for example, integrated into the genome of the host cell, for production of the encoded protein in the host cell.
  • the nucleic acid according to various embodiments disclosed herein may be delivered to a host cell via one or more non-viral delivery methods and/or using one or more non-viral vectors, including, but not limited to, physical/mechanical methods, inorganic particles, and synthetic or natural biodegradable particles.
  • physical/mechanical methods include needle injection, ballistic injection, gene gun, electroporation, sonoporation, photoporation, optoporation, magnetofection, and hydroporation.
  • Non-limiting examples of inorganic particles include calcium phosphate, silica, gold, and magnetic particles.
  • Non-limiting examples of synthetic or natural biodegradable particles include polymeric-based non-viral vectors (e.g., poly lactic-co- glycolic acid, poly lactic acid, polyethylene imine, chitosan, dendrimers, polymethacrylates), cationic lipid-based non-viral vectors (e.g., cationic liposomes, cationic emulsions, solid lipid nanoparticles), and peptide-based non-viral vectors (e.g., poly-L-lysine).
  • polymeric-based non-viral vectors e.g., poly lactic-co- glycolic acid, poly lactic acid, polyethylene imine, chitosan, dendrimers, polymethacrylates
  • cationic lipid-based non-viral vectors e.g., cationic liposomes, cationic emulsions, solid lipid nanoparticles
  • peptide-based non-viral vectors e.g.
  • the nucleic acid according to various embodiments disclosed herein may be operatively linked to certain regulatory elements of the vector.
  • expression vectors are typically engineered to contain polynucleotide sequences that are needed to affect the expression and processing of coding sequences to which they are operatively linked.
  • Expression control sequences may include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency; sequences that enhance protein stability; and possibly sequences that enhance protein secretion.
  • Expression control sequences may be operatively linked if they are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
  • the vector may comprise a promoter that drives constitutive gene expression in a host cell (e.g., a mammalian cell).
  • a promoter that drives constitutive gene expression in a host cell
  • a host cell e.g., a mammalian cell
  • Those frequently used promoters include, for example, elongation factor 1 alpha (EF1 a) promoter, cytomegalovirus (CMV) immediate-early promoter (Greenaway et al., Gene 18: 355-360 (1982)), simian vacuolating virus 40 (SV40) early promoter (Fiers et al., Nature 273:113-120 (1978)), spleen focus-forming virus (SFFV) promoter, phosphoglycerate kinase (PGK) promoter (Adra et al., Gene 60(1 ):65-74 (1987)), human beta actin promoter, polyubiquitin C gene (UBC) promoter, and CAG promote
  • the vector may comprise an inducible promoter.
  • inducible promoters can switch between an on and an off state in response to certain stimuli (e.g., chemical agents, temperature, light) and can be regulated in tissue- or cell-specific manners.
  • stimuli e.g., chemical agents, temperature, light
  • frequently used inducible promoters include the tetracycline On (Tet-On) system and the tetracycline Off (Tet-Off) system, which utilize tetracycline response elements (TRE) placed upstream of a minimal promoter (e.g., CMV promoter) (Gossen & Bujard, Proc. Natl. Acad. Sci.
  • the TRE is made of 7 repeats of a 19-nucleotide tetracycline operator (tetO) sequence and can be recognized by the tetracycline repressor (tetR).
  • tetO tetracycline operator
  • tetR tetracycline repressor
  • tTA tetracycline-controlled transactivator
  • the tTA In the absence of tetracycline or its analogs (e.g., doxycycline), the tTA will bind the tetO sequences of the TRE and drives expression; in the presence of tetracycline, the rTA will bind to tetracycline and not to the TRE, resulting in reduced gene expression.
  • tetracycline or its analogs e.g., doxycycline
  • a reverse transactivator (rtTA) was generated by mutagenesis of amino acid residues important for tetracycline-dependent repression, and the rtTA binds at the TRE and drives gene expression in the presence of tetracycline or doxycycline (Gossen et al., Science 268(5218):1766-1769 (1995)).
