Methods and compositions for reducing scar formation and treating dermatological wounds

Abstract

The present disclosure relates, in part, to compositions comprising anti-CD39 antibodies for use in methods for reducing wound healing inflammation / granulation cells or function as part of a means for preventing or reducing the severity of scar formation.

Description

[0001] Methods and Compositions for Reducing Scar Formation and Treating Dermatological Wounds

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application Serial No. 63 / 607,835, filed on December 8, 2023; the entire contents of said application are incorporated herein in their entirety by this reference.

[0004] BACKGROUND OF THE INVENTION

[0005] The skin and related epithelial tissues (including corneal epithelium and mucosal tissues) represent a set of complex organs that have numerous strategies to protect the body from external insults. The skin, for example, contains a highly specialized network of immune cells, crucial for the defense and repair, and also for the maintenance of tissue homeostasis. Following injury, the skin’s immune system plays a key role not only in preventing infections, but also in orchestrating the tissue-repair process.

[0006] Basic biologic processes involved in wound repair are epithelialization and granulation. Epithelialization covers ruptured epithelial surfaces. Hence, it creates a barrier to cover the wound and prevents the entering of microorganisms and other pathogenic substances. On the other hand, granulation forms new connective tissue and blood vessels to fill the wound completely. It occurs from the base of the wound, creating a structure to fill the gap or space of the wound, and consists of a tissue matrix with different types of cells, i.e., CD39highwound healing inflammation / granulation cells. It is the overproliferative or inappropriate remodeling of those cells in the granulation tissue that turns wound healing into problematic granulation structure - hypertrophic granulation, hypertrophic scar, or keloid.

[0007] Scarring is a major cause of many clinical problems. Post-burn contractures, postoperative adhesions and strictures causing intestinal obstruction, mid-facial contractures following cleft palate surgery and painful neuromas are a few examples of the problems caused by scarring. Scar tissue interferes with growth, causes deformities, impairs function and is aesthetically unsightly. Failure to regenerate tissue following iatrogenic or accidental trauma results in the formation of scar tissue.

[0008] A hypertrophic scar is a thick raised scar that’s an abnormal response to wound healing. Prevention and treatment of abnormal scarring represents a challenge in the medical field. Several therapeutic modalities have been described for the treatment and prevention of scars, but the optimal management approach has not yet been defined. Hypertrophic scar formation, in particular, is a major clinical problem in the resolution of severe bums and can give rise to exuberant scarring that result in permanent functional loss and the stigma of disfigurement. Annually, over 1 million people require treatment for burns in the United States. The incidence of hypertrophic scarring following burns is a common outcome that creates a problem of enormous magnitude.

[0009] The medical and economic burden of scars and their sequelae is extensive; in the United States alone more than 100 million new scars are formed every year as a physiological response to cutaneous injury. The resulting fibrotic scar tissue is abnormal in both form and function, and has the potential to cause devastating disfigurement and permanent functional loss. Despite the prevalence of scars and an abundance of treatment options, no current molecular therapies effectively prevent or reverse scarring. As such, there remains a critical need to elucidate the key mechanisms mediating scar formation. Currently, the limited effective clinical treatments of hypertrophic scar, such as local corticosteroid injections, postoperative pressure on the incision site, radiotherapy, and anti-angiogenesis therapy using monoclonal antibodies, are considered to be mediated through inhibition of neovascular buds and proliferating fibroblasts, which result in decreased collagen production.

[0010] SUMMARY OF THE INVENTION

[0011] The newly formed granulation tissue at the wound site consists of CD39highinflammatory cells, such as endothelial cells, monocytes / macrophages and fibroblasts, which are the crucial types of cells participating in granulation formation. Therefore, by administering a CD39-targeted wound healing inflammation / granulation cell-depleting agent to reduce the number of CD39highinflammatory cells at the site of the wound, thereby decreasing / limiting granulation formation, would eventually minimize scar formation. The present invention is based on the discovery that a group of wound healing inflammation / granulation cells plays an important role in scar formation and severity, and that selective ablation of those cell populations provides a means for reducing the severity of scarring.

[0012] Our invention is, through inhibiting granulation while not disturbing epithelialization, to reduce the granulation component and / or delay the granulation formation process during wound repair, thereby leaving space and time for epithelial cell regeneration. Such strategy can ultimately drive wound healing process to less scarring and / or faster healing. The present invention is based on the observation that a group of inflammatory cells in the granulation tissue having a CD39highphenotype are associated with the wound healing process and also play a role in scar formation and severity, and that selective ablation of those CD39highcell populations reduces the severity of scarring. As illustrated below, the use of ADCC competent anti-CD39 antibodies can be effective therapeutic modalities for reducing CD39highinflammation / granulation cells in epithelial tissues and can be used as part of a therapeutic or cosmetic use for reducing the severity of scar formation.

[0013] Exemplary scar reduction treatments utilizing ADCC competent anti-CD39 antibodies include the reduction in severity of post-operative scar formation on a skin or mucosal tissues. For instance, the subject anti-CD39 antibodies can be used to reduce the occurrence or severity of a keloid, which is a type of raised scar. Unlike other raised scars, keloids grow much larger than the wound that caused the scar.

[0014] The subject anti-CD39 antibodies can also be used to reduce the occurrence or severity of hypertrophic scars, atrophic scars and sclerodermas. This includes reducing the occurrence and / or severity of atrophic scarring that is otherwise often an unfortunate and permanent complication of acne vulgaris.

[0015] In still another embodiment, the subject anti-CD39 antibodies can also be used to reduce the occurrence or severity of scarring in the cornea and conjunctiva, including as part of a treatment in the case of, for example: alkali burn (e.g., alkali burn to the cornea), postcataract surgery, excess scarring in the tissue around the extraocular muscles in the strabismus surgery, and tractional retinal detachment in association with contraction of the tissue in diabetic retinopathy. Relatedly, the subject anti-CD39 antibodies can also be used to reduce the occurrence or severity of fibrosis in the corneal endothelium, post-cataract surgery fibrosis of the lens capsule, anterior segment fibrotic diseases of the eye, fibrosis of the corneal stroma (e.g., associated with corneal opacification), fibrosis of the trabecular network (e.g., associated with glaucoma), posterior segment fibrotic diseases of the eye, fibrovascular scarring (e.g., in retinal or choroidal vasculature of the eye), retinal fibrosis, epiretinal fibrosis, subretinal fibrosis (e.g., associated with age related macular degeneration), and fibrosis associated with post-retinal and glaucoma surgery.

[0016] And in yet another embodiment, the subject anti-CD39 antibodies can also be used to reduce the occurrence or severity of scarring as part of a treatment to promote periodontal wound healing. The present invention is based on the observation that CD39highinflammatory cells which infiltrate epithelial tissues during wound healing and which contribute to scar formation can be selectively targeted with CD39-targeted wound healing inflammation / granulation celldepleting agents, such as ADCC (antibody-dependent cellular cytotoxicityj-competent and / or ADCP (antibody-dependent cellular phagocytosis)-competent anti-CD39 antibodies or CD39- targeted cytotoxic drug conjugates, in order to reduce the level of inflammation / granulation cells and / or function at the wound site. These CD39-targeted wound healing inflammation / granulation cell-depleting agents thus have utilities in wound repair including, but not limited to: (1) prevent hypertrophic scar after surgery for patients with known predisposition to keloid formation; (2) as an adjuvant treatment after keloid surgical therapy; (3) demolish hypergranulation. Hypergranulation, usually presents by secondary intention in the wound healing process, is excessive granulation that rises above the wound surface, being referred to as hypergranulation, overgranulation, exuberant tissue or proud flesh. Too much granulation tissue deters epithelialization because the epithelial cells have difficulty climbing up the mountain of granular tissue, imposing a barrier to the inward- migrating epidermis; (4) minimize surgical incision scar after cosmetic surgery.

[0017] With the exception of the PSC22 antibody described herein, the current clinical use of anti-CD39 antibodies is in oncology and has focused on inhibiting the ectonucleotidase activity of CD39, that is, have been targeted to produce a decrease in the intratumoral level of the enzymatic activity associated with that protein, and, in doing so, reduce the intratumoral levels of the immunosuppressive agent, adenosine. Those antibodies have been chosen to not have any CD39-dependent cell depletion activity, i.e., the antibodies are purposefully selected to not include an ADCC and / or ADCP function. Again, the purpose of these prior art antibodies has been in the ability to inhibit the enzymatic activity of CD39 not to kill cells which express CD39, and in the formats those antibodies are used would not have the level of wound healing inflammation / granulation cell depletion of the antibodies contemplated for use in the present methods and formulations.

[0018] However, in the context of the current invention, those versions of the inventive methods and formulations utilizing anti-CD39 antibodies that retain ADCC and / or ADCP function - such that when bound to CD39highinflammation / granulation cells at the wound site results in the ADCC-mediated and / or ADCP-mediated clearance of those cells. Such anti- CD39 antibodies can be, to illustrate, monovalent or multivalent (including bivalent) for CD39. However, the invention also provides for bispecific antibodies which include, in addition to one or more CD39 binding moieties, additional binding moieties that bind to one or more cell surface epitopes expressed by wound healing inflammation / granulation cells.

[0019] Numerous embodiments are provided that may be applied to any aspect encompassed by the present invention and / or combined with any other embodiment described herein. For example, in one aspect, an anti-CD39 antibody, or antigen-binding fragment thereof, comprising (i) at least one antigen binding domain that binds ectonucleoside triphosphate diphosphohydrolase- 1 (CD39) at a site such that the anti-CD39 antibody forms a stable immune complex, and (ii) an FcyRIIIa binding moiety that binds FcyRIIIa receptor and confers ADCC and / or ADCP activity against CD39+ cells to the anti-CD39 antibody, is provided.

[0020] In some embodiments, the targeted CD39highwound healing inflammation / granulation cells (i) are CD45-CD31+ endothelial cells; (ii) are CD45+CDl lb+ monocytes / macrophages; and / or (iii) are CD45-CD90.2+ fibroblast cells.

[0021] In some embodiments, the anti-CD39 antibody, or antigen-binding fragment thereof, promotes: (i) stable immune complex formation when incubated with HCC1739BL cells as characterized by loss of less than 40% of the immune complex after 24 hours, or less than 35%, less than 30%, less than 25%, less than 20%, less than 15% or even less than 10% after 24 hours, optionally wherein the immune complex formation is detected by fluorescence intensity using a fluorescently labeled secondary antibody (e.g., merely to illustrate, the stability of an immune complex formed with an anti-CD39 antibody can be determined by incubating anti- CD39 monoclonal antibodies (mAbs) (e.g., at 2 pg / mL or greater) with HCC1739BL cells for different times and then detecting the presence of immune complex by fluorescent conjugated secondary antibody); (ii) depletion of CD39highwound healing inflammation / granulation cells; (iii) binding to a CD39 epitope having a sequence selected from the group of CD39 amino acid epitope sequences listed in Figure 21; and / or (iv) binding to CD39 in a manner that is noncompetitive or only partially competitive with monoclonal antibody Clone Al binding to CD39. In some embodiments, the anti-CD39 antibody, or antigen-binding fragment thereof, promotes depletion of CD39highwound healing inflammation / granulation cells via ADCC-mediated killing and / or ADCP-mediated killing. In some embodiments, the anti-CD39 antibody, or antigen-binding fragment thereof, promotes depletion of CD39highwound healing inflammation / granulation cells in the form of an antibody-drug conjugate that is taken up by and is toxic to the CD39highwound healing inflammation / granulation cells at the wound site. In another embodiment, the FcyRIIIa binding moiety is selected from the group consisting of an Fc domain, an antibody or fragment thereof that binds to FcyRIIIa, and an FcyRIIIa binding peptide.

[0022] In still another embodiment, the antigen binding domain is selected from the group consisting of a Fab, Fab', F(ab')2, Fv or single chain Fv (scFv), Fav, dsFv, sc(Fv)2, Fde, sdFv, single domain antibody (dAb), and diabodies fragments and / or wherein the anti-CD39 antibody, or antigen-binding fragment, is monoclonal. In some embodiments, the antigen-binding domain is an scFV comprising the sequence of SEQ ID NO: 40.

[0023] In another embodiment, the anti-CD39 antibody, or antigen-binding fragment thereof, has a VH domain with an amino acid sequence that can be encoded by the nucleic acid sequence of or a nucleic acid that hybridizes under stringent conditions to the nucleic acid of SEQ ID No. 1 and a VL domain with an amino acid sequence that can be encoded by the nucleic acid sequence of or a nucleic acid that hybridizes under stringent conditions to the nucleic acid of SEQ ID No. 3 (such as hybridization under 6x sodium chloride / sodium citrate (SSC) at 45°C, and washing in 0.2x SSC / 0.1% SDS at 50-65°C). In still another embodiment, the anti-CD39 antibody, or antigen-binding fragment thereof, comprises a heavy chain having CDRs at least 60% identical (e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) to the CDRs of SEQ ID No. 2, 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54, and a light chain having CDRs at least 60% identical (e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) to the CDRs of SEQ ID No. 4, 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56. In yet another embodiment, the anti- CD39 antibody, or antigen-binding fragment thereof, comprises a variable heavy (VH) chain at least 60% identical (e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) to SEQ ID No. 2, 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54, and a variable light (VL) chain at least 60% identical (e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) to SEQ ID No. 4, 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56. In another embodiment, the anti-CD39 antibody, or antigen-binding fragment thereof, comprises: (i) a heavy chain having a CDR1 amino acid sequence at least 80% identical (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) to SEQ ID No. 29, a CDR2 amino acid sequence at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) identical to SEQ ID No. 30, and a CDR3 amino acid sequence at least 80% identical (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) to SEQ ID No. 31; and (ii) a light chain having a CDR1 amino acid sequence at least 80% identical (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) to SEQ ID No. 32, a CDR2 amino acid sequence at least 80% identical (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) to SEQ ID No. 33, and a CDR3 amino acid sequence at least 80% identical (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) to SEQ ID No. 34. In still another embodiment, the anti-CD39 antibody, or antigen-binding fragment thereof, comprises a heavy chain having CDRs selected from the group consisting of CDRs of SEQ ID No. 6, 10, 14, 18, 22, 26, 42, 46, 50, and 54, a light chain having CDRs selected from the group consisting of CDRs of SEQ ID No. 8, 12, 16, 20, 24, 28, 44, 48, 52, and 56, and human framework sequences to form humanized heavy and light chains with an antigen binding site able to specifically bind human CD39. In still another embodiment, the anti-CD39 antibody, or antigen-binding fragment thereof, comprising (i) a heavy chain variable domain comprising a CDRH1 having the sequence of SEQ ID NO: 29, a CDRH2 having the amino acid sequence of SEQ ID NO: 30, and a CDRH3 having the sequence of SEQ ID NO: 31; and (ii) a light chain variable domain comprising a CDRL1 having the sequence of SEQ ID NO: 32, a CDRL2 having the sequence of SEQ ID NO: 33, and a CDRL3 having the sequence of SEQ ID NO: 34. In another embodiment, the anti-CD39 antibody, or antigenbinding fragment thereof, comprises a heavy chain having CDRs selected from the group consisting of CDRs of SEQ ID NO. 6, 10, 14, 18, 22, 26, 42, 46, 50, and 54, and a light chain having CDRs selected from the group consisting of CDRs of SEQ ID NO. 8, 12, 16, 20, 24, 28, 44, 48, 52, and 56, and human framework sequences to form humanized heavy and light chains with an antigen binding site able to specifically bind human CD39.

