WO2019166701A1 - Anticorps anti-voie de signalisation de l'adénosine conjugués ou fusionnés avec de l'adénosine désaminase ou capables de se lier à l'adénosine désaminase - Google Patents

Anticorps anti-voie de signalisation de l'adénosine conjugués ou fusionnés avec de l'adénosine désaminase ou capables de se lier à l'adénosine désaminase Download PDF

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WO2019166701A1
WO2019166701A1 PCT/FI2019/050169 FI2019050169W WO2019166701A1 WO 2019166701 A1 WO2019166701 A1 WO 2019166701A1 FI 2019050169 W FI2019050169 W FI 2019050169W WO 2019166701 A1 WO2019166701 A1 WO 2019166701A1
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human
adenosine
ecto
specifically binds
antibody
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PCT/FI2019/050169
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Anton Zavialov
Vladimir ZAVIYALOV
Gennady YEGUTKIN
Maksym SKALDIN
Andrey ZAVIALOV
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Turun Yliopisto
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Publication of WO2019166701A1 publication Critical patent/WO2019166701A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04004—Adenosine deaminase (3.5.4.4)
    • 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
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin

Definitions

  • the present invention relates to human medicine, and more specifical ly to the treatment or prevention of cancer and bacterial infections.
  • Adenosine is a strong immunosuppressant. Tumor cell-derived adeno sine protects the tumor from immune cells and enhances tumor growth. Adeno sine accumulates in the extracellular space as a product of degradation of adenine nucleotides, predominantly, adenosine triphosphate (ATP).
  • ATP adenosine triphosphate
  • ecto-phosphatases ecto-nucleoside triphosphate diphosphohy- drolase 1 (ecto-NTPDasel or CD39) and ecto -nucleotide pyrophosphatase 1 (ec- to-NPPl), which both convert ATP to AMP (adenosine monophosphate), and ecto- 5 '-nucleotidase (5’NDase or CD73), which converts AMP to adenosine.
  • CD39 and CD73 are present in most malignant and nonmalignant tissues.
  • adenosine may be produced by alkaline phosphatase (AP).
  • AP alkaline phosphatase
  • AP is present both on the cell surface and in soluble form and produces adenosine by dephosphorylation of extracellular ATP and AMP.
  • Adenosine deaminase (ADA) converts adenosine into signaling-inactive ino- sine and, hence, may decrease the immunosuppressive effect.
  • ADA1 is predomi nantly an intracellular enzyme. However, it leaks to extracellular space and may act as ecto-ADA. ln contrast, ADA2 encoded by the CECR1 gene is a secreted en zyme, which accumulates in significant amounts in the extracellular space and is the dominant ADA in the blood.
  • mAbs monoclonal antibodies
  • CD 73 the therapeutic ef fect of which is predominantly mediated through inhibition of tumor -derived adenosine production.
  • An additional benefit of anti-CD73 mAbs is the suppression of its pro-metastatic functions mediated by the stimulation of autocrine adeno sine receptor (ADOR) A2b.
  • ADOR autocrine adeno sine receptor
  • the therapeutic effect of mAb to CD39 is also predom inantly due to inhibition of tumor-derived adenosine production.
  • Anti-CD39 mAb A1 improves targeted therapy in ovarian cancer by blocking adenosine - dependent immune evasion.
  • A2a and A2b receptors are also promising targets for anti-tumor or anti-cancer therapy with mAbs.
  • W02016191283 discloses treatment of Her2 -positive breast cancer patients with a polypeptide comprising a fusion between the variable fragment (Fv) targeted to Her2 single chain (sc) (an artificial antibody (Ab) composed of variable domains of light, VL, and heavy, VH, chains), human ADA (hADA) and crys- tallizable fragment (Fc) region of an Ab. Also disclosed is treatment of melanoma patients with a polypeptide comprising a fusion between the single chain variable fragment (scFv) targeted to tumor associated antigens PD-1, T1M-3, LAG-3, and CTLA4, hADA and Fc region of an Ab.
  • L. pneumophila comprises the genes lpg0971 and lpgl905, which encode ecto- NTPDases that share sequence similarity with human CD39. Similar to human CD39, recombinant purified Lpgl905 exhibited ATPase and ADPase activities. As part of its pathogenesis, L.
  • pneumophila persists within human alveolar macro phages in non-acidified organelles that do not mature into phagolysosomes. The evidence was obtained that an Ipgl905 mutant was recovered in lower numbers from macrophages and alveolar epithelial cells compared with wild-type L. pneu mophila. Thus, a bacterial ecto-NTPDase is implicated in virulence.
  • Staphylococcus aureus a Gram-positive bacterium representing the most frequent cause of bacteremia, is complicated to treat due to the emergence of methicillin-resistant strains (mrsa).
  • AdsA adenosine synthase A
  • Synthesis of adenosine by S. aureus in blood allows bacteria to escape from phagocytic clearance with subsequent formation of organ abscesses.
  • An AdsA homologue was identified in the anthrax pathogen, and adenosine syn thesis also enabled an escape of Bacillus anthracis from phagocytic clearance.
  • Streptococcus sanguinis is the most common cause of infective endo carditis (IE). 1E is characterized by the formation of septic thrombi or vegetations on the heart valves.
  • the genome of S. sanguinis SK36 encompasses four genes containing LPxTG motifs that encode putative cell surface nucleotidases. The four predicted nucleotidase products are expected to be anchored to the cell wall by a sortase A-dependent mechanism.
  • Nt5e ecto-5’-nucleotidase
  • ADP extracellular ATP
  • AMP ecto-5’-nucleotidase
  • Streptococcus agalactiae (Group B Streptococcus ) is the leading cause of invasive infection in neonates.
  • a cell wall-localized ecto-5’-nucleoside diphos phate phosphohydrolase (NudP) was found in this Gram-positive pathogen and characterized lt was demonstrated that the absence of NudP activity decreases bacterial survival in mouse blood, a process dependent on extracellular adeno sine.
  • NudP activity is crit ical for virulence.
  • New and improved therapeutics for treating cancer as well as bacterial infections are needed.
  • An object of the present invention is to provide a method and a com position for implementing the method so as to provide a novel treatment regimen for treating or preventing diseases or conditions such as cancer and bacterial in fections.
  • Objects of the invention are achieved by a method and an arrangement which are characterized by what is stated in the independent claims.
  • the pre- ferred embodiments of the invention are disclosed in the dependent claims.
  • the invention is based on the unexpected realization that cancer ther apy by the present ADA-conjugated antibodies may be greatly improved in com parison with therapy by conventional antibodies targeting the adenosine path way, such as mAbs to CD73 or CD39, because the present antibodies do not only inhibit adenosine production with these enzymes but also degrade produced adenosine by converting it to inosine.
  • the invention is also based on the unexpected discovery that therapy of bacterial infections by the ADA-conjugated Abs to bacterial antigens, especially enzymes of the adenosine pathway, may be greatly improved in comparison with therapy by Abs to bacterial antigens alone, because such conjugates degrade adenosine by converting it to inosine and increase antibacterial immune re sponse.
  • the present invention provides a binding body, which specifically binds to a protein of an adenosine signaling pathway and is conjugated or fused with human adenosine deaminase (hADA) or is capable of binding hADA.
  • hADA human adenosine deaminase
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a binding body according to the present invention and a pharmaceutically acceptable carrier.
  • the present invention provides said binding bodies for use in cancer therapy and a method of treating cancer in a subject in need thereof by said binding bodies.
  • the present invention provides said binding bodies for use in treating bacterial infections and a method of treating bacterial infections in a subject in need thereof by said binding bodies.
  • Figure 1 is an exemplary scheme showing a conjugate consisting of hADAl and a human therapeutic mAb specific to an ecto-enzyme involved in adenosine production on tumor or cancer cells or an adenosine receptor on im mune cells.
  • Figure 2 is a scheme showing a possible mechanism of anti-cancer ac tion of the Ab-hADAl conjugate specific to CD73.
  • Figure 3 is a scheme showing a possible mechanism of anti-cancer ac tion of the Ab-hADAl conjugate specific to CD39.
  • Figure 4 is a scheme showing a possible mechanism of anti-cancer ac tion of the conjugate Ab-hADAl specific to ADORAz a -
  • Figure 5 depicts a bispecific antibody (bisAb), in which one site binds to hADA2 and another site binds to an ecto-enzyme involved in adenosine pro duction on tumor or cancer cells or an adenosine receptor on immune cells (Ag).
  • bisAb bispecific antibody
  • Figure 6 is a scheme showing a possible mechanism of anti-cancer ac tion of the bisAbs (anti-ADA, anti-CD 73).
  • Figure 7 is a scheme showing a possible mechanism of anti-cancer ac tion of the bisAbs (anti-ADA, anti-CD 39).
  • Figure 8 is a scheme showing a possible mechanism of anti-cancer ac tion of the bisAb (anti-ADA, anti-ADORA 2a ).
  • Figure 9 is a scheme showing bisAbs, one site of which binds to a hADA and the other binds to an antigen on bacterial cells (BA).