  • inducible promoters include, for example, AlcA, LexA, and Cre.
  • the vector further comprises a Kozak consensus sequence, usually upstream of the coding sequence.
  • a Kozak consensus sequence is a nucleic acid motif that functions as the protein translation initiation site in most eukaryotic mRNA transcripts and mediates ribosome assembly and translation initiation.
  • the Kozak consensus sequence comprises or consists of the sequence of (gcc)gccrccatgg (SEQ ID NO: 41 ), wherein r is a purine (i.e., a or g).
  • r is a purine (i.e., a or g).
  • the vector further comprises a Woodchuck hepatitis virus (WHV) posttranscriptional regulatory element (WPRE), optionally after the coding sequence.
  • WPRE Woodchuck hepatitis virus
  • a WPRE is a DNA sequence that, when transcribed, creates a tertiary structure enhancing expression.
  • the WPRE sequence is commonly used to increase expression of genes delivered by viral vectors.
  • the WPRE sequence comprises or consists of a nucleotide sequence of SEQ ID NO: 42, or a nucleotide sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 42: aatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttta atgcctttgtatcatgctattgctttcccgtatggcttttcattttctcctcttgtataaatcctggttgctgtctctttatgaggagttgtggccc ctgtcaggcaacgtggcgtggtggtgg
  • host cells such as bacterial, yeast, or mammalian cells, that contain the nucleic acid comprising a nucleotide sequence encoding an anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof, and/or express anti- ALPP/ALPPL2 antibody or antigen-binding fragment thereof, according to various embodiments disclosed herein.
  • the vector, virus, or host cell comprising a nucleotide sequence encoding an anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof according to various embodiments disclosed herein may be present in a composition.
  • the composition may further comprise one or more pharmaceutically acceptable carriers, excipients, preservatives, or a combination thereof.
  • a “pharmaceutically acceptable carrier or excipient” refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or fragment of the body to another tissue, organ, or fragment of the body.
  • the carrier or excipient may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or some combination thereof.
  • Each component of the carrier or excipient must be “pharmaceutically acceptable,” in that it must be compatible with the other ingredients of the formulation. It also must be suitable for contact with any tissue, organ, or fragment of the body that it may encounter, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.
  • Suitable excipients include water, saline, dextrose, glycerol, or the like and combinations thereof.
  • ADCs comprising (1 ) an antibody or antigenbinding fragment thereof that specifically recognize or bind ALPP and/or ALPPL2; and (2) one or more payloads, wherein the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof is connected to each of the one or more payloads by a linker.
  • the ADCs may have a general formula of formula (I) shown below: wherein Ab is an antibody or antigen-binding fragment thereof (e.g., an anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof), L is a linker, D is a payload, and z is an integer between 1 and 20.
  • the linker may be covalently bound to the antibody or antigen-binding fragment thereof, and the payload may be covalently bound to the linker, for example, via a nitrogen or oxygen present in the payload.
  • the L-D fragment of the ADC may be referred to as a “linker payload” in this disclosure.
  • the ADC has a structure of: wherein Ab is an antibody or antigen-binding fragment thereof (e.g., an anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof), D is a payload, n is an integer between 1 and 20, and z is an integer between 1 and 20.
  • the linker has a structure of L1A: Wherein D is a payload, and n is an integer between 1 and 20, e.g., 1 , 2, 3, 4, 5, or 6.
  • the ADC has a structure of: wherein Ab is an antibody or antigen-binding fragment thereof (e.g., an anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof), D is a payload, n is an integer between 1 and 20, and z is an integer between 1 and 20.
  • the linker has a structure of L2B: wherein D is a payload, and n is an integer between 1 and 20, e.g., 1 , 2, 3, 4, 5, or 6.
  • the ADC has a structure of:
  • the linker has a structure of L4: wherein D is a payload.
  • the ADC has a structure of: wherein Ab is an antibody or antigen-binding fragment thereof (e.g., an anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof), D is a payload, and z is an integer between 1 and 20.
