WO2019166701A1

WO2019166701A1 - Anti-adenosine signaling pathway antibodies conjugated or fused with adenosine deaminase or capable of binding adenosine deaminase

Info

Publication number: WO2019166701A1
Application number: PCT/FI2019/050169
Authority: WO
Inventors: Anton Zavialov; Vladimir ZAVIYALOV; Gennady YEGUTKIN; Maksym SKALDIN; Andrey ZAVIALOV
Original assignee: University of Turku
Current assignee: University of Turku
Priority date: 2018-03-02
Filing date: 2019-03-01
Publication date: 2019-09-06
Anticipated expiration: 2020-09-02

Abstract

The present disclosure relates to human medicine, more specifically to cancer therapy and treatment of bacterial infections. The invention provides novel binding bodies, such as antibodies, which specifically bind to a protein of an adenosine signaling pathway, and is conjugated or fused with human adenosine deaminase (hADA) or is capable of binding hADA. Also provided are pharmaceutical compositions comprising said binding bodies, and methods of treating cancer or bacterial infections by said binding bodies or by said pharmaceutical compositions.

Description

ANTI-ADENOSINE SIGNALING PATHWAY ANTIBODIES CONJUGATED OR FUSED

WITH ADENOSINE DEAMINASE OR CAPABLE OF BINDING ADENOSINE DEAMINASE

F1ELD OF THE INVENTION

The present invention relates to human medicine, and more specifical ly to the treatment or prevention of cancer and bacterial infections.

BACKGROUND OF THE INVENTION

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). This is achieved by an action of three 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. However, CD39 and, particularly, CD73 are overexpressed in the hypoxic tumor microenviron ment resulting in accumulation of high levels of adenosine that strongly suppress the anti-cancer immune response ln addition, adenosine may be produced by alkaline phosphatase (AP). 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.

Humans have two types of ADA, ADA1 and ADA2. 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.

A number of studies have investigated the potential of targeting the adenosine pathway to improve anti-tumor immunity in the tumor microenviron ment. These include monoclonal antibodies (mAbs) to 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. 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. Hypoxia, occurring in many cancers, has been shown to upregulate the expression of ADOR A2a and A2b thereby increasing cell re sponsiveness to adenosine. Therefore, 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. Although selective cancer treatment by tar geting tumor-associated antigens with anti-cancer agents is a well-recognized strategy, this approach is often difficult to implement in practice. Such treatments cannot be applied for a broad range of cancers, because different types of cancer possess different tumor associated antigens. Moreover, for many types of cancers, tumor-specific antigens are not known or are present in quantities too small to be useful for the treatment. Hence, finding of ubiquitous and abundant targets for cancer treatments, in particularly for the adenosine elimination approach, is of critical importance.

During the last decade the evidences were obtained that also bacterial pathogens exploit enzymes of adenosine pathway as a powerful virulence factors to escape host immune response. For the first time such a phenomenon was iden tified for Legionella pneumophila, a Gram-negative pathogen, the causative agent of legionnares’ disease or legionellosis, a severe form of acute pneumonia. 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). The study of the ability of S. aureus to es cape phagocytic clearance in blood led to the finding of adenosine synthase A (AdsA), a cell wall-anchored enzyme that converts AMP to adenosine, as a critical virulence factor. 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. One of them, ecto-5’-nucleotidase (Nt5e), is a putative surface-located enzyme that may hydrolyze extracellular ATP, ADP, AMP to adenosine ln animal model of endocarditis, it was shown that Nt5e deletion attenuates virulence. Cell-surface Nt5e activities have been described also in Heli cobacter pylori and Streptococcus gordonii. AMPases on pathogens like Staphylo coccus epidermidis and Enterococcus faecilis can also generate immunosuppres sive adenosine. Therefore, the nucleotidase activity of Nt5e is a common virulence factor for the survival of bacterial pathogens in the blood.

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. In vivo assays in animal models of infection showed that NudP activity is crit ical for virulence.

New and improved therapeutics for treating cancer as well as bacterial infections are needed.