[0024] In still another embodiment, the anti-CD39 antibody, or antigen-binding fragment thereof, comprises a heavy chain having CDRs each or cumulatively having no more than 1, 2, or 3 amino acid differences (e.g., insertions, deletions and / or substitutions) from the CDRs of SEQ ID No. 2, 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54, and a light chain having CDRs each or cumulatively having no more than 1, 2, or 3 amino acid differences (e.g., insertions, deletions and / or substitutions) from the CDRs of SEQ ID No. 4, 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56. In still another embodiment, the anti-CD39 antibody, or antigen-binding fragment thereof, comprises a heavy chain having CDRs cumulatively having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid differences from the CDRs of SEQ ID No. 2, 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54, and a light chain having CDRs cumulatively having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid differences from the CDRs of SEQ ID No. 4, 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56. In yet another embodiment, the anti-CD39 antibody, or antigen-binding fragment thereof, comprises a variable heavy (VH) chain having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid differences from SEQ ID No. 2, 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54, and a variable light (VL) chain having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid differences from SEQ ID No. 4, 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56. In another embodiment, the anti-CD39 antibody, or antigen-binding fragment thereof, comprises: (i) a heavy chain having a CDR1 amino acid sequence having no more than 1, 2, or 3 amino acid differences (e.g., insertions, deletions and / or substitutions) from SEQ ID No. 29, a CDR2 amino acid sequence having no more than 1, 2, or 3 amino acid differences (e.g., insertions, deletions and / or substitutions) from SEQ ID No. 30, and a CDR3 amino acid sequence having no more than 1, 2, or 3 amino acid differences (e.g., insertions, deletions and / or substitutions) from SEQ ID No. 31; and (ii) a light chain having a CDR1 amino acid sequence having no more than 1, 2, or 3 amino acid differences (e.g., insertions, deletions and / or substitutions) from SEQ ID No. 32, a CDR2 amino acid sequence having no more than 1, 2, or 3 amino acid differences (e.g., insertions, deletions and / or substitutions) from SEQ ID No. 33, and a CDR3 amino acid sequence having no more than 1, 2, or 3 amino acid differences (e.g., insertions, deletions and / or substitutions) from SEQ ID No. 34.

[0025] In yet another embodiment, the anti-CD39 antibody, or antigen-binding fragment thereof, comprises an Fc domain of an IgGl or IgG3 isotype, optionally wherein the Fc domain is human. In another embodiment, the anti-CD39 antibody, or antigen-binding fragment thereof, is hypo-fucosylated or afucosylated.

[0026] In still another embodiment, the anti-CD39 antibody, or antigen-binding fragment thereof, is human or is humanized.

[0027] In yet another embodiment, the anti-CD39 antibody, or antigen-binding fragment thereof, is a bispecific including at least one additional antigen binding site for a wound healing inflammation / granulation cell antigen. In certain embodiments, the present invention provides bispecific antibodies that bind to both CD39 and a wound healing inflammation / granulation cell surface marker selected from the group consisting of CD31, CD45, CD1 lb, F4 / 80, and CD90.2, and preferably cause wound healing inflammation / granulation cell depletion - such as by ADCC-mediated and / or ADCP- mediated killing or in the form of an antibody-drug conjugate that is preferentially taken up by and toxic to wound healing inflammation / granulation cells at the wound site.

[0028] In other embodiments, the bispecific is generated with binding domains against antigens that are upregulated on activated wound healing inflammation / granulation cells at the wound site, with the avidity of the bispecific for different antigens (CD39 and a second antigen) both expressed on those wound healing inflammation / granulation cells providing for the selectivity. Exemplary antigens for generation of bispecifics using the CD39 binders of the present invention include CD31, CD45, CDl lb, F4 / 80, and CD90.2. Such bispecifics bind to and preferably cause wound healing inflammation / granulation cell depletion - such as by ADCC-mediated and / or ADCP-mediated killing or in the form of an antibody-drug conjugate that is preferentially taken up by and toxic to wound healing inflammation / granulation cells. In some embodiments, the anti-CD39 antibody, or antigen-binding fragment thereof, reduces wound healing inflammation / granulation cells. In some embodiments, the anti-CD39 antibody, or antigen-binding fragment thereof, reduces CD39highwound healing inflammation / granulation cells.

[0029] In another aspect, a pharmaceutical preparation comprising a therapeutically effective amount of at least one anti-CD39 antibody, or antigen-binding fragment thereof, described herein, and one or more pharmaceutically acceptable excipients, buffers or solutions, is provided. For example, the pharmaceutical preparation can be for reducing wound healing inflammation / granulation cell levels, activity, and / or function and suitable for administration to a subject in order to prevent or reduce the severity of scarring (merely to illustrate) comprising an effective amount of the anti-CD39 antibody and one or more pharmaceutically acceptable excipients, buffers or solutions, wherein administration of the anti-CD39 antibody to the subject results in a reduction in numbers and / or function of CD39highwound healing inflammation / granulation cells at the site of wound.

[0030] BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows that a fucosylated PSCNP22 and PSCAF22 counterparts exhibit higher ADCC activity (NK cytotoxicity) toward CD39highHCC1739BL cells than their parental fully- fucosylated clone PSCWT22. Two different afucosylation methods through chemical modifications were employed using the fully human anti-CD39 monoclonal antibody PSCWT22 to produce its ADCC-enhanced counterparts PSCAF22 and PSCNP22, without genetic modifications. To evaluate their ADCC profiles, target cells (CFSE-labeled HCC1739BL, an Epstein-Barr virus (EBV)-transformed human B lymphoblastoid cell line) were incubated with serially diluted human IgGl isotype control antibody (Isotype Ctrl) or PSCWT22, PSCAF22 or PSCNP22 for 30 minutes at 37°C in 5% CO2. Cells were then cocultured with NK-92-CD16 V / V effector cells (E:T=1:8) for 6 hours at 37°C. Target cell death was analyzed by flow cytometry and cytotoxicity was determined by the % of CFSE+P / I+cells.

[0031] Figure 2 shows that IgGl Fc fraction confers ADCC activity to antibodies. The hCD39 Ref antibody shares the antigen binding sites with an antibody in the art. However, the Ref antibody used in the current examples was generated with an Fc portion specifically designed to have ADCC function. Therefore, both hCD39 Ref antibody and our PSCWT22 contain the same ADCC-competent human IgGl Fc fraction. NK cytotoxicity toward HCC1739BL cells were performed as described in Figure 1 and ADCC cell killing profiles of hCD39 Ref and PSCWT22 were determined. Note that hCD39 Ref and PSCWT22 exert similar ADCC activity profiles.

[0032] Figure 3 shows that a further optimized afucosylated counterpart PSC22 exerts higher ADCC activity than PSCNP22. PSC22 is a further ADCC-enhanced version by optimizing the afucosylation process for the parental clone PSCWT22. NK cytotoxicity toward HCC1739BL cells were performed as described in Figure 1 and ADCC cell killing profiles of PSC22 and PSCNP22 were determined.

[0033] Figure 4 shows that PSCWT22 elicited ADCC activity is selective against cells highly expressing human CD39. Various Raji cell lines expressing different levels of human CD39 including Raji cells (Raji-hCD39neg), hCD39-transfected Raji cells that highly express human CD39 (Raji-hCD39hi), or hCD39-transfected Raji cells that express low level of human CD39 (Raji-hCD391o) were used as target cells and were pre-incubated with serially diluted PSCWT22 for 30 minutes at 37 °C in 5% CO2. Afterwards, effector cells (Jurkat cells stably expressing luciferase and hCD16a-158V) were added into the culture (E:T=6:1) and incubated for 6 hours. ADCC activity was indicated by an increase of luciferase activity over background (RLU). RLU: Relative Luminescence Unit. Figure 5 shows that PSCWT22 does not exert ADCC activity toward CD39-low normal endothelial cells (HUVEC), suggestive of its low potential for systemic side-effects. Both human melanoma cells (SK-MEL-28; endogenously expressing intermediate level of CD39) and human umbilical vein endothelial cells (HUVEC; endogenously expressing low level of CD39) were used as target cells and were pre-incubated with serially diluted PSCWT22 for 30 minutes at 37°C in 5% CO2. Afterwards, effector cells (i.e., Jurkat cells stably expressing luciferase and hCD 16a- 158V) were added into the culture (E:T=6:1) and incubated for 6 hours. ADCC activity was indicated by an increase of luciferase activity over background. RLU: Relative Luminescence Unit.

[0034] Figure 6 shows that most of Human / Rabbit chimeric anti-human CD39 monoclonal antibodies target the same epitope as the reference anti-human CD39 monoclonal antibody Clone Al: Epitope competition assay using HCC1739BL cells. HCC1739BL cells were incubated with a panel of 18 anti-human CD39 monoclonal antibodies (Human / Rabbit chimeric clones; unconjugated, 2 pg / mL) at 4°C for 30 minutes, followed by washing and staining with PE-conjugated mouse anti-human CD39 monoclonal antibody (Clone Al) for 30 minutes at 4°C. Cells were then analyzed by flow cytometry and PE median fluorescence intensity (MFI) was detected. Cells incubated with media instead of chimeric antibody were used as control.

[0035] Figure 7 shows that human / rabbit chimeric antibodies which do not compete for the same epitope of Clone Al have high ADCC activity. HCC1739BL cells were used as target cells and were pre-incubated with serially diluted chimeric antibodies for 30 minutes at 37°C in 5% CO2. Afterwards, effector cells (i.e., Jurkat cells stably expressing luciferase and hCD16a-158V) were added into the culture (E:T=6:1) and incubated for 6 hours. ADCC activity was indicated by an increase of luciferase activity over background. RLU: Relative Luminescence Unit. Note that among these high ADCC clones, only PSC23 antibody competes for the same epitope of Clone Al (See Figure 6).

[0036] Figure 8 shows that human / rabbit chimeric antibodies which completely compete for the same epitope of Clone Al have low or no ADCC activity. Luc-reporter ADCC assay was performed as described above in Figure 7. PSC18 serves as a positive control.

[0037] Figure 9 shows that antibodies with high ADCC activity form stable immune complex on cell membrane while antibodies with low or no ADCC activity do not. Exemplary antihuman CD39 antibodies presenting high, low and no ADCC activity (2 pg / mL) were incubated with HCC1739BL cells for 24 hours at 37°C in 5% CO2or 20 minutes at 4°C, followed by secondary antibody staining (anti-human IgG (Fc specific), Alexa Fluor® 488) for 30 minutes at 4°C. Cells were then washed and analyzed by flow cytometry. The difference in AF488 MFI between 20 minutes and 24 hours treatment represents the loss of human CD39 on cell membrane that was calculated as described in Materials and Methods. Note that clone PSC26 (low-ADCC activity) and clones PSC27, PSC28 and PSC29 (no-ADCC activity) do not form a stable immune complex on cell membrane after 24 hours incubation (e.g., the loss of CD39 is greater than 40%), whereas clones PSCNP22, PSC19, PSC20, PSC21 (high-ADCC activity) form a stable antibody-antigen immune complex (e.g., the loss of CD39 is lower than 30%). Hu / Ra: Human / Rabbit chimeric antibody; hlgGl: humanized rabbit antibody, IgGl isotype.

[0038] Figure 10 shows that endothelial cells and macrophages from scar tissue express high levels of CD39 on their membrane. Single-cell suspensions from granulation / scar tissues of h(uman) CD39KI mice wound healing by secondary intention model were prepared and analyzed for CD39 expression on fibroblasts (FB), endothelial cells (EC), and macrophages (MAC) by flow cytometry. Viable cells were gated as FB: LinTCD45’CDllb’CD3TCD202b’ CD90.2+cells; EC: CD45’CD31+cells; and MAC: CD45+CDl lb+Grl-Ly6CF4 / 80highcells. Human CD39 (hCD39) expression levels on each subpopulation were calculated and expressed as mean fluorescence intensity (MFI). Note that both EC and MAC express CD39, while FB barely express the protein on their membrane.

[0039] Figure 11 shows that PSC22 strongly binds to both endothelial cells and macrophages from scar tissue. Single-cell suspensions of granulation / scar tissue from hCD39KI mice wound healing by secondary intention model were incubated with different concentrations of PSC22 or isotype control antibody (Isotype Ctrl), followed by staining with fluorophore-conjugated primary antibodies against cell surface markers as well as anti-human IgGl secondary antibody. Viable EC and MAC were analyzed by flow cytometry and gated as CD45'CD31+cells and CD45+CDl lb+Grl'Ly6C'F4 / 80hlghcells, respectively. Anti-human IgGl secondary antibody mean fluorescence intensity (MFI) in each cell subpopulation surface was determined and EC50 was calculated as 60.7 ng / mL and 35.1 ng / mL for EC and MAC, respectively.

[0040] Figure 12 shows that PSC22 exhibits ADCC activity towards endothelial cells and macrophages from scar tissue. Single-cell suspensions of granulation / scar tissue (including target cells EC and MAC) from hCD39KI mice wound healing by secondary intention model were incubated with different concentrations of PSC22, followed by co-culture with effector cells (human NK-92-CD16 V / V cells) (E:T=1:2) for 6 hours at 37°C. Target cell death was analyzed by flow cytometry and the percentage of 7-ADD+EC and MAC (% of cytotoxicity) were determined. The EC50 was calculated as 5.9X1 O'4ng / mL and 1.7X1 O'4ng / mL for EC and MAC, respectively.

[0041] Figure 13 shows that PSC22 depletes granulation tissue cells in hCD39KI mice wound healing model. hCD39KI mice subjected to open excisional wound healing by secondary intention model induction were treated with saline or PSC22 (3 mg / kg) i.p. every two days for two doses (on days 5 and 7). Scar tissues were collected one day after the last dosing and dissociated into single-cell suspensions for further analysis of FB, EC, and MAC by flow cytometry. Absolute cell number per scar tissue was calculated for each cell subpopulation. n=8 mice per group.

[0042] Figure 14 shows that PSC22 reduces scar fiber and granular tissue formation in hCD39KI mice wound healing model. hCD39KI mice subjected to open excisional wound healing by secondary intention model induction were treated with saline or PSC22 (3 mg / kg) i.p. every two days for two doses (on days 10 and 12). Scar fiber and granular tissue formation were visualized one day after the last dosing by exposing the underlying connective tissues after surgical incision and wound scab removal. Representative images of one animal per group were shown. Solid arrow shows subcutaneous scar fiber and dashed arrow points to subcutaneous granular tissue. Note that animals treated with PSC22 have minimal granular tissue and scar fiber formation when compared to saline treated group.

[0043] Figure 15 shows that PSC22 reduces inflammation cell density, scarring and recovered epidermis area in hCD39KI mice wound healing model. hCD39KI mice wound healing by secondary intention model induction and treatment were performed as described in Figure 5. Scar tissues were collected one day after the last dosing, stained with H&E and analyzed by pathology. Pictures were taken at 40X. Representative images of one animal per group were shown. Double sided arrows mark the edges of restored epidermis and dashed arrow points to inflammation cell density. Note that in the PSC22 treated group, the inflammation cell density and restored epidermis area are significantly reduced concurrent with emerging hair follicles, when compared to saline treated group (which has no hair follicles in the wound area).

[0044] Figure 16 shows that PSC22 diminishes collagen deposition area in hCD39KI mice wound healing model. hCD39KI mice wound healing by secondary intention model induction and treatment were performed as described in Figure 14. Scar tissues were collected one day after the last dosing, stained with Masson's trichrome and analyzed by pathology. Pictures were taken at 40X. Representative images of one animal per group were shown. Note that the collagen deposition area is significantly reduced in the PSC22 treated group in comparison to saline treated group.

[0045] Figure 17 shows that PSC22 promotes faster wound healing in hCD39KI mice wound healing model. hCD39KI mice subjected to open excisional wound healing by secondary intention model induction were treated with saline or PSC22 (3 mg / kg) i.p. every two days for a total of four doses (on days 7, 9, 11, and 13). Gross pictures of scar tissue wound healing were taken over time, when wound size was measured in two dimensions using a digital caliper. Wound areas (A) were calculated and expressed in mm2, using the formula: A = (L / 2) x (W / 2) x 7t, where L and W were the long and short dimensions of the wound, respectively. The percentage of wound closure over time was calculated as [(wound area on day 7- wound area on day 13) / wound area on day 7] x 100 and were compared between study groups. Note that animals which received PSC22 3 mg / kg treatment have significantly smaller wound area and faster tissue healing (as indicated by higher percentage of wound closure). n=2 for saline group and n=3 for PSC22 group. *P<0.05, **P<0.01 in relation to saline control group (unpaired, t- test, one-tailed).

