WO2023278463A1 - Anticorps anti-enpp1 et leurs utilisations - Google Patents

Anticorps anti-enpp1 et leurs utilisations Download PDF

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WO2023278463A1
WO2023278463A1 PCT/US2022/035335 US2022035335W WO2023278463A1 WO 2023278463 A1 WO2023278463 A1 WO 2023278463A1 US 2022035335 W US2022035335 W US 2022035335W WO 2023278463 A1 WO2023278463 A1 WO 2023278463A1
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antibody
enppl
amino acid
seq
enpp1
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Sarah Bettigole
Rumin Zhang
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Volastra Therapeutics, Inc.
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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2121/00—Preparations for use in therapy
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/04—Phosphoric diester hydrolases (3.1.4)
    • C12Y301/04001—Phosphodiesterase I (3.1.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
    • C12Y306/01—Hydrolases acting on acid anhydrides (3.6) in phosphorus-containing anhydrides (3.6.1)
    • C12Y306/01009—Nucleotide diphosphatase (3.6.1.9), i.e. nucleotide-pyrophosphatase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)

Definitions

  • the present disclosure relates to anti-ENPPl antibodies and uses, including therapeutic uses thereof.
  • Ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPP1) is a transmembrane enzyme that is expressed in many tissues and functions in extracellular signaling via purines (e.g., adenosine and ATP). Such purinergic signaling plays an important role in the regulation of cardiovascular, neurological, immunological, musculoskeletal, hormonal, and hematological functions in mammals.
  • ENPP1 has been implicated in diseases including cancer.
  • ENPP1 has been found to play an important role in immunological responses to various stimuli through the cyclic GMP-AMP synthase (cGAS)- stimulator of interferon genes (STING) pathway.
  • cGAS cyclic GMP-AMP synthase
  • STING interferon genes
  • ENPP1 has been shown to promote metastasis by selectively degrading extracellular cGAMP, an immune-stimulatory metabolite whose breakdown products include the immune suppressor adenosine (Li et ah, Cancer Discov, 11(5): 1212-1227, 2011).
  • ENPP1 expression correlates with reduced immune cell infiltration, increased metastasis, and resistance to anti-PD-l/PD-Ll treatment.
  • ENPP1 has been suggested as a therapy for cancer (Onyedibe et al., Molecules, 24(22):4192, 2019).
  • ENPP1 has also been implicated in bacterial or viral infections, insulin resistance and type II diabetes, chondrocalcinosis, calcium pyrophosphate deposition disorder (CPPD), or hypophosphatasia.
  • CPPD calcium pyrophosphate deposition disorder
  • compositions that include antibodies, e.g., monoclonal antibodies, antibody fragments, etc., that specifically bind to a human ENPP1 protein, and use of such compositions in preventing, reducing risk, or treating diseases or conditions associated with aberrant ENPP1 activity in an individual in need thereof.
  • antibodies e.g., monoclonal antibodies, antibody fragments, etc.
  • FIG. 1 shows a dose response binding analysis by flow cytometry of recombinant anti-ENPPl antibodies against CHO-K1 cells expressing human ENPP1.
  • a commercially available sheep anti-hENPPl polyclonal antibody (Biovendor, Catalogue No. RD184124100) was used as a positive control for binding.
  • FIG. 2 shows a dose response inhibition curve of recombinant anti-ENPPl antibodies in a 2’3’-cGAMP hydrolysis assay with human ENPP1 protein.
  • FIG. 3 shows a dose response inhibition curve of recombinant anti-ENPPl antibodies in a 2’3’-cGAMP hydrolysis assay with the MDA-MB-231 cell line.
  • the present disclosure is based, at least in part, on the discovery of anti-ENPPl antibodies inhibit 2’3’-cGAMP hydrolysis by human ENPP1, and do not inhibit pNP-AMP hydrolysis by human ENPP1. Without wishing to be bound by theory, it is believed that such antibodies are useful for inducing STING-dependent anti-tumor immune responses while avoiding deleterious soft tissue calcification side effects, which may result from inhibiting ENPP1 -mediated extracellular ATP hydrolysis. Further, described herein are anti-ENPPl antibodies that bind human ENPP1 and cynomolgus monkey ENPP1.
  • anti-ENPPl antibodies e.g., monoclonal antibodies
  • methods of making and using such antibodies pharmaceutical compositions containing such antibodies; nucleic acids encoding such antibodies; and host cells containing nucleic acids encoding such antibodies.
  • immunoglobulin is used interchangeably with “ antibody ” herein.
  • antibody herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
  • the basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. The pairing of a VH and VL together forms a single antigen-binding site.
  • L light
  • H heavy
  • immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated alpha (“a”), delta (“d”), epsilon (“s”), gamma (“g”) and mu (“m”), respectively.
  • the g and a classes are further divided into subclasses (isotypes) on the basis of relatively minor differences in the CH sequence and function, e.g., humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.
  • subclasses immunoglobulins
  • the subunit structures and three dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al. , Cellular and Molecular Immunology, 4 th ed. (W.B. Saunders Co., 2000).
  • An ⁇ isolated’" antibody such as an isolated anti-ENPPl antibody of the present disclosure, is one that has been identified, separated and/or recovered from a component of its production environment (e.g., naturally or recombinantly).
  • the isolated polypeptide is free of association with all other contaminant components from its production environment.
  • Contaminant components from its production environment such as those resulting from recombinant transfected cells, are materials that would typically interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-pro teinaceous solutes.
  • the polypeptide will be purified: (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated antibody includes the antibody in situ within recombinant T-cells since at least one component of the antibody’s natural environment will not be present. Ordinarily, however, an isolated polypeptide or antibody will be prepared by at least one purification step.
  • variable region or “ variable domain” of an antibody, such as an anti-
  • ENPP1 antibody of the present disclosure refers to the amino-terminal domains of the heavy or light chain of the antibody.
  • the variable domains of the heavy chain and light chain may be referred to as “V H ” and “V L ”, respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites.
  • variable refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies, such as anti-ENPPl antibodies of the present disclosure.
  • the V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen.
  • CDRs complementarity-determining regions
  • FR framework regions
  • variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies ( see Rabat et al., Sequences of Immunological Interest , Fifth Edition, National Institute of Health, Bethesda, MD (1991)).
  • the constant domains are not involved directly in the binding of antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody- dependent-cellular toxicity.
  • monoclonal antibody refers to an antibody, such as a monoclonal anti-ENPPl antibody of the present disclosure, obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translation modifications (e.g ., isomerizations, amidations, etc.) that may be present in minor amounts.
  • Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3):253-260 (1995), Harlow et al., Antibodies:
  • full-length antibody “full-length antibody ,” “ intact antibody ” or “ whole antibody ” are used interchangeably to refer to an antibody, such as an anti-ENPPl antibody of the present disclosure, in its substantially intact form, as opposed to an antibody fragment.
  • whole antibodies include those with heavy and light chains including an Fc region.
  • the constant domains may be native sequence constant domains ( e.g ., human native sequence constant domains) or amino acid sequence variants thereof.
  • the intact antibody may have one or more effector functions.
  • an “antibody fragment” comprises a portion of an intact antibody, preferably the antigen binding and/or the variable region of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab') 2 and Fv fragments; diabodies; linear antibodies (see U.S. Patent 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); single-chain antibody molecules and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily.
  • the Fab fragment consists of an entire L chain along with the variable region domain of the H chain (V H ), and the first constant domain of one heavy chain (CHI).
  • V H variable region domain of the H chain
  • CHI first constant domain of one heavy chain
  • Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen -binding site.
  • Pepsin treatment of an antibody yields a single large F(ab') 2 fragment which roughly corresponds to two disulfide linked Fab fragments having different antigen-binding activity and is still capable of cross-linking antigen.
  • Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • the Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides.
  • the effector functions of antibodies are determined by sequences in the Fc region, the region which is also recognized by Fc receptors (FcR) found on certain types of cells.
  • Fv is the minimum antibody fragment which contains a complete antigen- recognition and -binding site. This fragment consists of a dimer of one heavy- and one light- chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • Single-chain Fv also abbreviated as “sFv ” or “scFv ” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain.
  • the sFv polypeptide further comprises a polypeptide linker between the V H and V L domains, which enables the sFv to form the desired structure for antigen binding.
  • “Functional fragments” of antibodies comprise a portion of an intact antibody, generally including the antigen binding or variable region of the intact antibody or the F region of an antibody which retains or has modified FcR binding capability.
  • antibody fragments include linear antibody, single-chain antibody molecules and multispecific antibodies formed from antibody fragments.
  • diabodies refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10) residues) between the V H and V L domains such that inter-chain but not intra-chain pairing of the V domains is achieved, thereby resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites.
  • Bispecific diabodies are heterodimers of two “crossover” sFv fragments in which the V H and VL domains of the two antibodies are present on different polypeptide chains.
  • Diabodies are described in greater detail in, for example, EP 404,097; WO 93/11161; Hollinger et ah, Proc. Nat’lAcad. Sci. USA 90:6444-48 (1993).
  • a “chimeric antibody ” refers to an antibody (immunoglobulin), such as a chimeric anti-ENPPl antibody of the present disclosure, in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is(are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Nat’lAcad. Sci.
  • Chimeric antibodies of interest herein include PRIMATIZED ® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with an antigen of interest.
  • PRIMATIZED ® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with an antigen of interest.
  • humanized antibody is used a subset of “chimeric antibodies.”
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and/or capacity.
  • donor antibody such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and/or capacity.
  • FR residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, and the like.
  • the number of these amino acid substitutions in the FR is typically no more than 6 in the H chain, and in the L chain, no more than 3.
  • the humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • a “human antibody ” is one that possesses an amino-acid sequence corresponding to that of an antibody, such as an anti-ENPPl antibody of the present disclosure, produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991).
  • Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Patent Nos.
  • human antibodies can also be prepared by employing yeast libraries and methods as disclosed in, for example,
  • complementarity-determining region refers to the regions of an antibody- variable domain, such as that of an anti-ENPPl antibody of the present disclosure, that bind to an epitope, such as ENPP1.
  • antibodies comprise six CDRs; three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3).
  • H3 and L3 display the most diversity of the six CDRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies.
  • the CDRs may be Rabat CDRs, which are based on sequence variability and are the most commonly used (Rabat et ah, supra).
  • the CDRs may be Chothia CDRs. Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
  • the CDRs may be AbM CDRs. The AbM CDRs represent a compromise between the Rabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody-modeling software.