  • Figure 10 is scheme showing a possible mechanism of anti -bacterial action of an antibody (Ab) conjugated with hADA (ADA1 or ADA2), which specifi cally binds to a bacterial antigen (BA) (ecto-5’NDase is selected as example).
  • Abs antibody conjugated with hADA (ADA1 or ADA2)
  • BA bacterial antigen
  • Figure 11 is a scheme showing a possible mechanism of anti -bacterial action of a bispecific antibody (bisAb), one site of which specifically binds to a hADA (ADA1 or ADA2) and the other binds to bacterial antigen (BA) (ecto- 5’NDase is selected as example).
  • bisAb bispecific antibody
  • BA bacterial antigen
  • Figure 12 is scheme showing a possible mechanism of anti -bacterial action of an antibody (Ab) conjugated with hADA (ADA1 or ADA2), which specifi cally binds to a bacterial antigen (BA) (ecto-NTPDase is selected as example).
  • Abs antibody conjugated with hADA (ADA1 or ADA2)
  • BA bacterial antigen
  • Figure 13 is a scheme showing a possible mechanism of anti -bacterial action of a bispecific antibody (bisAb), one site of which specifically binds to a hADA (ADA1 or ADA2) and the other binds to bacterial antigen (BA) (ecto- NTPDase is selected as example).
  • bisAb bispecific antibody
  • ADA1 or ADA2 hADA
  • BA bacterial antigen
  • Figure 14 demonstrates the expression and purification of human and mouse recombinant ADA1.
  • Figure 15 demonstrates the production and purification of hADAl- anti-CD73 antibody conjugates.
  • A Analysis of biotinylation of hADAl and anti- CD73 antibody using streptavidin beads.
  • B Gel filtration purification of biotin- streptavidin-linked hADAl-anti-CD73 conjugate.
  • Figure 16 demonstrates the binding of biotinylated anti-CD73 anti- body to cancer cells.
  • Figure 17 demonstrates the production of covalently-linked mADAl- anti-CD73 antibody conjugates for mouse experiments.
  • Figure 18 demonstrates the generation of 3 H-labeled adenosine from the [ 3 H]AMP substrate in the presence of human PC-3 prostate or MDA-MB-231 (MDA) breast cancer cells (but not medium alone; "Blank").
  • Figure 19 demonstrates the generation of 3 H-labeled adenosine from the [ 3 H]AMP substrate in the presence of human PC-3 prostate or MDA-MB-231 (MDA) breast cancer cells treated with anti-CD73 (4G4) antibody alone or in combination with human ADA (hADA) or bovine ADA (bADA).
  • Figure 20 demonstrates the deamination of adenosine to inosine in human PC-3 prostate and MDA-MB-231 breast cancer cells treated with anti- CD73 (4G4, h5NT) antibody alone or in combination with human ADA (hADA) or bovine ADA (bADA).
  • Figure 21 is a scheme showing (A) the suggested mode-of-action of the mAb-hADAl conjugate specific to CD73 and (B) the in vitro assay used to ban gate the immune cell reactivation by the hADAl-anti-CD73 antibody conjugate.
  • Figure 22 demonstrates that CD73 on MDA-MB-231 human breast cancer cells dephosphorylates AMP to adenosine, resulting in inhibition of TNF- alpha secretion by monocytes.
  • Figure 23 demonstrates that the hADAl-anti-CD73 antibody conju gates are able to the restore the cancer cell-mediated suppression of immune cell activity.
  • Figure 24 demonstrates (A) the production and purification of the an- ti-ADA2 part of the bispecific antibody (bisAb), anti-ADA2 single chain antibody (HI 1), and (B) its capability to harvest AD A2 from human blood.
  • bisAb bispecific antibody
  • HI 1 anti-ADA2 single chain antibody
  • Figure 25 shows exemplary ways of generation of ADA-binding body fusion proteins and ADA-small molecule-binding body conjugates.
  • the present invention provides a binding body specific for a member of an anti-adenosine signaling pathway, conjugated or fused with adenosine de aminase (ADA).
  • ADA adenosine de aminase
  • An alternative to the conjugation or fusion is that the binding body is capable of binding also ADA, i.e. is a binding body bispecific to the mem ber of the adenosine signaling pathway and ADA.
  • the term “or” has the meaning of both “and”' and “or” (i.e. "and/or”). Furthermore, the meaning of a singular noun includes that of a plural noun and thus a singular term, unless otherwise specified, may also carry the meaning of its plural form ln other words, the term “a” or “an” may mean one or more.
  • binding body refers to any molecule, pref erably a protein or a peptide or a small molecule, having the desired binding properties, such as high affinity to its target(s).
  • the term includes, but is not lim ited to, antibodies and antibody mimetics such as affibody molecules and small affinity molecules such as enzymatic inhibitors.
  • Non-limiting examples of binding bodies of the invention are illustrated in Figure 25.
  • antibody refers to an immunoglobulin structure comprising two heavy (H) chains and two light (L) chains inter connected by disulfide bonds.
  • Antibodies can exist as intact immunoglobulins or as any of a number of well-characterized antigen-binding fragments or single chain variants thereof, all of which are herein encompassed by the term "anti body".
  • Non-limiting examples of said antigen-binding fragments include Fab fragments, Fab' fragments, F(ab') 2 fragments, and Fv fragments. Said fragments and variants may be produced by recombinant DNA techniques, or by enzymatic or chemical separation of intact immunoglobulins as is well known in the art.
  • antibody also includes, but is not limited to, polyclonal, monoclonal, and recombinant antibodies of isotype classes immunoglobulin A, D, E, G and M (lgA, lgD, lgE, lgG, and IgM, respectively) and subtypes thereof.
  • antibody also includes bispecific antibodies (bisAb), i.e. artificial protein that can simulta neously bind to two different types of antigen. Means and methods for producing bispecific antibodies are readily available in the art. Unless otherwise stated, any thing disclosed herein with respect to antibodies applies to other binding bodies, such as antibody mimetics, as well. Preferably, the present binding bodies are human or humanized anti bodies.
  • Humanized antibodies are antibodies wherein the variable region may be murine-derived but which has been mutated so as to more resemble a human antibody and may contain a constant region of human origin.
  • Fully human anti bodies are antibodies wherein both the variable region and the constant region are of human origin. Means and methods for producing human and humanized antibodies are readily available in the art.
  • adenosine signaling pathway refers to the signal transduction pathway of the immunosuppressive metabolite adenosine within a cell, such as a mammalian, preferably human cell, or a bacterial cell ln some preferred embodiments, said adenosine pathway is a pathway relevant to the accumulation of extracellular adenosine.
  • adenosine signaling pathway include, but are not limited to, four subtypes of specific G- protein-coupled adenosine receptors (Al, A2a, A2b and A3 receptors, ADORs), nucleoside transporters (ENT1 and ENT2), CD73, CD39 and alkaline phosphatase (AP).
  • AP is over-expressed in many cancer types. Such as CD73, it is present both on the cell surface and in soluble form and produces adenosine by dephosphory lation of extracellular nucleotides. AP is more cancer type specific than CD73.
  • Members of the bacterial adenosine signaling pathway include but are not limited to ecto-enzymes, more specifically homologues of the ecto -enzymes of the mam malian adenosine signaling pathway.
  • the members of the bacterial adenosine signaling pathway include ecto-nucleoside triphosphate di- phosphohydrolase (ecto-NTPDase) and ecto-5'-nucleotidase (ecto-5’NDase).
  • a preferred member of the adenosine signaling pathway is an enzyme involved in the adenosine production, such as CD73, its bacterial homologue Nt5e, CD39, its bacterial homologue NTPDase, or AP. ln some further embodiments, a preferred member of the adenosine signaling pathway is an ecto-enzyme involved in the adenosine production, such as CD73, its bacterial homologue Nt5e, CD39, or its bacterial homologue NTPDase.
  • the present invention is based on the realization that therapy by the hADA conjugated or fused with binding bodies specific to a member of the adeno sine signaling pathway, such as AP, ecto-5’-nucleotidase CD73, ecto-nucleoside triphosphate phosphorylase CD39 or A2a/A2b receptors, may be greatly im proved in comparison with therapy by binding bodies alone.
  • a member of the adeno sine signaling pathway such as AP, ecto-5’-nucleotidase CD73, ecto-nucleoside triphosphate phosphorylase CD39 or A2a/A2b receptors
  • the present invention provides an adenosine deaminase (ADA) conjugated or fused with a therapeutic antibody to any adenosine signaling pathway-related antigen on tumor or cancer cells or on bacterial cells to improve the treatment of a tumor or cancer or a bacterial infection.
  • ADA adenosine deaminase conjugated or fused with a therapeutic antibody to any adenosine signaling pathway-related antigen on tumor or cancer cells or on bacterial cells to improve the treatment of a tumor or cancer or a bacterial infection.