[0046] Figure 18 shows that PSC22 decreases inflammation / granulation area underneath the healed epidermis in hCD39KI mice wound healing model. hCD39KI mice subjected to open excisional wound healing by primary intention model induction were treated with saline or PSC22 (3 mg / kg) i.p. every two days for a total of four doses (on days 7, 9, 11, and 13). Scar tissues were collected one day after the last dosing, stained with H&E, and analyzed by pathology. Pictures of scar area were taken at 100X (bar 100 pm). Representative images of one animal per group were shown. Draw lines show granular / scar tissue areas in each group. Note that granular / scar tissue area is markedly smaller in the PSC22 group when compared to saline.

[0047] Figure 19 shows that PSC22 reduces inflammatory cell infiltration and fibrosis in foreign body granuloma of hCD39KI mice wound healing model. hCD39KI mice wound healing by primary intention model induction, treatment, and scar tissue collection and analysis were performed as described in Figure 18. Herein, pictures of synthetic suture area were taken at 400X (bar 10 pm) and 1000X (bar 2 pm). Representative images of one animal per group were shown. Note that inflammatory cells (chiefly macrophages) that are responsible for foreign body response and tissue fibrous encapsulation are markedly decreased in the PSC22 treated group in comparison to the saline counterpart. Figure 20 shows that PSC22 depletes granulation tissue cells in hCD39KI mice mesh implantation model. hCD39KI mice subjected to mesh implantation model induction were treated with saline or PSC22 (3 mg / kg) i.p. every two days for four doses (on days 10, 12, 14 and 16). Meshes with underlying granular / connective tissues were collected one day after the last dosing, weighted, photographed, and dissociated into single-cell suspensions for further analysis of FB, EC, and MAC by flow cytometry. Absolute cell number per mesh was calculated for each cell subpopulation. n=8 mice per group. *P<0.05, **P<0.01 in relation to saline control group (unpaired, t-test, one-tailed). Note that meshes removed from PSC22 treatment group are lighter and clearer than the saline control group. Moreover, flow cytometry results are in agreement with previous data observed with the open wound healing model shown on Figure 13.

[0048] Figure 21 shows conformational epitope mapping and a list of main putative epitope candidates.

[0049] DETAILED DESCRIPTION OF THE INVENTION

[0050] I. Overview

[0051] The term "wound" generally refers to both open and closed wounds, as defined below. A wound can be further classified as an acute or chronic wound. An acute wound is one that does not have an underlying healing defect, and usually occurs secondarily to surgery or trauma in a healthy individual, healing quickly and completely. In contrast, a chronic wound is one that has a loss in tissue integrity, produced by insult or injury that is of extended duration or frequent recurrence. A hypertrophic scar, a thick raised scar that’s an abnormal response to wound healing, frequently developed in both acute and chronic wound.

[0052] As used herein, the term "skin wound" refers to a break in the skin.

[0053] The term "open wound" is usually classified according to the object that caused the wound. This includes bums, incisions, lacerations, abrasions, puncture wounds, penetration wounds, gunshot wounds and the like. Incisions or incised wounds may be caused by a clean, sharp- edged object such as a knife, a razor, or a glass splinter. Incisions involving only the epidermis can be classified as cuts. Lacerations are irregular wounds caused by a blunt impact to soft tissue that lies over hard tissue (such as laceration of the skin covering the skull) or tearing of skin and other tissues (such as caused by childbirth). Lacerations may show bridging, as connective tissue or blood vessels are flattened against the underlying hard surface. Abrasions (grazes) are superficial wounds in which the topmost layer of the skin (the epidermis) is scraped off, and are often caused by a sliding fall onto a rough surface. Puncture wounds may be caused by an object puncturing the skin, such as a nail or needle. Penetration wounds may be caused by an object such as a knife entering the body. Gunshot wounds are caused by a bullet or similar projectile driving into or through the body. As such, there may be two wounds, one at the site of entry and one at the site of exit, which is generally known as a through- and through.

[0054] The term "closed wound" refers to contusions, more commonly known as bruises, caused by blunt force trauma that damages tissue under the skin; hematomas, also called blood tumors, caused by damage to a blood vessel that in turn causes blood to collect under the skin; and crushing injuries, which may be caused by a great or extreme amount of force applied over a long period of time.

[0055] The term "scar" refers to an abnormal morphological structure resulting from a previous injury or wound (e.g., an incision, excision or trauma). Scars are composed of a connective tissue that is predominately a matrix of collagen types 1 and 3 and fibronectin. A scar may consist of collagen fibers in an abnormal organization (as seen in normal scars of the skin) or may be an abnormal accumulation of connective tissue (as seen in scars of the central nervous system or pathological scarring of the skin). The types of scars include, but are not limited to, atrophic, hypertrophic and keloidal scars, as well as scar contractures. Atrophic scars are flat and depressed below the surrounding skin as a valley or hole. Hypertrophic scars are elevated scars that remain within the boundaries of the original lesion, and often contain excessive collagen arranged in an abnormal pattern. Keloidal scars are elevated scars that spread beyond the margins of the original wound and invade the surrounding normal skin in a way that is site specific, and often contain whorls of collagen arranged in an abnormal fashion. Scar contractures are scars that cross joints or skin creases at right angles, and are prone to developing shortening or contracture. Scar contractures occur when the scar is not fully matured, often tend to be hypertrophic, and are typically disabling and dysfunctional.

[0056] Scars differ from normal skin in three key ways: (1) they are devoid of any dermal appendages (hair follicles, sweat glands, etc.); (2) their collagen structure is fundamentally different, with dense, parallel fibers rather than the “basketweave” pattern that lends normal skin its flexibility and strength; and (3) as a result of their inferior matrix structure, they are weaker than skin. A variety of conditions may cause scarring, including surgical wounds, burns, cuts, gunshot, etc. Scars commonly form as a result of facial plastic surgery, which includes, but is not limited to, rhytidectomy, blepharoplasty, rhinoplasty, otoplasty, mentoplasty, face lift, forehead lift, brow lift, facial scar revision, facial scar removal, laser surgery, skin resurfacing, wrinkle treatment, plasma skin regeneration, facial fat grafting, skin tightening, tattoo removal and hair replacement. Thus, this disclosure is advantageous to patients who undergo facial plastic surgery, particularly to aid with scarring and bruising, by speeding up wound healing and reducing scar formation. Scars also commonly form as a result of full-body plastic surgery, which includes, but is not limited to abdominoplasty, breast reduction, breast enhancement, body lift procedures, spider vein treatment, stretch mark treatment, liposuction, excess skin removal surgery, cellulite reduction treatment, body contouring, body resurfacing and body implants.

[0057] Effective treatment for preventing or reducing the severity of scarring is often challenging owing to their heterogeneous pathophysiology. Understanding of the underlying disease mechanisms is improving and it is now clear from the present invention that CD39highwound healing inflammation / granulation cells play a complex pathophysiological role in a broad range of scar formation events and that targeting CD39highwound healing inflammation / granulation cells for ablation can be an effective means for controlling scar formation and severity.

[0058] A group of CD39highinflammatory cells are involved in the structure formation of granulation tissue at the wound site. Granulation tissue contains a number of important types of cells: (1) majorly fibroblasts, which are the cells that form during the early stages of granulation to assist in the creation of collagen; (2) endothelial cells, which are the main cells responsible for creating vessels through a process called angiogenesis, which ultimately supplies the wound with nutrients. In addition to those structural cells, there are a number of neutrophils and macrophages within the granulation tissue. These are the types of cells responsible for fighting pathogens and preventing infection. Macrophage modulation is also the central axis of the exacerbation or control of fibrosis. M2 macrophages are intended to create an anti-inflammatory environment and promote healing and regeneration of wounds. However, when the lesion is persistent, M2 macrophages take an important pro-fibrotic role.

[0059] The present invention is based at least in part on the discovery that certain CD39- targeted agents, such as ADCC-competent and / or ADCP-competent anti-CD39 antibodies are capable of selectively targeting and ablating CD39 expressing wound healing inflammation / granulation cells, and as a consequence causing a decrease in the number of wound healing inflammation / granulation cells located in the target epithelial tissue at the wound site. The resulting reduction in numbers and / or function of CD39highwound healing inflammation / granulation cells can lead to changes in the scarring severity and characteristics of the scar in the target epithelial tissue.

[0060] Accordingly, in some aspects, the present invention relates to CD39-targeted wound healing inflammation / granulation cell-depleting agents. The term "CD39-targeted wound healing inflammation / granulation cell-depleting agents" refers to any agent (e.g., antibodies, small molecules, aptamers, etc.) that specifically binds to CD39 on the surface of wound healing inflammation / granulation cells and induces cell death of those inflammation / granulation cells at the site of wound. In some embodiments, the CD39-targeted wound healing inflammation / granulation cell-depleting agent is anti-CD39 antibodies, such as ADCC-competent and / or ADCP-competent anti-CD39 antibodies.

[0061] To illustrate the use of anti-CD39 antibodies as an example of CD39-targeted wound healing inflammation / granulation cell-depleting agents, antibodies which can be selected for use in the methods of the present invention are capable of forming more stable immune complexes with CD39 in order to produce a more potent ADCC killing efficacy. Antibodies that are not able to form stable immune complexes with CD39, the inventors have observed, result in reduction of CD39 from the surface but through a mechanism of increased shedding of CD39 (antigenic modulation) or cytosis (internalization) and do not have the same efficacy in terms of being able to ablate the CD39 expressing cells by antibody-dependent cellular cytotoxicity. Without being bound by theory, it is further believed that the use of anti-CD39 antibodies as an example of CD39-targeted wound healing inflammation / granulation celldepleting agents, antibodies which can be selected for use in the methods of the present invention are capable of forming more stable immune complexes with CD39 in order to produce a more potent ADCP killing efficacy. ADCP is another major Fc effector function whose mechanism, by which antibody-opsonized target cells activate FcyRs (such as FcyRIIIa receptors described herein on cells, such as those expressed on NK cells and conferring NK cells' ADCC killing function) on the surface of macrophages to induce phagocytosis, resulting in internalization and degradation of the target cell and, ultimately, killing of target cells. Hypo- fucosylation or afucosylation of therapeutic monoclonal antibodies has been shown to result in enhanced FcyRIIIa receptor binding and subsequent ADCC-mediated target cell depletion activity by NK cells, as well as ADCP-mediated target cell depletion by macrophages (see Figures 1, 3, 12, 13, and 20 herein).

[0062] Exemplary features of certain preferred anti-CD39 monoclonal antibodies, features which are taught away from for use in therapeutic anti-CD39 antibodies described in the literature, are summarized as below.

[0063] The subject antibodies reduce CD39highwound healing inflammation / granulation cell populations through FcyRIIIa receptor-dependent activity e.g. ADCC.

[0064] As examples, Figures 1 and 3 show that decreased fucosylation (a.k.a. hypo- fucosylation or afucosylation) of clone PSCWT22, a fully human anti-CD39 monoclonal antibody, either by using a fucosylation inhibitor (PSCAF22) or by optimizing the production process (PSCNP22 and PSC22), dramatically boosts its ADCC activity against CD39+ cells in vitro - ADCC activity ranking: PSC22 > PSCNP22 > PSCWT22. Such phenomenon is also valid when using human NK cells (as the effector cells) and granulation tissue cells obtained from human CD39 knock-in (hCD39KI) mice scar models (as the target cells) in an in-vitro ADCC assay, viz., PSC22 shows markedly enhanced NK cytotoxicity toward granulation tissue endothelial cells (EC50 = 5.9E-04 pg / mL) and macrophages (EC50 = 1.7E-04 pg / mL) (Figure 12). As expected, all these in vitro CD39-targeted wound healing inflammation / granulation cell-depleting activities are concurrent with enhancement of anti- inflammation / granulation cells activity of the afucosylated antibody PSC22 in vivo (Figures 13 and 20).

[0065] ADCC activity of the subject anti-CD39 antibodies is selective toward CD39highinflammatory cells in the granulation tissue at the wound site, e.g., CD39highendothelial cells and macrophages.

[0066] As examples, Figures 4 and 5 show that ADCC activity of PSCWT22 is selective against CD39highcells in vitro (i.e., Raji-hCD39hi cells in Figure 4 and SK-MEL-28 cells in Figure 5).

[0067] As further examples shown in Figures 11 and 12, PSC22 is capable of selectively targeting and ablating CD39highinflammatory cells in the granulation tissue in vitro; and as a consequence causing a decrease in the number of these CD39highinflammation / granulation cells in vivo (Figures 13 and 20). The resulting reduction in numbers and / or function of CD39highwound healing inflammation / granulation cells lead to decreased scar formation and severity as well as faster wound healing, associated with diminished granulation tissue and collagen deposition at the wound site (Figures 14-20).

[0068] In summary, this functional trait should confer specificity to the subject antibodies and result in a safer, highly selective, and more effective anti-CD39 antibody for the prevention and treatment of scars.

[0069] Formation of a stable immune complex of anti-CD39 antibodies with the antigen on target cell membrane confers high ADCC activity to the antibodies.

[0070] As an example shown in Figure 9, the stability of the antibody-antigen immune complex on target cell surface was examined using antibodies selected from three groups: ADCC -high (i.e. PSCNP22, hCD39 Ref, and Human / Rabbit chimeric clones PSC19, PSC20, PSC21 and PSC25), ADCC-low (Human / Rabbit chimeric clone PSC26), or ADCC-negative (Human / Rabbit chimeric clones PSC27, PSC28 and PSC29). A strong and positive correlation between the stability of such immune complex and the antibody's ADCC activity is clearly seen, viz., the more stable the antibody-antigen immune complex is, the higher the antibody's ADCC activity.

[0071] Different epitopes of anti-CD39 antibodies directly link to antibodies' ADCC activity.

[0072] As an example, by comparing epitopes of the subject Human / Rabbit chimeric anti- hCD39 antibodies against the commercially available anti-hCD39 monoclonal antibody Clone Al, Figures 6-8 show anti-CD39 antibodies that bind to CD39 in a manner that is noncompetitive or only partially competitive with Clone Al binding to CD39 have a high likelihood of containing high ADCC activity, e.g., five (PSC18, PSC19, PSC20, PSC21 and PSC24,) out of six ADCC-high antibodies, except PSC23, display such trait (Figures 6 and 7). In contrast, all antibodies in the ADCC-low (PSC26, PSC30, PSC31 and PSC32) and ADCC- negative (PSC27, PSC28, PSC29, PSC33, PSC34, PSC35, PSC36 and PSC37) groups display epitopes that completely overlap with Clone Al's (Figures 6 and 8).

[0073] In certain embodiments, rather than be focused on direct inhibition of CD39 NTPase activity, e.g., as is the focus for anti-CD39 therapeutic antibodies in the oncological use of the prior art (Perrot et al., 2019, Cell Reports 27:2411-2425; Li et al., 2019, Cancer Discovery 9(12): 1754-1773; and WO / 2017 / 089334), anti-CD39 antibodies useful in the present invention (whether or not inhibitory of the NTPase activity) can be specifically designed to have human constant regions with an IgGl Fc domain. This design confers FcyRIIIa receptor-dependent cellular activities, e.g., antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and (optionally) complement dependent cytotoxicity (CDC) against CD39+ cells. Consequently, such cellular activities result in ablation and reduction of CD39highinflammation / granulation cells at the site of wound.

[0074] In certain embodiments, certain antibodies encompassed by the present invention have been shown to bind to epitopes on CD39 that are non-competitive with or only partially competitive with the binding of the monoclonal antibody Clone Al to CD39.

[0075] II. Definitions

[0076] To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

[0077] "CD39", also referred to as "Cluster of Differentiation 39", "ectonucleoside triphosphate diphosphohydrolase- 1" or (gene) "ENTPD1" and (protein) "NTPDasel" is a cell surface-located ectonucleotidase with an extracellularly facing catalytic site that catalyses the hydrolysis of y- and P-phosphate residues of triphospho- and diphosphonucleosides to the monophosphonucleoside derivative (ENZYME entry: EC 3.6.1.5), such as to hydrolyze P2 receptor ligands such as ATP, ADP, UTP and UDP (Junger et al., 2011, Nat. Rev. Immunol. 11:201-212). A representative human NTPDasel protein sequence is provided in the UniProtKB entry "P49961 (ENTP1_HUMAN)", and a representative human coding sequence for the enzyme is provided in GenBank Accession S73813.