  • the CDRs may be “contact” CDRs. The “contact” CDRs are based on an analysis of the available complex crystal structures. The residues from each of these CDRs are noted below.
  • CDRs may comprise “extended CDRs” as follows: 24-36 or 24-34 (LI), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 (HI), 50-65 or 49-65 (a preferred embodiment) (H2), and 93-102, 94-102, or 95-102 (H3) in the VH.
  • the variable-domain residues are numbered according to Rabat et al., supra, for each of these extended-CDR definitions.
  • Framework ” or ”FR residues are those variable-domain residues other than the
  • variable-domain residue-numbering as in Rabat or “ amino-acid- position numbering as in Rabat,” and variations thereof, refers to the numbering system used for heavy-chain variable domains or light-chain variable domains of the compilation of antibodies in Rabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain.
  • a heavy-chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g ., residues 82a, 82b, and 82c, etc.
  • the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.
  • the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • EU or, Kabat numbering system or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al, supra).
  • EU index as in Kabat refers to the residue numbering of the human IgGl EU antibody.
  • References to residue numbers in the variable domain of antibodies means residue numbering by the Kabat numbering system.
  • References to residue numbers in the constant domain of antibodies means residue numbering by the EU or, Kabat numbering system (e.g., see United States Patent Publication No. 2010- 280227).
  • acceptor human framework is a framework comprising the amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework or a human consensus framework.
  • An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.
  • VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.
  • a “human consensus framework” is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences.
  • the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences.
  • the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples include for the VL, the subgroup may be subgroup kappa I, kappa II, kappa III or kappa IV as in Kabat et al, supra. Additionally, for the VH, the subgroup may be subgroup I, subgroup II, or subgroup III as in Kabat et al, supra.
  • amino-acid modification at a specified position, e.g., of an anti-ENPPl antibody of the present disclosure, refers to the substitution or deletion of the specified residue, or the insertion of at least one amino acid residue adjacent the specified residue. Insertion “adjacent” to a specified residue means insertion within one to two residues thereof. The insertion may be N-terminal or C-terminal to the specified residue.
  • the preferred amino acid modification herein is a substitution.
  • the term “ specifically recognizes” or “ specifically binds” refers to measurable and reproducible interactions such as attraction or binding between a target and an antibody, such as between an anti-ENPPl antibody and ENPP1 that is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules.
  • an antibody such as an anti-ENPPl antibody of the present disclosure, that specifically or preferentially binds to a target or an epitope is an antibody that binds this target or epitope with greater affinity, avidity, more readily, and/or with greater duration than it binds to other targets or other epitopes of the target.
  • an antibody (or a moiety) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target.
  • specific binding or preferential binding does not necessarily require (although it can include) exclusive binding.
  • An antibody that specifically binds to a target may have an association constant of at least about 10 3 M 1 or 10 4 M _1 , sometimes about 10 5 M 1 or 10 6 M _1 , in other instances about 10 6 M 1 or 10 7 M _1 , about 10 8 M 1 to 10 9 M _1 , or about 10 10 M 1 to 10 11 M 1 or higher.
  • immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • Antibody ffector functions refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype.
  • Fc region herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native- sequence Fc regions and variant Fc regions.
  • the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof.
  • the C-terminal lysine (residue 447 according to the EU or, Kabat numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody.
  • composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.
  • Suitable native-sequence Fc regions for use in the antibodies of the present disclosure include human IgGl, IgG2, IgG3 and IgG4.
  • a “ native sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature.
  • Native sequence human Fc regions include a native sequence human IgGl Fc region (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region as well as naturally occurring variants thereof.
  • a “ variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s).
  • the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g. from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide.
  • the variant Fc region herein will preferably possess at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.
  • Fc receptor or “ FcR ” describes a receptor that binds to the Fc region of an antibody.
  • the preferred FcR is a native sequence human FcR.
  • a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors, FcyRII receptors include FcyRIIA (an “activating receptor”) and FcyRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
  • Activating receptor FcyRIIA contains an immunoreceptor tyrosine- based activation motif (“IT AM”) in its cytoplasmic domain.
  • Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine -based inhibition motif (“ITIM”) in its cytoplasmic domain.
  • ITIM immunoreceptor tyrosine -based inhibition motif
  • Binding to FcRn in vivo and serum half-life of human FcRn high-affinity binding polypeptides can be assayed, e.g., in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which the polypeptides having a variant Fc region are administered.
  • WO 2004/42072 (Presta) describes antibody variants with improved or diminished binding to FcRs. See also, e.g., Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).
  • percent (%) amino acid sequence identity and “ homology ” with respect to a peptide, polypeptide or antibody sequence refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms known in the art needed to achieve maximal alignment over the full-length of the sequences being compared.
  • An “isolated ’ nucleic acid molecule encoding an antibody is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced. Preferably, the isolated nucleic acid is free of association with all components associated with the production environment.
  • the isolated nucleic acid molecules encoding the polypeptides and antibodies herein is in a form other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from nucleic acid encoding the polypeptides and antibodies herein existing naturally in cells.
  • vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA into which additional DNA segments may be ligated.
  • phage vector refers to a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • viral vector is capable of autonomous replication in a host cell into which they are introduced (e.g ., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as “recombinant expression vectors,” or simply, “expression vectors.”
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector.
  • Polynucleotide refers to polymers of nucleotides of any length, and include DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may comprise modification(s) made after synthesis, such as conjugation to a label.
  • modifications include, for example, “caps,” substitution of one or more of the naturally occurring nucleotides with an analog, intemucleotide modifications such as, for example, those with uncharged linkages (e.g ., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metal
  • any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid or semi-solid supports.
  • the 5’ and 3’ terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms.
  • Other hydroxyls may also be derivatized to standard protecting groups.
  • Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2’-0-methyl-, 2’-0-allyl-, 2’-fluoro- or 2’-azido-ribose, carbocyclic sugar analogs, a-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs, and basic nucleoside analogs such as methyl riboside.
  • One or more phosphodiester linkages may be replaced by alternative linking groups.
  • linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(0)S (“thioate”), P(S)S (“dithioate”), (0)NR2 (“amidate”), P(0)R, P(0)0R’, CO, or CH2 (“formacetal”), in which each R or R’ is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (-0-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.
  • a “host cell ” includes an individual cell or cell culture that can be or has been a recipient for vector(s) for incorporation of polynucleotide inserts.
  • Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation.
  • a host cell includes cells transfected in vivo with a polynucleotide(s) of this invention.
  • Carriers as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution.
  • physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • proteins such as serum albumin,
  • preventing includes providing prophylaxis with respect to occurrence or recurrence of a particular disease, disorder, or condition in an individual.
  • An individual may be predisposed to, susceptible to a particular disease, disorder, or condition, or at risk of developing such a disease, disorder, or condition, but has not yet been diagnosed with the disease, disorder, or condition.
  • treatment refers to clinical intervention designed to alter the natural course of the individual being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of progression, ameliorating or palliating the pathological state, and remission or improved prognosis of a particular disease, disorder, or condition.
  • An individual is successfully “treated”, for example, if one or more symptoms associated with a particular disease, disorder, or condition are mitigated or eliminated.
  • an “effective amount ” refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • An effective amount can be provided in one or more administrations.
  • An effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the treatment to elicit a desired response in the individual.
  • An effective amount is also one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects.
  • beneficial or desired results include results such as eliminating or reducing the risk, lessening the severity, or delaying the onset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease.
  • beneficial or desired results include clinical results such as decreasing one or more symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication such as via targeting, delaying the progression of the disease, and/or prolonging survival.
  • An effective amount of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly.
  • an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition.
  • an “effective amount” may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.
  • An “individual ” for purposes of treatment, prevention, or reduction of risk refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport, or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, and the like. In some embodiments, the individual is human.
  • administration “in conjunction” with another compound or composition includes simultaneous administration and/or administration at different times.
  • Administration in conjunction also encompasses administration as a co-formulation or administration as separate compositions, including at different dosing frequencies or intervals, and using the same route of administration or different routes of administration.
  • the present disclosure provides antibodies that interact with or otherwise bind to a region, such as an epitope, within an ENPP1 protein.
  • ENPP1 also known as “Ectonucleotide pyrophosphatase/phosphodiesterase family member 1”
  • ENPP1 has broad specificity and cleaves a variety of substrates, including phosphodiester bonds of nucleotides and nucleotide sugars, and pyrophosphate bonds of nucleotides and nucleotide sugars.
  • ENPP1 is involved in purinergic signaling (i.e., extracellular signaling mediated by purine nucleotides and nucleosides such as adenosine and ATP). In humans, ENPP1 is encoded by the ENPP1 gene.
  • ENPP1 is variously referred to as membrane component chromosome 6 surface marker 1, M6S1, phosphodiesterase I/nuclcotidc pyrophosphatase 1, NPP1, NPPS, plasma-cell membrane glycoprotein 1, PC-1, PCA1, ARHR2, COLED, PDNP1, Ly-41 antigen, alkaline phosphodiesterase 1, or CD203a.
  • an antibody that binds human ENPP1 (NCBI Reference Sequence: NP_006199.2).
  • the amino acid sequence of human ENPP1 is set forth below as SEQ ID NO:26:
  • an antibody that binds cynomolgus ENPP1 (Uniprot Accession No. A0A2K5WPZ1).
  • the amino acid sequence of cynomolgus ENPP1 is set forth below as SEQ ID NO:27:
  • ENPP1 is a homodimer made up of two identical disulfide-bonded subunits. Each subunit comprises, from N- to C-terminus, a cytosolic domain, a transmembrane domain, and an extracellular domain (ECD).
  • the extracellular domain is made up of two N-terminal somatomedin B (SMB)-like domains (SMB1 and SMB2), two linker regions (LI and L2), a catalytic domain, and a nuclease-like domain (Kato, K, et ah, Proc Natl Acad Sci USA. 2012 Oct 16; 109(42): 16876-81).
  • SMB N-terminal somatomedin B
  • L2 linker regions
  • a catalytic domain a catalytic domain
  • nuclease-like domain Kato, K, et ah, Proc Natl Acad Sci USA. 2012 Oct 16; 109(42): 16876-81).