  • the physical link be tween the counterparties (the therapeutic antibody and the ADA enzyme) allows targeting ADA to the site where the extracellular adenosine is produced (e.g. re leased by a surface enzyme such as CD73), before diffusion and dilution into the surroundings. This is beneficial particularly because the ADA enzyme functions efficiently only when the concentration of adenosine is high.
  • the therapeutic antibody is employed to decrease the amount of adenosine produced
  • the ADA enzyme is employed to inactivate the amount of adenosine that escapes the blockade by the therapeutic antibody, or that is produced by an other pathway.
  • the secreted sub- type of human ADA, human adenosine deaminase 2 (hADA2) is used.
  • the inven tors have discovered that hADA2 can be produced cost-efficiently by purifying it from commercially available medical immunoglobulin preparations.
  • one aspect of the invention relates to a method of producing ADA2, in which method the raw material is a medical human immunoglobulin preparation.
  • the present binding body is an anti-ADOR antibody, an anti -CD 73 -antibody, an anti-CD39 antibody, an anti-AP antibody, or a mimetic thereof, which is conjugated or fused with ADA or is capable of binding ADA.
  • the present binding body is specific for a bacterial ecto-NTPDase, such as Lpgl905 or Lpg0971, or for a bacterial ecto-5’NDase.
  • the present binding body is an antibody or mi metic thereof conjugated, e.g.
  • the present binding body (e.g. an antibody or a mimetic thereof) may be conjugated with one or more hADA molecules.
  • Means and meth ods for conjugating a binding body, such as an antibody or a mimetic thereof, with ADA are readily available in the art, including those set forth below. Accordingly, the term "conjugate” as used herein includes any synonyms for the protein conju gate of the invention including e.g. complex, protein complex, and fusion protein.
  • the conjugates/complexes can be established employing a covalent or a non- covalent (e.g. streptavidin-biotin) bond.
  • the complexes can also be produced as fusion proteins with both the ADA enzyme and the antibody present in the same transcript.
  • the conjugates/complexes can comprise linkers to separate the two active proteins from each other e.g. to prevent steric hindrances that could affect the functioning of the proteins.
  • the present binding body (e.g. an antibody or mimetic thereof) is provided as a fusion with ADA, preferably human ADA (hA DA), more preferably ADA1 and/or ADA2.
  • ADA preferably human ADA
  • ADA1 and/or ADA2 preferably ADA1 and/or ADA2.
  • Means and methods for fusing the binding body with said ADA are readily available and known to those skilled in the art.
  • ADA- conjugated binding bodies such as Abs, particularly those to the ectoenzymes (CD73 and CD39), alkaline phosphatase and adenosine receptors (Al, A2a, A2b and A3), are significantly more effective for therapy of cancer than ADA- conjugated Abs to tumor specific antigens, such as PD-1, CTLA-4, Her2/neu, T1M- 3, or LAG-3, because the present binding bodies do not only degrade adenosine by converting it to inosine but also inhibit a tumor-derived adenosine production or binding of adenosine to its receptors.
  • Abs particularly those to the ectoenzymes (CD73 and CD39), alkaline phosphatase and adenosine receptors (Al, A2a, A2b and A3)
  • ADA-conjugated binding bodies such as Abs, particularly those specific for various members of the bacterial adenosine pathway, and more particularly anti-ecto-5-NDase antibodies (e.g. those specific for Staphylococcus aureus or Staphylococcus agalactiae ) and anti-ecto-NTPDase Lpgl905 antibodies (e.g. those specific for Legionella pneumophila ) are more effective in inhibiting bacterial survival or replication that corresponding antibodies without the ADA- conjugation.
  • the present binding body is an antibody or a mimetic thereof which is capable of binding ADA, i.e.
  • it may be a bispecific binding body, one site of which specifically binds to a member of the adenosine signaling pathway, while the other site specifically binds to ADA, preferably to hADA, more preferably to ADA1 and/or ADA2.
  • ADA a member of the adenosine signaling pathway
  • bispecific binding bodies particularly those that have one one antigen binding site is specific to a hADA (ADA1 or ADA2) and the other antigen binding site is specific to CD73 or CD39 on tumor or cancer cells, may exhibit im proved therapeutic activity in comparison to the therapy using Abs to CD73 or CD39 alone, because the present bispecific binding bodies do not only inhibit a tumor-derived adenosine production but also degrade adenosine by converting it to inosine.
  • ADA-mAb conjugates As used herein, the expressions “ADA-mAb conjugates” and “mAb-ADA conjugates” and the like are interchangeable.
  • bispecific binding bodies whose one antigen binding site is specific to a hADA (ADA1 or ADA2) and the other antigen binding site is specific for a bacterial ecto-5’NDase or ecto-NTPDase Lpgl905 are more effective in in hibiting bacterial survival or replication that corresponding antibodies without the ADA-bispecificity.
  • One advantage of the present bispecific binding bodies over ADA- conjugated antibodies disclosed in W02016191283 is the targeted delivery into a site of endogenous ADA production in tumor or cancer cells. This may prevent side effects of any over-reactive ADA-antibody fusion proteins as well as the in duction of an immune response to the fusion protein.
  • Among other benefits are the stronger bivalent binding to antigens on cancer or tumor cells and delivery of two molecules of hADA by a single molecule of bisAbs instead of only one by a fusion between scFv, hADA and Fc region of an Ab disclosed in W02016191283.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable excipient, carrier or diluent, and, as an active ingredient, a binding body (e.g. an antibody or a mimetic thereof) according to the present invention.
  • the pharmaceutical composition may be formulated as desired, for example as a solution, dispersion or suspension, using means and methods readily available in the art.
  • Amounts and regimens for the administration of a binding body (e.g. an antibody or a mimetic thereof) or a pharmaceutical composition according to the present invention can be determined readily by those with ordinary skill in the clinical art of treating the disease in question, such as cancer and bacterial infections.
  • the dosage of the present binding body treatment will vary depending on considerations such as: age, gender and general health of the pa tient to be treated; kind of concurrent treatment, if any; frequency of treatment and nature of the effect desired; severity and type of disease in question; causa tive agent of the disease and other variables to be adjusted by the individual phy sician.
  • a desired dose can be administered in one or more applications to obtain the desired results.
  • Pharmaceutical compositions according to the present inven tion may be provided in unit dosage forms.
  • the present invention is directed to a method of treating or preventing a disease such as cancer or a bacterial infection in a subject in need thereof by administering an efficient amount of a binding body (e.g. an antibody or a mimetic thereof) according to the present invention.
  • a binding body e.g. an antibody or a mimetic thereof
  • treatment or “treating” is intended to include the administration of the present binding body or a pharmaceutical composition comprising the same to a subject for purposes which may include ameliorating, lessening, inhibiting, or curing the disease; whereas the term “prevention” or “preventing” refers to any action re sulting in suppression or delay of the onset of the disease by the administration of the present binding body or a pharmaceutical composition comprising the same.
  • the present conjugates or bispecific binding bodies e.g. antibodies or mimetic thereof
  • a desired target such as a tumor tissue.
  • the term "efficient amount” refers to an amount by which harmful effects of the disease are, at a minimum, ameliorated.
  • the term "subject” refers to an animal, preferably to a mammal, more preferably to a human.
  • human subject refers to an animal, preferably to a mammal, more preferably to a human.
  • human subject refers to an animal, preferably to a mammal, more preferably to a human.
  • cancer refers to any type of cancer, includ ing but not limited to solid tumors and hematological malignancies, such as can cers of the breast, respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid, and their dis tant metastases. Those disorders also include lymphomas, sarcomas and leuke mias.
  • bacterial infection refers to any infection caused by a bacterial pathogen, including both Gram-negative and Gram-positive pathogens ln some specific embodiments, said infection is caused by Staphylo coccus aureus, Staphylococcus agalactiae or Legionella pneumophila.
  • the present binding body or a pharmaceutical composition comprising the same may be used in combination with checkpoint inhibitors, including but not limited to those targeting cytotoxic T-lymphocyte- associated protein 4 (CTLA4), programmed death-1 (PD-1) or programmed death-ligand 1 (PD-L1).
  • CTL4 cytotoxic T-lymphocyte-associated protein 4
  • PD-1 programmed death-1
  • PD-L1 programmed death-ligand 1
  • mAbs monoclonal antibodies
  • the disclosure applies to other types of binding bodies as well, as is readily understood by those skilled in the art.
  • mAb-ADA chemical conjugates may be generated by attaching ADA to lysine (Lys) or cysteine (Cys) residues exposed on the mAb’s surface as is well known in the art. Attachment to Lys is nonspecific and leads to a heterogeneous mixture of mAb-ADA complexes with modifications in both the Fab and Fc do mains. Attachment to Cys is more specific and can be conducted using either sol- vent-exposed Cys after partial or full reduction of interchain disulfide bonds (such as those in the hinge region) or engineered Cys in the Fab or Fc domains.
  • site-specific chemical approaches include introducing rare (selenocysteine) or nonnatural amino acids into the mAb and linking them to ADA using chemistries that do not modify common amino acids.