[0078] Representative human CD39 cDNA and protein sequences are well-known in the art and are publicly available from the National Center for Biotechnology Information (NCBI). For example, at least seven human CD39 transcript variants are known encoding six different human CD39 isoforms. Human CD39 isoform 1 is available under accession numbers NM_001776.5 and NP_001767.3. The transcript variant represents the longest transcript and encodes isoform 1. Human CD39 isoform 2, available under accession numbers NM_001098175.1 and NP_001091645.1, uses an alternate 5' exon than transcript variant 1 that results in a distinct 5' untranslated region (UTR) and causes translation initiation at an alternate start codon leading to a longer and distinct N-terminus. Human CD39 isoform 3, available under accession numbers NM_001164178.1 and NP_001157650.1, uses an alternate 5' exon than transcript variant 1 that results in a distinct 5' UTR and causes translation initiation at an alternate start codon leading to a longer and distinct N-terminus. Human CD39 isoform 4, available under accession numbers NM_001164179.1 and NP_001157651.1, uses an alternate in-frame splice site as compared with transcript variant 1 resulting in a shorter isoform. Human CD39 isoform 5, available under accession numbers NM_001164181.1 and NP_001157653.1, uses an alternate exon in the 5' region that results in a distinct 5' UTR and translation initiation at a downstream start codon relative to transcript variant 1 resulting in a shorter isoform. Human CD39 isoform 6, available under accession numbers NM_001164182.1 and NP_001157654.1, lacks an alternate exon that results in a distinct 5 ' UTR and causes translation initiation at a downstream start codon relative to transcript variant 1 resulting in a shorter isoform. Human CD39 isoform 6 is also encoded by another transcript variant, available under accession numbers NM_001164183.1 and NP_001157655.1, which lacks two alternate internal exons that results in a distinct 5' UTR and causes translation initiation at a downstream start codon relative to transcript variant 1 resulting in a shorter isoform.

[0079] Nucleic acid and polypeptide sequences of CD39 orthologs in organisms other than humans are well known and include, for example, mouse CD39 (NM_009848.3 and NP_033978.1), rat CD39 (NM_022587.1 and NP_072109.1), cow CD39 (NM_174536.2 and NP_776961.1), frog CD39 (NM_001006795.1 and NP_001006796.1), and zebrafish CD39 (NM_001003545.1 and NP_001003545.1).

[0080] The extensive glycosylation of CD39 is associated with its cell surface expression and activity such that deletion of glycosylated residues or mutations to non-glycosylatable residues results in significantly reduced CD39 activity (see, for example, deletion or mutation of glycosylatable residues 73 at the N terminus, 333 in the middle, and / or 429 and / or 458 at the C terminus of rat CD39 or corresponding residues in orthologs thereof (Wu et al., 2005, Mol. Biol. Cell. 16: 1661-1672). Similarly, mutations of conserved residues in the apyrase conserved region (ACR) of any one or more of ACRs 1-5 causes a reduction in CD39 activity (Schulte am Esch et al., 1999, Biochem. 38:2248-2258; Yang et al., 2001, Biochem. 40:3943-4940; Wang and Guidotti, 1998, J. Biol. Chem. 273:11392-11399).

[0081] The modulation (e.g., decrease) in CD39 activity can be measured in any number of ways (e.g., according to measures described herein, including using controls, ratios, comparisons to baselines, and the like). For example, a CD39 activity modulator can decrease the catalytic activity of the ectonucleotidase or overall CD39 activity as compared to the level of such ectonucleotidase in the presence of a test agent. In one embodiment, CD39 activity is determined by analyzing the concentration of adenosine in a sample. The concentration can be assessed over time. In another embodiment, ATP is added in the sample tested and the concentration of remaining ATP, AMP or adenosine is determined or assessed. A modulation in this context, such as a decrease, can mean a decrease of 1%, 5%, 10%>, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120%, 150%, 200%, 500%, 1000%, or more. In an embodiment, said increase is detected over time.

[0082] In certain embodiments, cells, such as granulation tissue endothelial cells and macrophages, exhibit expression of a gene (e.g., CD39) or other biomarker of interest (e.g., flow cytometry side scatter known as SSC) at a high level. In one embodiment, any method described herein to determine the level of expression of the gene or other biomarker may be used to determine the high level. For example, a representative, non-limiting method to define CD39highwound healing inflammation / granulation cells involves analysis of granulation tissue cells, such as granulation tissue cells obtained from hCD39KI mice scar models (obtained from the Purinomia Animal Facility), under an antibody-based flow cytometry assay described herein in the Exemplary Materials and Methods using the detection antibodies as listed in Table 1, such as using a Cytek® Aurora flow cytometer.

[0083] In certain embodiments, a CD39highwound healing inflammation / granulation cell can be identified as a granulation tissue cell which, in addition to expressing markers characteristic of a granulation tissue cell and / or having functional characteristics of a granulation tissue cell, expresses CD39 at a high enough level such that by flow cytometry has CD39 detection antibody fluorescence intensity of at least 104, e.g., such as detected using an antibody-based flow cytometry assay described herein in the Exemplary Materials and Methods using the detection antibodies as listed in Table 1, such as using a Cytek® Aurora flow cytometer.

[0084] In some embodiments, a CD39highpopulation of cells, such as wound healing inflammation / granulation cells, comprises a population of cells in which at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or greater, or any range in between, inclusive, such as 60-99%, 65-95%, 70-90% 70-80%, and the like, expresses high levels of CD39 (e.g., merely to illustrate, CD39 detection antibody fluorescence intensity is at least 104under this specific flow cytometry assay, such as at least 104, 105, 106, 107, or any range in between, inclusive, such as 104-107, 104-106, and the like). In some embodiments, a CD39lowpopulation of cells, such as wound healing inflammation / granulation cells, comprises a population of cells in which less than 41, 40, 39, 38, 37, 36, 35, 30, 25, 20, 15, 10, 5, or even lower, or any range in between, inclusive, such as 4-41%, 10-35%, 15-25%, and the like, expresses low levels of CD39 (e.g., merely to illustrate, CD39 detection antibody fluorescence intensity is less than 104under this specific flow cytometry assay, such as less than 104, 103, 102, 101, 10°, or any range in between, inclusive, such as less than 100-103, 101-102, 101-103, 101-104, and the like). In some embodiments, CD39highwound healing inflammation / granulation cells, such as CD45-CD31+ endothelial cells, account for 60-99% or any range in between, inclusive, such as 60-95%, 60- 80%, 70-80%, 75-80%, etc. of a total population, such as a CD45-CD31+CD39highendothelial cell population. In some embodiments, CD39highwound healing inflammation / granulation cells, such as CD45+CDl lb+ macrophages, account for 60-99% or any range in between, inclusive, such as 60-95%, 60-80%, 70-80%, 75-80%, etc. of a total population, such as a CD45+CDl lb+CD39highmacrophage population.

[0085] A "CD39 Antibody" (alternatively an "anti-CD39 antibody") refers to an antibody that selectively binds to one or more epitopes of the NTPDasel protein, and includes monoparatopic antibodies, as well as biparatopic and other multiparatopic format antibodies.

[0086] An "immune complex" (also known as an antigen-antibody complex or antigen-bound antibody) may refer, in some embodiments, to a composition formed through binding of an antigen (e.g. expressed on a medium such as a cell, or alone) to an antibody. A "stable immune complex" may be formed when loss of the interaction between the antigen and the antibody is less than 40% of the immune complex after 24 hours, less than 35%, less than 30%, less than 25%, less than 20%, less than 15% or less than 10% after 24 hours, or any range in between, inclusive, such as 40%-35%, 35%-30%, 30%-25%, 25%-20% , 20%-15%, 15%-10%, and the like (e.g., when incubating the antibody with cells expressing an antigen, such as HCC1739BL cells). In some embodiments, the immune complex formation is detected by fluorescence intensity using a fluorescently labeled secondary antibody (e.g., merely to illustrate, the stability of an immune complex formed with an anti-CD39 antibody can be determined by incubating anti-CD39 monoclonal antibodies (mAbs) (e.g., at 2 pg / mL or greater) with HCC1739BL cells for different times and then detecting the presence of immune complex by fluorescent conjugated secondary antibody). a. Antibodies and other Polypeptides

[0087] The term "antibody" as used herein refers to an immunoglobulin molecule that recognizes and specifically binds a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of any of the foregoing, through at least one antigenbinding site wherein the antigen-binding site is usually within the variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) antibodies provided those fragments have been formatted to include an Fc or other FcyRIII binding domain, multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen-binding site of an antibody (formatted to include an Fc or other FcyRIII binding domain), and any other modified immunoglobulin molecule comprising an antigen-binding site as long as the antibodies exhibit the desired biological activity.

[0088] The term "antigen-binding portion" or antigen-binding fragment" of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human CD39). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody, e.g., an anti- CD39 antibody described herein, include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., 1989, Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) or (vii) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These and other potential constructs are described at Chan and Carter (2010) Nat. Rev. Immunol. 10:301. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0089] The term "variable region" of an antibody refers to the variable region of an antibody light chain, or the variable region of an antibody heavy chain, either alone or in combination. Generally, the variable region of heavy and light chains each consist of four framework regions (FR) and three complementarity determining regions (CDRs), also known as "hypervariable regions". The CDRs in each chain are held together in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigenbinding sites of the antibody. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, National Institutes of Health, Bethesda Md.); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al Lazikani et al., 1997, J. Mol. Biol. 273:927-948). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.

[0090] While the antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively, the preferred CD39 antibody is an IgGl and IgG3 isotype in order to engage FcyRIII most effectively (i.e., with a Kd of 10'7or smaller).

[0091] In certain embodiments, the antibody is "hypo-fucosylated" and may even be "afucosylated". A "hypo-fucosylated" antibody preparation refers to an antibody preparation in which less than 50% of the oligosaccharide chains contain oc-l,6-fucose. Typically, less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than 5% or less than 1% of the oligosaccharide chains contain oc-l,6-fucose in a "hypo-fucosylated" antibody preparation. An "afucosylated" antibody lacks oc-l,6-fucose in the carbohydrate attached to the CH2 domain of the IgG heavy chain.

[0092] The term "monoclonal antibody" as used herein refers to an antibody that displays a single binding specificity and affinity for a particular epitope or a composition of antibodies in which all antibodies display a single binding specificity and affinity for a particular epitope. Typically such monoclonal antibodies will be derived from a single cell or nucleic acid encoding the antibody, and will be propagated without intentionally introducing any sequence alterations. Accordingly, the term "human monoclonal antibody" refers to a monoclonal antibody that has variable and optional constant regions derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by a hybridoma, for example, obtained by fusing a B cell obtained from a transgenic or transchromosomal non-human animal (e.g., a transgenic mouse having a genome comprising a human heavy chain transgene and a light chain transgene), to an immortalized cell.

[0093] The term "humanized antibody" as used herein refers to forms of non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human sequences. Typically, humanized antibodies are human immunoglobulins in which residues of the CDRs are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, or hamster) that have the desired specificity, affinity, and / or binding capability. In some instances, the Fv framework region residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species. The humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and / or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and / or binding capability. The humanized antibody may comprise variable domains containing all or substantially all of the CDRs that correspond to the non-human immunoglobulin whereas all or substantially all of the framework regions are those of a human immunoglobulin sequence. In some embodiments, the variable domains comprise the framework regions of a human immunoglobulin sequence. In some embodiments, the variable domains comprise the framework regions of a human immunoglobulin consensus sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. A humanized antibody is usually considered distinct from a chimeric antibody.

[0094] The term "human antibody" as used herein refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any of the techniques known in the art.

[0095] The term "chimeric antibody" as used herein refers to an antibody wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammals (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and / or binding capability, while the constant regions are homologous to the sequences in antibodies derived from another species (usually human) to avoid eliciting an immune response in that species.

[0096] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind to an IgG antibody comprise receptors of the FcyR family, including allelic variants and alternatively spliced forms of these receptors. The FcyR family consists of three activating (FcyRI, FcyRIII, and FcyRIV in mice; FcyRIA, FcyRIIA, and FcyRIIIA in humans) and one inhibitory (FcyRIIB) receptor.

[0097] An "FcyRIII binding moiety" is a peptide, protein, nucleic acid or other moiety which, when associated with an antigen binding site of an anti-CD39 antibody, is able to bind to FcyRIII (CD 16) and, optionally, mediate antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP). The heavy chain Fc fragment containing the CH2 and CH3 domains of IgGl and IgG3 isotypes are FcyRIII binding moiety.

[0098] The terms "epitope" and "antigenic determinant" are used interchangeably herein and refer to that portion of an antigen capable of being recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids (also referred to as linear epitopes) are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding (also referred to as conformational epitopes) are typically lost upon protein denaturing. An epitope typically includes at least 3, and more usually, at least 5, 6, 7, or 8-10 amino acids in a unique spatial conformation.

[0099] As use herein, the term "specifically binds to" or is "specific for" refers to measurable and reproducible interactions such as binding between a target and an antibody, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules. For example, an antibody that specifically binds to a target (which can be an epitope) is an antibody that binds this target with greater affinity, avidity, more readily, and / or with greater duration than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of less than or equal to 1 |lM, 100 nM, 10 nM, 1 nM, or even 0.1 nM. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among the protein from different species. In another embodiment, specific binding can include, but does not require exclusive binding.

[0100] The terms "polypeptide" and "peptide" and "protein" are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art. It is understood that, because the polypeptides encompassed by the present invention may be based upon antibodies or other members of the immunoglobulin superfamily, in certain embodiments, the polypeptides can occur as single chains or as associated chains.

[0101] The terms "identical" or percent "identity" in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity may be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that may be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two nucleic acids or polypeptides encompassed by the invention are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the amino acid sequences that is at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 residues, such as at least about 80-100 residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a target protein or an antibody. In some embodiments, identity exists over a region of the nucleotide sequences that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as a nucleotide sequence encoding a protein of interest.

[0102] A "conservative amino acid substitution" is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. Generally, conservative substitutions in the sequences of the polypeptides, soluble proteins, and / or antibodies encompassed by the present invention do not abrogate the binding of the polypeptide, soluble protein, or antibody containing the amino acid sequence, to the target binding site. Methods of identifying amino acid conservative substitutions which do not eliminate binding are well-known in the art.

[0103] A polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is "isolated" is a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature. Isolated polypeptides, soluble proteins, antibodies, polynucleotides, vectors, cells, or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.

[0104] The term "substantially pure" as used herein refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0105] The term "fusion protein" or "fusion polypeptide" as used herein refers to a hybrid protein expressed by a nucleic acid molecule comprising nucleotide sequences of at least two genes.

[0106] The term "linker" or "linker region" as used herein refers to a linker inserted between a first polypeptide (e.g., an anti-CD39 antibody) and a second polypeptide (e.g., an Fc or other FcyRIII binding moiety; an scFV, Vhh domain or the like the binds a different protein to create a bispecific antibody format maintaining the bivalency for CD39). In some embodiments, the linker is a peptide linker. Linkers should not adversely affect the expression, secretion, or bioactivity of the polypeptides. Preferably, linkers are not antigenic and do not elicit an immune response. b. Treatments

[0107] The term "effective amount" as used herein refers to an amount to provide therapeutic or prophylactic benefit with respect to the size and / or severity of scarring.

[0108] The term "treatment" as used herein refers to action by an individual to change the process of a clinical disease, which may be either preventive or an intervention to alter a course of clinical pathology. The term includes, but is not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, reducing the direct or indirect pathological consequences of any disease, preventing metastasis, slowing the rate of disease progression, ameliorating or remitting disease remission, and improving prognosis - in the instance invention, the size and / or severity of a scar or adhesion.