  • the linker has a structure of L6: wherein D is a payload.
  • D is a payload
  • linkers commonly used and/or appropriate for linking a payload to an antibody or antigen-binding fragment thereof may be used in the present technology, including, for example, the structures provided in Table 3: Table 3. Additional exemplary linker structures
  • Non-limiting examples of a saccharide or a monosaccharide include p-D- galactose, N-acetyl-p-D-galactosamine, N-acetyl-a-D-galactosamine, N-acetyl-p-D- glucosamine, p-D-glucuronic acid, a-L-iduronic acid, a-D-galactose, a-D-glucose, p-D- glucose, a-D-mannose, (3-D-mannose, a-L-fucose, p-D-xylose, neuraminic acid, and any of the foregoing with a sulfate, phosphate, carboxyl, amino, or O-acetyl modification.
  • the MMAE or derivative thereof has a structure of P1 or P2 provided in Table 4.
  • the payload comprises lurbinectedin or a derivative thereof.
  • Lurbinectedin is a synthetic tetrahydropyrrolo [4,3,2-de]quinolin-8(1 H)-one alkaloid analogue with antineoplastic activity. Lurbinectedin covalently binds to residues lying in the minor groove of DNA, which may result in delayed progression through S phase, cell cycle arrest in the G2/M phase, and cell death. Lurbinectedin is sold under the brand name ZEPZELCA®, which is a medication approved for the treatment of small cell lung cancer.
  • R4 is -H or a C1-4 alkyl, optionally wherein the following combinations of R2, R3, and R4 are excluded from formula (III):
  • R2 is -H, R3 is -H, and R4 is -H;
  • R2 is -OCH3, R3 is -H, and R4 is -H.
  • alkyl refers to an unsubstituted and saturated hydrocarbon group (branched or unbranched). Where indicated, the number of carbons refers to all carbons present in that group, including in sidechains.
  • C1-4 alkyl includes -CH3, -CH2CH3, -CH2CH2CH3 (n-propyl), -CH(CHs)2 (isopropyl), -CH2CH2CH2CH3 (n-butyl), -CH 2 CH2CH(CH 3 )2 (iso-butyl), -CH(CH 3 )CH2CH 3 (secbutyl), and -CH(CHs)3 (tert-butyl).
  • lurbinectedin and derivatives thereof have a general formula of formula (IV) shown below: wherein
  • R9 and R10 are independently H or a C1-4 alkyl
  • R4 is -H or -CH2NHR11, wherein Rn is H or a Ci 4 alkyl.
  • the lurbinectedin or derivative thereof has a structure of P6 provided in Table 4.
  • the payload comprises trabectedin or a derivative thereof.
  • Trabectedin is a chemotherapy agent, sold under the brand name YONDELIS® for the treatment of advanced soft-tissue sarcoma and ovarian cancer.
  • Trabectedin and derivatives thereof have a general formula of formula (V) shown below: wherein
  • R2, R3, R4, and Rs is excluded from formula (V):
  • the pharmaceutical composition can be formulated (e.g., injectable, lyophilized, liquid, or oral formulations) to be compatible with their intended route of administration.
  • routes of administration include oral administration, extracorporeal administration, parenteral administration, intravenous administration, subcutaneous administration, intralesional administration (e.g., injection into tumors), and by administration into biological spaces infiltrated by tumors (e.g., intraspinal administration, intracerebel lar administration, intraperitoneal administration, intralymphatic administration, intranodal administration, and/or pleural administration).
  • a pharmaceutical composition provided herein can be administered systemically by oral administration or by intravenous administration (e.g., injection or infusion).
  • kits for treating and/or preventing a disease in a subject in need thereof entail administering to the subject a therapeutically effective amount or a clinically effective amount of an anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof, an ADC comprising an anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof connected to a payload by a linker, or a pharmaceutical composition containing the same, according to various embodiments disclosed herein.
  • the cancer is a solid cancer.