  • ENPP1 has been implicated in diseases including cancer. Specifically, the hydrolysis of cGAMP by ENPP1 leads to the production of AMP, which eventually contributes to a more profound immunosuppression via the subsequent dephosphorylation of AMP to adenosine by CD73 (Onyedibe, K.I. et al, Molecules. 2019 Nov; 24(22): 4192). The role of ENPP1 in cancer is exemplified by the observations of enhanced tumor metastasis to the bone from breast cancer, for example, by over-expression of ENPP1.
  • ENPP1 and CD73-mediated production of adenosine are further demonstrated by several reports of resistance to carcinogenesis or metastasis in mice deficient in either CD73 or ENPP1.
  • elevated ENPP1 expression has been associated with ovarian cancer and breast cancer.
  • the location of the gene for ENPP1 is the 6q22-q23 position, which is a region that has been found to be amplified in many tumors, including breast and neural brain cancers.
  • anti-ENPPl antibodies are anti-ENPPl antibodies.
  • anti-ENPPl antibodies that inhibit 2’3’-cGAMP hydrolysis.
  • the anti- ENPPl antibody inhibits 2’3’-cGAMP hydrolysis by purified human ENPP1.
  • the anti-ENPPl antibody inhibits 2’3’-cGAMP hydrolysis by purified human ENPPlwith a half maximal inhibitory concentration of antibody (IC50) that is about 15, 20, 25, 30, 25, 40, 45, 50, 55, 60, or 100 nM, including any value or range between these values.
  • IC50 half maximal inhibitory concentration of antibody
  • the anti-ENPPl antibody inhibits 2’3’-cGAMP hydrolysis by purified human ENPP1 with an IC 50 that is less than about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
  • the anti-ENPPl antibody inhibits 2’3’-cGAMP hydrolysis by purified human ENPP1 with an IC50 that is about 20.90, 50.21, 34.83, 47.96, 44.85, 46.45, or 36.61 nM.
  • Exemplary methods for measuring the inhibition of 2’3’-cGAMP hydrolysis by purified human ENPP1 are provided in Example 1.
  • the purified human ENPP1 is tagged, e.g., His-tagged.
  • the purified human ENPP1 comprises the extracellular domain of human ENPP1.
  • the purified human ENPP1 consists of the extracellular domain of human ENPP 1.
  • the anti-ENPPl antibody inhibits 2’3’-cGAMP hydrolysis by human ENPP1 in a cell-based assay. In some embodiments, the anti-ENPPl antibody inhibits 2’3’-cGAMP hydrolysis in a cell-based assay with an IC50 that is about 10, 15, 20, 25, 30, 25, 40, 45, or 50 nM, including any value or range between these values.
  • the anti- ENPPl antibody inhibits 2’3’-cGAMP hydrolysis in a cell-based assay with an IC 50 that is less than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nM, including any value or range between these values. In some embodiments, the anti-ENPPl antibody inhibits 2’3’-cGAMP hydrolysis in a cell-based assay with an IC 50 that is about 14.56, 23.71, 33.68, 29.37 , 13.42, or 34.25 nM.
  • the cell based assay comprises measuring the ability of a cell line to undergo 2’3’-cGAMP hydrolysis in the presence or absence of an anti-ENPPl antibody.
  • the cell based assay uses cancer cells, e.g., breast cancer cells. In some embodiments, the cell based assay uses MDA-MB-231 cells.
  • anti-ENPPl antibodies that do not inhibit pNP-AMP hydrolysis.
  • the anti-ENPPl antibody does not inhibit pNP-AMP hydrolysis by human ENPP1 in a cell-based assay.
  • the anti- ENPPl antibody does not inhibit pNP-AMP hydrolysis in a cell-based assay with an IC 50 that is about 666 nM or greater. Exemplary methods for measuring the inhibition of pNP-AMP hydrolysis in a cell based assay are provided in Example 1.
  • the anti- ENPPl antibody does not inhibit pNP-AMP hydrolysis by cancer cells, e.g., breast cancer cells.
  • the anti-ENPPl antibody does not inhibit pNP-AMP hydrolysis by MDA- MB-231 cells.
  • anti-ENPPl antibodies that do not induce internalization of human ENPP1 expressed on a cell surface.
  • ENPP1 internalization is indirectly measured by pre-incub ating MDA-MB-231 cells with an anti-ENPPl antibody for different amounts of time (e.g., 1, 4 and 24 hrs) at 37oC and then hydrolysis of pNP-AMP is measured. Inhibition of pNP-AMP hydrolysis in the presence of the anti-ENPPl antibody is indicative of ENPP1 internalization. Said another way, lack of inhibition of pNP-AMP hydrolysis in the presence of the anti-ENPPl antibody is indicative of lack of ENPP1 internalization.
  • anti-ENPPl antibodies that bind human ENPP1 and cynomolgus monkey ENPP1.
  • the anti-ENPPl antibody binds human ENPP1 as set forth in SEQ ID NO:26.
  • the anti- ENPPl antibody binds cynomolgus ENPP1 as set forth in SEQ ID NO:27.
  • the anti-ENPPl antibody binds human ENPP1 as set forth in SEQ ID NO:26 and cynomolgus ENPP1 as set forth in SEQ ID NO:27.
  • the anti-ENPPl antibody binds to human ENPP1 with half-maximal binding at a concentration of antibody (EC50) that is less than about 0.01, 0.1, or 1 mM. In some embodiments, the anti-ENPPl antibody binds to human ENPP1 with an ECsothat is about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 pM, including any value or range between these values. In some embodiments, the anti-ENPPl antibody binds to human ENPP1 with an EC50 that is less than about 1, 10, 100, or 1000 nM.
  • the anti-ENPPl antibody binds to human ENPP1 with an EC50 that is about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 100, 250, 500, 750, or 1000 nM, including any value or range between these values.
  • the anti- ENPP1 antibody binds to human ENPP1 with an EC 50 of less than 100 nM.
  • Methods of calculating EC50S are known in the art and include, for example, enzyme-linked immunosorbent assays (ELIS As), fluorescence activated cell sorting (FACS) assays, and bio-layer interferometry (BLI).
  • the anti-ENPPl antibody binds to human ENPP1 with a dissociation constant (KD) that is less than about 1, 0.1, 0.01, 0.001, or 0.0001 mM, including any value or range between these values. In some embodiments, the anti-ENPPl antibody binds to human ENPP1 with a KD that is less than about 0.001, 0.01, 0.1, 1, or 10 nM, including any value or range between these values.
  • KD dissociation constant
  • the anti-ENPPl antibody binds to human ENPP1 with a KD that is about 0.001, 0.01, 0.1, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1,
  • the anti-ENPPl antibody binds to human ENPP1 with a K that is about 2.77, 2.49, 4.47, 2.61, 3.66, 1.93, or 0.001 nM.
  • the KD is measuring using bio-layer interferometry.
  • Exemplary methods of measuring the binding of anti-ENPPl antibodies to human ENPP1 are provided in Example 1.
  • binding of anti-ENPPl antibodies to human ENPP1 is measured using bio-layer interferometry, e.g., using the Octet system.
  • binding of anti- ENPPl antibodies to human ENPP1 is measured using an ELISA.
  • binding of anti-ENPPl antibodies to human ENPP1 is measured using a FACS assay.
  • binding of anti-ENPPl antibodies to human ENPP1 is measured using flow cytometry.
  • the anti-ENPPl antibody is an allosteric inhibitor of human ENPP1.
  • the anti-ENPPl antibody is a monoclonal anti-ENPPl antibody. In some embodiments, the anti-ENPPl antibody is a mouse anti-ENPPl antibody. In some embodiments, the anti-ENPPl antibody is a mouse monoclonal anti-ENPPl antibody.
  • the anti-ENPPl antibody comprises one, two, three, four, five, or six CDRs of antibody 1F12 as shown in Table 1-1. In some preferred embodiments, the anti-ENPPl antibody comprises all six CDRs of antibody 1F12 as shown in Table 1-1. In some embodiments, the anti-ENPPl antibody comprises the VH and/or the VL of antibody 1F12 as shown in Table 1-1. In some preferred embodiments, the anti-ENPPl antibody comprises the VH and the VL of antibody 1F12 as shown in Table 1-1.
  • the anti-ENPPl antibody comprises a heavy chain variable domain (VH) comprising three heavy chain complementarity determining regions (VH-CDRs), and a light chain variable domain (VL) comprising three light chain complementarity determining regions (VL-CDRs).
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the anti-ENPPl antibody comprises one, two, three, four, five, or six CDRs selected from the group consisting of a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:3, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:5, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:7; a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 15.
  • the anti-ENPPl antibody comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:3, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:5, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:7; a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 15.
  • the anti-ENPPl antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:l.
  • VH heavy chain variable domain
  • a VH sequence contains substitutions (e.g ., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO:l, but retains the ability to bind ENPP1 as the anti-ENPPl antibody comprising SEQ ID NO:l.
  • a total of 1 to 13 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:l.
  • the VH comprises one, two or three CDRs selected from the group consisting of: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:3, (b) a CDR- H2 comprising the amino acid sequence of SEQ ID NO:5, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:7.
  • an anti-ENPPl antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:9.
  • VL light chain variable domain
  • a VL sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO:9, but retains the ability to bind ENPP1 as the anti-ENPPl antibody comprising SEQ ID NO:9.
  • a total of 1 to 11 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:9.
  • the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs).
  • the VL comprises one, two or three CDRs selected from the group consisting of (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 11; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15.
  • the anti-ENPPl antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:l and a VL comprising the amino acid sequence of SEQ ID NO:9.
  • an anti-ENPPl antibody comprising a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO:3, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:5, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:7; and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 11, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15.
  • an anti-ENPPl antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3, respectively comprising the amino acid sequences of a CDR1, a CDR2, and a CDR3 within a VH having the sequence set forth in SEQ ID NO:l; and a VL CDR1, a VL CDR2, and a VL CDR3, respectively comprising the amino acid sequences of a CDR1, a CDR2, and a CDR3 within a VL having the sequence set forth in SEQ ID NO:9.
  • the heavy chain variable region comprises one, two, three or four framework regions (FRs) selected from VH FR1, VH FR2, VH FR3, and VH FR4, wherein the VH FR1 comprises the amino acid sequence of SEQ ID NO: 2, the VH FR2 comprises the amino acid sequence of SEQ ID NO: 4, the VH FR3 comprises the amino acid sequence of SEQ ID NO: 6, and the VH FR4 comprises the amino acid sequence of SEQ ID NO: 8.