  • Multiple site-specific enzymatic ap proaches that activate sugars in the Fc portion of mAb may be used for the gener ation of homogeneous mAb-ADA conjugates.
  • a-amino groups of the N-terminal amino acids.
  • the a-amino groups are less basic than Lys and are reactive at pH ⁇ 7.0. Some of them can be selectively modified in the pres ence of Lys. Since either N-terminal amines or Lys are always present in mAbs, and since they are easily reacted, these aliphatic amines provide the most com monly employed method of mAb modification.
  • Cys contains a free thiol group, which is more nucleophilic than amines and is generally the most reactive functional group in a protein.
  • Thiols unlike most amines, are generally reactive at neutral pH, and therefore can be coupled to other molecules selectively in the presence of amines (Eq. 2). This selectivity makes the thiol group the linker of choice for coupling mAb and ADA:
  • lgM disulfide bridge lmmunoglobulin M
  • DTT dithiothreitol
  • thiolating crosslinking reagents such as Tra- ut's reagent (2-iminothiolane), succinimidyl (acetylthio) acetate (SATA), and sul- fosuccinimidyl 6-[3-(2-pyridyldithio) propionamidojhexanoate (Sulfo-LC-SPDP) provide efficient ways of introducing multiple sulfhydryl groups via reactive ami no groups.
  • Carboxylic acids aspartic acid, Asp, glutamic acid, Glu
  • mAbs contain carboxylic acid groups at the C -terminal position and within the side chains of Asp and Glu.
  • carboxylic acids in water usually makes it difficult to use these groups to selectively modify mAbs.
  • carboxylic acid group is usually converted to a reac tive ester by the use of a water-soluble carbodiimide and reacted with a nucleo philic reagent such as an amine, hydrazide, or hydrazine.
  • a nucleo philic reagent such as an amine, hydrazide, or hydrazine.
  • the amine-containing reagent should be weakly basic in order to react selectively with the activated carboxylic acid in the presence of other amines on the protein.
  • mAb-ADA cross- linking can occur when the pH is raised above 8.0.
  • Sodium periodate can be used to oxidize the alcohol part of the sugar within the carbohydrate moiety of mAbs to an aldehyde.
  • Each group can be react ed with an amine, hydrazide, or hydrazine as described for carboxylic acids. Since the carbohydrate moiety is predominantly found on the Fc region of the mAbs, conjugation can be achieved through site-directed modification of the carbohy drate away from the antigen-binding site.
  • amine-reactive reagents react primarily with lysines and the a-amino groups of proteins. Some amine-reactive reagents are more reactive, and therefore, less sensitive, than others lt is necessary to consider reactivity when choosing the best reagent for modification of a specific protein. Table 1 lists commercially available amine-reactive reagents.
  • Reactive esters particularly N-hydroxy-succinimide (NHS) esters
  • NHS N-hydroxy-succinimide
  • They are moderately reactive toward amines, with high selectivity toward ali phatic amines. Their reaction rate with aromatic amines, alcohols, phenols, and histidine is relatively low.
  • the optimum pH for reaction in an aqueous environ- ment is 8.0 to 9.0, and they form very stable aliphatic amine products.
  • the NHS esters are slowly hydrolyzed by water but are stable if stored well desiccated. Virtually any molecule that contains a carboxylic acid, or that can be chemically modified to contain a carboxylic acid, can be converted into its NHS ester.
  • lsothiocyanates behave like NHS esters. They are amine-modification reagents of intermediate reactivity and form thiourea bonds with proteins. They are somewhat more stable in water than NHS esters and react with protein amines in aqueous solution (optimally at pH 9.0 to 9.5). Since this is a higher pH than optimal for NHS esters (which undergo competing hydrolysis at pH 9.0 to 9.5), isothiocyanates may not be as suitable as NHS esters when modifying pro teins that are sensitive to alkaline conditions.
  • Aldehyde groups react under mild aqueous conditions with aliphatic and aromatic amines, hydrazines, and hydrazides to form an imine intermediate (Schiffs base).
  • a Schiffs base can be selectively reduced with mild or strong re ducing agents (such as sodium boro hydride or sodium cyanoboro hydride) to de rive a stable alkyl amine bond.
  • mild or strong re ducing agents such as sodium boro hydride or sodium cyanoboro hydride
  • This method modification of amine can successful ly be employed in situations in which the mAb is modified away from the antigen - binding site via the oxidation (typically with sodium periodate) of the alcohols on the carbohydrate moiety of the Fc region.
  • reagents that have been used to modify amines are acid anhy drides.
  • diethylenetriaminepentaacetic anhydride DTPA
  • DTPA diethylenetriaminepentaacetic anhydride
  • the anhydride rings open to create multivalent, metal -chelating arms able to bind tightly to metals in a coordination complex. This type of reaction is particularly useful in the preparation of radiolabeled immunoconjugates.
  • Thiol-reactive reagents are those that will couple to thiol groups on proteins, forming thioether-coupled products. These reagents react rapidly at slight acidic to neutral pH and therefore can be reacted selectively in the presence of amine groups. 2.2.1. Haloacetyl derivatives (formation of a thioether bond)
  • reagents usually iodoacetamides
  • thiol modification ln mAbs the reaction takes place at cysteines that are either intrinsically present or that result from the reduction of cystine's disulfides at various positions of the mAb.
  • the thioether linkages formed from any reaction of haloacetamides are very stable.
  • iodoacetamide modification reagents are unstable in light, especially in solution. They must be protected from light during reaction and in storage. The level of control to achieve reproducibility re quired for large scale manufacturing conditions may be difficult to achieve.
  • maleimides react rapidly at slight acidic to neu tral pH. Above pH 8.0, maleimides can undergo hydrolysis to form nonreactive maleamic acids.
  • Amines, hydrazides, and hydrazines can be coupled to carboxylic acids of proteins after the activation of the carboxyl group by a water-soluble car- bodiimide.
  • the amine-containing reagent must be weakly basic so that it reacts selectively with the carbodiimide -activated protein in the presence of the more highly basic e-amines of lysine to form a stable amide bond.
  • Amines, hydrazides, and hydrazines also can react with aldehyde groups, which can be generated on mAbs by periodate oxidation of the carbohy drate residues on the mAb. ln this scenario, a Schiff s base intermediate is formed, which can be reduced to an alkyl amine through the reduction of the intermediate with sodium cyanoborohydride (mild and selective) or sodium borohydride (strong) water-soluble reducing agents.
  • Bifunctional crosslinking reagents are specialized reagents that will form a bond between different groups, either on the same molecule or two differ ent molecules. These reagents can be divided into two kinds, homobifunctional reagents (those with the same reactive group at each end of the molecule) and heterobifunctional reagents (those with different reactive groups at each end of the molecule). Recent trends appear to strongly favor the use of heterobifunc tional cross-linkers where the bifunctional reagent has two reactive sites, each with selectivity toward different functional groups (for example, an amine reac tive and a thiol reactive). Many bifunctional reagents are commercially available with variable chain lengths and water solubility.
  • ADA can be fused to the carboxy-terminus (C-terminus) of the heavy (A), light (B) or both (C) chains of an antibody.
  • ADA can be fused with a single chain antibody fragment consisting of the variable fragment (Fv) and frag ment crystallizable region (Fc) (D).
  • ADA can be fused with a single chain Fv as a monomer (E) or dimer through ADA1 crosslinking (F).
  • ADA can be fused with tandem format Fv stabilized by disulfide bond (G).
  • Antigen-binding fragment (Fab) can be linked to one (H) or two (1) ADA molecules.
  • ADA1 can be located be tween single chain Fv and Fc (J).
  • ADA1 can be fused to the CHI domain which is linked by lgG3 hinge region to single chain Fv (K). ADA1 can be fused with af- fibody or similar peptides (L) In all these constructs convenient linker sequences can be introduced to facilitate construction and/or to avoid steric problems. ADA1 can also be cross-linked with some non-protein compounds, capable of binding to ecto-enzymes, such as enzymatic inhibitors (M).
  • antibodies are used as non-limiting examples of the present binding bodies.
  • the exam ples apply to other types of binding bodies as well, as is readily understood by those skilled in the art.
  • adenosine deaminase 1 conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds proteins of adenosine signaling pathway overexpressed on tumor or cancer cells.
  • adenosine deaminase 1 conjugated or fused with human or humanized antibody, or antigen binding portion thereof, which specifically binds the human ecto-5'nucleotidase CD73.
  • adenosine deaminase 1 conjugated with or fused with human or humanized antibody, or antigen binding portion thereof, which specifically binds the human ecto- nucleoside triphosphate phosphohydrolase CD39.
  • adenosine deaminase 1 conjugated or fused with human or humanized antibody, or antigen binding portion thereof, which specifically binds a human adenosine receptor.
  • adenosine deaminase 1 conjugated or fused with or human ized human antibody, or antigen binding portion thereof, which specifically binds the human adenosine receptor Al.
  • adenosine deaminase 1 conjugated or fused with or human ized human antibody, or antigen binding portion thereof, which specifically binds the human adenosine receptor A2a.