[0109] The term "subject" refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, canines, felines, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[0110] The term "pharmaceutically acceptable" refers to a substance approved or approvable by a regulatory agency of the Federal government or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.

[0111] The terms "pharmaceutically acceptable excipient, carrier or adjuvant" or "acceptable pharmaceutical carrier" refer to an excipient, carrier or adjuvant that can be administered to a subject, together with at least one agent of the present disclosure, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic effect. In general, those of skill in the art and the U.S. FDA consider a pharmaceutically acceptable excipient, carrier, or adjuvant to be an inactive ingredient of any formulation.

[0112] The terms "effective amount" or "therapeutically effective amount" or "therapeutic effect" refer to an amount of an anti-CD39 antibody effective to "treat" a disease or disorder in a subject such as, a mammal.

[0113] The terms "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to both (1) therapeutic measures that cure, slow down, lessen symptoms of, and / or halt progression of scar formation and (2) prophylactic or preventative measures that prevent or slow the development of a scar or the reduction in the severity of the scar. c. Miscellaneous

[0114] It is understood that wherever embodiments are described herein with the language "comprising" otherwise analogous embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided. It is also understood that wherever embodiments are described herein with the language "consisting essentially of" otherwise analogous embodiments described in terms of "consisting of" are also provided.

[0115] As used herein, reference to "about" or "approximately" a value or parameter includes (and describes) embodiments that are directed to that value or parameter. For example, description referring to "about X" includes description of "X".

[0116] The term "and / or" as used in a phrase such as "A and / or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0117] III. Anti-CD39 Antibodies a. Monoclonal Antibodies

[0118] The anti-CD39 antibodies useful in the methods and pharmaceutical preparations of the present invention may be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, 1975, Nature 256:495. In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro. In some embodiments, monoclonal antibodies, e.g., rabbit monoclonal antibodies, may be produced using single B cell cloning technology, such as those described in Rashidian and Lloyd, 2020, Methods Mol. Biol. 2070:423-441, the content of which is incorporated by reference herein in its entirety.

[0119] The immunizing agent will typically include the CD39 polypeptide or a fusion protein thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.

[0120] Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif, and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor et al., 1984, J. Immunol. 133:3001; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).

[0121] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the polypeptide. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, 1980, Anal. Biochem. 107:220.

[0122] After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPML 1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.

[0123] The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0124] The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies encompassed by the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells encompassed by the present invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison et al., supra) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a nonimmunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody encompassed by the present invention, or can be substituted for the variable domains of one antigen-combining site of an antibody encompassed by the present invention to create a chimeric bivalent antibody. b. Human and Humanized Antibodies

[0125] The anti-CD39 antibodies encompassed by the present invention may further comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non- human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988, Nature 332:323-329; and Presta, 1992, Curr. Op. Struct. Biol. 2:593- 596).

[0126] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988, Nature 332:323-327; Verhoeyen et al., 1988, Science 239:1534-1536), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0127] Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, 1991, J. Mol. Biol. 227:381; Marks et al., 1991, J. Mol. Biol. 222:581), and yeast display (Chao et al., 2006, Nat. Protoc. l(2):755-68). The techniques of Cole et al. and Boemer et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77 (1985) and Boerner et al., 1991, J. Immunol. 147(l):86-95). Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; and in the following scientific publications: Marks et al., 1992, Bio / Technology 10:779-783; Lonberg et al., 1994, Nature 368:856-859; Morrison, 1994, Nature 368:812-13; Fishwild et al., 1996, Nature Biotechnology 14:845-51; Neuberger, 1996, Nature Biotechnology 14:826; Lonberg and Huszar, 1995, Intern. Rev. Immunol. 13:65-93.

[0128] The antibodies may also be affinity matured using known selection and / or mutagenesis methods as described above. Preferred affinity matured antibodies have an affinity which is five times, more preferably 10 times, even more preferably 20 or 30 times greater than the starting antibody (generally murine, humanized or human) from which the matured antibody is prepared. c. Bispecific Antibodies

[0129] Anti-CD39 antibodies described herein include bispecific molecules. An anti-CD39 antibody, or antigen-binding portions thereof, can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibody described herein may in fact be derivatized or linked to more than one other functional molecule to generate multispecific molecules that bind to more than two different binding sites and / or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule described herein, an antibody described herein can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, such that a bispecific molecule results.

[0130] Accordingly, provided herein are bispecific molecules comprising at least one first binding specificity for CD39 and a second binding specificity for a second target epitope. In an embodiment described herein in which the bispecific molecule is multi- specific, the molecule can further include a third binding specificity.

[0131] In certain embodiments, the present invention provides bispecific antibodies that bind to both CD39 and a granulation tissue cell surface antigen selected from the group consisting of CD31, CD45, CDl lb, F4 / 80, and CD90.2, and preferably causes wound healing inflammation / granulation cell depletion - such as by ADCC-mediated and / or ADCP-mediated killing or in the form of an antibody-drug conjugate that is preferentially taken up by and toxic to inflammation / granulation cells at the site of wound. In one embodiment, the bispecific molecules described herein comprise as a binding specificity at least one antibody, or an antibody fragment thereof, including, e.g., an Fab, Fab', F(ab')2, Fv, or a single chain Fv. The antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof such as a Fv or a single chain (scFv) construct.

[0132] Binding of the bispecific molecules to their specific targets can be confirmed using art- recognized methods, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western Blot assay. Each of these assays generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest.

[0133] Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain / light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, 1983, Nature 305:537-539). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93 / 08829, published 13 May 1993, and in Traunecker et al., 1991, EMBO J. 10:3655-3659.

[0134] Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., 1986, Methods in Enzymology 121:210.

[0135] According to another approach described in WO 96 / 27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

[0136] Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared can be prepared using chemical linkage. Brennan et al., 1985, Science 229:81 describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

[0137] Fab' fragments may be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., 1992, J. Exp. Med. 175:217-225 describe the production of a fully humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.

[0138] Various technique for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers (Kostelny et al., 1992, J. Immunol. 148(5): 1547- 1553). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448 has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., 1994, J. Immunol. 152:5368.

[0139] Antibodies with more than two valencies are contemplated. As one nonlimiting example, trispecific antibodies can be prepared. See, e.g., Tutt et al., 1991, J. Immunol. 147:60. d. Heteroconjugate Antibodies

[0140] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91 / 00360; WO 92 / 200373; EP 03089). It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980. e. Effector Function Engineering

[0141] It may be desirable to modify the antibody encompassed by the present invention with respect to effector function, so as to further enhance, e.g., the effectiveness of the anti-CD39 antibody in depleting wound healing inflammation / granulation cells. For example, cysteine residue(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability, increased complement-mediated cell killing, improved antibodydependent cellular cytotoxicity (ADCC), and / or improved antibody-dependent cellular phagocytosis (ADCP). See Caron et al., 1992, J. Exp Med. 176:1191-1195 and Shopes, 1992, J. Immunol., 148:2918-2922. f. Representative anti-CD39 Antibody Sequences

[0142] In certain embodiments, the anti-CD39 antibody is a fully human antibody, such as generated from a human antibody library. An exemplary fully human anti-CD39 antibody is clone PSCWT22, the heavy and light variable domains (VH and VL) sequences provided as follows:

[0143] For the PSCWT22 clone, the CDRs for each of the VH and VL domains are:

[0144] The sequences for an exemplary full-length antibody and an exemplary single chain antibody (scFV) utilizing the VH and VL domains above are provided as follows:

[0145] In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof comprises at least one heavy chain variable domain is at least 60% identical to SEQ ID No. 2 and even more preferably at least 65%, 70%, 75%, 80%, 85% or even 90% identical to SEQ ID No. 2, and able to specifically bind human CD39.

[0146] In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof comprises at least one light chain variable domain is at least 60% identical to SEQ ID No. 4, and even more preferably at least 65%, 70%, 75%, 80%, 85% or even 90% identical to SEQ ID No. 4 and able to specifically bind human CD39.

[0147] In certain embodiments, the anti-CD39 antibody is a humanized antibody comprising a VH domain having human framework sequences associated with CDRs of a VH domain shown in SEQ ID Nos. 29, 30 and 31, and the CDRs of the corresponding VL domain shown in SEQ ID Nos. 32, 33 and 34. The CDRs are preferably identical, but may vary by 1, 2 or 3 amino acids across each CDR so long as the resulting antibody specifically binds human CD39.

[0148] In certain embodiments, the heavy and light chains of the anti-CD39 antibody have variable domains that can be encoded by a nucleic acid which is identical to, or hybridizes under stringent conditions (such as the 6x sodium chloride / sodium citrate (SSC) at 45°C, and washing in 0.2x SSC / 0.1% SDS at 50-65°C) to the VH and VL domain (correspondingly) coding sequences shown in SEQ ID No. 1 (VH) and SEQ ID No. 3 (VL).

[0149] In some embodiments, anti-CD39 antibodies were generated in rabbits, and the variable domains of the heavy and light chains of these antibodies are rabbit sequence while the constant domains are human sequence. Exemplary sequences for the VH and VL domains of rabbit anti- CD39 antibodies are:

[0150]

[0151] In some embodiments, anti-CD39 antibodies were generated in rabbits, and then humanized by CDR grafting. Exemplary sequences for the VH and VL domains of rabbit anti-

[0152] CD39 antibodies are:

[0153]

[0154] In some embodiments, anti-CD39 antibodies provided herein promote: (i) stable immune complex formation when incubated with HCC1739BL cells as characterized by loss of less than 30% of the immune complex after 24 hours, optionally wherein the immune complex formation is detected by fluorescence intensity using a fluorescently labeled secondary antibody; (ii) antibody-dependent cellular cytotoxicity (ADCC) and / or antibodydependent cellular phagocytosis (ADCP) activity against CD39+ cells; (iii) depletion of CD39highwound healing inflammation / granulation cells; (iv) binding to a CD39 epitope having a sequence selected from the group of CD39 amino acid epitope sequences listed in Figure 21 (for example, binding one or more linear or conformational CD39 epitopes, such as selected from the group consisting of 1) IYLTDCMERAR, 2) LRMESEELADR, 3) RVKGPGISKFV, 4) DCMERAREVIPR, 5) LTDCMERAREVIPR, 6) SLSNYPFDFQGAR, 7) CRVKGPGISKF, 8) GAYGWITINYLLGKFSQK, 9) ILRDPCFHPGYKK, and any combination thereof, such as RVKGPGISKFV and DCMERAREVIPR, LTDCMERAREVIPR and SLSNYPFDFQGAR, or any combination of CRVKGPGISKF, GAYGWITINYLLGKFSQK, and ILRDPCFHPGYKK); and / or (v) binding to CD39 in a manner that is non-competitive or only partially competitive with monoclonal antibody Clone Al binding to CD39.

[0155] Representative anti-CD39 antibody sequences described above according to sequence identification number correspond to the following:

[0156] SEQ ID No. 1 (Clone PSCWT22 vH nucleic acid sequence) gag gtg caa ctg gtg gag tct ggg gga ggt gtg gta agg cct ggg ggg 48

[0157] Glu Vai Gin Leu Vai Glu Ser Gly Gly Gly Vai Vai Arg Pro Gly Gly 1 5 10 15 tcc ctg aga etc tcc tgt gca gcc tct gga ttc acc ttc agt age tat 96 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser

[0158] Tyr

[0159] 20 25 30 get atg cac tgg gtc ege cag get cca ggc aag ggg etg gag tgg gtg 144

[0160] Ala Met His Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Vai

[0161] 35 40 45 gca gtt ata tea tat gat gta age aat aaa tac tac gca gac tee gtg 192

[0162] Ala Vai l ie Ser Tyr Asp Vai Ser Asn Lys Tyr Tyr Ala Asp Ser Vai

[0163] 50 55 60 aag ggc ega ttc ace ate tee aga gac aat tee aag aac acg etg tat 240

[0164] Lys Gly Arg Phe Thr l ie Ser Arg Asp Asn Ser Lys Asn Thr Leu

[0165] Tyr 65 70 75 80 etg caa atg aac age etg aga get gag gac acg get gtg tat tac tgt 288

[0166] Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Vai Tyr Tyr

[0167] Cys

[0168] 85 90 95 geg aga tet tac tac tac tac tac ggt atg gac gtc tgg ggc caa ggg 336

[0169] Ala Arg Ser Tyr Tyr Tyr Tyr Tyr Gly Met Asp Vai Trp Gly Gin

[0170] Gly

[0171] 100 105 110 acc acg gtc ace gtc tee tea 357

[0172] Thr Thr Vai Thr Vai Ser Ser

[0173] 115

[0174] SEQ ID No. 2 (Clone PSCWT22 vH amino acid sequence)

[0175] Glu Vai Gin Leu Vai Glu Ser Gly Gly Gly Vai Vai Arg Pro Gly Gly 1 5 10 15

[0176] Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

[0177] Ala Met His Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Vai 35 40 45

[0178] Ala Vai l ie Ser Tyr Asp Vai Ser Asn Lys Tyr Tyr Ala Asp Ser Vai 50 55 60

[0179] Lys Gly Arg Phe Thr l ie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

[0180] 65 70 75 80

[0181] Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Vai Tyr Tyr Cys

[0182] 85 90 95 Ala Arg Ser Tyr Tyr Tyr Tyr Tyr Gly Met Asp Vai Trp Gly Gin Gly 100 105 110

[0183] Thr Thr Vai Thr Vai Ser Ser 115

[0184] SEQ ID No. 3 (Clone PSCWT22 vL domain nucleic acid sequence) gat gtt gtg atg acc cag tct cca tcc tcc ctg tct gca tct gta gga 48

[0185] Asp Vai Vai Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Vai Gly 1 5 10 15 gac aga gtc acc ate act tgc egg gca agt cag age att age agg tac 96

[0186] Asp Arg Vai Thr T ie Thr Cys Arg Ala Ser Gin Ser T ie Ser Arg Tyr 20 25 30 tta gee tgg tac caa cag aaa cct ggc cag get ccc agg etc etc ate 144

[0187] Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu T ie

[0188] 35 40 45 tat gat gca tcc aac agg gcc act ggc ate cca gtc agg ttc agt ggc 192

[0189] Tyr Asp Ala Ser Asn Arg Ala Thr Gly l ie Pro Vai Arg Phe Ser Gly 50 55 60 agt ggg tet ggg aca gac ttc act etc ace ate age aga etg gag cca 240

[0190] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr l ie Ser Arg Leu Glu Pro 65 70 75 80 gaa gat ttt gca gtg tat tac tgt cag cag ttt ggt agg tea cct egg 288

[0191] Glu Asp Phe Ala Vai Tyr Tyr Cys Gin Gin Phe Gly Arg Ser Pro Arg 85 90 95 acg ttc ggc caa ggg aca ega etg gag att aaa 321 Thr Phe Gly Gin Gly Thr Arg Leu Glu l ie Lys 100 105

[0192] SEQ ID No. 4 (Clone PSCWT22 vL domain amino acid sequence)

[0193] Asp Vai Vai Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Vai Gly 1 5 10 15

[0194] Asp Arg Vai Thr l ie Thr Cys Arg Ala Ser Gin Ser l ie Ser Arg Tyr

[0195] 20 25 30

[0196] Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu l ie

[0197] 35 40 45

[0198] Tyr Asp Ala Ser Asn Arg Ala Thr Gly l ie Pro Vai Arg Phe Ser Gly

[0199] 50 55 60

[0200] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr l ie Ser Arg Leu Glu Pro 65 70 75 80

[0201] Glu Asp Phe Ala Vai Tyr Tyr Cys Gin Gin Phe Gly Arg Ser Pro Arg

[0202] 85 90 95

[0203] Thr Phe Gly Gin Gly Thr Arg Leu Glu l ie Lys

[0204] 100 105 SEQ ID No. 5 (Clone PSC18 vH domain nucleic acid sequence) cag tea gtg aag gag gcc ggg ggt ege etg gta acg cct gga gga tee 48

[0205] Gin Ser Vai Lys Glu Ala Gly Gly Arg Leu Vai Thr Pro Gly Gly Ser 1 5 10 15 etg aca etc ace tgc aca gtc tet gga ttc tee etc agt geg tat gga 96