BR1EF DESCRIPTION OF THE INVENTION

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.

Accordingly, in one aspect 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.

ln another aspect, the present invention provides a pharmaceutical composition comprising a binding body according to the present invention and a pharmaceutically acceptable carrier.

ln further aspects, 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.

ln still further aspects, 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.

Other objectives, embodiments, details and advantages of the present invention will become apparent from the following figures, detailed description, exemplary aspects, examples, and dependent claims.

BR1EF DESCRIPTION OF THE DRAW1NGS

ln the following, the invention will be described in greater detail by means of preferred embodiments with reference to the attached drawings, in which

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).

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).

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).

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).

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).

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 ³H-labeled adenosine from the [³H]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 ³H-labeled adenosine from the [³H]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 investi 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.

Figure 25 shows exemplary ways of generation of ADA-binding body fusion proteins and ADA-small molecule-binding body conjugates. DETA1LED DESCRIPTION OF THE INVENTION

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). 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.

As used herein, 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.

As used herein, the term "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.

As used herein, the term "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. The term "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. The term "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.

As used herein, the term "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. Members of the human 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. Non-limiting examples the members of the bacterial adenosine signaling pathway include ecto-nucleoside triphosphate di- phosphohydrolase (ecto-NTPDase) and ecto-5'-nucleotidase (ecto-5’NDase).

ln some embodiments, 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.

Accordingly, 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. 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. This way the accu mulation of adenosine can be blocked by two simultaneous approaches. Although the overall release of adenosine would be decreased due to the blockage of the surface enzyme by the antibody, the local concentration of adenosine at the very site of the cell surface enzyme is yet high. Therefore, on one hand, the therapeutic antibody is employed to decrease the amount of adenosine produced, and on the other hand, 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. This makes the system very effective compared to the therapeutic antibodies alone lndeed, as demonstrated in Example 5, hADA-conjugated with an antibody specific for an enzyme involved in adenosine production was more effective in eliminating extracellular adenosine in vitro than either of the compo nents alone. Furthermore, if the ADA enzymes are administered non-linked with the antibodies, very large doses need to be used to obtain concentrations high enough for effective blockage of immune suppression locally in the tumor, as they would spread in the body and not seek to or remain in the tumor/cancer tissue. For the same reason, severe side effects would be expectable.

ln a preferred embodiment of the present invention, 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. Thus, one aspect of the invention relates to a method of producing ADA2, in which method the raw material is a medical human immunoglobulin preparation.

Accordingly, in some preferred embodiments, 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. ln some further embodiments, the present binding body is specific for a bacterial ecto-NTPDase, such as Lpgl905 or Lpg0971, or for a bacterial ecto-5’NDase. ln some embodiments, the present binding body is an antibody or mi metic thereof conjugated, e.g. chemically or by genetic engineering, to ADA, pref erably to human ADA (hADA), more preferably to ADA1 and/or ADA2. ln some further embodiments, 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.

ln some embodiments, 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. Means and methods for fusing the binding body with said ADA are readily available and known to those skilled in the art.

Without being limited to any theory, it is envisaged that 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.

Moreover, 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. ln some embodiments, 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. Those skilled in the art can know how to pre pare such binding bodies.

Notably, 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.

As used herein, the expressions "ADA-mAb conjugates" and "mAb-ADA conjugates" and the like are interchangeable.

Moreover, 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 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. Generally, 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.

ln another aspect, 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. The term "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.

ln some preferred embodiments, the present conjugates or bispecific binding bodies (e.g. antibodies or mimetic thereof) can be administered e.g. intra venously or as injections in a desired target, such as a tumor tissue.

As used herein, the term "efficient amount" refers to an amount by which harmful effects of the disease are, at a minimum, ameliorated.

As used herein, the term "subject" refers to an animal, preferably to a mammal, more preferably to a human. Herein, the terms "human subject", "pa tient" and "individual" are interchangeable.

As used herein, the term "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.