  • adenosine deaminase 2 conjugated or fused with human or humanized antibody, or antigen binding portion thereof, which specifically binds proteins of adenosine signaling pathway overexpressed on tumor or cancer cells.
  • adenosine deaminase 2 conjugated or fused with human or humanized antibody, or antigen binding portion thereof, which specifically binds the human ecto-5'nucleotidase CD73.
  • adenosine deaminase 2 conjugated or fused with human or humanized antibody, or antigen binding portion thereof, which specifically binds the human ecto- nucleoside triphosphate phosphohydrolase CD39.
  • adenosine deaminase 2 conjugated or fused with human or humanized antibody, or antigen binding portion thereof, which specifically binds a human adenosine receptor.
  • adenosine deaminase 2 conjugated or fused with human or humanized antibody, or antigen binding portion thereof, which specifically binds the human adenosine receptor A2a.
  • cancer antigen selected from the group consisting of colorectal cancer, pancreatic cancer, bladder cancer, leu kemia, lymphoma, glioma, glioblastoma, melanoma, ovarian cancer, thyroid can cer, esophageal cancer, prostate cancer, and breast cancer.
  • bisAb A human or humanized bispecific antibody (bisAb), one site of which specifically binds a human adenosine deaminase (hADA) and another spe cifically binds an antigen overexpessed on tumor or cancer cells (cancer antigen, CA).
  • a human or humanized bisAb one site of which specifically binds a hADA and another specifically binds the human ecto- 5 'nucleotidase CD73.
  • a human or humanized bisAb one site of which specifically binds a hADA and another specifically binds the human ecto- nucleoside triphosphate phosphohydrolase CD39.
  • a human or humanized bisAb one site of which specifically binds a hADA and another specifically binds a human adenosine receptor.
  • a human or humanized bisAb one site of which specifically binds a hADA and another specifically binds the human adenosine receptor Al.
  • a human or humanized bisAb one site of which specifically binds a hADA and another specifically binds the human adenosine receptor A2a.
  • a human or humanized bisAb one site of which specifically binds a hADA and another specifically binds the human adenosine receptor A2b.
  • a human or humanized bisAb one site of which specifically binds a hADA and another specifically binds the human adenosine receptor A3.
  • a human or humanized bisAb one site of which specifically binds the hADAl and another specifically binds the human ecto -5 'nucleotidase CD73.
  • a human or humanized bisAb one site of which specifically binds the hADAl and another specifically binds the human ecto- nucleoside triphos phate phosphohydrolase CD39.
  • a human or humanized bisAb one site of which specifically binds the hADAl and another specifically binds a human adenosine receptor.
  • a human or humanized bisAb one site of which specifically binds the hADAl and another specifically binds the human adenosine receptor Al.
  • a human or humanized bisAb one site of which specifically binds the hADAl and another specifically binds the human adenosine receptor A2a.
  • a human or humanized bisAb one site of which specifically binds the hADAl and another specifically binds the human adenosine receptor A2b.
  • a human or humanized bisAb one site of which specifically binds the hADAl and another specifically binds the human adenosine receptor A3.
  • a human or humanized bisAb one site of which specifically binds the hADA2 and another specifically binds the human ecto -5 'nucleotidase CD73.
  • a human or humanized bisAb one site of which specifically binds the hADA2 and another specifically binds the human ecto- nucleoside triphos phate phosphohydrolase CD39.
  • a human or humanized bisAb one site of which specifically binds the hADA2 and another specifically binds a human adenosine receptor.
  • a human or humanized bisAb one site of which specifically binds the hADA2 and another specifically binds the human adenosine receptor Al.
  • a human or humanized bisAb one site of which specifically binds the hADA2 and another specifically binds the human adenosine receptor A2a.
  • a human or humanized bisAb one site of which specifically binds the hADA2 and another specifically binds the human adenosine receptor A2b.
  • a human or humanized bisAb one site of which specifically binds the hADA2 and another specifically binds the human adenosine receptor A3.
  • a human or humanized bisAb according to any one of aspects 27 to 48, where bisAb is of the human lgG4 subtype to avoid recruitment of undesir able pro-inflammatory activities and the human lgG4 is with the S228P mutation to prevent the lgG4 arm exchange.
  • cancer selected from the group consisting of colorectal cancer, pancreatic cancer, bladder cancer, leu kemia, lymphoma, glioma, glioblastoma, melanoma, ovarian cancer, thyroid can cer, esophageal cancer, prostate cancer, and breast cancer.
  • a human or humanized bispecific antibody, or antigen binding por tions thereof one site of which specifically binds a human adenosine deaminase and another specifically binds to an ecto-enzyme, homologue of one of ecto- enzymes of mammalian adenosine signaling pathway, on a surface of bacterial pathogen.
  • a human or humanized bispecific antibody, or antigen binding por tions thereof one site of which specifically binds to a human adenosine deami nase and another specifically binds to an ecto-enzyme, homologue of one of ecto- enzymes of mammalian adenosine signaling pathway, on a surface of Gram negative pathogen.
  • a human or humanized bispecific antibody, or antigen binding por tions thereof one site of which specifically binds a human adenosine deaminase and another specifically binds to an ecto-enzyme, homologue of one of ecto- enzymes of mammalian adenosine signaling pathway, on a surface of Gram positive pathogen.
  • a human or humanized bispecific antibody, or antigen binding por tions thereof one site of which specifically binds to a human adenosine deami nase and another specifically binds to the ecto -nucleoside triphosphate diphos- phohydrolase on a surface of Gram-negative pathogen.
  • a human or humanized bispecific antibody, or antigen binding por tions thereof one site of which specifically binds to a human adenosine deami nase and another specifically binds to the ecto-5'-nucleotidase on a surface of Gram-positive pathogen.
  • a human or humanized bispecific antibody, or antigen binding por tions thereof one site of which specifically binds to a human adenosine deami nase 1 and another specifically binds to an ecto -enzyme, homologue of one of ec- to-enzymes of mammalian adenosine signaling pathway, on a surface of bacterial pathogen.
  • a human or humanized bispecific antibody, or antigen binding por tions thereof one site of which specifically binds to a human adenosine deami nase 1 and another specifically binds to an ecto -enzyme, homologue of one of ec- to-enzymes of mammalian adenosine signaling pathway, on a surface of Gram negative pathogen.
  • a human or humanized bispecific antibody, or antigen binding por tions thereof one site of which specifically binds to a human adenosine deami nase 1 and another specifically binds to an ecto-enzyme, homologue of one of ec- to-enzymes of mammalian adenosine signaling pathway, on a surface of Gram positive pathogen.
  • a human or humanized bispecific antibody, or antigen binding por tions thereof one site of which specifically binds to a human adenosine deami nase 1 and another specifically binds to the ecto -nucleoside triphosphate diphos- phohydrolase on a surface of Gram-negative pathogen.
  • a human or humanized bispecific antibody, or antigen binding por tions thereof one site of which specifically binds to a human adenosine deami nase 1 and another specifically binds to the ecto-5'-nucleotidase on a surface of Gram-positive pathogen.
  • a human or humanized bispecific antibody, or antigen binding por tions thereof one site of which specifically binds to a human adenosine deami nase 2 and another specifically binds to a bacterial antigen on a surface of Gram positive pathogen.
  • a human or humanized bispecific antibody, or antigen binding por tions thereof one site of which specifically binds to a human adenosine deami nase 2 and another specifically binds to an ecto -enzyme, homologue of one of ec- to-enzymes of mammalian adenosine signaling pathway, on a surface of bacterial pathogen.
  • a human or humanized bispecific antibody, or antigen binding por tions thereof one site of which specifically binds to a human adenosine deami nase 2 and another specifically binds to an ecto -enzyme, homologue of one of ec- to-enzymes of mammalian adenosine signaling pathway, on a surface of Gram negative pathogen.
  • a human or humanized bispecific antibody, or antigen binding por tions thereof one site of which specifically binds to a human adenosine deami nase 2 and another specifically binds to an ecto -enzyme, homologue of one of ec- to-enzymes of mammalian adenosine signaling pathway, on a surface of Gram positive pathogen.
  • a human or humanized bispecific antibody, or antigen binding por tions thereof one site of which specifically binds to a human adenosine deami nase 2 and another specifically binds to the ecto -nucleoside triphosphate diphos- phohydrolase on a surface of Gram-negative pathogen.
  • a human or humanized bispecific antibody, or antigen binding portions thereof one site of which specifically binds to a human adenosine deam inase 2 and another specifically binds to the ecto-5'-nucleotidase on a surface of Gram-positive pathogen.
  • a human or humanized bispecific antibody, or antigen binding portions thereof one site of which specifically binds to a human adeno sine deaminase and another specifically binds to a bacterial antigen on a surface of bacterial pathogen according to any one of aspects 77 to 100 in the manufac ture of a medicament for treating a bacterial infection.
  • Method of producing ADA2 in which method the raw material is a medical human immunoglobulin preparation.