  • solid cancers include pancreatic cancer, glioma, glioblastoma, colorectal cancer, thyroid cancer, gastric cancer, ovarian cancer, melanoma, endometrial cancer, lung cancer, renal cancer, cervical cancer, prostate cancer, breast cancer, urothelial cancer, testicular cancer, head and neck cancer, liver cancer, esophageal cancer, and other types of solid cancers.
  • the cancer is mesothelioma, ovarian, pancreatic, endometrial, non- small cell lung, stomach, gastroesophageal junction, cervical, colorectal, or testicular cancer.
  • the schedule of administration is a hybrid of these periods, for example, the anti-ALPP/ALPPL2 antibody or antigen-binding fragment thereof, ADC, or a pharmaceutical composition containing the same, is administered a number of times a week and that pattern is repeated a number of times a month every month or every second, third, fourth, fifth, or sixth month, and treatment according to that pattern is continued for part of a year to several years, as set out above.
  • FIG. 13 shows exemplary linker payloads used in ADCs generated below (top structure, see Table 8, LP26; bottom structure, see Table 8, LP14).
  • HPLC instrument Agilent 1260 Bio-Inert High Performance Liquid Chromatography system. [0179] Column: AdvanceBio HIC column, 4.6 x 100 mm, 3.5 urn (Manufacturer: Agilent).
  • Mobile phase mobile phase A (MPA): 1.5 M ammonium sulphate in 50 mM phosphate buffer pH 7);
  • Mobile phase B 50 mM phosphate buffer pH 7.0 / isopropyl alcohol (75:25 V/V); the following gradient elution procedure was followed, in which the composition of mobile phase B was 30%-100% in 0-30 min. Mobile phase B was kept at 100% at 30-44.99 min and brought to 30% at 45 min. The post-run time was 10 min.
  • HPLC instrument Agilent 1260 Bio-Inert High Performance Liquid Chromatography system.
  • Mobile phase 1x PBS / isopropyl alcohol (90:10 V/V), isocratic flow.
  • Detection conditions Flow rate of mobile phase was set at 0.86 mL/min, detection wavelength was set at 280 nm, and column temperature was set at 35°C.
  • ADCs made from antibody AT04-10A (a) Preparation of ADC-1
  • AT04-10A (0.45 mg, 6.39 mg/mL, 1 .0 eq.) was partially reduced by adding TCEP (2 mg/mL, 3.0 eq.) aqueous solution. The reduction was conducted at 37°C for 2.0 hrs, and then conjugated with maleimidocaproyl (mc)-vc-PAB-MI ⁇ /IAE (10 mg/mL, 10.0 eq.) in DMSO at 25°C for 1.0 hr. The mixture was purified as described to afford ADC-1 (CADC (mg/mL): 2.52, V (mL): 0.10, yield: 56.0%).
  • TCEP 2 mg/mL, 3.0 eq.
  • ADC-1 has the following structure (see also FIG. 13, bottom; Table 8, LP14):
  • AT04-10A (0.45 mg, 6.39 mg/mL, 1 .0 eq.) was partially reduced by adding TCEP (2 mg/mL, 9.0 eq.) aqueous solution. The reduction was conducted at 37°C for 2.0 hrs, and then conjugated with mc-vc-PAB-MMAE (10 mg/mL, 12.0 eq.) in DMSO at 25°C for 1.0 hr. The mixture was purified as described to afford ADC-2 (CADC (mg/mL): 2.18, V (mL): 0.10, yield: 48.4%).
  • TCEP 2 mg/mL, 9.0 eq.
  • ADC-2 has the following structure (see also FIG. 13, bottom; Table 8, LP14):
  • AT04-10A (0.45 mg, 6.39 mg/mL, 1 .0 eq.) was partially reduced by adding TCEP (2 mg/mL, 3.0 eq.) aqueous solution. The reduction was conducted at 37°C for 2.0 hrs. An equal volume of 0.5 M Tris pH 8.8 buffer was added. Conjugation with allenamidocaproyl (ac)-vc-MMAE (10 mg/mL, 10.0 eq.) in DMSO was performed at 37°C for 16.0 hrs. The mixture was purified as described to afford ADC-3 (CADC (mg/mL): 2.87, V (mL): 0.10, yield: 63.7%).