[0206] Leu Thr Leu Thr Cys Thr Vai Ser Gly Phe Ser Leu Ser Ala Tyr Gly 20 25 30 ata agt tgg gtc ege cag get cca ggg aag gga etg gaa tgg ate gga 144

[0207] T ie Ser Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp T ie Gly 35 40 45

[0208] ate att tat agt agt ggt agg act tac tac geg aac tgg geg aaa ggc 192

[0209] T ie T ie Tyr Ser Ser Gly Arg Thr Tyr Tyr Ala Asn Trp Ala Lys Gly 50 55 60 ega ttc acc ate tcc aaa acc teg teg acc acg gtg gat etg aaa atg 240 Arg Phe Thr T ie Ser Lys Thr Ser Ser Thr Thr Vai Asp Leu Lys Met

[0210] 65 70 75 80 acc agt etg aca acc gag gac acg gee gee tat ttc tgt gee aga tea

[0211] 288 Thr Ser Leu Thr Thr Glu Asp Thr Ala Ala Tyr Phe Cys Ala Arg Ser 85 90 95 egg get ggt att agt agt ggt gat ggt ttt gat tcc tgg ggc cca ggc 336 Arg Ala Gly T ie Ser Ser Gly Asp Gly Phe Asp Ser Trp Gly Pro Gly 100 105 110 acc etg gtc acc gtc tcc tea 357

[0212] Thr Leu Vai Thr Vai Ser Ser

[0213] 115

[0214] SEQ ID No. 6 (Clone PSC18 vH domain amino acid sequence)

[0215] Gin Ser Vai Lys Glu Ala Gly Gly Arg Leu Vai Thr Pro Gly Gly Ser 1 5 10 15

[0216] Leu Thr Leu Thr Cys Thr Vai Ser Gly Phe Ser Leu Ser Ala Tyr Gly

[0217] 20 25 30

[0218] T ie Ser Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp T ie Gly

[0219] 35 40 45

[0220] T ie T ie Tyr Ser Ser Gly Arg Thr Tyr Tyr Ala Asn Trp Ala Lys Gly

[0221] 50 55 60

[0222] Arg Phe Thr T ie Ser Lys Thr Ser Ser Thr Thr Vai Asp Leu Lys Met 65 70 75 80

[0223] Thr Ser Leu Thr Thr Glu Asp Thr Ala Ala Tyr Phe Cys Ala Arg Ser

[0224] 85 90 95 Arg Ala Gly l ie Ser Ser Gly Asp Gly Phe Asp Ser Trp Gly Pro Gly

[0225] 100 105 110

[0226] Thr Leu Vai Thr Vai Ser Ser 115

[0227] SEQ ID No. 7 (Clone PSC18 vL domain nucleic acid sequence) gcc ctt gtg atg acc cag act cca tcc tcc gtg tct gca get gtg gga 48

[0228] Ala Leu Vai Met Thr Gin Thr Pro Ser Ser Vai Ser Ala Ala Vai Gly 1 5 10 15

[0229] ggc aca gtc acc ate aat tgc cag gee agt cag aac att tac age aat 96

[0230] Gly Thr Vai Thr l ie Asn Cys Gin Ala Ser Gin Asn l ie Tyr Ser Asn

[0231] 20 25 30 tta gee tgg tat cag cag aaa cca ggg cag cgt ccc cag etc etg ate 144

[0232] Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Arg Pro Gin Leu Leu l ie

[0233] 35 40 45 tac agg gca tee act etg gca tet ggg gtc cca teg egg ttc aaa ggc 192

[0234] Tyr Arg Ala Ser Thr Leu Ala Ser Gly Vai Pro Ser Arg Phe Lys Gly

[0235] 50 55 60 agt gca tet ggg aca gaa tac act etc acc ate age ggt gtg cag tgt 240

[0236] Ser Ala Ser Gly Thr Glu Tyr Thr Leu Thr T ie Ser Gly Vai Gin

[0237] Cys 65 70 75 80 gac gat get gee act tac tat tgt caa cag ggt ttt gat agt agt aac 288

[0238] Asp Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Gly Phe Asp Ser Ser Asn

[0239] 85 90 95 att gat aat act ttc ggc gga ggg acc gag gtg gtg gtc aca 330

[0240] T ie Asp Asn Thr Phe Gly Gly Gly Thr Glu Vai Vai Vai Thr

[0241] 100 105 110

[0242] SEQ ID No. 8 (Clone PSC18 vL domain amino acid sequence)

[0243] Ala Leu Vai Met Thr Gin Thr Pro Ser Ser Vai Ser Ala Ala Vai Gly 1 5 10 15

[0244] Gly Thr Vai Thr T ie Asn Cys Gin Ala Ser Gin Asn T ie Tyr Ser Asn

[0245] 20 25 30

[0246] Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Arg Pro Gin Leu Leu T ie

[0247] 35 40 45 Tyr Arg Ala Ser Thr Leu Ala Ser Gly Vai Pro Ser Arg Phe Lys Gly

[0248] 50 55 60

[0249] Ser Ala Ser Gly Thr Glu Tyr Thr Leu Thr He Ser Gly Vai Gin

[0250] Cys

[0251] 65 70 75 80

[0252] Asp Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Gly Phe Asp Ser Ser

[0253] Asn

[0254] 85 90 95

[0255] T ie Asp Asn Thr Phe Gly Gly Gly Thr Glu Vai Vai Vai Thr

[0256] 100 105 110

[0257] SEQ ID No. 9 (Clone PSC19 vH domain nucleic acid sequence) cag teg gtg gag gag tcc ggg ggt ege etg gtc acg cct ggg aca cac 48 Gin Ser Vai Glu Glu Ser Gly Gly Arg Leu Vai Thr Pro Gly Thr His 1 5 10 15 etg aca etc acc tgc aca gtc tet gga ttc tec etc agt aag agt ata 96 Leu Thr Leu Thr Cys Thr Vai Ser Gly Phe Ser Leu Ser Lys Ser l ie 20 25 30 ata agt tgg gtc ege cag get cca ggg aag ggg etg gaa tac ate gga 144 l ie Ser Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Tyr l ie Gly 35 40 45 ate att ggt agt agt ggt age aca tac tac geg aac tgg geg aaa ggc 192 l ie l ie Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Asn Trp Ala Lys Gly 50 55 60 ega ttc acc ate tec aaa acc teg teg acc acg gtg gat etg aga atg 240 Arg Phe Thr l ie Ser Lys Thr Ser Ser Thr Thr Vai Asp Leu Arg Met 65 70 75 80 acc agt etg aca ccc gag gac acg gee acc tat ttc tgt gee aga gga 288 Thr Ser Leu Thr Pro Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly 85 90 95 ett ett tat tet ggt aat aaa teg tgg ggc ccg ggc acc etg gtc acc 336 Leu Leu Tyr Ser Gly Asn Lys Ser Trp Gly Pro Gly Thr Leu Vai Thr 100 105 110 gtc tec tea 345 Vai Ser Ser 115

[0258] SEQ ID No. 10 (Clone PSC19 vH domain amino acid sequence)

[0259] Gin Ser Vai Glu Glu Ser Gly Gly Arg Leu Vai Thr Pro Gly Thr His 1 5 10 15 Leu Thr Leu Thr Cys Thr Vai Ser Gly Phe Ser Leu Ser Lys Ser lie

[0260] 20 25 30 lie Ser Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Tyr lie Gly

[0261] 35 40 45 lie lie Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Asn Trp Ala Lys Gly

[0262] 50 55 60

[0263] Arg Phe Thr lie Ser Lys Thr Ser Ser Thr Thr Vai Asp Leu Arg Met

[0264] 65 70 75 80

[0265] Thr Ser Leu Thr Pro Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly

[0266] 85 90 95

[0267] Leu Leu Tyr Ser Gly Asn Lys Ser Trp Gly Pro Gly Thr Leu Vai Thr

[0268] 100 105 110

[0269] Vai Ser Ser

[0270] 115

[0271] SEQ ID No. 11 (Clone PSC19 vL domain nucleic acid sequence) gcc att gat atg acc cag act cca tcc tcc gtg tct gca get gtg gga 48

[0272] Ala T ie Asp Met Thr Gin Thr Pro Ser Ser Vai Ser Ala Ala Vai Gly 1 5 10 15 ggc aca gtc acc ate aac tgc cag tcc agt cag agt gtt tta etg aac 96

[0273] Gly Thr Vai Thr T ie Asn Cys Gin Ser Ser Gin Ser Vai Leu Leu Asn

[0274] 20 25 30 aac caa tta tcc tgg ttt cag cag aaa cca ggg cag cct ccc aag etc 144

[0275] Asn Gin Leu Ser Trp Phe Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu

[0276] 35 40 45 etg ate tat gat gca tcc act etg gaa tct ggg gtc cca tct egg ttc 192

[0277] Leu T ie Tyr Asp Ala Ser Thr Leu Glu Ser Gly Vai Pro Ser Arg Phe

[0278] 50 55 60 aca ggc agt gga tct ggg aca cag ttc act etc acc ate age gac etg 240

[0279] Thr Gly Ser Gly Ser Gly Thr Gin Phe Thr Leu Thr T ie Ser Asp Leu 65 70 75 80 gag tgt gac gat get gcc act tac tat tgt tta ggc ggt tat agt ggg 288

[0280] Glu Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Gly Tyr Ser Gly 85 90 95 aac ett tat get ttc ggc gga ggg acc gag gtg eta gtc aaa 330 Asn Leu Tyr Ala Phe Gly Gly Gly Thr Glu Vai Leu Vai Lys 100 105 110

[0281] SEQ ID No. 12 (Clone PSC19 vL domain amino acid sequence)

[0282] Ala l ie Asp Met Thr Gin Thr Pro Ser Ser Vai Ser Ala Ala Val Gly 1 5 10 15

[0283] Gly Thr Vai Thr T ie Asn Cys Gin Ser Ser Gin Ser Vai Leu Leu Asn

[0284] 20 25 30

[0285] Asn Gin Leu Ser Trp Phe Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu

[0286] 35 40 45

[0287] Leu l ie Tyr Asp Ala Ser Thr Leu Glu Ser Gly Vai Pro Ser Arg Phe

[0288] 50 55 60

[0289] Thr Gly Ser Gly Ser Gly Thr Gin Phe Thr Leu Thr T ie Ser Asp

[0290] Leu 65 70 75 80

[0291] Glu Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Gly Tyr Ser Gly

[0292] 85 90 95

[0293] Asn Leu Tyr Ala Phe Gly Gly Gly Thr Glu Vai Leu Vai Lys 100 105 110

[0294] SEQ ID No. 13 (Clone PSC20 vH domain nucleic acid sequence) cag teg gtg gag gag tcc ggg ggt ege etg gtc acg cct ggg aca ccc 48

[0295] Gin Ser Vai Glu Glu Ser Gly Gly Arg Leu Vai Thr Pro Gly Thr Pro 1 5 10 15 etg aca etc acc tgc aca gtc tet gga ttc tcc etc agt age tat gca 96

[0296] Leu Thr Leu Thr Cys Thr Vai Ser Gly Phe Ser Leu Ser Ser Tyr Ala 20 25 30 ata agt tgg gtc ege cag get cca ggg aag ggg etc gaa tat ate geg 144 T ie Ser Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Tyr T ie Ala 35 40 45 ate att aat agt tat ggt acc aca tac tac geg age tgg geg aaa ggc 192 T ie T ie Asn Ser Tyr Gly Thr Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 ega gtc acc ate tcc aaa acc teg age acg gtg gat etg aaa ate tcc 240

[0297] Arg Vai Thr T ie Ser Lys Thr Ser Ser Thr Vai Asp Leu Lys T ie Ser

[0298] 65 70 75 80 agt ccg aca acc gag gac acg gcc acc tat ttc tgt gcc aga ggc gat

[0299] 288 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Asp agt tat ggt agt ggt gtt ggt ttg ggc ttg tgg ggc cca ggc acc etg 336

[0300] Ser Tyr Gly Ser Gly Vai Gly Leu Gly Leu Trp Gly Pro Gly Thr Leu 100 105 110 gtc acc gtc tcc tea 351

[0301] Vai Thr Vai Ser Ser

[0302] 115

[0303] SEQ ID No. 14 (Clone PSC20 vH domain amino acid sequence)

[0304] Gin Ser Vai Glu Glu Ser Gly Gly Arg Leu Vai Thr Pro Gly Thr Pro 1 5 10 15

[0305] Leu Thr Leu Thr Cys Thr Vai Ser Gly Phe Ser Leu Ser Ser Tyr Ala

[0306] 20 25 30 l ie Ser Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Tyr l ie Ala

[0307] 35 40 45 l ie l ie Asn Ser Tyr Gly Thr Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

[0308] 50 55 60

[0309] Arg Vai Thr T ie Ser Lys Thr Ser Ser Thr Vai Asp Leu Lys T ie Ser 65 70 75 80

[0310] Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Asp

[0311] 85 90 95

[0312] Ser Tyr Gly Ser Gly Vai Gly Leu Gly Leu Trp Gly Pro Gly Thr Leu

[0313] 100 105 110

[0314] Val Thr Vai Ser Ser

[0315] 115

[0316] SEQ ID No. 15 (Clone PSC20 vL domain nucleic acid sequence) gcc tat gat atg acc cag act cca gcc tct gtg gag gta get gtg gga 48

[0317] Ala Tyr Asp Met Thr Gin Thr Pro Ala Ser Vai Glu Vai Ala Vai Gly 1 5 10 15 ggc aca gtc acc ate aag tgc cag gcc agt cag aac att tac age aat 96

[0318] Gly Thr Vai Thr T ie Lys Cys Gin Ala Ser Gin Asn T ie Tyr Ser Asn

[0319] 20 25 30 tta gcc tgg tat cag cag aaa cca ggg cag cgt ccc aag etc etg ate 144

[0320] Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Arg Pro Lys Leu Leu T ie

[0321] 35 40 45 tac agg gca tec agt etg gca tct ggg gtc ccg teg egg ttc agt ggc 192 Tyr Arg Ala Ser Ser Leu Ala Ser Gly Vai Pro Ser Arg Phe Ser Gly

[0322] 50 55 60 agt gga tct ggg aca gag ttc act etc ace ate age ggt gtg cag tgt 240

[0323] Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr T ie Ser Gly Vai Gin

[0324] Cys

[0325] 65 70 75 80 gac gat get gcc act tac tac tgt caa cag ggt ttt agt agt aat aat 288

[0326] Asp Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Gly Phe Ser Ser Asn Asn

[0327] 85 90 95

[0328] gtt gat aat act ttc ggc gga ggg acc gag gtg gtg gtc aaa 330

[0329] Vai Asp Asn Thr Phe Gly Gly Gly Thr Glu Vai Vai Vai Lys 100 105 110

[0330] SEQ ID No. 16 (Clone PSC20 vL domain amino acid sequence)

[0331] Ala Tyr Asp Met Thr Gin Thr Pro Ala Ser Vai Glu Vai Ala Vai Gly 1 5 10 15

[0332] Gly Thr Vai Thr T ie Lys Cys Gin Ala Ser Gin Asn T ie Tyr Ser Asn

[0333] 20 25 30

[0334] Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Arg Pro Lys Leu Leu T ie

[0335] 35 40 45

[0336] Tyr Arg Ala Ser Ser Leu Ala Ser Gly Vai Pro Ser Arg Phe Ser Gly

[0337] 50 55 60

[0338] Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr T ie Ser Gly Vai Gin Cys 65 70 75 80

[0339] Asp Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Gly Phe Ser Ser Asn

[0340] Asn 85 90 95

[0341] Vai Asp Asn Thr Phe Gly Gly Gly Thr Glu Vai Vai Vai Lys 100 105 110

[0342] SEQ ID No. 17 (Clone PSC21 vH domain nucleic acid sequence) cag teg gtg gag gag tcc ggg ggt ege etg gtc acg cct ggg aca ccc 48

[0343] Gin Ser Vai Glu Glu Ser Gly Gly Arg Leu Vai Thr Pro Gly Thr Pro 1 5 10 15 etg aca etc acc tgc acc gtc tcc gga ttc tcc etc agt age tat gca 96