  • FRs framework regions
  • the light chain variable region comprises one, two, three or four FRs selected from VL FR1, VL FR2, VL FR3, and VL FR4, wherein the VL FR1 comprises the amino acid sequence of SEQ ID NO: 10, the VL FR2 comprises the amino acid sequence of SEQ ID NO:
  • the VL FR3 comprises the amino acid sequence of SEQ ID NO: 14
  • the VL FR4 comprises the amino acid sequence of SEQ ID NO: 16.
  • humanized anti-ENPPl antibodies also provided herein are humanized anti-ENPPl antibodies.
  • human/mouse chimeric anti-ENPPl antibodies Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fab, Fab’-SH, Fv, scFv, F(ab’)2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementarity determining region
  • donor antibody non-human species
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323- 329 (1988) and Presta, Curr. Opin. Struct. Biol. 2: 593-596 (1992).
  • Fc immunoglobulin constant region
  • the humanized anti-ENPPl antibody may be an antibody fragment, such as an Fab.
  • the humanized anti-ENPPl antibody may be an intact antibody, such as an intact IgGl antibody.
  • the anti-ENPPl antibody comprises a heavy chain variable region comprising substitutions (e.g., conservative substitutions), insertions, or deletions in one, two, three or four framework regions (FRs) selected from VH FR1, VH FR2, VH FR3, and VH FR4, wherein the VH FR1 comprises the amino acid sequence of SEQ ID NO: 2, the VH FR2 comprises the amino acid sequence of SEQ ID NO: 4, the VH FR3 comprises the amino acid sequence of SEQ ID NO: 6, and the VH FR4 comprises the amino acid sequence of SEQ ID NO: 8.
  • FRs framework regions
  • the anti-ENPPl antibody comprises a light chain variable region comprising substitutions (e.g., conservative substitutions), insertions, or deletions in one, two, three or four FRs selected from VL FR1, VL FR2, VL FR3, and VL FR4, wherein the VL FR1 comprises the amino acid sequence of SEQ ID NO: 10, the VL FR2 comprises the amino acid sequence of SEQ ID NO: 12, the VL FR3 comprises the amino acid sequence of SEQ ID NO: 14, and the VL FR4 comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, a total of 1 to 11 amino acids have been substituted, inserted and/or deleted in the VL FR1, VL FR2, VL FR3, and/or VL FR4.
  • the anti-ENPPl antibody comprises a VH comprising an amino acid sequence according to Formula I: Q V QLV QS G AE VKKPG AS VKV S CKAS G YTFTD YWMHW VKQRPGQGLEWMG YINPTS D YPRYN QNFKDKX i TX 2 T ADKS T S T V YMELS S LTS EDT A V Y Y CASS NWD YW GQGTT VT V SS (SEQ ID NO:22), wherein Xi is V or A; and X2 is M or L.
  • the anti-ENPPl antibody comprises a VL comprising an amino acid sequence according to Formula II:
  • the anti-ENPPl antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 17.
  • VH heavy chain variable domain
  • a VH sequence contains substitutions (e.g ., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 17, but retains the ability to bind ENPP1 as the anti-ENPPl antibody comprising SEQ ID NO: 17.
  • a total of 1 to 13 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 17.
  • the VH comprises one, two or three CDRs selected from the group consisting of: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NOG, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NOG, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NOG.
  • the anti-ENPPl antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 19.
  • VH heavy chain variable domain
  • a VH sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 19, but retains the ability to bind ENPP1 as the anti-ENPPl antibody comprising SEQ ID NO: 19.
  • a total of 1 to 13 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 19.
  • the VH comprises one, two or three CDRs selected from the group consisting of: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:3, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:5, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:7.
  • an anti-ENPPl antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 18.
  • VL light chain variable domain
  • a VL sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 18, but retains the ability to bind ENPP1 as the anti-ENPPl antibody comprising SEQ ID NO: 18.
  • a total of 1 to 11 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 18.
  • the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs).
  • the VL comprises one, two or three CDRs selected from the group consisting of (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 11; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15.
  • an anti-ENPPl antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:20.
  • VL light chain variable domain
  • a VL sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO:20, but retains the ability to bind ENPP1 as the anti-ENPPl antibody comprising SEQ ID NO:20.
  • a total of 1 to 11 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:20.
  • the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs).
  • the VL comprises one, two or three CDRs selected from the group consisting of (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 11; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15.
  • an anti-ENPPl antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:21.
  • VL light chain variable domain
  • a VL sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO:21, but retains the ability to bind ENPP1 as the anti-ENPPl antibody comprising SEQ ID NO:21.
  • a total of 1 to 11 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:21.
  • the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs).
  • the VL comprises one, two or three CDRs selected from the group consisting of (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 11; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15.
  • the anti-ENPPl antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 18.
  • an anti-ENPPl antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3, respectively comprising the amino acid sequences of a CDR1, a CDR2, and a CDR3 within a VH having the sequence set forth in SEQ ID NO:17; and a VL CDR1, a VL CDR2, and a VL CDR3, respectively comprising the amino acid sequences of a CDR1, a CDR2, and a CDR3 within a VL having the sequence set forth in SEQ ID NO: 18.
  • the anti-ENPPl antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising the amino acid sequence of SEQ ID NO: 18.
  • an anti-ENPPl antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3, respectively comprising the amino acid sequences of a CDR1, a CDR2, and a CDR3 within a VH having the sequence set forth in SEQ ID NO: 19; and a VL CDR1, a VL CDR2, and a VL CDR3, respectively comprising the amino acid sequences of a CDR1, a CDR2, and a CDR3 within a VL having the sequence set forth in SEQ ID NO: 18.
  • the anti-ENPPl antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 20.
  • an anti-ENPPl antibody is provided, wherein the antibody comprises a VH CDR1, a VH CDR2, and a VH CDR3, respectively comprising the amino acid sequences of a CDR1, a CDR2, and a CDR3 within a VH having the sequence set forth in SEQ ID NO:17; and a VL CDR1, a VL CDR2, and a VL CDR3, respectively comprising the amino acid sequences of a CDR1, a CDR2, and a CDR3 within a VL having the sequence set forth in SEQ ID NO:20.
  • the anti-ENPPl antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising the amino acid sequence of SEQ ID NO: 20.
  • an anti-ENPPl antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3, respectively comprising the amino acid sequences of a CDR1, a CDR2, and a CDR3 within a VH having the sequence set forth in SEQ ID NO: 19; and a VL CDR1, a VL CDR2, and a VL CDR3, respectively comprising the amino acid sequences of a CDR1, a CDR2, and a CDR3 within a VL having the sequence set forth in SEQ ID NO:20.
  • the anti-ENPPl antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 21.
  • an anti-ENPPl antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3, respectively comprising the amino acid sequences of a CDR1, a CDR2, and a CDR3 within a VH having the sequence set forth in SEQ ID NO:17; and a VL CDR1, a VL CDR2, and a VL CDR3, respectively comprising the amino acid sequences of a CDR1, a CDR2, and a CDR3 within a VL having the sequence set forth in SEQ ID NO:21.
  • the anti-ENPPl antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising the amino acid sequence of SEQ ID NO: 21.
  • an anti-ENPPl antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3, respectively comprising the amino acid sequences of a CDR1, a CDR2, and a CDR3 within a VH having the sequence set forth in SEQ ID NO: 19; and a VL CDR1, a VL CDR2, and a VL CDR3, respectively comprising the amino acid sequences of a CDR1, a CDR2, and a CDR3 within a VL having the sequence set forth in SEQ ID NO:21.
  • the anti-ENPPl antibody comprises an Fc.
  • the Fc comprises one or more conservative amino acid substitutions (e.g., one, two, three, four, five or more substitutions).
  • the anti-ENPPl antibody is of the IgG class, the IgM class, or the IgA class. In some embodiments, the anti-ENPPl antibody the antibody is of the IgG class and has an IgGl, IgG2, IgG3, or IgG4 isotype. In some embodiments, the anti-ENPPl antibody is an IgGl isotype.
  • the anti-ENPPl antibody has an IgG2 isotype.
  • the anti-ENPPl antibody contains a human IgG2 constant region.
  • the human IgG2 constant region includes an Fc region.
  • the antibody binds an inhibitory Fc receptor.
  • the inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB (FcyllB).
  • the Fc region contains one or more modifications. For example, in some embodiments, the Fc region contains one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype).
  • the one or more amino acid substitutions are selected from V234A (Alegre et al., (1994) Transplantation 57:1537-1543. 31; Xu et al., (2000) Cell Immunol, 200:16-26), G237A (Cole et al. (1999) Transplantation, 68:563-571), H268Q, V309L, A330S, P331S (US 2007/0148167; Armour et al. (1999) Eur J Immunol 29: 2613-2624; Armour et al. (2000) The Haematology Journal l(Suppl.l):27; Armour et al.
  • the anti-ENPPl antibody has an IgG2 isotype with a heavy chain constant domain that contains a C127S amino acid substitution, where the amino acid position is according to the EU or, Rabat numbering convention (White et al, (2015) Cancer Cell 27, 138-148; Lightle et al., (2010) PROTEIN SCIENCE 19:753-762; and W02008079246).
  • the anti-ENPPl antibody has an IgG2 isotype with a Kappa light chain constant domain that contains a C214S amino acid substitution, where the amino acid position is according to the EU or, Kabat numbering convention (White et ah, (2015) Cancer Cell 27, 138-148; Lightle et ah, (2010) PROTEIN SCIENCE 19:753-762; and W02008079246).
  • the anti-ENPPl antibody has an IgGl isotype. In some embodiments, the anti-ENPPl antibody contains a mouse IgGl constant region. In some embodiments, the anti-ENPPl antibody contains a human IgGl constant region. In some embodiments, the human IgGl constant region includes an Fc region. In some embodiments, the antibody binds an inhibitory Fc receptor. In certain embodiments, the inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB (FcyllB). In some embodiments, the Fc region contains one or more modifications.
  • the Fc region contains one or more amino acid substitutions (e.g relative to a wild-type Fc region of the same isotype).
  • the one or more amino acid substitutions are selected from N297A (Bolt S et al. (1993) Eur J Immunol 23:403-411), D265A (Shields et al. (2001) R. J. Biol. Chem. 276, 6591 — 6604), F234A, F235A (Hutchins et al. (1995) Proc Natl Acad Sci USA, 92:11980-11984; Alegre et al., (1994) Transplantation 57:1537-1543.
  • the anti-ENPPl antibody includes an IgG2 isotype heavy chain constant domain 1(CH1) and hinge region (White et al., (2015) Cancer Cell 27, 138-148).