  • TCEP 2 mg/mL, 3.0 eq.
  • ADC-3 has the following structure (see also FIG. 13, top; Table 8, LP26; Table 3, L11):
  • AT04-10A (0.35 mg, 6.39 mg/mL, 1 .0 eq.) was partially reduced by adding TCEP (2 mg/mL, 9.0 eq.) aqueous solution. The reduction was conducted at 37°C for 2.0 hrs. An equal volume of 0.5 M Tris pH 8.8 buffer was added. Conjugation with ac-vc-MMAE in DMSO was performed at 37°C for 16.0 hrs. The mixture was purified as described to afford ADC-4 (CADO (mg/mL): 2.04, V (mL): 0.10, yield: 58.2%).
  • CADO CADO (mg/mL): 2.04, V (mL): 0.10, yield: 58.2%).
  • ADC-4 has the following structure (see also FIG. 13, top; Table 8, LP26; Table 3, L11):
  • AT04-13A (0.45 mg, 6.49 mg/mL, 1 .0 eq.) was partially reduced by adding TCEP (2 mg/mL, 3.0 eq.) aqueous solution. The reduction was conducted at 37°C for 2.0 hrs, and then conjugated with mc-vc-PAB-MMAE (10 mg/mL, 10.0 eq.) in DMSO at 25°C for 1.0 hr. The mixture was purified as described to afford ADC-5 (CADC (mg/ml): 2.38, V (ml): 0.10, Yield: 52.9%).
  • TCEP 2 mg/mL, 3.0 eq.
  • ADC-5 has the following structure (see also FIG. 13, bottom; Table 8, LP14):
  • AT04-13A (0.355 mg, 4.15 mg/mL, 1 .0 eq.) was partially reduced by adding TCEP (2 mg/mL, 9.0 eq.) aqueous solution. The reduction was conducted at 37°C for 2.0 hrs, and then conjugated with mc-vc-PAB-MMAE (10 mg/mL, 12.0 eq.) in DMSO at 25°C for 1 .0 hr. The mixture was purified as described to afford ADC-6 (CADO (mg/mL): 1 .66, V (mL): 0.10, yield: 46.7%).
  • CADO CADO
  • ADC-6 has the following structure (see also FIG. 13, bottom; Table 8, LP14):
  • AT04-13A (0.4 mg, 4.15 mg/mL, 1.0 eq.) was partially reduced by adding TCEP (2 mg/mL, 3.0 eq.) aqueous solution. The reduction was conducted at 37°C for 2.0 hrs. An equal volume of 0.5 M Tris pH 8.8 buffer was added. Conjugation with ac-vc-MMAE (10 mg/mL, 10.0 eq.) in DMSO was performed at 37°C for 16.0 hrs. The mixture was purified as described to afford ADC-7 (CADC (mg/mL): 2.14, V (mL): 0.10, yield: 53.6%).
  • TCEP 2 mg/mL, 3.0 eq.
  • ADC-7 has the following structure (see also FIG. 13, top; Table 8, LP26; Table 3, L11):
  • AT04-13A (0.4 mg, 4.15 mg/mL, 1.0 eq.) was partially reduced by adding TCEP (2 mg/mL, 9.0 eq.) aqueous solution. The reduction was conducted at 37°C for 2.0 hrs. An equal volume of 0.5 M Tris pH 8.8 buffer was added. Conjugation with ac-vc-MMAE (10 mg/mL, 12.0 eq.) in DMSO was performed at 37°C for 16.0 hrs. The mixture was purified as described to afford ADC-8 (CADC (mg/mL): 1.91 , V (mL): 0.10, yield: 47.8%).
  • TCEP 2 mg/mL, 9.0 eq.