As used herein, the term "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.

ln some embodiments, 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). Temporary inhibition of both checkpoint and adenosine signaling pathways may result in particularly powerful activation of the immune system against cancers and infectious diseases.

lt will be obvious to a person skilled in the art that, as technology ad vances, the inventive concept can be implemented in various ways. The invention and its embodiments are not limited to the exemplary aspects or examples de scribed below but may vary within the scope of the claims.

EXEMPLARY WAYS OF ADA-CONJUGATION

ln the following disclosure, monoclonal antibodies (mAbs) are used as examples of the present binding bodies. However, 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. Other 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.

1. THE MOST CHEMICALLY REACTIVE GROUPS IN mAbs

1.1. Amines (lysines, a-amino groups)

One of the most common reactive groups of mAbs is the aliphatic e- amine of the Lys. Lys amines are reasonably good nucleophiles above pH 8.0 (pKa= 9.18) and therefore react easily and cleanly with a variety of reagents to form stable bonds (Eq. 1): mAb-NH2 + Z-B ® mAb-NHB + Z-H

(1)

Other reactive amines that are found in proteins are the 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.

1.2. Thiols (cystines, cysteine, Cys, methionine, Met)

Another common reactive group in mAbs is thiol residue from the sul fur-containing amino acid cystine and its reduction product cysteine (Cys or half cystine). 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:

NH2-mAb-SH + Z-B ® NH2- mAb-SZ + BH

(2)

Since free sulfhydryl groups are relatively reactive, proteins with these groups often exist in their oxidized form as disulfide bridge lmmunoglobulin M (lgM) is an example of a disulfide-linked pentamer, while the subunits of lgG are bonded by internal disulfide bridges ln such proteins, reduction of the disulfide bonds with a reagent such as dithiothreitol (DTT) is required to generate the re active free thiol ln addition to cystine and Cys, mAbs also have the Met containing sulfur in a thioether linkage. Several 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. 1.3. 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. The relatively low reactivity of carboxylic acids in water usually makes it difficult to use these groups to selectively modify mAbs. When this is done, the 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. 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.

1.4. Sugar alcohols

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.

2. REAGENTS

2.1. Amine-reactive reagents

These 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.

Table 1. Some commonly used homo- and heterobifunctional crosslinking and chelating reagents

2.1.1. Reactive esters (formation of an amide bond)

Reactive esters, particularly N-hydroxy-succinimide (NHS) esters, are among the most commonly employed reagents for modification of amine groups. 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. That explains why these reagents are among the most powerful protein -modification reagents available. A new class of NHS esters is now commercially available with sulfonate groups that have improved water solubility. 2.1.2. Isothiocyanates (formation of a thiourea bond)

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.

2.1.3. Aldehydes (formation of imine, Schiffs base, reduction to secondary amine bond)

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. 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.

2.1.4. Miscellaneous amine-reactive reagents (anhydrides)

Other reagents that have been used to modify amines are acid anhy drides. For example, diethylenetriaminepentaacetic anhydride (DTPA) is a bifunc tional chelating agent that contains two amine-reactive anhydride groups lt can react with N-terminal and e-amine groups of the proteins to form amide linkages. 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.

2.2. Thiol-reactive reagents

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)

These reagents (usually iodoacetamides) are common reagents for 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. However, 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.

2.2.2. Maleimides (formation of a thioether bond)

The reaction of maleimides with thiol -reactive reagents is essentially the same as with iodoacetamides. Maleimides react rapidly at slight acidic to neu tral pH. Above pH 8.0, maleimides can undergo hydrolysis to form nonreactive maleamic acids.

2.3. Aldehyde and carboxylic acid-reactive reagents

2.3.1 Amines, hydrazides, and hydrazines (formation of amide, hydrazone, or alkyl amine bonds)

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.

2.4. Bifunctional reagents

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.

EXEMPLARY WAYS OF GENERATION OF ADA-BINDING BODY FUSION PROTEINS AND ADA-SMALL MOLECULE-BINDING BODY CONJUGATES

As shown in Fig. 25, 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).

EXEMPLARY ASPECTS OF THE INVENTION

ln the following exemplary aspects of the invention, antibodies are used as non-limiting examples of the present binding bodies. However, the exam ples apply to other types of binding bodies as well, as is readily understood by those skilled in the art.