[0344] Leu Thr Leu Thr Cys Thr Vai Ser Gly Phe Ser Leu Ser Ser Tyr Ala 20 25 30 atg age tgg gtc ege cag get cca ggg aag ggg etg gaa tac ate gga 144

[0345] Met Ser Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Tyr l ie Gly 35 40 45 ate att agt agt agt ggt age aca tac tac geg age tgg geg aaa ggc 192 l ie l ie Ser Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 ega ttc ace ate tee aaa ace teg acc acg gtg gat etg aaa ate tee 240

[0346] Arg Phe Thr l ie Ser Lys Thr Ser Thr Thr Vai Asp Leu Lys l ie Ser 65 70 75 80 agt ccg aca acc gag gac acg gee acc tat ttc tgt gee aga gat cgt 288

[0347] Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asp Arg

[0348] 85 90 95 gtt att tat agt att ggt ccg tat tat ttt aat ttg tgg ggc cca ggc 336

[0349] Vai l ie Tyr Ser l ie Gly Pro Tyr Tyr Phe Asn Leu Trp Gly Pro Gly 100 105 110 acc ctg gtc acc gtc too tea 357

[0350] Thr Leu Vai Thr Vai Ser Ser

[0351] 115

[0352] SEQ ID No. 18 (Clone PSC21 vH domain amino acid sequence)

[0353] Gin Ser Vai Glu Glu Ser Gly Gly Arg Leu Vai Thr Pro Gly Thr Pro 1 5 10 15

[0354] Leu Thr Leu Thr Cys Thr Vai Ser Gly Phe Ser Leu Ser Ser Tyr Ala

[0355] 20 25 30

[0356] Met Ser Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Tyr T ie Gly

[0357] 35 40 45

[0358] T ie T ie Ser Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

[0359] 50 55 60

[0360] Arg Phe Thr T ie Ser Lys Thr Ser Thr Thr Vai Asp Leu Lys T ie Ser 65 70 75 80

[0361] Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asp Arg

[0362] 85 90 95

[0363] Vai T ie Tyr Ser T ie Gly Pro Tyr Tyr Phe Asn Leu Trp Gly Pro Gly

[0364] 100 105 110

[0365] Thr Leu Vai Thr Vai Ser Ser 115

[0366] SEQ ID No. 19 (Clone PSC21 vL domain nucleic acid sequence) gcc tat gat atg acc cag act cca tcc tcc gtg tet gca act gtg gga 48

[0367] Ala Tyr Asp Met Thr Gin Thr Pro Ser Ser Vai Ser Ala Thr Vai Gly 1 5 10 15 ggc aca gtc acc ate aat tgc cag gee agt gag ate att tat age aat 96 Gly Thr Vai Thr l ie Asn Cys Gin Ala Ser Glu l ie l ie Tyr Ser Asn

[0368] 20 25 30 tta gee tgg tat cag cag aaa cca ggg cag cct ccc aag etc etg ate 144 Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu l ie

[0369] 35 40 45

[0370] tat ggc gca tcc act etg gca tct ggg gtc cca teg egg ttc aaa ggc 192

[0371] Tyr Gly Ala Ser Thr Leu Ala Ser Gly Vai Pro Ser Arg Phe Lys

[0372] Gly

[0373] 50 55 60 agt gga tct ggg aca gag tac act etc ace ate age gac etg cag tgt 240

[0374] Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr l ie Ser Asp Leu Gin

[0375] Cys

[0376] 65 70 75 80 gac gat get gcc act tac tac tgt caa cag agt ttt agt agt aat aat 288

[0377] Asp Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Ser Phe Ser Ser Asn

[0378] Asn

[0379] 85 90 95 gtt ggg aat att ttc ggc gga ggg ace gag gtg gtg gtc aaa

[0380] 330

[0381] Vai Gly Asn l ie Phe Gly Gly Gly Thr Glu Vai Vai Vai Lys

[0382] 100 105 110

[0383] SEQ ID No. 20 (Clone PSC21 vL domain amino acid sequence)

[0384] Ala Tyr Asp Met Thr Gin Thr Pro Ser Ser Vai Ser Ala Thr Vai

[0385] Gly 1 5 10 15

[0386] Gly Thr Vai Thr He Asn Cys Gin Ala Ser Glu He He Tyr Ser

[0387] Asn

[0388] 20 25 30

[0389] Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu He

[0390] 35 40 45

[0391] Tyr Gly Ala Ser Thr Leu Ala Ser Gly Vai Pro Ser Arg Phe Lys

[0392] Gly

[0393] 50 55 60

[0394] Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr l ie Ser Asp Leu Gin

[0395] Cys

[0396] 65 70 75 80 Asp Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Ser Phe Ser Ser Asn

[0397] Asn 85 90 95

[0398] Vai Gly Asn l ie Phe Gly Gly Gly Thr Glu Vai Vai Vai Lys 100 105 110

[0399] SEQ ID No. 21 (Clone PSC23 vH domain nucleic acid sequence) cag teg gtg gag gag tcc ggg ggt ege etg gtc acg cct ggg aca ccc 48

[0400] Gin Ser Vai Glu Glu Ser Gly Gly Arg Leu Vai Thr Pro Gly Thr Pro 1 5 10 15 etg aca etc acc tgc aca gee tet gga ttc tcc etc agt ace cat gca 96

[0401] Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Thr His Ala 20 25 30

[0402] ata aac tgg gtc cgc cag get cca ggg aag ggg etg gaa tgg ate ggg 144 l ie Asn Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp l ie Gly 35 40 45 ate act tat get agt ggt agg aca tat tac geg age tgg geg aaa ggc 192 l ie Thr Tyr Ala Ser Gly Arg Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 ega ttc acc ate tee aaa acc teg acc acg gtg gat etg aaa ate acc 240

[0403] Arg Phe Thr l ie Ser Lys Thr Ser Thr Thr Vai Asp Leu Lys l ie Thr 65 70 75 80 agt ccg aca acc gag gac acg gee acc tat ttc tgt gee aga aat ggg 288

[0404] Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asn Gly

[0405] 85 90 95 get gat gaa aca ttt tac tac ttt gac ttg tgg ggc cca ggc acc etg 336

[0406] Ala Asp Glu Thr Phe Tyr Tyr Phe Asp Leu Trp Gly Pro Gly Thr Leu

[0407] 100 105 110 gtc acc gtc tee tea 351

[0408] Vai Thr Vai Ser Ser 115

[0409] SEQ ID No. 22 (Clone PSC23 vH domain amino acid sequence)

[0410] Gin Ser Vai Glu Glu Ser Gly Gly Arg Leu Vai Thr Pro Gly Thr Pro 1 5 10 15

[0411] Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Thr His Ala

[0412] 20 25 30 l ie Asn Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp l ie Gly

[0413] 35 40 45 l ie Thr Tyr Ala Ser Gly Arg Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

[0414] 50 55 60

[0415] Arg Phe Thr l ie Ser Lys Thr Ser Thr Thr Vai Asp Leu Lys l ie Thr 65 70 75 80 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asn Gly 85 90 95

[0416] Ala Asp Glu Thr Phe Tyr Tyr Phe Asp Leu Trp Gly Pro Gly Thr Leu

[0417] 100 105 110

[0418] Vai Thr Vai Ser Ser

[0419] 115

[0420] SEQ ID No. 23 (Clone PSC23 vL domain nucleic acid sequence) gcc tat gat atg acc cag act cca gcc tcc gtg gag gca get gtg gga 48

[0421] Ala Tyr Asp Met Thr Gin Thr Pro Ala Ser Vai Glu Ala Ala Vai Gly 1 5 10 15 ggc aca gtc acc ate aag tgc cag gcc agt cag aat att aat act tgg 96

[0422] Gly Thr Vai Thr T ie Lys Cys Gin Ala Ser Gin Asn T ie Asn Thr Trp

[0423] 20 25 30 tta tcc tgg tat cag cag aag gca ggg cag cct ccc aag etc etg ate 144

[0424] Leu Ser Trp Tyr Gin Gin Lys Ala Gly Gin Pro Pro Lys Leu Leu T ie

[0425] 35 40 45 tac agg gca tcc act etg gca tet ggg gtc tea teg egg ttc aaa ggc 192

[0426] Tyr Arg Ala Ser Thr Leu Ala Ser Gly Vai Ser Ser Arg Phe Lys Gly

[0427] 50 55 60 agt gga tet ggg aca cag ttc act etc acc ate age ggc gtg gag tgt 240

[0428] Ser Gly Ser Gly Thr Gin Phe Thr Leu Thr T ie Ser Gly Vai Glu Cys 65 70 75 80 gcc gat get gcc act tac tac tgt caa caa tat gat get agt att aat 288

[0429] Ala Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Tyr Asp Ala Ser T ie Asn

[0430] 85 90 95 att gat aat get ttc ggc gga ggg acc gag gtg gtg gtc aaa 330

[0431] T ie Asp Asn Ala Phe Gly Gly Gly Thr Glu Vai Vai Vai Lys 100 105 110

[0432] SEQ ID No. 24 (Clone PSC23 vL domain amino acid sequence)

[0433] Ala Tyr Asp Met Thr Gin Thr Pro Ala Ser Vai Glu Ala Ala Vai Gly 1 5 10 15 Gly Thr Vai Thr l ie Lys Cys Gin Ala Ser Gin Asn l ie Asn Thr Trp

[0434] 20 25 30

[0435] Leu Ser Trp Tyr Gin Gin Lys Ala Gly Gin Pro Pro Lys Leu Leu l ie

[0436] 35 40 45

[0437] Tyr Arg Ala Ser Thr Leu Ala Ser Gly Vai Ser Ser Arg Phe Lys Gly

[0438] 50 55 60

[0439] Ser Gly Ser Gly Thr Gin Phe Thr Leu Thr T ie Ser Gly Vai Glu

[0440] Cys 65 70 75 80

[0441] Ala Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Tyr Asp Ala Ser l ie Asn

[0442] 85 90 95 l ie Asp Asn Ala Phe Gly Gly Gly Thr Glu Vai Vai Vai Lys 100 105 110

[0443] SEQ ID No. 25 (Clone PSC24 vH domain nucleic acid sequence) cag teg gtg gag gag tcc ggg ggt ege etg gtc acg cct ggg aca ccc 48

[0444] Gin Ser Vai Glu Glu Ser Gly Gly Arg Leu Vai Thr Pro Gly Thr Pro 1 5 10 15 etg aca etc acc tgc aca gtc tet gga ate gac etc agt age aat gca 96

[0445] Leu Thr Leu Thr Cys Thr Vai Ser Gly T ie Asp Leu Ser Ser Asn Ala 20 25 30 atg age tgg gtc ege cag get cca ggg aag ggg etg gaa tat ate gga 144 Met Ser Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Tyr T ie Gly 35 40 45 att att agg aat aat gat ate aca tac tac geg age tgg geg aaa ggc 192 T ie T ie Arg Asn Asn Asp T ie Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 ega ttc acc ate tec aaa acc teg acc acg gtg gat etg ata ate acc 240 Arg Phe Thr T ie Ser Lys Thr Ser Thr Thr Vai Asp Leu T ie T ie Thr

[0446] 65 70 75 80 agt ccg aca acc gag gac acg gee acc tat ttc tgt gee aga ggg ggt

[0447] 288 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Gly 85 90 95 ggt tet tac agt att gtc ttc tgg aac tta tgg ggc cca ggc acc etg 336 Gly Ser Tyr Ser T ie Vai Phe Trp Asn Leu Trp Gly Pro Gly Thr Leu 100 105 110 gtc acc gtc tec tea 351

[0448] Vai Thr Vai Ser Ser 115

[0449] SEQ ID No. 26 (Clone PSC24 vH domain amino acid sequence) Gin Ser Vai Glu Glu Ser Gly Gly Arg Leu Vai Thr Pro Gly Thr Pro 1 5 10 15

[0450] Leu Thr Leu Thr Cys Thr Vai Ser Gly He Asp Leu Ser Ser Asn Ala

[0451] 20 25 30

[0452] Met Ser Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Tyr He Gly 35 40 45 l ie l ie Arg Asn Asn Asp He Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

[0453] 50 55 60

[0454] Arg Phe Thr l ie Ser Lys Thr Ser Thr Thr Vai Asp Leu l ie l ie Thr 65 70 75 80

[0455] Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Gly

[0456] 85 90 95

[0457] Gly Ser Tyr Ser T ie Vai Phe Trp Asn Leu Trp Gly Pro Gly Thr Leu

[0458] 100 105 110

[0459] Vai Thr Vai Ser Ser

[0460] 115

[0461] SEQ ID No. 27 (Clone PSC24 vL domain nucleic acid sequence) gcc tat gat atg acc cag act cca gcc tct gtg gag gta get gtg gga 48

[0462] Ala Tyr Asp Met Thr Gin Thr Pro Ala Ser Vai Glu Vai Ala Vai Gly 1 5 10 15 ggc aca gtc acc ate aat tgc cag gcc agt gag agg att tat age aat 96

[0463] Gly Thr Vai Thr T ie Asn Cys Gin Ala Ser Glu Arg T ie Tyr Ser Asn

[0464] 20 25 30 tta gcc tgg tat cag cag aaa cca ggg cag cgt ccc aaa etc etg ate 144

[0465] Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Arg Pro Lys Leu Leu T ie

[0466] 35 40 45 tat tat gca tec act etg gca tct ggg gtc tea teg egg ttc aaa ggc 192

[0467] Tyr Tyr Ala Ser Thr Leu Ala Ser Gly Vai Ser Ser Arg Phe Lys Gly

[0468] 50 55 60 agt gga tct ggg aca cag ttc act etc acc ate age ggc gtg cag tgt 240

[0469] Ser Gly Ser Gly Thr Gin Phe Thr Leu Thr T ie Ser Gly Vai Gin Cys 65 70 75 80 gcc gat get gcc act tac tac tgt cag cag ggt tat agt aat aat aat 288

[0470] Ala Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Gly Tyr Ser Asn Asn Asn 85 90 95 gtt gac aat act ttc ggc gga ggg acc gag gtg gtg gtc aga 330

[0471] Vai Asp Asn Thr Phe Gly Gly Gly Thr Glu Vai Vai Vai Arg 100 105 110

[0472] SEQ ID No. 28 (Clone PSC24 vL domain amino acid sequence)

[0473] Ala Tyr Asp Met Thr Gin Thr Pro Ala Ser Vai Glu Vai Ala Val Gly 1 5 10 15

[0474] Gly Thr Vai Thr T ie Asn Cys Gin Ala Ser Glu Arg T ie Tyr Asn

[0475] 20 25 30

[0476] Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Arg Pro Lys Leu Leu l ie

[0477] 35 40 45

[0478] Tyr Tyr Ala Ser Thr Leu Ala Ser Gly Vai Ser Ser Arg Phe Lys Gly

[0479] 50 55 60

[0480] Ser Gly Ser Gly Thr Gin Phe Thr Leu Thr T ie Ser Gly Vai Gin

[0481] Cys

[0482] 65 70 75 80

[0483] Ala Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Gly Tyr Ser Asn Asn

[0484] Asn

[0485] 85 90 95

[0486] Vai Asp Asn Thr Phe Gly Gly Gly Thr Glu Vai Vai Vai Arg

[0487] 100 105 110

[0488] SEQ ID No. 29 (Clone PSCWT22 vH domain CDR1 amino acid sequence)

[0489] Gly Phe Thr Phe Ser Ser Tyr Ala 1 5

[0490] SEQ ID No. 30 (Clone PSCWT22 vH domain CDR2 amino acid sequence)

[0491] T ie Ser Tyr Asp Vai Ser Asn Lys 1 5

[0492] SEQ ID No. 31 (Clone PSCWT22 vH domain CDR3 amino acid sequence)

[0493] Ala Arg Ser Tyr Tyr Tyr Tyr Tyr Gly Met Asp Vai 1 5 10

[0494] SEQ ID No. 32 (Clone PSCWT22 vL domain CDR1 amino acid sequence)