  • ADC-8 has the following structure (see also FIG. 13, top; Table 8, LP26; Table 3, L11):
  • AT04-13A (0.63 mg, 4.36 mg/mL, 1 .0 eq.) was partially reduced by adding TCEP (1 mg/mL, 4.5 eq.) aqueous solution. The reduction was conducted at 20°C for 1.5 hrs, and then conjugated with LP4 (see Table 8) (10 mg/mL, 5.0 eq.) in DMSO at 20°C for 0.5 hr. The mixture was purified as described to afford ADC-9 (CADC (mg/mL): 1 .63, V (mL): 0.24, yield: 61.8%).
  • TCEP 1 mg/mL, 4.5 eq.
  • ADC-9 has the following structure (see also Table 8, LP4):
  • ADC-10 has the following structure (see also Table 8, LP1 ):
  • AT04-23A (0.45 mg, 7.72 mg/mL, 1 .0 eq.) was partially reduced by adding TCEP (2 mg/mL, 9.0 eq.) aqueous solution. The reduction was conducted at 37 °C for 2.0 hrs, and then conjugated with mc-vc-PAB-MMAE (10 mg/mL, 12.0 eq.) in DMSO at 25 °C for 1.0 hr. The mixture was purified as described to afford ADC-11 (CADC (mg/mL): 1.78, V (mL): 0.10, yield: 39.5%).
  • TCEP 2 mg/mL, 9.0 eq.
  • ADC-11 has the following structure (see also FIG. 13, bottom; Table 8, LP14): [0223] According to general conjugation procedure (allenamide conjugation) for preparing ADCs, AT04-23A (0.45 mg, 7.72 mg/mL, 1 .0 eq.) was partially reduced by adding TCEP (2 mg/mL, 9.0 eq.) aqueous solution. The reduction was conducted at 37°C for 2.0 hrs. An equal volume of 0.5M Tris pH 8.8 buffer was added. Conjugation with ac-vc-MMAE (10 mg/mL, 12.0 eq.) in DMSO was performed at 37 °C for 16.0 hrs. The mixture was purified as described to afford ADC-12 (CA DC (mg/mL): 2.20, V (mL): 0.10, yield: 48.8%).
  • ADC-12 has the following structure (see also FIG. 13, top; Table 8, LP26; Table 3, L11):
  • ADCs made from antibodies AT04-25A and AT04-26 A
  • Antibodies engineered with reactive cysteine residues for drug conjugation were generated and subjected to conjugation with various linkers to make ADC-13 to ADC-16 using the following general procedure.
  • monoclonal antibodies were buffer exchanged into 2 mM EDTA and 100 mM Tris-HCI (pH 8.0). 1 mg of antibody was added to a 1 .5 ml_ centrifuge tube and mixed with 200 equivalent DTT. The antibody was incubated at 22°C overnight for reduction. The reduced antibody was buffer exchanged into 2 mM EDTA, 150 mM NaCI, 50 mM Tris-HCI (pH 7.5), and 20 equivalent of DHAA was added. Reaction was incubated at 22°C for 2 hrs after pipetting and mixing.
  • DHAA was removed with a Zeba spin desalting column into buffer containing 150 mM NaCI, 50 mM Tris-HCI (pH 7.5). Six equivalents of linker-payload were added. Incubation was performed at 20°C for 4 hrs with agitation. At the end of the reaction, the free small molecules were removed, and ADC storage buffer was replaced with 50 mM His-HAC (pH 5.5). Conjugated ADCs were stored at -80°C for later use.
  • ADC-13 prepared from AT04-25A has the following characteristic values obtained according to the common characterization protocols for ADCs: HIC-DAR: 3.9, SEC purity: 97.0%.
  • ADC-13 has the following structure (see also FIG. 13, bottom; Table 8, LP14):
  • ADC-14 prepared from AT04-26A has the following characteristic values obtained according to the common characterization protocols for ADCs: HIC-DAR: 3.9, SEC purity: 94.0%.