1. An adenosine deaminase 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.

2. An adenosine deaminase conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds the human ecto-5'nucleotidase CD73.

3. An adenosine deaminase conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds the human ectonucleoside triphosphate phosphohydrolase CD39. 4. An adenosine deaminase conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds to a human adenosine receptor.

5. An adenosine deaminase conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds the human adenosine receptor Al.

6. An adenosine deaminase conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds the human adenosine receptor A2a.

7. An adenosine deaminase conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds the human adenosine receptor A2b.

8. An adenosine deaminase conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds the human adenosine receptor A3.

9. The 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.

10. The 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.

11. The 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.

12. The adenosine deaminase 1 conjugated or fused with human or humanized antibody, or antigen binding portion thereof, which specifically binds a human adenosine receptor.

13. The 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.

14. The 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.

15. The adenosine deaminase 1 conjugated or fused with human or humanized antibody, or antigen binding portion thereof, which specifically binds the human adenosine receptor A2b. 16. The adenosine deaminase 1 conjugated or fused with or human ized human antibody, or antigen binding portion thereof, which specifically binds the human adenosine receptor A3.

17. The 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.

18. The 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.

19. The 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.

20. The adenosine deaminase 2 conjugated or fused with human or humanized antibody, or antigen binding portion thereof, which specifically binds a human adenosine receptor.

21. The adenosine deaminase 2 conjugated or fused with human or humanized antibody, or antigen binding portion thereof, which specifically binds the human adenosine receptor Al.

22. The adenosine deaminase 2 conjugated or fused with human or humanized antibody, or antigen binding portion thereof, which specifically binds the human adenosine receptor A2a.

23. The adenosine deaminase 2 conjugated or fused with human or humanized antibody, or antigen binding portion thereof, which specifically binds the human adenosine receptor A2b.

24. The adenosine deaminase 2 conjugated or fused with human or humanized antibody, or antigen binding portion thereof, which specifically binds the human adenosine receptor A3.

25. Use of the adenosine deaminase conjugated or fused with human or humanized antibody, or antigen binding portion thereof according to any one of aspects 1 to 24 in the manufacture of a medicament for treating a tumor or cancer.

26. The use according to aspect 25, wherein the cancer is 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. 27. 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).

28. A human or humanized bisAb, one site of which specifically binds a hADA and another specifically binds the human ecto- 5 'nucleotidase CD73.

29. A human or humanized bisAb, one site of which specifically binds a hADA and another specifically binds the human ecto- nucleoside triphosphate phosphohydrolase CD39.

30. A human or humanized bisAb, one site of which specifically binds a hADA and another specifically binds a human adenosine receptor.

31. A human or humanized bisAb, one site of which specifically binds a hADA and another specifically binds the human adenosine receptor Al.

32. A human or humanized bisAb, one site of which specifically binds a hADA and another specifically binds the human adenosine receptor A2a.

33. A human or humanized bisAb, one site of which specifically binds a hADA and another specifically binds the human adenosine receptor A2b.

34. A human or humanized bisAb, one site of which specifically binds a hADA and another specifically binds the human adenosine receptor A3.

35. A human or humanized bisAb, one site of which specifically binds the hADAl and another specifically binds the human ecto -5 'nucleotidase CD73.

36. 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.

37. A human or humanized bisAb, one site of which specifically binds the hADAl and another specifically binds a human adenosine receptor.

38. A human or humanized bisAb, one site of which specifically binds the hADAl and another specifically binds the human adenosine receptor Al.

39. A human or humanized bisAb, one site of which specifically binds the hADAl and another specifically binds the human adenosine receptor A2a.

40. A human or humanized bisAb, one site of which specifically binds the hADAl and another specifically binds the human adenosine receptor A2b.

41. A human or humanized bisAb, one site of which specifically binds the hADAl and another specifically binds the human adenosine receptor A3.

42. A human or humanized bisAb, one site of which specifically binds the hADA2 and another specifically binds the human ecto -5 'nucleotidase CD73. 43. 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.