  • the antibody Fc region contains a S267E amino acid substitution, a L328F amino acid substitution, or both, and/or a N297A or N297Q amino acid substitution, where the amino acid position is according to the EU or, Kabat numbering convention.
  • the anti-ENPPl antibody has an IgG4 isotype.
  • the antibody contains a human IgG4 constant region.
  • the human IgG4 constant region includes an Fc region.
  • the antibody binds an inhibitory Fc receptor.
  • the inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB (FcyllB).
  • the Fc region contains one or more modifications.
  • the Fc region contains one or more amino acid substitutions (. e.g ., relative to a wild-type Fc region of the same isotype).
  • the one or more amino acid substitutions are selected from L235A, G237A, S228P, L236E (Reddy et al., (2000) J Immunol, 164:1925-1933), , where the amino acid position is according to the EU or, Kabat numbering convention.
  • the anti-ENPPl antibody has a hybrid IgG2/4 isotype.
  • the antibody includes an amino acid sequence containing amino acids 118 to 260 according to EU or, Kabat numbering of human IgG2 and amino acids 261-447 according to EU or, Kabat numbering of human IgG4 (WO 1997/11971; WO 2007/106585).
  • the anti-ENPPl antibody contains a mouse IgG4 constant region (Bartholomaeus, et al. (2014). J. Immunol. 192, 2091-2098).
  • an IgG4 variant of the present disclosure may be combined with an S228P mutation according to the EU or, Kabat numbering convention (Angal et al., (1993) Mol Immunol, 30:105-108) and/or with one or more mutations described in Peters et al., (2012) J Biol Chem. 13;287(29):24525-33) to enhance antibody stabili ation.
  • the anti-ENPPl antibody comprises an ENPP1 -binding fragment.
  • the antibody fragment is an Fab, Fab’, Fab’-SH, F(ab’)2, Fv or scFv fragment.
  • the anti-ENPPl antibody a diabody or a triabody.
  • the anti-ENPPl antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:24.
  • the anti- ENPPl antibody comprises a heavy chain constant region having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:24.
  • the heavy chain constant region contains substitutions ( e.g ., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO:24.
  • the anti-ENPPl antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO:25.
  • the anti- ENPPl antibody comprises a light chain constant region having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:25.
  • the light chain constant region contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO:25.
  • isolated antibodies that contact one or more residues of human ENPP1 that are contacted by any one of the monoclonal anti-ENPPl antibodies described herein.
  • the anti-ENPPl antibody is a multi- specific antibody.
  • the multi- specific antibody recognizes a first antigen and a second antigen, wherein the first antigen is human ENPP1.
  • the multi- specific antibody is a bispecific antibody that recognizes a first antigen and a second antigen, wherein the first antigen is human ENPP1.
  • the second antigen is a protein, a polysaccharide, a glycoprotein, a lipid, or a glycolipid.
  • the second antigen comprises a protein or a fragment thereof that is at least 8 amino acids in length.
  • the second antigen is a surface antigen express on immune cells (e.g., CD3 expressed on T lymphocytes, or CD16 expressed on NK cells). In some embodiments, the second antigen is a cancer antigen expressed by a cancer cell.
  • Anti-ENPPl antibodies of the present disclosure may be produced using recombinant methods and compositions, e.g., as described in U.S. Patent No. 4,816,567.
  • isolated nucleic acid(s) having a nucleotide sequence encoding any one of the anti-ENPPl antibodies of the present disclosure are provided.
  • the isolated nucleic acid(s) encode the anti-ENPPl antibody 1F12, 1F12-04, 1F12-05, 1F12-09, 1F12-10, 1F12-14, or 1F12-15, as described herein.
  • nucleic acids may encode an amino acid sequence containing the VF and/or an amino acid sequence containing the VH of the anti- ENPPl antibody (e.g., the light and/or heavy chains of the antibody).
  • expression cassettes comprising such nucleic acids are provided (e.g., an expression cassette comprising a nucleic acid encoding an anti-ENPPl antibody in operable combination with a promoter, and/or an expression cassette comprising a nucleic acid encoding an anti-ENPPl antibody and a selectable marker).
  • one or more vectors e.g., expression vectors
  • a host cell containing such nucleic acid is also provided.
  • the host cell contains (e.g., has been transduced with): (1) a vector containing a nucleic acid that encodes an amino acid sequence containing the VL of the antibody and an amino acid sequence containing the VH of the antibody, or (2) a first vector containing a nucleic acid that encodes an amino acid sequence containing the VL of the antibody and a second vector containing a nucleic acid that encodes an amino acid sequence containing the VH of the antibody.
  • the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NSO, Sp20 cell).
  • Host cells of the present disclosure also include, without limitation, isolated cells, in vitro cultured cells, and ex vivo cultured cells.
  • the host cell is a mammalian cell.
  • the mammalian cell is a CHO cell.
  • the host cell is an insect cell.
  • the insect cell is a Spodoptera frugiperda cell, or a Trichoplusiani cell.
  • the cell is a prokaryotic cell.
  • the prokaryotic cell is an E. coli cell.
  • Methods of making an anti-ENPPl antibody of the present disclosure include culturing a host cell of the present disclosure containing a nucleic acid encoding the anti-ENPPl antibody, under conditions suitable for expression of the antibody. In some embodiments, the antibody is subsequently recovered from the host cell (or host cell culture medium).
  • nucleic acid encoding the anti-ENPPl antibody is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell.
  • nucleic acid may be readily isolated and sequenced using conventional procedures (e.g ., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
  • Suitable vectors containing a nucleic acid sequence encoding any of the anti- ENPP1 antibodies of the present disclosure, or fragments thereof polypeptides (including antibodies) described herein include, without limitation, cloning vectors and expression vectors.
  • Suitable cloning vectors can be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors generally have the ability to self-replicate, may possess a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in selecting clones containing the vector.
  • Suitable examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColEl, pCRl, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28.
  • Bluescript e.g., pBS SK+
  • mpl8 mpl9 mpl9
  • pBR322 mpl9
  • ColEl ColEl
  • pCRl pCRl
  • RP4 phage DNAs
  • shuttle vectors such as pSA3 and pAT28.
  • Expression vectors generally are replicable polynucleotide constructs that contain a nucleic acid of the present disclosure.
  • the expression vector may replicable in the host cells either as episomes or as an integral part of the chromosomal DNA.
  • Suitable expression vectors include but are not limited to plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and expression vector(s) disclosed in PCT Publication No. WO 87/04462.
  • Vector components may generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcriptional controlling elements (such as promoters, enhancers and terminator).
  • suitable transcriptional controlling elements such as promoters, enhancers and terminator
  • one or more translational controlling elements are also usually required, such as ribosome binding sites, translation initiation sites, and stop codons.
  • the vectors containing the nucleic acids of interest can be introduced into the host cell by any of a number of appropriate means, including electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (e.g., where the vector is an infectious agent such as vaccinia vims).
  • electroporation employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances
  • microprojectile bombardment e.g., where the vector is an infectious agent such as vaccinia vims.
  • infection e.g., where the vector is an infectious agent such as vaccinia vims.
  • the vector contains a nucleic acid containing one or more amino acid sequences encoding an anti-ENPPl antibody of the present disclosure.
  • Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells.
  • anti-ENPPl antibodies of the present disclosure may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed.
  • For expression of antibody fragments and polypeptides in bacteria e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523; and Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Fo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody fragments in E. coli.).
  • the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • eukaryotic microorganisms such as filamentous fungi or yeast
  • suitable cloning or expression hosts for antibody-encoding vectors including fungi and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern (e.g., Gerngross, Nat. Biotech. 22:1409-1414 (2004); and Fi et ah, Nat. Biotech. 24:210-215 (2006)).
  • Suitable host cells for the expression of glycosylated antibody can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts (e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429, describing PFANTIBODIESTM technology for producing antibodies in transgenic plants.).
  • Vertebrate cells may also be used as hosts.
  • mammalian cell lines that are adapted to grow in suspension may be useful.
  • useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et ah, J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod.
  • monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HEFA); canine kidney cells (MDCK; buffalo rat liver cells (BRF 3A); human lung cells (W 138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells.
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub et al., Proc. Natl.
  • Anti-ENPPl antibodies of the present disclosure, or antibody fragments thereof can be further modified to contain additional non-pro teinaceous moieties that are known in the art and readily available, or to contain different types of drug conjugates that are known in the art and readily available (e.g., a detectable marker, a toxin, or a therapeutic agent).
  • the moieties suitable for derivatization of the antibody are water-soluble polymers.
  • Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
  • PEG polyethylene glycol
  • copolymers of ethylene glycol/propylene glycol carboxymethylcellulose
  • dextran polyvinyl alcohol
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
  • Such techniques and other suitable formulations are disclosed in Remington: The Science and Practice of Pharmacy, 20th Ed., Alfonso Gennaro, Ed., Philadelphia College of Pharmacy and Science (2000).
  • Drug conjugation involves coupling of a biological active cytotoxic (anticancer) payload or drug to an antibody that specifically targets a certain tumor marker (e.g. a protein that, ideally, is only to be found in or on tumor cells).
  • a tumor marker e.g. a protein that, ideally, is only to be found in or on tumor cells.
  • Antibodies track these proteins down in the body and attach themselves to the surface of cancer cells.
  • the biochemical reaction between the antibody and the target protein (antigen) triggers a signal in the tumor cell, which then absorbs or internalizes the antibody together with the cytotoxin.
  • the cytotoxic drug is released and kills the cancer. Due to this targeting, ideally the drug has lower side effects and gives a wider therapeutic window than other chemotherapeutic agents.
  • Anti-ENPPl antibodies of the present disclosure can be incorporated into a variety of formulations for therapeutic administration by combining the antibodies with appropriate pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms.
  • suitable pharmaceutically acceptable carriers or diluents include, without limitation, tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
  • Pharmaceutical compositions can include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers of diluents, which are vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.
  • the diluent is selected so as not to affect the biological activity of the combination.
  • examples of such diluents include, without limitation, distilled water, buffered water, physiological saline, PBS, Ringer’s solution, dextrose solution, and Hank’s solution.
  • a pharmaceutical composition or formulation of the present disclosure can further include other carriers, adjuvants, or non-toxic, nontherapeutic, nonimmunogenic stabilizers, excipients and the like.
  • the compositions can also include additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents and detergents.
  • a pharmaceutical composition of the present disclosure can also include any of a variety of stabilizing agents, such as an antioxidant for example.