  • adenosine deaminase 1 conjugated or fused with or human ized human antibody, or antigen binding portion thereof, which specifically binds alkaline phosphatase.
  • adenosine deaminase 2 conjugated or fused with human or humanized antibody, or antigen binding portion thereof, which specifically binds alkaline phosphatase.
  • adenosine deaminase conjugated or fused with human or humanized antibody, or antigen binding portion thereof according to any one of aspects 103 to 105 in the manufacture of a medicament for treating a tumor or cancer.
  • cancer is selected from the group consisting of colorectal cancer, pancreatic cancer, bladder cancer, leukemia, lymphoma, glioma, glioblastoma, melanoma, ovarian cancer, thyroid cancer, esophageal cancer, prostate cancer, and breast cancer.
  • a human or humanized bisAb one site of which specifically binds a hADA and the other specifically binds alkaline phosphatase.
  • a human or humanized bisAb one site of which specifically binds the hADAl and another specifically binds alkaline phosphatase.
  • a human or humanized bisAb one site of which specifically binds the hADA2 and another specifically binds alkaline phosphatase.
  • cancer is selected from the group consisting of colorectal cancer, pancreatic cancer, bladder cancer, leukemia, lymphoma, glioma, glioblastoma, melanoma, ovarian cancer, thyroid cancer, esophageal cancer, prostate cancer, and breast cancer.
  • the open reading frames (ORFs] of the genes were PCR amplified with the following primers: (hADAl-F] 5'- AGCTCGAGACCGGTCCACCATGGCCCAGACGCCCGCCT-3' (SEQ 1D NO:l), (hADAl - R) 5'-ATGGATCCGCTAGCTCAGAGGTTCTGCCCTGCAG-3' (SEQ 1D NO:2), (mADAl- F] 5’- AGCTCGAGACCGGTCCACCATGGCCCAGACACCCGCAT-3’ (SEQ 1D NO:3] and (mADAIR] 5’- TCAGATCTATTGGTATTCTCTGTAGAGC-3’ (SEQ 1D N0:4).
  • PCR products were then subcloned into the pCR2.1-TOPO plasmid, excised by re striction digest with Xhol and BamH 1, and ligated into a Xho ⁇ / BamHl-digested self-inactivating (SIN] transfer plasmid (pHR-cPPT-hB7-S!N).
  • HEK-293T cells were transfected with the transfer, VSV-G envelope and AR 8.2 packaging plas mids using the calcium phosphate transfection reagent. Subsequently, lentiviral particles were purified by ultracentrifugation from the cell culture medium of HEK-293T cells and used to infect a new patch of HEK-293T cells.
  • the cells were grown in complete Dulbecco's Modified Eagle Medium (DMEM) medium (Sigma- Aldrich], supplemented with 5% FBS, lOO U/ml penicillin, 100 pg/ml streptomy cin, and 2 mM L-glutamine.
  • DMEM Dulbecco's Modified Eagle Medium
  • the cells were trypsinized with trypsin/EDTA (Sigma- Aldrich], centrifuged for 5 min at 300 g, and the cell pellet was frozen in liquid nitrogen and kept at -80 °C.
  • the recombinant human and mouse ADA1 were puri fied from the HEK-293T cell lysates.
  • Frozen HEK-293T cells expressing hA- DAl/mADAl were lysed through three freeze/thaw cycles, using liquid nitrogen and a 42 °C water bath.
  • the cell lysate was re-suspended in 50 mL of ice-cold 50 mM Tris-HCl buffer (pH 6.8], 50 mM NaCl, 10 mM Zn(AcO] 2 , and 0.02% NaN 3 (Buffer A1 for hADAl] or 50 mM Tris-HCl buffer pH 6.8, 100 mM NaCl, 10 mM ZnCl2, 0.02% NaN 3 (Buffer A2 for mADAl].
  • the supernatant was separated from the cell debris by centrifugation at 4,000 g for 20 min, filtered using 0.45 pm fil ters (Sigma-Aldrich], and applied onto a DEAE Sepharose (GE Healthcare] equili brated with Buffer A1 (hADAl] or Buffer A2 (mADAl].
  • the flow through contain ing ADA1 was collected, and the pH was adjusted to 8.4 with Tris base (hADAl] or 8.5 with MQ and 1 M Tris-HCl buffer, pH 9.5 (mADAl].
  • the hADA and mADA en zymes were applied onto a DEAE Sepharose columns equilibrated with Buffer B (50 mM Tris-HCl pH 8.5, 10 mM Zn(AcO] 2 , 0.02% NaN 3 ].
  • Buffer B 50 mM Tris-HCl pH 8.5, 10 mM Zn(AcO] 2 , 0.02% NaN 3 ].
  • the ADA1 bound to the column was eluted using 0-500 mM NaCl gradient.
  • the fractions containing ADA activity were pooled, concentrated using 10 kDa centrifuge ultraconcentrators (Millipore), and further purified on a Superdex 200 column (GE Healthcare), equilibrated with phosphate-buffered saline (PBS) containing 10 mM Zn(AcO) 2 and 0.02% NaN 3 .
  • PBS phosphate-buffered saline
  • Concentrations of purified ADA were determined spectroscopi cally by measuring the absorbance at 280 nm.
  • the kinetic parameters of the re combinant human ADA1 and its activity were determined as described before (Zavialov A. & Engstrom A., 2005, The Biochem. J. 391, pp. 51-57; Zhou Q. et al., 2014, N. Engl. J. Med. 370, pp. 911-920).
  • SDS-PAGE analysis confirmed the high purity of the human and mouse ADA1 enzymes (Fig. 14).
  • hADAl and a human mAb specific to human ecto-enzymes involved in adenosine production as exemplified in Fig. 1 (AgBS is the antigen binding site)
  • hADAl and an anti-CD73 monoclonal antibody (mAb) were biotinylated.
  • the purified hADAl and the 4G4 mouse anti- human CD73 mAb were biotinylated using EZ-LinkTM NHS-PEG4-Biotin, No-WeighTM Format (TermoFisher, cat#A39259).
  • the samples were dialyzed in Spectra-Por® Float-A- Lyzer® G2 (MWCO 20kDa) against PBS in order to remove traces of primer amines and adjusted to about 5 mg/mL concentration lmmediately before use, ultrapure water was added to 2 mg of NHS-PEG4-Biotin to prepare a 20 mM stock solution. Next, NHS-PEG4-Biotin solution was added at 31 -fold excess compared to lgG or hADAl. The reaction mix was incubated at RT for 30 minutes and dia lyzed to remove unbound labels.
  • biotinylated anti-CD73, non-biotinylated anti-CD73 (negative con trol) and biotinylated hADAl were analyzed for biotinylation.
  • Dynabeads® M- 280 Streptavidin beads were washed twice with PBS using a magnet. The beads were resuspended in PBS and divided in four aliquots into 1.5 mL tubes. 5 gg of samples were added to each tube. The mixtures were incubated for 30 min at RT with slow shacking. The beads were collected at the bottom using a magnet. The supernatants were collected and prepared for SDS-PAGE analysis. The beads were washed twice with PBS and resuspended in fresh PBS.
  • Fig. 15B Complex formation was checked by size-exclusion chromatography on Superdex ® 200 lncrease 10/300 GL (GE Healthcare, 28-9909-44) (Fig. 15B).
  • Corresponding strategy is used to conjugate anti-human CD39 or anti-human ADORA 2a with human ADA1.
  • Anti-human antibodies are generated using phage display technology.
  • Fig. 2, Fig. 3 and Fig. 4 show possible mechanisms of anti cancer action of the conjugates consisting of hADAl and a human mAb specific to for CD73, CD39 or ADORA 2a , respectively.
  • Example 3 The mAb-hADAl conjugates specifically target CD73 on breast and prostate cancer cells
  • 4G4-biotin and h5’NT-2L!5-biotin were added to the cells with a final concentration 20 gg/mL, incubated on ice for 1 hour and washed 3x with PBS, 2% FCS, 0.02% NaN 3 .
  • Corresponding secondary antibodies (1:200, mouse-488 to 4G4, rabbit-633 to h5’NT-2L!5) and SA-488, 20 gg/mL (both 4G4 and h5’NT-2L!5) were added, incubated on ice for 1 hour and washed 3x with PBS, 2% FCS, 0.02% NaN 3 .
  • the cells were fixed with PBS, 1% formaldehyde on ice for 15 min and analyzed by flow cytometry.
  • This example describes the generation of a mouse counterpart of the antibody-ADAl conjugate to be used in mouse studies in vivo.
  • the crosslinking was performed via an inverse-electron demand Diels-Alder cycloaddition reaction between trans-cyclooctene (TCO) and tetrazine (Tz).
  • TCO trans-cyclooctene
  • Tz tetrazine
  • Tz- PEG5-NHS or TC0-PEG4-NHS reagent were dissolved in dry DMSO to 10 mM concentration. 20-fold Tz-PEG5-NHS molar excess was added to antibody and 20-fold TC0-PEG4-NHS molar excess was added to ADA1 solution, and incu bated at room temperature for 60 min. Excess reagent was removed using two buffer exchange cycles against BupH (pH 7.5) with ZebaTM Spin Desalting Columns (ThermoFisher) (buffer was exchanged twice in order to minimize the amount of non-reacted reagent).