44. A human or humanized bisAb, one site of which specifically binds the hADA2 and another specifically binds a human adenosine receptor.

45. A human or humanized bisAb, one site of which specifically binds the hADA2 and another specifically binds the human adenosine receptor Al.

46. A human or humanized bisAb, one site of which specifically binds the hADA2 and another specifically binds the human adenosine receptor A2a.

47. A human or humanized bisAb, one site of which specifically binds the hADA2 and another specifically binds the human adenosine receptor A2b.

48. A human or humanized bisAb, one site of which specifically binds the hADA2 and another specifically binds the human adenosine receptor A3.

49. 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.

50. Use of a human or humanized bisAb according to any one of as pects 27 to 49 in the manufacture of a medicament for treating a tumor or cancer.

51. The use according to aspect 50 wherein the cancer is 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.

52. An adenosine deaminase conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds to a bacterial antigen on a surface of bacterial pathogen.

53. An adenosine deaminase conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds to a bacterial antigen on a surface of Gram-negative pathogen.

54. An adenosine deaminase conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds to a bacterial antigen on a surface of Gram-positive pathogen.

55. An adenosine deaminase conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds to an ecto-enzyme, homologue of one of ecto-enzymes of mammalian adenosine sig naling pathway, on a surface of bacterial pathogen. 56. An adenosine deaminase conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds to an ecto-enzyme, homologue of one of ecto-enzymes of mammalian adenosine sig naling pathway, on a surface of Gram-negative pathogen.

57. An adenosine deaminase conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds to an ecto-enzyme, homologue of one of ecto-enzymes of mammalian adenosine sig naling pathway, on a surface of Gram-positive pathogen.

58. An adenosine deaminase conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds to the ecto-nucleoside triphosphate diphosphohydrolase on a surface of Gram- negative pathogen.

59. An adenosine deaminase conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds to the ecto-5'-nucleotidase on a surface of Gram-positive pathogen.

60. An adenosine deaminase 1 conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds to a bacterial antigen on a surface of bacterial pathogen.

61. An adenosine deaminase 1 conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds to a bacterial antigen on a surface of Gram-negative pathogen.

62. An adenosine deaminase 1 conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds to a bacterial antigen on a surface of Gram-positive pathogen.

63. An adenosine deaminase 1 conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds to an ecto-enzyme, homologue of one of ecto-enzymes of mammalian adenosine sig naling pathway, on a surface of bacterial pathogen.

64. An adenosine deaminase 1 conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds to an ecto-enzyme, homologue of one of ecto-enzymes of mammalian adenosine sig naling pathway, on a surface of Gram-negative pathogen.

65. An adenosine deaminase 1 conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds to an ecto-enzyme, homologue of one of ecto-enzymes of mammalian adenosine sig naling pathway, on a surface of Gram-positive pathogen. 66. An adenosine deaminase 1 conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds to the ecto-nucleoside triphosphate diphosphohydrolase on a surface of Gram negative pathogen.

67. An adenosine deaminase 1 conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds to the ecto-5'-nucleotidase on a surface of Gram-positive pathogen.

68. An adenosine deaminase 2 conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds to a bacterial antigen on a surface of bacterial pathogen.

69. An adenosine deaminase 2 conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds to a bacterial antigen on a surface of Gram-negative pathogen.

70. An adenosine deaminase 2 conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds to a bacterial antigen on a surface of Gram-positive pathogen.

71. An adenosine deaminase 2 conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds to an ecto-enzyme, homologue of one of ecto-enzymes of mammalian adenosine sig naling pathway, on a surface of bacterial pathogen.

72. An adenosine deaminase 2 conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds to an ecto-enzyme, homologue of one of ecto-enzymes of mammalian adenosine sig naling pathway, on a surface of Gram-negative pathogen.

73. An adenosine deaminase 2 conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds to an ecto-enzyme, homologue of one of ecto-enzymes of mammalian adenosine sig naling pathway, on a surface of Gram-positive pathogen.