  • the pharmaceutical composition includes a polypeptide
  • the polypeptide can be complexed with various well-known compounds that enhance the in vivo stability of the polypeptide, or otherwise enhance its pharmacological properties (e.g., increase the half-life of the polypeptide, reduce its toxicity, and enhance solubility or uptake).
  • modifications or complexing agents include, without limitation, sulfate, gluconate, citrate and phosphate.
  • the polypeptides of a composition can also be complexed with molecules that enhance their in vivo attributes. Such molecules include, without limitation, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.
  • the active ingredient can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions.
  • the active component(s) can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate.
  • additional inactive ingredients that may be added to provide desirable color, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, and edible white ink.
  • Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract.
  • Fiquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.
  • Formulations suitable for parenteral administration include aqueous and non- aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • compositions intended for in vivo use are usually sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic agents, particularly any endotoxins, which may be present during the synthesis or purification process.
  • compositions for parental administration are also sterile, substantially isotonic and made under GMP conditions.
  • Formulations may be optimized for retention and stabilization in the brain or central nervous system.
  • Stabilization techniques include cross-linking, multimerizing, or linking to groups such as polyethylene glycol, polyacrylamide, neutral protein carriers, etc. in order to achieve an increase in molecular weight.
  • Other strategies for increasing retention include the entrapment of the antibody, such as an anti-ENPPl antibody of the present disclosure, in a biodegradable or bioerodible implant.
  • the rate of release of the therapeutically active agent is controlled by the rate of transport through the polymeric matrix, and the biodegradation of the implant.
  • the transport of drug through the polymer barrier will also be affected by compound solubility, polymer hydrophilicity, extent of polymer cross-linking, expansion of the polymer upon water absorption so as to make the polymer barrier more permeable to the drug, geometry of the implant, and the like.
  • the implants are of dimensions commensurate with the size and shape of the region selected as the site of implantation. Implants may be particles, sheets, patches, plaques, fibers, microcapsules and the like and may be of any size or shape compatible with the selected site of insertion.
  • the implants may be monolithic, i.e. having the active agent homogenously distributed through the polymeric matrix, or encapsulated, where a reservoir of active agent is encapsulated by the polymeric matrix.
  • the selection of the polymeric composition to be employed will vary with the site of administration, the desired period of treatment, patient tolerance, the nature of the disease to be treated and the like. Characteristics of the polymers will include biodegradability at the site of implantation, compatibility with the agent of interest, ease of encapsulation, a half-life in the physiological environment.
  • Biodegradable polymeric compositions which may be employed may be organic esters or ethers, which when degraded result in physiologically acceptable degradation products, including the monomers. Anhydrides, amides, orthoesters or the like, by themselves or in combination with other monomers, may find use.
  • the polymers will be condensation polymers.
  • the polymers may be cross-linked or non-cross-linked.
  • polymers of hydroxyaliphatic carboxylic acids either homo- or copolymers, and polysaccharides. Included among the polyesters of interest are polymers of D-lactic acid, L-lactic acid, racemic lactic acid, glycolic acid, polycaprolactone, and combinations thereof.
  • a slowly biodegrading polymer is achieved, while degradation is substantially enhanced with the racemate.
  • Copolymers of glycolic and lactic acid are of particular interest, where the rate of biodegradation is controlled by the ratio of glycolic to lactic acid.
  • the most rapidly degraded copolymer has roughly equal amounts of glycolic and lactic acid, where either homopolymer is more resistant to degradation.
  • the ratio of glycolic acid to lactic acid will also affect the brittleness of in the implant, where a more flexible implant is desirable for larger geometries.
  • polysaccharides of interest are calcium alginate, and functionalized celluloses, particularly carboxymethylcellulose esters characterized by being water insoluble, a molecular weight of about 5 kD to 500 kD, etc.
  • Biodegradable hydrogels may also be employed in the implants of the subject invention. Hydrogels are typically a copolymer material, characterized by the ability to imbibe a liquid. Exemplary biodegradable hydrogels which may be employed are described in Heller in: Hydrogels in Medicine and Pharmacy, N. A. Peppes ed., Vol. Ill, CRC Press, Boca Raton, Fla., 1987, pp 137-149.
  • compositions of the present disclosure containing an anti-ENPPl antibody of the present disclosure may be administered to an individual in need of treatment with the anti-ENPPl antibody, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, intracranial, intraspinal, subcutaneous, intra- articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
  • Dosages and desired drug concentration of pharmaceutical compositions of the present disclosure may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary artisan.
  • normal dosage amounts may vary from about 10 ng/kg up to about 100 mg/kg of an individual’s body weight or more per day, preferably about 1 mg/kg/day to 10 mg/kg/day, depending upon the route of administration.
  • the treatment is sustained until a desired suppression of symptoms is achieved.
  • An exemplary dosing regimen may include administering an initial dose of an anti-ENPPl antibody, of about 2 mg/kg, followed by a weekly maintenance dose of about 1 mg/kg every other week.
  • Other dosage regimens may be useful, depending on the pattern of pharmacokinetic decay that the physician wishes to achieve. For example, dosing an individual from one to twenty-one times a week is contemplated herein. In certain embodiments, dosing ranging from about 3 pg/kg to about 2 mg/kg (such as about 3 pg/kg, about 10 pg/kg, about 30 pg/kg, about 100 pg/kg, about 300 pg/kg, about 1 mg/kg, and about 2/mg/kg) may be used.
  • dosing frequency is three times per day, twice per day, once per day, once every other day, once weekly, once every two weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, or once monthly, once every two months, once every three months, or longer. Progress of the therapy is easily monitored by conventional techniques and assays.
  • the dosing regimen, including the anti-ENPPl antibody administered, can vary over time independently of the dose used.
  • Dosages for a particular anti-ENPPl antibody may be determined empirically in individuals who have been given one or more administrations of the anti-ENPPl antibody. Individuals are given incremental doses of an anti-ENPPl antibody.
  • a clinical symptom of any of the diseases, disorders, or conditions of the present disclosure e.g ., cancer, bacterial and/or viral infection, insulin resistance, type II diabetes, chondrocalcinosis, a calcium pyrophosphate deposition disorder (CPPD), or hypophosphatasia
  • CPPD calcium pyrophosphate deposition disorder
  • Administration of an anti-ENPPl antibody of the present disclosure can be continuous or intermittent, depending, for example, on the recipient’s physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners.
  • the administration of an anti-ENPPl antibody may be essentially continuous over a preselected period of time or may be in a series of spaced doses.
  • dosages and methods of delivery are provided in the literature; see, for example, U.S. Patent Nos. 4,657,760; 5,206,344; or 5,225,212. It is within the scope of the present disclosure that different formulations will be effective for different treatments and different disorders, and that administration intended to treat a specific organ or tissue may necessitate delivery in a manner different from that to another organ or tissue. Moreover, dosages may be administered by one or more separate administrations, or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • anti-ENPPl antibodies of the present disclosure may be used for inhibiting cGAMP hydrolysis, or preventing, reducing risk, or treating cancer, bacterial and/or viral infection, insulin resistance, type II diabetes, chondrocalcinosis, a calcium pyrophosphate deposition disorder (CPPD), and/or hypophosphatasia.
  • CPPD calcium pyrophosphate deposition disorder
  • the present disclosure provides methods of inhibiting cGAMP hydrolysis by ENPP1.
  • the method comprises contacting a cell expressing ENPP1 with an effective amount of any one of the anti-ENPPl antibodies disclosed herein in the presence of cGAMP to inhibit hydrolysis of the cGAMP.
  • the contacting is in vitro.
  • the contacting is in vivo.
  • the contacting in vivo comprises administering the antibody to a subject such that pyrophosphate concentration in blood of the subject is reduced.
  • the subject is a human patient.
  • an anti-ENPPl antibody as described herein in the manufacture of a medicament for inhibiting cGAMP hydrolysis by ENPP1.
  • provided herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any one of the anti-ENPPl antibodies described herein. Also provided herein is the use of an anti- ENPPl antibody as described herein in the manufacture of a medicament for treatment of cancer in a subject in need thereof. In some embodiments, the subject is a human patient.
  • a method for treating or preventing a disease or condition associated with ENPP1 activity comprises administering an effective amount of any one of the anti-ENPPl antibodies described herein to a subject in need thereof.
  • the disease or condition is cancer or abnormal cell proliferation responsive to inhibition of ENPP1 activity.
  • the disease or condition is selected from the group consisting of bacterial and/or viral infection, insulin resistance, type II diabetes, chondrocalcinosis, a calcium pyrophosphate deposition disorder (CPPD), or hypophosphatasia.
  • the subject is a human patient.
  • administration of an anti-ENPPl antibody of the present disclosure results in pyrophosphate concentration in blood of the subject to be reduced.
  • an anti-ENPPl antibody as described herein in the manufacture of a medicament for treating a disease or condition associated with ENPP1 activity.
  • the disease or condition is cancer or abnormal cell proliferation responsive to inhibition of ENPP1 activity.
  • the disease or condition is selected from the group consisting of bacterial and/or viral infection, insulin resistance, type II diabetes, chondrocalcinosis, a calcium pyrophosphate deposition disorder (CPPD), or hypophosphatasia. VII. Diagnostic Uses
  • the isolated antibodies of the present disclosure also have diagnostic utility.
  • This disclosure therefore provides for methods of using the antibodies of this disclosure, or functional fragments thereof, for diagnostic purposes, such as the detection of ENPP1 in an individual or in tissue samples derived from an individual.
  • the method comprises (a) contacting human ENPP1 in a sample with any one of the anti-ENPPl antibodies described herein under conditions suitable for binding human ENPP1 with the antibody; and (b) detecting binding of the antibody to human ENPP1 thereby detecting human ENPP1 in the sample.
  • the sample is derived from an individual.
  • the sample is a biological sample comprising human cells or a human cell lysate.
  • the individual is a human.
  • the individual is a human patient suffering from, or at risk for developing, cancer.
  • the diagnostic methods involve detecting ENPP1 in a biological sample, such as a biopsy specimen, a tissue, or a cell.
  • an anti-ENPPl antibody is conjugated to a detectable marker and binding of the antibody to human ENPP1 is detected by measuring the detectable marker, wherein the detectable marker is selected from the group consisting of a radioisotope, a metal chelator, an enzyme, a fluorescent compound, a bioluminescent compound, and a chemiluminescent compound.
  • binding of the antibody to human ENPP1 is detected by measuring binding of a secondary antibody to the anti-ENPPl antibody.