  • the desired antibody- ADAl stoichiometry of 1:3 was chosen for the conjugation reaction.
  • TCO-modified ADA1 was mixed with Tz-labeled antibody.
  • the conjugation reaction was performed for 60 min at room temperature and analyzed with gel electrophoresis (SDS-PAGE) (Fig. 17). Protein-protein conju gates were stored at 4°C before purification by size-exclusion chromatography.
  • Example 5 The mAb-hADAl conjugate specific to CD73 converts cancer cell- produced adenosine to inosine
  • the ability of mAb-hADAl conjugate to abolish adenosine produced by cancer cells was examined.
  • the PC-3 human prostate carcinoma and MDA-MB- 231 human breast cancer cell lines were obtained from ATCC.
  • the cells were maintained at 37°C in a humidified atmosphere of 5% C0 2 in DMEM containing 10% fetal bovine serum (FBS), 0.03 mg/mL penicillin and 0.05 mg/mL strepto mycin.
  • the cells were harvested with trypsin/EDTA and seeded onto appropriate tissue culture flasks for 24-48 hours until confluent.
  • the medium was replaced by RPM1-1640 containing 1% heat-inactivated FCS (RPM1/FCS).
  • PC-3 and MDA-MB-231 cells were seeded overnight onto 96- well clear plates (8,000 cells per well). The cells were incubated for 1 hour in RPM1/FCS medium with biotinylated anti-CD73 antibody 4G4 (10 gg/mL) with out (control) or with streptavidin-conjugated recombinant human or intestine- purified bovine ADA1 (20-30 gg/mL). The treated cells were incubated for addi tional 60 min with 300 mM [3HJAMP in a final volume of 120 gL RPM1/FCS.
  • the amount of radioactivity was quantified by b-counting after addition of HiSafe-3 scintillation cocktail. To obtain qualitative images (Fig. 18- 19), autoradiographic analysis was performed. The TLC sheets were covered with X-ray films and incubated for several weeks.
  • Example 6 The mAb-hADAl conjugate specific to CD73 restores the anti tumor activity of immune cells
  • Adenosine acts as a strong immunosuppressant preventing the im mune attack on the tumor and therefore, promotes tumor growth.
  • MDA-MB-231 cells were co cultured with human peripheral blood mononuclear cells (PBMC) and the secre tion of TNF-alpha by the immune cells was determined by enzyme-linked im munosorbent assay (EL1SA) (Fig. 21).
  • TNF-alpha is an important signaling protein known to inhibit tumorigenesis and to trigger acute immune response against various infections.
  • Adenosine suppresses the TNF-alpha secretion and measure ment of TNF-alpha concentration allowed to us to estimate immunosuppressive potency of the adenosine.
  • MDA-MB-231 cells TCGA were seeded on 48- well plate (75,000 cells/well) and on the next day, cells were pre-treated with either 1) biotinylated 4G4 anti-CD73 (10 gg/mL), 2) biotinylated hADA (20 gg/n lL) and streptavidin (40 gg/m L) or 3) biotinylated 4G4 anti-CD73, biotinyl ated hADA and streptavidin in DMEM, 5% FBS.
  • Cells without pre-treatment served as controls. The cells were incubated for 10 min at 37°C and washed with DMEM, 5% FBS. On the following day, MDA-MB-231 cultures were supplemented with PBMCs (4xl0 6 cells/mL in RPM1, 5% FBS) and adenosine monophosphate (AMP) and treated with 10 ng/mL lipopolysaccharide (LPS) to stimulate T cells. The cells without AMP substrate served as a negative control. The cells were incubated at 37°C and the supernatants were collected at 15, 30, 60, 75 90 and 120 min and analyzed by EL1SA using the TNF-alpha EL1SA Kit (Sinobiological).
  • the anti-CD73-ADAl antibody conjugate was found to restore the cancer cell-mediated suppression of immune cell activity (Fig. 22 and Fig. 23).
  • this example demonstrates that the mAb-hADAl conjugate is able to reactivate immune cells against cancer cells by reducing adenosine concentra tion in the tumor.
  • Corresponding strategy is used with various types of immune cells such as purified T cells and tumor infiltrating lymphocytes and with the mAb-hADAl conjugate specific to CD39.
  • the immune cells in this experi ment were activated by LPS, which is commonly present on the surface of bacte rial pathogens.
  • the mAb-hADAl conjugate should also be able to reactivate immune cells against bacterial infections.
  • Example 7 Therapy with the ADA-anti-CD73 or ADA-anti-CD39 antibody conjugate inhibits tumor growth more effectively than the anti-CD73 mAh alone
  • This example describes the investigation of whether administration of the mAb-ADAl and mAb-ADA2 conjugates specific to CD73 or CD39 inhibit tumor growth more effectively than the respective mAbs alone and whether they show any additive effects in combination with immune checkpoint inhibitors.
  • the mAb- ADA conjugates are tested using mouse models that recapitulate the complexity of immune contexture within the tumor microenvironment.
  • Mouse cancer models can be classified as immunologically "hot”, “warm”, or "cold” defining the extent to which the immune infiltration of the tumor allows for immune system engage ment and are therefore, applicable for testing therapies acting via the immune system.
  • tumor volumes are measured three times weekly and mean, median and relative tumor volumes (therapy/control, T/C, RTV %) are calculated. The behavior is monitored daily and general condition after each treatment. The body weight is measured twice weekly, and mean body weights and body weight change (BWC %) are calculated.
  • mice C57B1/6 are injected subcutaneously (s.c.) with lxlO 6 MC38 mouse colorectal cancer cells and treated with the mAb-ADA conjugates specific to CD73 or CD39, anti-CD73 or anti-CD39 mAbs, isotype con trol lg or vehicle.
  • the mAb-hADAl and mAb-hADA2 conjugates specific to CD73 or CD39 clearly inhibit tumor growth in MC38 "hot" mouse colorectal cancer model and treatment with the ADA-mAb conjugates delays primary tumor growth more effectively than administration of mAb alone. Similar results are obtained with CT26 "warm" colorectal mouse cancer cell model after s.c. inoculation (lxlO 5 cells) into Balb/C mice.
  • mice immune-competent mice (Balb/C) are injected or- thotopically into the mammary fat pad with lxlO 5 4T1 "cold” mouse mammary carcinoma cells and treated with mAb-ADA conjugates specific to CD73 or CD39, control lg, anti-PD-Ll or mAb-ADA conjugates in combination with anti-PD-Ll.
  • the mAb-hADAl and mAb-hADA2 conjugates specific to CD73 or CD39 clearly inhibit tumor growth in 4T1 "cold" mouse mammary carcinoma model and the combination with immunocheckpoint inhibitor anti-PD-Ll shows additive effects.
  • the conjugates change the orthotopic 4T1 "cold" mouse mammary carcinoma model more responsive to immunocheckpoint inhibition with anti-PD- Ll.
  • mAb-ADA conjugates specific to CD73 or CD39 inhibit lung me tastasis and the groups receiving the combination of ADA-anti-CD73 and anti-PD- 1 have less lung metastases than the respective monotherapy groups. All treat ments are well-tolerated.
  • Figures 3 and 4 illustrate a possible mechanism of anti cancer action of the ADA-anti-CD73 and ADA-anti-CD39 antibody conjugates, re spectively.
  • Corresponding strategy is used in combination therapies with other checkpoint inhibitors, including but not limited to antibodies against cytotoxic T - lymphocyte-associated protein 4 (CTLA4) and programmed death-1 (PD-1).
  • CTLA4 cytotoxic T - lymphocyte-associated protein 4
  • PD-1 programmed death-1
  • Example 8 The anti-ADA2 part of the bispecific antibody (bisAb) is capable of extracting ADA2 from blood
  • the Hll anti-ADA2 single chain monoclonal Ab (generated by Applicants using phage display technology) was immobilized on Ni-NTA Sepharose resin. Briefly, the Ni-NTA Sepharose resin was washed twice with 0.05% Tween 20 in PBS using centrifugation (1 min, 10,000 RPM) and resuspended in 1% bovine serum albumin (BSA) in PBS. The Hll Ab (1.9 gg) was added to the resin and the tube was rotated for 30 min at room temperature. The resin was washed twice with 0.05% Tween 20 in PBS and once with PBS.
  • BSA bovine serum albumin
  • the supernatant was discarded.
  • the Ni-NTA Sepharose pellet was resuspended in human plasma and the tube was rotated for 1 h at room tempera ture. The tube was centrifuged (1 min, 10,000 RPM), the supernatant was collect ed (SI) and the pellet was resuspended in 200 mM imidazole in PBS, pH 7.3 and spun down (1 min, 10,000 RPM). The supernatant was collected and diluted in PBS (S2). On a 96-well plate, SI and S2 were mixed with 10 mM adenosine in 50 mM Tris HC1 pH 6.8 and incubated for 4 h at 37°C.