74. An adenosine deaminase 2 conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds to the ecto-nucleoside triphosphate diphosphohydrolase on a surface of Gram- negative pathogen.

75. An adenosine deaminase 2 conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds to the ecto-5'-nucleotidase on a surface of Gram-positive pathogen. 76. Use of the adenosine deaminase conjugated or fused with human or humanized antibody, or antigen binding portion thereof according to any one of claims 52 to 75 in the manufacture of a medicament for treating a bacterial in fection.

77. 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 a bacterial antigen on a surface of bacterial pathogen.

78. 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 a bacterial antigen on a surface of Gram-negative pathogen.

79. 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 a bacterial antigen on a surface of Gram-positive pathogen.

80. 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.

81. 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.

82. 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.

83. 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. 84. 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.

85. 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 a bacterial antigen on a surface of bacte rial pathogen.

86. 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 a bacterial antigen on a surface of Gram negative pathogen.

87. 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 a bacterial antigen on a surface of Gram positive pathogen.

88. 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.

89. 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.

90. 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.

91. 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. 92. 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.

93. 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 bacte rial pathogen.

94. 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 negative pathogen.

95. 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.

96. 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.

97. 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.

98. 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.

99. 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. 100. 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.

101. Use of 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.

102. Method of producing ADA2, in which method the raw material is a medical human immunoglobulin preparation.

103. An adenosine deaminase conjugated or fused with human or hu manized antibody, or antigen binding portion thereof, which specifically binds alkaline phosphatase.

104. The adenosine deaminase 1 conjugated or fused with or human ized human antibody, or antigen binding portion thereof, which specifically binds alkaline phosphatase.

105. The adenosine deaminase 2 conjugated or fused with human or humanized antibody, or antigen binding portion thereof, which specifically binds alkaline phosphatase.

106. Use of the 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.

107. The use according to aspect 106, wherein the 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.

108. A human or humanized bisAb, one site of which specifically binds a hADA and the other specifically binds alkaline phosphatase.

109. A human or humanized bisAb, one site of which specifically binds the hADAl and another specifically binds alkaline phosphatase.

110. A human or humanized bisAb, one site of which specifically binds the hADA2 and another specifically binds alkaline phosphatase. 111. Use of a human or humanized bisAb according to any one of as pects 108 to 110 in the manufacture of a medicament for treating a tumor or can cer.

112. The use according to aspect 111 wherein the 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.

EXAMPLES

Example 1. Cloning, expression and purification of human and mouse ADA1

To stably express human and mouse ADA1 in cells transfected with a replication-defective lentiviral vector, 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). The 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. 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% NaN₃ (Buffer A1 for hADAl] or 50 mM Tris-HCl buffer pH 6.8, 100 mM NaCl, 10 mM ZnCl2, 0.02% NaN₃ (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% NaN₃]. 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% NaN₃. 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). Enzymatic analysis showed that hADAl had similar catalytic parameters (k_cat=190 s ¹ and K_m=26 mM) to the recombinant hADAl expressed in bacteria (Gracia et al., 2008, J. Neuro- chem.107, 161-170).

Example 2. Conjugation of human ADA1 with mAh specific to human ecto- enzymes involved in adenosine production

To form a conjugate consisting of 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. Briefly, the purified hADAl and the 4G4 mouse anti- human CD73 mAb (provided by Professor Sirpa Jalkanen, University of Turku, Finland) were biotinylated using EZ-Link™ NHS-PEG4-Biotin, No-Weigh™ 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.