  • An isolated antibody of the present disclosure is contacted with the biological sample and antigen-bound antibody is detected.
  • a tumor sample e.g., a biopsy specimen
  • an anti-ENPPl antibody described herein in order to detect and/or quantify tumor-associated macrophages (e.g., M2-type macrophages).
  • the detection method may involve quantification of the antigen-bound antibody.
  • Antibody detection in biological samples may occur with any method known in the art, including immunofluorescence microscopy, immunocytochemistry, immunohistochemistry, ELISA, FACS analysis, immunoprecipitation, or micro-positron emission tomography.
  • the antibody is radiolabeled, for example with 18 F and subsequently detected utilizing micro-positron emission tomography analysis.
  • Antibody-binding may also be quantified in a patient by non-invasive techniques such as positron emission tomography (PET), X-ray computed tomography, single-photon emission computed tomography (SPECT), computed tomography (CT), and computed axial tomography (CAT).
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • CT computed tomography
  • CAT computed axial tomography
  • kits containing an isolated antibody of the present disclosure e.g ., an anti-ENPPl antibody as described herein, or a functional fragment thereof.
  • Kits of the present disclosure may include one or more containers comprising a purified antibody of the present disclosure.
  • the kits further include instructions for use in accordance with the methods of this disclosure.
  • these instructions comprise a description of administration of the isolated antibody of the present disclosure (e.g., an anti-ENPPl antibody described herein) to prevent, reduce risk, or treat an individual having a disease, disorder, or injury selected from cancer, bacterial and/or viral infection, insulin resistance, type II diabetes, chondrocalcinosis, a calcium pyrophosphate deposition disorder (CPPD), or hypophosphatasia, according to any methods of this disclosure.
  • a disease, disorder, or injury selected from cancer, bacterial and/or viral infection, insulin resistance, type II diabetes, chondrocalcinosis, a calcium pyrophosphate deposition disorder (CPPD), or hypophosphatasia, according to any methods of this disclosure.
  • CPPD calcium pyrophosphate deposition disorder
  • the instructions comprise a description of how to detect ENPP1, for example in an individual, in a tissue sample, or in a cell.
  • the kit may further comprise a description of selecting an individual suitable for treatment based on identifying whether that individual has the disease and the stage of the disease.
  • the instructions generally include information as to dosage, dosing schedule, and route of administration for the intended treatment.
  • the containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses.
  • Instructions supplied in the kits of the present disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
  • kits of this disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g ., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump.
  • a specific device such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump.
  • a kit may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the container may also have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is an isolated antibody of the present disclosure (e.g., an anti- ENPP1 antibody described herein).
  • the container may further comprise a second pharmaceutically active agent.
  • Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container.
  • An isolated anti-ENPPl antibody wherein the anti-ENPPl antibody binds human ENPP1, and comprises a heavy chain variable domain (VH) comprising three heavy chain complementarity determining regions (VH-CDRs) of SEQ ID NO:l, and a light chain variable domain (VL) comprising three light chain complementarity determining regions (VL-CDRs) of SEQ ID NO:9.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • an isolated anti-ENPPl antibody wherein the anti-ENPPl antibody binds human ENPP1, and comprises a heavy chain variable domain (VH) comprising three heavy chain complementarity determining regions (VH-CDRs), and a light chain variable domain (VL) comprising three light chain complementarity determining regions (VL-CDRs), wherein the VH- CDRs comprise VH-CDR1 comprising the amino acid sequence of SEQ ID NO:3, VH-CDR2 comprising the amino acid sequence of SEQ ID NO:5, and VH-CDR3 comprising the amino acid sequence of SEQ ID NO:7; and wherein the VL-CDRs comprise VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 11, VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 15.
  • the anti-ENPPl antibody is an IgGl isotype.
  • the anti-ENPPl antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:24, and a light chain constant region comprising the amino acid sequence of SEQ ID NO:25.
  • anti-ENPPl antibody is a conjugated antibody in which the antibody is conjugated to a detectable marker, a toxin, or a therapeutic agent.
  • nucleic acid comprising a nucleotide sequence encoding the antibody of any one of the preceding embodiments.
  • 31. A vector comprising the nucleic acid of embodiment 30.
  • eukaryotic cell is a mammalian cell
  • the mammalian cell is a Chinese hamster ovary (CHO) cell.
  • the host cell of embodiment 33 wherein the eukaryotic cell is an insect cell, optionally wherein the insect cell is a Spodoptera frugiperda cell, or a Trichoplusiani cell.
  • a method of producing an antibody that binds to human ENPP1, comprising culturing the host cell of any one of claims 32-36 in culture medium under conditions suitable for expression of the antibody.
  • a pharmaceutical composition comprising the antibody of any one of embodiments 1-29 and a pharmaceutically acceptable excipient.
  • a method for detecting human ENPP1 comprising:
  • the sample is a biological sample comprising human cells or a human cell lysate.
  • the anti-ENPPl antibody is conjugated to a detectable marker and binding of the antibody to human ENPP1 is detected by measuring the detectable marker, wherein the detectable marker is selected from the group consisting of a radioisotope, a metal chelator, an enzyme, a fluorescent compound, a bioluminescent compound, and a chemiluminescent compound.
  • a method of inhibiting cGAMP hydrolysis by ENPP1, comprising contacting a cell expressing ENPP1 with an effective amount of the antibody of any one of embodiments 1-29 in the presence of cGAMP to inhibit hydrolysis of the cGAMP.
  • a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the antibody of any one of embodiments 1-29.
  • hematologic cancer is a lymphoma, a leukemia, or a myeloma.
  • This example described the generation of a murine anti-ENPPl monoclonal antibody that binds human ENPP1 and cynomolgus monkey ENPP1. This example also described the humanization of the anti-ENPPl monoclonal antibody and characterization of recombinant anti-ENPPl antibodies.
  • ELISA plates (BeaverBio, #40301) were coated with 100 pi of 1 pg/ml huENPPl ECD-his and mENPPl-ECD-his in PBS and incubated at 4°C overnight. The plates were blocked with 200 m ⁇ blocking buffer at 37°C for 2 hours and washed 3 times with 200 m ⁇ Phosphate-Buffered Saline, 0.1% Tween® 20 Detergent (PBST). The serum was serially diluted ten-fold from 1:100 diluent and added to the plate, then incubated at 37°C for 1 hour before being washed 3 times with PBST.
  • PBST Phosphate-Buffered Saline
  • Serially diluted serum (from 1:100 serum diluent, 10 fold dilution, and total 2 concentrations used) was prepared in FACS buffer starting from 1:50. 50 m ⁇ /well of resuspended cell and 50 m ⁇ /well of serum were seeded into the assay plate. The serum and cell mixture was incubated at 4 °C for 1 hour. Cells were washed twice with 200 m ⁇ /well FACS buffer by centrifuging at 400g for 5 minutes and discarding the supernatant.
  • Cells were then re-suspended with 100 m ⁇ FACS buffer/well diluted 2 nd antibody (Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor® 488 conjugate or Alexa Fluor® 488 Donkey anti-rat IgG (H+L) Antibody (Invitrogen A21202; 1:500, original stock 2mg/ml). Cells were incubated at 4°C for 1 hour in the dark. Cells were washed twice, re-suspended in 100 m ⁇ /well cold FACS buffer and analyzed on a BD FACSCaliburTM Flow Cytometer.
  • Splenocyte harvest and culture Mice with a strong immune response against ENPP1 and potent inhibitory activity in biochemical AMP-glo assay when cGAMP used as substrate as determined by serum titer were selected and used for hybridoma generation. Mice selected for fusion and were given a final boost by intraperitoneal injection of HuENPPl-ECD- his (25 pg). Three days later, the mice were euthanized by carbon dioxide asphyxiation following an approved institutional animal care and use committee protocol, and a blood sample and splenocytes were collected as established methods. Serum was generated from the blood and is used as a positive control (designated as final bleed (FB)) at the hybridoma screening stage. Splenocytes were centrifuged at 400 g (or 1000 rpm) for 5 minutes and the supernatant was discarded.
  • FB final bleed
  • Splenocyte fusion Splenocytes were resuspended in medium (DMEM) by inverting the centrifuge tube 3-5 times, centrifuged at 400 g (or 1000 rpm) for 5 minutes and supernatant was discarded. Splenocytes were re-suspended in 5 ml Red blood cell lysis buffer, incubated for 5 minutes at 4 °C, the reaction was stopped by the addition of DMEM. Splenocytes were then centrifuged at 400 g for 5 minutes, re-suspended in DMEM. Cell count was performed.
  • DMEM medium
  • SP2/0 cells (2.5 x 10 7 SP2/0 cells per 10 8 splenocytes) were added to the splenocytes to give a final ratio of splenocytes: SP2/0 of 4:1.
  • the cells were centrifuged at 400 g for 5 minutes, and the medium was discarded.
  • the cell pellet was washed twice using electro-fusion (EF) solution and resuspended in EF solution.
  • EF electro-fusion
  • the mixture of cells was placed in the fusion slot and the fusion was effectuated by cell fusion generator using an optimized program (VI :50V, tl: 15S, V2: 600V, t2: 20S). After electro-fusion, the fused cells were left in slot for an additional 10 min.
  • hybridoma supernatant was collected and screened for binding to human ENPP1 and activity in biochemical 2’3’-cGAMP assay.
  • Hybridoma clones that exhibited binding to human ENPP1 or mouse ENPP1 by EFISA were expanded into 24- well plates.
  • Hybridoma supernatant was collected from 24- well cultures and tested again for their binding activity with ENPP1 of human, mouse and cyno by either FACS or EFISA approach.
  • supernatant was also tested in a biochemical AMP-glo assay to determine the ability to inhibit enzyme activity. Clones showing potent inhibition in the AMP- glo assay were selected for subcloning.
  • ENPP1 extracellular domain ECD
  • mouse ENPP1 ECD His tag recombinant protein ECD His tag recombinant protein
  • chimeric ENPP1 protein with catalytic domain ECD
  • Recombinant proteins were diluted with coating buffer to 1 pg/ml, and 100 m ⁇ of diluted recombinant protein was coated onto 96-well EFISA plates overnight at 4°C. The plates were washed 3 times with washing buffer (PBST-PBS, 0.05% Tween-20, pH 7.4). 200 pl/well blocking buffer (1% BSA in PBST, pH 7.4) was added to each well and incubated for 2 hours at 37°C. Thereafter, 100 m ⁇ of hybridoma supernatant was diluted in blocking buffer (PBS, 0.05% Tween-20, pH 7.4), added to each well of the 96-well ELISA plate, and incubated at 37°C for 1 hr.