  • ADC-14 has the following structure (see also FIG. 13, bottom; Table 8, LP14): [0232] ADC-15 prepared from AT04-25A has the following characteristic values obtained according to the common characterization protocols for ADCs: HIC-DAR: 4.0, SEC purity: 94.0%.
  • ADC-15 has the following structure (see also Table 8, LP8):
  • ADC-16 prepared from AT04-26A has the following characteristic values obtained according to the common characterization protocols for ADCs: HIC-DAR: 4.0, SEC purity: 92.0%
  • ADC-16 has the following structure (see Table 8, LP8):
  • N87 cells were seeded, allowed to adhere overnight, and then treated for 120 hours with at least 9 different concentrations of each of ADC-1 to ADC-16, prepared through serial dilution.
  • Cell viability of the NCI-N87 cells was determined by CellTitre-Glo Cell Viability Assay per manufacturer instructions. Dose response curves were generated for ADC-1 to ADC-4 (FIG. 14A), ADC- 5 to ADC-8 (FIG. 14B), ADC-9 to ADC-10 (FIG. 14C), ADC-11 to ADC-12 (FIG.
  • ADC-14 was then tested in mouse xenograft models of pancreatic (HPAC) and gastric (NCI-N87) tumor cells.
  • Tumor cells were injected into BALB/c nude mice. After tumors became palpable, mice were treated intravenously with 3mg/kg of ADC-14 on day 0 and day 7.
  • Non-binding lgG1 anti-DT conjugated to maleimidocaproyl (mc)-vc-MMAE (DAR 3.8) was used as an isotype ADC control, and PBS was used as vehicle control.
  • Tumor size was determined from measurements taken with calipers at least twice a week.
  • ADC-14 inhibited tumor growth and induced tumor regression at 3 mg/kg (QWx2) in pancreatic (HPAC) and gastric (NCI-N87) models expressing ALPP/ALPPL2, while not significantly affecting body weight.
  • ADC-14 was tested in patient-derived xenograft (PDX) mouse model of cancer.
  • Tumor cells isolated from tissue biopsies of patient with gastric (FIG. 16) or gastroesophageal junction (GEJ) cancer (FIG. 17) were implanted into BALC/c nude mice. After tumors became palpable, mice were treated with either 3mg/kg (FIG. 16) or 6mg/kg (FIG. 17) of ADC-14 on day 0, day 7, and day 14 (QWx3).
  • TGI tumor growth inhibition ratio

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Abstract

La présente technologie concerne de nouveaux anticorps spécifiques de la phosphatase alcaline placentaire (PALP) et/ou de la phosphatase alcaline germinale 2 (PALPL2), des fragments de liaison à l'antigène de ceux-ci, et des conjugués anticorps-médicaments (ADC) qui peuvent être utilisés en tant qu'agents thérapeutiques potentiels pour le traitement de maladies notamment le cancer.
PCT/SG2024/050541 2023-08-25 2024-08-23 Anticorps anti-palp/palpl2, conjugués anticorps-médicaments et leurs utilisations WO2025048716A1 (fr)

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US5786464A (en) 1994-09-19 1998-07-28 The General Hospital Corporation Overexpression of mammalian and viral proteins
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WO2019240934A1 (fr) * 2018-06-12 2019-12-19 Promab Biotechnologies, Inc. Cellules effectrices plap-car
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EP0429242B1 (fr) * 1989-11-17 1995-06-14 Unilever Plc Agents attachant spécifiques
US5786464A (en) 1994-09-19 1998-07-28 The General Hospital Corporation Overexpression of mammalian and viral proteins
US5786464C1 (en) 1994-09-19 2012-04-24 Gen Hospital Corp Overexpression of mammalian and viral proteins
US6114148A (en) 1996-09-20 2000-09-05 The General Hospital Corporation High level expression of proteins
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WO2019240934A1 (fr) * 2018-06-12 2019-12-19 Promab Biotechnologies, Inc. Cellules effectrices plap-car
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