The 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. Loading buffer was added and samples were heated at 95°C. The beads were collected by centrifugation and aliquots pipetted from the top of solutions were analyzed by SDS-PAGE. The anti- CD73 antibody and hADAl were successfully biotinylated and were shown to bind streptavidin (SA; Dynabeads® MyOne™ Streptavidin Cl, ThermoFisher cat# 65001) as determined by the analysis of beads and solutions by gel electrophore sis (SDS-PAGE) (Fig. 15A). Conjugates of hADAl and anti-human CD73 were suc cessfully reconstructed by mixing biotinylated hADAl, biotinylated CD73 and streptavidin. 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

This example demonstrates that the mAb-hADAl conjugates specifical ly target human ectoenzymes on cancer cells. To this end, the androgen- independent PC-3 human prostate cancer and MDA-MB-231 human breast cancer cells obtained from American Type Culture Collection (ATCC) were incubated with mouse monoclonal 4G4 or rabbit polyclonal h5’NT-2L!5 anti-human CD73 antibody and analyzed by flow cytometry. Briefly, the PC-3 cells were trypsinized, washed with PBS, 2% fetal calf serum (FCS), 0.02% NaN₃, and lxlO⁵ cells were added into a 1.5 mL tube. 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% NaN₃. 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% NaN₃. The cells were fixed with PBS, 1% formaldehyde on ice for 15 min and analyzed by flow cytometry. Both biotinylated antibodies were able to bind to CD73 on cell surface of both MDA-MB-231 breast and PC-3 pros tate cancer cells and conjugate with streptavidin or streptavidin -hADAl mixture (Fig. 16). This example shows that the mAb-hADAl conjugate efficiently binds to CD73 expressed on cancer cells of various types, demonstrating the generality of the approach. Example 4. Chemical conjugation of mouse ADA1 with anti-mouse CD73 and CD39 antibodies

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). First, the protein solutions were dialyzed against BupH buffer. A BupH dry-blend buffer pack was dissolved in 480 mL ultrapure water and pH was adjusted to 7.5 ± 0.05 with 6N NaOH. The final volume was adjusted to 500 mL with water and the solution was used for buffer exchange through dialysis. For antibody (TY/23) and ADA1 labeling, Tz- PEG5-NHS or TC0-PEG4-NHS reagent, respectively, was 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 Zeba™ Spin Desalting Columns (ThermoFisher) (buffer was exchanged twice in order to minimize the amount of non-reacted reagent). For protein-protein conjugation, the desired antibody- ADAl stoichiometry of 1:3 (approximately 1:1 mass ratio) 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% C0₂ 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). For thin-layer chromatographic (TLC) analysis of purine-converting pathways (adenosine de aminase assays), 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. Ali quots of the mixture were applied onto Alugram S1L G/UV254 TLC sheets (Ma- cherey-Nagel, Duren, Germany). 3H-labeled AMP and nucleosides were separated using the appropriate solvent system. Aliquots of the medium were applied onto Alugram S1L G/UV235 sheets (Macherey-Nagel) and separated by TLC. Radioac tive areas that co-migrated with respective nucleotide/nucleoside standards were visualized under reflective UV-light, circled and scraped into scintillation vials followed by 30-min extraction of the radiolabeled purines from the silica using 1 mL of 0.1 N HC1. 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.

Human and bovine ADA (bADA) targeted to CD73 on PC-3 and MDA- MB-231 cells eliminated extracellular adenosine via its rapid conversion into ino- sine or hypoxanthine (Fig. 18, Fig. 19 and Fig. 20). The conjugate consisting of hADAl/bADAl and the anti-CD73 mAb was dramatically more efficacious than either of its components alone (Fig. 19 and 20). Corresponding strategy to con vert adenosine to inosine is used with the mAb-hADAl conjugate specific to CD39.

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. To investigate if the hADAl-anti-CD73 antibody conjugate is capable of restoring adenosine- mediated suppression of immune cell activity, 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. Briefly, 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 (only RPM1 added) 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 (4xl0⁶cells/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). Taken together, 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. Of note, the immune cells in this experi ment were activated by LPS, which is commonly present on the surface of bacte rial pathogens. Hence, 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. "Hot" tumors are readily engaging, "cold" tumors are less likely to engage the immune system, and "warm" tumors have elements of each ln the mouse ex periments, 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.

lmmune-competent mice (C57B1/6) are injected subcutaneously (s.c.) with lxlO⁶ 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 (lxlO⁵ cells) into Balb/C mice.

ln another study, immune-competent mice (Balb/C) are injected or- thotopically into the mammary fat pad with lxlO⁵ 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. Furthermore, the conjugates change the orthotopic 4T1 "cold" mouse mammary carcinoma model more responsive to immunocheckpoint inhibition with anti-PD- Ll. ln this model, 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).