  • blocking buffer PBS, 0.05% Tween-20, pH 7.4
  • a commercially available anti-ENPPl antibody (Sino Biological, 10208-MM02) and an mouse IgGl isotype control (generated in-house) were used as positive and negative controls, respectively.
  • the plates were washed 3 times with washing buffer.
  • 50 pl/well of anti-mouse IgG- HRP (Sigma, A1068; 1:1000 in washing buffer) were added to each well and incubated for 50 min at room temperature, and the plates were washed 3 times with washing buffer.
  • 50 pl/well of 3,3',5,5'-tctramcthylbcnzidinc (TMB) substrate was added to each well and incubated at room temperature for 10 min.
  • 50 m ⁇ /well of 1 N HC1 was added to terminate the reaction, and the absorbance at 450 nm was measured with a microplate reader.
  • Hybridoma supernatant was added and the cell mixture was incubated at 4°C for 1 hour.
  • the cells were washed twice with 200 m ⁇ /well FACS buffer by centrifuging at 400g for 5 minutes and the supernatant was discarded.
  • the cells were resuspended with 100 m ⁇ /well diluted secondary antibody (1:500) and incubated at 4°C for 1 hour in the dark.
  • the cells were washed twice by using the condition in last step.
  • the cells were resuspended in 100 m ⁇ /well cold FACS buffer.
  • the cells were kept in the dark for FACS analysis and data were processed by FlowJo 7.6 software.
  • Hybridoma scale up Hybridoma cells were cultured with 1 mL culture medium of 24 well plate before transferred to T-75 flask when the cell number reached to 1-2 x 10 4 . Hybridoma cells were seeded to T-75 flasks with 25ml culture medium (DMEM + 10% FBS + lx HT) and cultured at 37 °C, 5% CO2 condition. Cell conditions were monitored with an inverted phase microscope every 2 days. Upon reaching 75% confluence or 5 xlO 5 cells/ml and over 85% cell viability, cells were transferred to roller bottle.
  • DMEM + 10% FBS + lx HT 25ml culture medium
  • Hybridoma Antibody Purification The culture supernatant was loaded onto a pre equilibrated Protein A affinity column, and the column was then washed with 10 CV of equilibration buffer (lx PBS, pH 7.2) until the OD of the flow-through sample turned to zero. Antibody was eluted from the column with 5 CV of elution buffer (0.1 M citrate sodium buffer, pH 3.0). Only one elution was performed without gradient elution. The eluted solution was collected in a clean tube and neutralized with neutralizing buffer to final pH 7.0. (1 M Trizma base, pH 9.0).
  • the collected antibody was dialyzed against 100-fold of elution volume of PBS, pH 7.4, at 2-8°C overnight with 3 buffer exchanges to ensure the complete buffer exchange.
  • the dialyzed antibody was transferred into a clean tube and sterile filtered with a 0.22 pm syringe filter in a biological safety cabinet.
  • Antibody concentrations were determined by Nanodrop at A280nm (Thermofisher, Nanodrop 2000C Spectrophotometer). Each sample was measured three times, then average value was taken.
  • Antibody purity was assessed by SDS-PAGE and SEC- HPLC. Endotoxin levels were determined with the Limulus Amebocyte Lysate (LAL) method following the kit manual. The purified antibody was then aliquoted and stored at -20°C until use.
  • LAL Limulus Amebocyte Lysate
  • the protein was isolated from the supernatant by Protein A chromatography (HiTrap MabSelect SuRe, GE, 11-0034-95).
  • the ultraviolet (UV) absorption (A280nm) was monitored with a UV detector, and washed the column with 100 ml Buffer A (1.5M glycine, 3M NaCl, pH 8.6) until the A280nm absorption returned to baseline levels.
  • Buffer A 1.5M glycine, 3M NaCl, pH 8.6
  • Antibody was eluted from the Protein A affinity column with 10 ml Buffer B (lOOmM sodium citrate, 150mM NaCl, pH 3.0) and neutralized immediately with 1 ml Buffer C (1M arginine, 400mM succinic acid, pH 9.0), to bring the pH to pH 5-6.
  • the elution fraction was dialyzed with PBS at 4°C overnight, and the molecular weight cutoff of Slide- A-LyzerTM Dialysis Cassettes was 20KD.
  • the final ratio of elution volume to dialysis volume was 1:100 at each dialysis step and 3 rounds of dialysis were performed to ensure the complete buffer exchange.
  • Condensation of the diluents was performed by Amicon Ultra- 15 Centrifugal Filter Unit centrifugation (Eppendorf, 5810R, 5811000398) at 4000 rpm, for 10 minutes.
  • the dialyzed protein was filtered with a 0.22 micron sterile filter (Acrodisc Syringe Filters with Supor Membrane, PALL, 4612) and then aliquoted and stored at - 30°C.
  • SEC-HPEC 20 mg of each purified antibody was injected into the column (TSK gel G3000SWXL with size 7.8 mm I.D. x 30 cm), and 1 x PBS buffer was used as flow phase.
  • the column temperature was 30°C, and flow rate was set at 0.8 ml/min.
  • the purity of the antibody preparation was determined based on the peak area corresponding to the antibody.
  • Endotoxin level ⁇ .
  • the kit used was Charles River Cat. R1708K.
  • the substrate 2’3’-cGAMP was prepared at a 2x concentration (24 mM) in phenol red-free medium with 20 mM AB-680 without FBS. The reaction was incubated at 37°C in 1% CO2 for 3 hours. After incubation, 5 microliters of supernatant were used in the AMP-glo assay according to the manufacturer’s recommended protocol. Luminescence was measured on a Spectramax M5e.
  • MDA-MB-231 cells were plated at 50,000 cells/well in 96- well plates in Leibovitz’s L-15 Medium+10% FBS. Cells were incubated in a 37°C incubator with 1% CO2 for 24 hours. After 24 hours, the cell culture medium was aspirated, then antibodies were serially diluted in phenol red-free medium without FBS and added to the cells. The substrate pNP-AMP was added to the reaction at a final concentration of 250 pM. The reaction was incubated at 37°C in 1% CO2 for 3 hours. Formed pNP was then quantified by measuring the absorbance at a wavelength of 405 nm on Envision.
  • Octet assay To determine the binding affinity of antibody to human ENPP1 (hENPPl), test antibodies were diluted to a working concentration of 1 pg/mL and loaded onto Anti-hlgG Fc Capture Biosensors for 600 seconds. Next, the biosensors were transferred to running buffer containing analyte His-hENPPl solution. The top concentration of analyte was 300 nM, with six additional threefold serial dilutions tested. The signals with coupled analyte samples subtracted from that without coupled analyte samples were calculated with Octet data analysis software (10.0 version). Results
  • a mouse with a high serum antibody titer was selected for splenocyte fusion and hybridoma generation.
  • a total of 188 hybridoma clones were screened by ELISA for binding to human ENPP1 (hENPPl).
  • hENPPl human ENPP1
  • Fifteen clones showed binding to hENPPl, and one (antibody 1F12) showed reproducible inhibition of 2’3’-cGAMP hydrolysis by a recombinant hENPPl protein.
  • 27 humanized antibodies, including 1F12-4, 1F12-5, 1F12-9, 1F12-10, 1F12-14, and 1F12-15, were derived from IF 12.
  • the amino acid sequences of the recombinant anti-ENPPl antibodies and portions thereof are shown in Table 1-1.
  • a dose response binding analysis of recombinant anti-ENPPl antibodies against CHO-K1 cells expressing human ENPP1 was performed using flow cytometry. As shown in FIG. 1, the antibody 1F12 bound the CHO-K1 cells expressing human ENPP1. Furthermore, six of twenty-seven humanized variants of 1F12 bound (ECso less than 67 nM) the CHO-K1 cells expressing human ENPP1 as shown in Table 1-2. No binding was observed with the isotype control antibody.
  • Table 1-5 IC50 values of anti-ENPPl antibodies in pNP-AMP hydrolysis assay with MDA- MB-231 cell line [0200] An Octet binding analysis was performed to measure the ability of the anti- ENPP1 antibodies to bind recombinant human ENPP1.
  • Table 1-6 below, provides the dissociation constant (KD), on-rate (k on ), and dissociation rate (k dis ) for the anti-ENPPl antibodies.

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Abstract

La présente divulgation concerne de manière générale des compositions qui comprennent des anticorps, par exemple, des anticorps monoclonaux, des fragments d'anticorps, etc., qui se lient spécifiquement à une protéine ENPP1 humaine, et l'utilisation desdites compositions dans la prévention, la réduction du risque, ou le traitement de maladies ou d'états associés à une activité ENPP1 aberrante chez un individu en ayant besoin.
PCT/US2022/035335 2021-06-29 2022-06-28 Anticorps anti-enpp1 et leurs utilisations WO2023278463A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995019570A1 (fr) * 1994-01-14 1995-07-20 Genentech, Inc. Antagonistes de l'inhibiteur de l'activite tyrosine kinase du recepteur de l'insuline

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995019570A1 (fr) * 1994-01-14 1995-07-20 Genentech, Inc. Antagonistes de l'inhibiteur de l'activite tyrosine kinase du recepteur de l'insuline

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
COGAN D ET AL.: "Re-awakening Innate Immune Signaling in Cancer: The Development of Highly Potent ENPP1 Inhibitors", CELL CHEM BIO L, vol. 27, no. 11, 19 November 2020 (2020-11-19), pages 1327 - 1328, XP086358988, DOI: 10.1016/j.chembiol.2020.11.001 *
GODING JAMES W.: "Ecto-enzymes: physiology meets pathology", JOURNAL OF LEUKOCYTE BIOLOGY, JOHN WILEY & SONS LTD., GB, vol. 67, no. 3, 1 March 2000 (2000-03-01), GB , pages 285 - 311, XP093024219, ISSN: 0741-5400, DOI: 10.1002/jlb.67.3.285 *
KATO KAZUKI, NISHIMASU HIROSHI, OIKAWA DAISUKE, HIRANO SEIICHI, HIRANO HISATO, KASUYA GO, ISHITANI RYUICHIRO, TOKUNAGA FUMINORI, N: "Structural insights into cGAMP degradation by Ecto-nucleotide pyrophosphatase phosphodiesterase 1", NATURE COMMUNICATIONS, vol. 9, no. 1, XP093022308, DOI: 10.1038/s41467-018-06922-7 *

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