  • the concentrations were measured using a standard curve with serial 1.5 dilutions. The 265/245 ratios were measured using the UV Costar plate. An initial concentration of 128.8 ⁇ 7.4 ng/mL ADA2 in plasma was detected. After pull-down with the Hll anti-ADA2, the remaining ADA2 concentration in plasma was 5.0 ⁇ 4.2 ng/mL, which was below the detection limit. The concentration of ADA2 bound to Hll was 104.4 ⁇ 9.0 ng/mL (Fig. 24).
  • Example 9 Therapy with the bisAb (anti-ADA2, anti-CD73) inhibits tumor growth in mice more effectively than anti-CD73 mAh alone
  • the Ab specific to purified hADAl is chemically conjugated with Abs specific to CD73, CD39 or AD0RA 2a as exemplified in Fig. 5.
  • mice are injected s.c. with cancer cells and treated with bisAb (anti-ADAl, anti-CD73), anti-CD73 mAb or control lg. Treatment with the bisAb (anti-ADAl, anti-CD73) delays primary tumor growth more effectively than administration of anti-CD73 mAb alone.
  • Fig. 6, Fig. 7 and Fig. 8 show possible mechanisms of anti-cancer action of the bisAbs specific for ADA1 and CD73, CD39 or ADORA 2a , respectively.
  • the Ab-ADAl conjugate specific to 5'NDase of Staphylococcus aureus or the bisAb (anti-ADAl, anti-5'NDase) specific to 5'NDase of S. aure us inhibits survival of S. aureus in blood obtained from human volunteers more effectively than anti-5'NDase Ab alone
  • the Ab produced to purified bacterial antigen (BA) is chemically con jugated with hADAl.
  • the design of bisAb, one site of which specifically binds to a hADA (ADA1 or ADA2) and another site of which binds to bacterial antigens (BA), is shown at Fig. 9.
  • This example describes how addition of the Ab-ADAl conjugate specif ic to 5’NDase of S. aureus or the bisAb (anti-ADAl, anti-5’NDase) inhibits survival of S. aureus in blood from human volunteers more effectively than anti-5’NDase Ab alone.
  • 10 5 colony forming units (CFU) of S. aureus Newman are incubated with 1 mL of blood from human volunteers, and Giemsa-stained samples were viewed by microscopy before and after incubation. Before incuba tion, only extracellular staphylococci are detected, whereas after incubation staphylococci are mostly associated with neutrophils.
  • Fig. 10 shows a possible mechanism of anti-bacterial action of an antibody (Ab) conjugated with hADA (hADAl or hADA2), which specifically binds to a bacterial antigen (BA) (ecto-5’NDase is selected as example).
  • Fig. 11 shows a possible mechanism of anti -bacterial action of a bisAb, one site of which specifically binds to hADA (hADAl or hADA2) and another specifically binds to an BA (ecto-5’NDase is selected as example).
  • Example 11 The Ab-ADAl conjugate specific to 5'NDase of Streptococcus agalactiae or the bisAb (anti-ADAl, anti-5'NDase) specific to 5'NDase of S. agalactiae inhibits survival of S. agalactiae in blood obtained from mice more effectively than anti-5'NDase Ab alone
  • This example describes how addition of the Ab-ADAl conjugate specif ic to 5’NDase of S. agalactiae or the bisAb (anti-ADAl, anti-5’NDase) inhibits sur vival of S. agalactiae in mice more effectively than anti-5’NDase Ab alone.
  • Whole blood is collected by cardiac puncture of mice into tubes containing anticoagulant (Vacuette Premium, Lithium Heparin Ridged, Greiner Bio -One). The blood from 10 mice is pooled and used for the experiment within 15 min. Overnight cultures of S. agalactiae are diluted 1:100 into fresh TH broth and grown at 37°C to midexponential phase.
  • Bacterial cells are centrifuged, washed, and diluted in PBS. Bacteria are mixed with mouse blood and the solution is incubated at 37°C under constant gentle agitation. Giemsa-stained samples are viewed by microscopy.
  • the addition of the 10 gg Ab-ADAl conjugate specific to 5’NDase of S. agalactiae or the 10 gg bisAb (anti-ADAl, anti-5’NDase) leads to the phagocytic killing of bacte ria after incubation, while after the addition of 10 gg of anti-5’NDase Ab alone a notable proportion of streptococci survive.
  • Example 12 The Ab-ADAl conjugate specific to ecto-NTPDase Lpgl905 of Legionella pneumophila or the bisAb (anti-ADAl, anti-ecto-NTPDase Lpgl905) specific to ecto-NTPDase Lpgl905 inhibits replication of L. pneu mophila in THP-1 macrophages more effectively than anti-ecto-NTPDase Lpgl905 Ab alone
  • This example describes how addition of the Ab-ADAl conjugate specif ic to ecto-NTPDase Lpgl905 or the bisAb (anti-ADAl, anti-ecto-NTPDase Lpgl905) specific to ecto-NTPDase Lpgl905 inhibits replication of L. pneumophi la in THP-1 macrophages more effectively than anti-ecto-NTPDase Lpgl905 Ab alone.
  • the human monocytic cell line, THP-1 is maintained in RPM1 1640 medium, supplemented with 10% fetal bovine serum at 37°C, 5% C0 2 .
  • THP-1 cells Prior to infection, THP-1 cells are seeded into 24-well tissue culture trays (Sarstedt, Leicestershire, UK) and pretreated with 10 8 M phorbol 12-myristate 13 -acetate for 36-48 h to induce differentiation into adherent macrophage-like cells.
  • L. pneumophila strain is grown for 72 h at 37°C on BCYE agar, resuspended in tissue culture media and added to THP-1 cells at a multiplicity of infection (moi) of 5. After incubation, cells are treated with 100 gg/mL gentamicin to kill extracellular bacteria lnfected macrophages are then washed three times with PBS before incubation with tissue culture maintenance media.
  • THP-1 cells are washed at different timepoints and lysed with 0.05% digitonin. lntracellular bacteria are enumerated by serial dilu tion and plating onto BCYE agar.
  • the addition of the 10 gg Ab-ADAl conjugate specific to ecto-NTPDase Lpgl905 of L. pneumophila or the 10 gg bisAb (anti- ADAl, anti- ecto-NTPDase Lpgl905) leads to almost complete inhibition of repli cation of L. pneumophila in THP-1 macrophages and shows improved efficacy as compared to 10 gg anti-ecto-NTPDase Lpgl905 Ab alone.
  • Figure 12 shows a pos sible mechanism of anti-bacterial action of an antibody (Ab) conjugated with hA- DA (hADAl or hADA2) which specifically binds to a bacterial antigen (BA) (ecto- NTPDase is selected as example).
  • Figure 13 shows a possible mechanism of anti bacterial action of a bisAb, one site of which specifically binds to hADA (hADAl or hADA2) and another specifically binds to an BA (ecto-NTPDase is selected as ex ample).

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Abstract

La présente invention concerne la médecine humaine, plus spécifiquement la thérapie anticancéreuse et le traitement d'infections bactériennes. L'invention concerne de nouveaux corps de liaison, tels que des anticorps, qui se lient spécifiquement à une protéine d'une voie de signalisation de l'adénosine, et qui sont conjugués ou fusionnés avec l'adénosine désaminase humaine (hADA) ou qui sont capables de se lier à hADA. L'invention concerne également des compositions pharmaceutiques comprenant lesdits corps de liaison, et des procédés de traitement du cancer ou d'infections bactériennes au moyen desdits corps de liaison ou desdites compositions pharmaceutiques.
PCT/FI2019/050169 2018-03-02 2019-03-01 Anticorps anti-voie de signalisation de l'adénosine conjugués ou fusionnés avec de l'adénosine désaminase ou capables de se lier à l'adénosine désaminase WO2019166701A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2023201267A1 (fr) 2022-04-13 2023-10-19 Gilead Sciences, Inc. Polythérapie pour le traitement de cancers exprimant trop-2

Citations (2)

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WO2016061286A2 (fr) * 2014-10-14 2016-04-21 Halozyme, Inc. Compositions d'adénosine désaminase-2 (ada2), variants de cette dernière et leurs procédés d'utilisation
WO2016191283A2 (fr) 2015-05-22 2016-12-01 University Of Houston System Immunomodulation enzymatique de tumeurs solides et utilisateur associé

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WO2016061286A2 (fr) * 2014-10-14 2016-04-21 Halozyme, Inc. Compositions d'adénosine désaminase-2 (ada2), variants de cette dernière et leurs procédés d'utilisation
WO2016191283A2 (fr) 2015-05-22 2016-12-01 University Of Houston System Immunomodulation enzymatique de tumeurs solides et utilisateur associé

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023201267A1 (fr) 2022-04-13 2023-10-19 Gilead Sciences, Inc. Polythérapie pour le traitement de cancers exprimant trop-2

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