Example 8. The anti-ADA2 part of the bispecific antibody (bisAb) is capable of extracting ADA2 from blood

To investigate if the anti -ADA part of the bispecific antibody (bisAb) approach is able to bind to endogenous human ADA2, 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. 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.

To investigate whether or not administration of the bisAb (anti-ADAl, anti-CD73) will inhibit tumor growth in immune-competent mice more effectively than anti-CD73 mAb alone, 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. Example 10. 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. For this purpose, 10⁵ 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. The addition of 10 gg Ab- ADAl conjugate specific to 5’NDase of S. aureus or 10 gg bisAb (anti-ADAl, anti- 5’NDase) leads to almost complete phagocytic killing of bacteria after incubation, while after the addition of 10 gg of anti-5’NDase Ab alone a notable proportion of staphylococci survive. 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% C0₂. 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).

Claims

CLA1MS

1. A human or humanized binding body, which

i) specifically binds to a protein of an adenosine signaling pathway, and is conjugated or fused with human adenosine deaminase (hADA), or

ii) is a bispecific binding body which specifically binds both to a pro tein of an adenosine signaling pathway and to hADA.

2. The binding body according to claim 1, wherein said binding body targets the site of adenosine production.

3. The binding body according to claim 1 or 2, wherein said pathway is a pathway relevant to the accumulation of extracellular adenosine.

4. The binding body according to any one of claims claim 1-3, wherein the protein of the adenosine signaling pathway is an enzyme involved in adeno sine production.

5. The binding body according to any one of claims 1 -4, wherein the protein of the adenosine signaling pathway is an ecto-enzyme.

6. The binding body according to any one of claims 1-5, wherein said protein is selected from a group consisting of ecto-5'nucleotidase CD73 and ecto- nucleoside triphosphate phosphohydrolase CD 39.

7. The binding body according to any one of claims 1-5, wherein said protein is a bacterial ecto-enzyme selected from a group consisting of ecto- nucleoside triphosphate diphosphohydrolase (ecto-NTPDase) and ecto-5'- nucleotidase (ecto-5’NDase).

8. The binding body according to any one of claims 1 -4, wherein the protein is alkaline phosphatase (AP) .

9. The binding body according to any one of claims 1 -8, wherein said hADA is selected from a group consisting of hADAl and hADA2.

10. The binding body according to any one of claims 1-9, wherein said binding body is an antibody or an antigen-biding fragment thereof.

11. The binding body according to any one of claims 1-6 and 8-10 for use in cancer therapy.

12. The binding body for use according to claim 11, wherein said can cer is selected from a group consisting of cancers of the breast, respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, and parathyroid, as well as lymphomas, sarcomas and leukemias, and distant metastases thereof.

13. The binding body according to any one of claims 1-5 and 7-10 for use in treating a bacterial infection.

14. The binding body for use according to claim 13, wherein said infec tion is caused by a Gram-negative or a Gram-positive bacterial pathogen.

15. The binding body for use according to claim 13, wherein said infec tion is caused by Staphylococcus aureus, Staphylococcus agalactiae or Legionella pneumophila.

16. A pharmaceutical composition comprising antibody binding body according to any one of claims 1-10 and a pharmaceutically acceptable carrier.

17. A method of treating or preventing cancer or a bacterial infection in a subject in need thereof, said method comprising administering to said subject an antibody according anyone of claims 1-10 or a pharmaceutical composition according to claim 16.

18. The method according to claim 17, wherein said cancer is selected from a group consisting of cancers of the breast, respiratory tract, brain, repro ductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thy roid, and parathyroid, as well as lymphomas, sarcomas and leukemias, and dis- tant metastases thereof.

19. The method according to claim 17, wherein said bacterial infection is caused by a Gram-negative or a Gram-positive bacterial pathogen, such as Staphylococcus aureus, Staphylococcus agalactiae or Legionella pneumophila.