[0495] Gin Ser T ie Ser Arg Tyr 1 5

[0496] SEQ ID No. 33 (Clone PSCWT22 vL domain CDR2 amino acid sequence)

[0497] Asp Ala Ser 1 SEQ ID No. 34 (Clone PSCWT22 vL domain CDR3 amino acid sequence)

[0498] Gin Gin Phe Gly Arg Ser Pro Arg Thr 1 5

[0499] SEQ ID No. 35 (Clone PSCWT22 full-length vH chain nucleic acid sequence) gag gtg caa ctg gtg gag tet ggg gga ggt gtg gta agg cct ggg ggg 48

[0500] Glu Vai Gin Leu Vai Glu Ser Gly Gly Gly Vai Vai Arg Pro Gly Gly 1 5 10 15 tcc ctg aga etc tcc tgt gca gee tet gga ttc acc ttc agt age tat 96

[0501] Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

[0502] 20 25 30 get atg cac tgg gtc ege cag get cca ggc aag ggg ctg gag tgg gtg 144

[0503] Ala Met His Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Vai 35 40 45 gca gtt ata tea tat gat gta age aat aaa tac tac gca gac tcc gtg 192

[0504] Ala Vai l ie Ser Tyr Asp Vai Ser Asn Lys Tyr Tyr Ala Asp Ser Vai 50 55 60 aag ggc ega ttc ace ate tcc aga gac aat tcc aag aac acg ctg tat 240

[0505] Lys Gly Arg Phe Thr T ie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 ctg caa atg aac age ctg aga get gag gac acg get gtg tat tac tgt 288

[0506] Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Vai Tyr Tyr Cys 85 90 95 geg aga tet tac tac tac tac tac ggt atg gac gtc tgg ggc caa ggg 336

[0507] Ala Arg Ser Tyr Tyr Tyr Tyr Tyr Gly Met Asp Vai Trp Gly Gin Gly

[0508] 100 105 110 ace acg gtc ace gtc tcc tea gee tcc act aag ggc cca tcc gtg ttc 384

[0509] Thr Thr Vai Thr Vai Ser Ser Ala Ser Thr Lys Gly Pro Ser Vai Phe

[0510] 115 120 125 cca ctg gca ccc tet agt aag age aca tet ggg ggt act gcc get ctg

[0511] 432

[0512] Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

[0513] 130 135 140 gga tgt ctg gtg aag gat tac ttc cca gag cca gtc acc gtg tcc tgg

[0514] 480

[0515] Gly Cys Leu Vai Lys Asp Tyr Phe Pro Glu Pro Vai Thr Vai Ser Trp 145 150 155 160 aac age ggg gee etg act tee ggt gtc cat acc ttt cca get gtg etg 528

[0516] Asn Ser Gly Ala Leu Thr Ser Gly Vai His Thr Phe Pro Ala Vai Leu 165 170 175 cag tea tee ggc etg tac age etg age tet gtg gtc acc gtc ccc agt 576

[0517] Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Vai Vai Thr Vai Pro Ser

[0518] 180 185 190 tea tee etg gga aca cag act tat ate tgc aac gtg aat cac aag cca 624

[0519] Ser Ser Leu Gly Thr Gin Thr Tyr T ie Cys Asn Vai Asn His Lys Pro

[0520] 195 200 205 tee aat aca aaa gtc gac aag aaa gtg gaa ccc aag age tgt gat aaa

[0521] 672

[0522] Ser Asn Thr Lys Vai Asp Lys Lys Vai Glu Pro Lys Ser Cys Asp Lys

[0523] 210 215 220 ace cat aca tgc ccc cct tgt cct get cca gag etg etg gga gga cca 720

[0524] Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

[0525] 225 230 235 240 tcc gtg ttc ctg ttt cca ccc aag cct aaa gac act ctg atg att tet

[0526] 768

[0527] Ser Vai Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met T ie Ser 245 250 255 cga acc ccc gaa gtc aca tgc gtg gtc gtg gac gtg tcc cac gag gat 816

[0528] Arg Thr Pro Glu Vai Thr Cys Vai Vai Vai Asp Vai Ser His Glu Asp

[0529] 260 265 270 cct gaa gtc aag ttc aac tgg tac gtg gat ggc gtc gag gtg cat aat

[0530] 864

[0531] Pro Glu Vai Lys Phe Asn Trp Tyr Vai Asp Gly Vai Glu Vai His Asn

[0532] 275 280 285 gcc aag aca aaa cca cga gag gaa cag tac aac agt acc tat cgt gtc

[0533] 912

[0534] Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Vai

[0535] 290 295 300 gtg tea gtc ctg aca gtg ctg cac cag gac tgg ctg aac ggg aag gaa

[0536] 960

[0537] Vai Ser Vai Leu Thr Vai Leu His Gin Asp Trp Leu Asn Gly Lys Glu

[0538] 305 310 315 320 tat aag tgc aaa gtg age aat aag gca ctg ccc gcc cct ate gag aaa 1008

[0539] Tyr Lys Cys Lys Vai Ser Asn Lys Ala Leu Pro Ala Pro T ie Glu Lys 325 330 335 aca att tet aag get aaa gga cag cct agg gaa cca cag gtg tac act 1056

[0540] Thr T ie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Vai Tyr Thr

[0541] 340 345 350 ctg cct cca tea egg gac gag ctg aca aag aac cag gtc agt ctg act 1104

[0542] Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Vai Ser Leu Thr

[0543] 355 360 365 tgt ctg gtg aaa ggg ttc tat cct tet gat ate gcc gtg gag tgg gaa 1152

[0544] Cys Leu Vai Lys Gly Phe Tyr Pro Ser Asp T ie Ala Vai Glu Trp Glu 370 375 380 agt aat ggt cag cca gag aac aat tac aag acc aca ccc cct gtc ctg 1200

[0545] Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Vai Leu 385 390 395 400 gac tct gat ggg agt ttc ttt etg tat tee aag etg acc gtg gat aaa 1248

[0546] Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Vai Asp Lys 405 410 415 age egg tgg cag cag ggt aat gtc ttt agt tgt tea gtg atg cac gag 1296

[0547] Ser Arg Trp Gin Gin Gly Asn Vai Phe Ser Cys Ser Vai Met His Glu

[0548] 420 425 430 gca etg cac aat cac tac acc cag aaa tea etg tea etg tea cca ggt 1344

[0549] Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly

[0550] 435 440 445 aaa tga 1350

[0551] Lys

[0552] SEQ ID No. 36 (Clone PSCWT22 full-length vH chain amino acid sequence)

[0553] Glu Vai Gin Leu Vai Glu Ser Gly Gly Gly Vai Vai Arg Pro Gly Gly

[0554] 1 5 10 15

[0555] Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

[0556] 20 25 30

[0557] Ala Met His Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Vai

[0558] 35 40 45

[0559] Ala Vai l ie Ser Tyr Asp Vai Ser Asn Lys Tyr Tyr Ala Asp Ser Vai

[0560] 50 55 60

[0561] Lys Gly Arg Phe Thr T ie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80

[0562] Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Vai Tyr Tyr Cys

[0563] 85 90 95

[0564] Ala Arg Ser Tyr Tyr Tyr Tyr Tyr Gly Met Asp Vai Trp Gly Gin Gly

[0565] 100 105 110

[0566] Thr Thr Vai Thr Vai Ser Ser Ala Ser Thr Lys Gly Pro Ser Vai Phe

[0567] 115 120 125

[0568] Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

[0569] 130 135 140

[0570] Gly Cys Leu Vai Lys Asp Tyr Phe Pro Glu Pro Vai Thr Vai Ser Trp 145 150 155 160

[0571] Asn Ser Gly Ala Leu Thr Ser Gly Vai His Thr Phe Pro Ala Vai Leu

[0572] 165 170 175

[0573] Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Vai Vai Thr Vai Pro Ser

[0574] 180 185 190

[0575] Ser Ser Leu Gly Thr Gin Thr Tyr T ie Cys Asn Vai Asn His Lys Pro

[0576] 195 200 205

[0577] Ser Asn Thr Lys Vai Asp Lys Lys Vai Glu Pro Lys Ser Cys Asp Lys

[0578] 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

[0579] 225 230 235 240

[0580] Ser Vai Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met l ie Ser

[0581] 245 250 255

[0582] Arg Thr Pro Glu Vai Thr Cys Vai Vai Vai Asp Vai Ser His Glu Asp

[0583] 260 265 270

[0584] Pro Glu Vai Lys Phe Asn Trp Tyr Vai Asp Gly Vai Glu Vai His Asn

[0585] 275 280 285

[0586] Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Vai

[0587] 290 295 300

[0588] Vai Ser Vai Leu Thr Vai Leu His Gin Asp Trp Leu Asn Gly Lys Glu

[0589] 305 310 315 320

[0590] Tyr Lys Cys Lys Vai Ser Asn Lys Ala Leu Pro Ala Pro T ie Glu Lys

[0591] 325 330 335

[0592] Thr T ie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Vai Tyr Thr

[0593] 340 345 350

[0594] Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Vai Ser Leu Thr

[0595] 355 360 365

[0596] Cys Leu Vai Lys Gly Phe Tyr Pro Ser Asp T ie Ala Vai Glu Trp Glu

[0597] 370 375 380

[0598] Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Vai Leu

[0599] 385 390 395 400

[0600] Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Vai Asp Lys

[0601] 405 410 415

[0602] Ser Arg Trp Gin Gin Gly Asn Vai Phe Ser Cys Ser Vai Met His Glu

[0603] 420 425 430

[0604] Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly

[0605] 435 440 445

[0606] Lys SEQ ID No. 37 (Clone PSCWT22 full-length vL chain nucleic acid sequence) gat gtt gtg atg acc cag tct cca tcc tcc ctg tct gca tct gta gga 48

[0607] Asp Vai Vai Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Vai Gly 1 5 10 15 gac aga gtc acc ate act tgc egg gca agt cag age att age agg tac 96

[0608] Asp Arg Vai Thr l ie Thr Cys Arg Ala Ser Gin Ser T ie Ser Arg Tyr

[0609] 20 25 30 tta gcc tgg tac caa cag aaa cct ggc cag get ccc agg etc etc ate 144

[0610] Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu l ie

[0611] 35 40 45 tat gat gca tcc aac agg gcc act ggc ate cca gtc agg ttc agt ggc

[0612] 192

[0613] Tyr Asp Ala Ser Asn Arg Ala Thr Gly l ie Pro Vai Arg Phe Ser Gly

[0614] 50 55 60 agt ggg tct ggg aca gac ttc act etc acc ate age aga etg gag cca

[0615] 240

[0616] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr l ie Ser Arg Leu Glu Pro 65 70 75 80 gaa gat ttt gca gtg tat tac tgt cag cag ttt ggt agg tea cct egg

[0617] 288

[0618] Glu Asp Phe Ala Vai Tyr Tyr Cys Gin Gin Phe Gly Arg Ser Pro Arg 85 90 95 acg ttc ggc caa ggg aca ega etg gag att aaa ega act gtg get gca 336

[0619] Thr Phe Gly Gin Gly Thr Arg Leu Glu l ie Lys Arg Thr Vai Ala Ala

[0620] 100 105 110 cca tct gtc ttc ate ttc ccg cca tct gat gag cag ttg aaa tct gga 384

[0621] Pro Ser Vai Phe l ie Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly

[0622] 115 120 125 act gcc tct gtt gtg tgc etg etg aat aac ttc tat ccc aga gag gcc

[0623] 432

[0624] Thr Ala Ser Vai Vai Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

[0625] 130 135 140 aaa gta cag tgg aag gtg gat aac gcc etc caa teg ggt aac tcc cag

[0626] 480

[0627] Lys Vai Gin Trp Lys Vai Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160 gag agt gtc aca gag cag gac age aag gac age acc tac age etc age

[0628] 528

[0629] Glu Ser Vai Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

[0630] 165 170 175 age acc etg acg etg age aaa gca gac tac gag aaa cac aaa gtc tac

[0631] 576

[0632] Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Vai Tyr

[0633] 180 185 190 gcc tgc gaa gtc acc cat cag ggc ctg age teg ccc gtc aca aag age 624

[0634] Ala Cys Glu Vai Thr His Gin Gly Leu Ser Ser Pro Vai Thr Lys Ser 195 200 205 ttc aac agg gga gag tgt tag 645

[0635] Phe Asn Arg Gly Glu Cys

[0636] 210

[0637] SEQ ID No. 38 (Clone PSCWT22 full-length vL chain amino acid sequence)

[0638] Asp Vai Vai Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Vai Gly 1 5 10 15

[0639] Asp Arg Vai Thr l ie Thr Cys Arg Ala Ser Gin Ser T ie Ser Arg Tyr 20 25 30

[0640] Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu T ie

[0641] 35 40 45

[0642] Tyr Asp Ala Ser Asn Arg Ala Thr Gly l ie Pro Vai Arg Phe Ser Gly

[0643] 50 55 60

[0644] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr T ie Ser Arg Leu Glu Pro 65 70 75 80

[0645] Glu Asp Phe Ala Vai Tyr Tyr Cys Gin Gin Phe Gly Arg Ser Pro Arg

[0646] 85 90 95

[0647] Thr Phe Gly Gin Gly Thr Arg Leu Glu T ie Lys Arg Thr Vai Ala Ala

[0648] 100 105 110

[0649] Pro Ser Vai Phe T ie Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly

[0650] 115 120 125

[0651] Thr Ala Ser Vai Vai Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

[0652] 130 135 140

[0653] Lys Vai Gin Trp Lys Vai Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin

[0654] 145 150 155 160

[0655] Glu Ser Vai Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

[0656] 165 170 175

[0657] Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Vai Tyr

[0658] 180 185 190

[0659] Ala Cys Glu Vai Thr His Gin Gly Leu Ser Ser Pro Vai Thr Lys Ser

[0660] 195 200 205

[0661] Phe Asn Arg Gly Glu Cys

[0662] 210

[0663] SEQ ID No. 39 (Clone PSCWT22 scFv nucleic acid sequence) gat gtt gtg atg acc cag tet cca tee tee etg tet gca tet gta gga 48

[0664] Asp Vai Vai Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Vai Gly

[0665] 1 5 10 15 gac aga gtc acc ate act tgc egg gca agt cag age att age agg tac 96

[0666] Asp Arg Vai Thr l ie Thr Cys Arg Ala Ser Gin Ser T ie Ser Arg Tyr

[0667] 20 25 30 tta gee tgg tac caa cag aaa cct ggc cag get ccc agg etc etc ate 144

[0668] Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu T ie

[0669] 35 40 45

[0670] tat gat gca tcc aac agg gee act ggc ate cca gtc agg ttc agt ggc 192

[0671] Tyr Asp Ala Ser Asn Arg Ala Thr Gly l ie Pro Vai Arg Phe Ser Gly 50 55 60 agt ggg tct ggg aca gac ttc act etc ace ate age aga etg gag cca 240

[0672] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr l ie Ser Arg Leu Glu Pro 65 70 75 80 gaa gat ttt gca gtg tat tac tgt cag cag ttt ggt agg tea cct egg 288

[0673] Glu Asp Phe Ala Vai Tyr Tyr Cys Gin Gin Phe Gly Arg Ser Pro Arg 85 90 95 acg ttc ggc caa ggg aca ega etg gag att aaa ggc gga tcc tct agg 336

[0674] Thr Phe Gly Gin Gly Thr Arg Leu Glu l ie Lys Gly Gly Ser Ser Arg

[0675] 100 105 110 tea agt tcc age ggc ggc ggt ggc age gga ggc ggc ggt gag gtg caa 384

[0676] Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Vai Gin

[0677] 115 120 125 etg gtg gag tct ggg gga ggt gtg gta agg cct ggg ggg tcc etg aga

[0678] 432

[0679] Leu Vai Glu Ser Gly Gly Gly Vai Vai Arg Pro Gly Gly Ser Leu Arg

[0680] 130 135 140 etc tcc tgt gca gcc tct gga ttc ace ttc agt age tat get atg cac

[0681] 480

[0682] Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met His 145 150 155 160 tgg gtc ege cag get cca ggc aag ggg etg gag tgg gtg gca gtt ata 528

[0683] Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Vai Ala Vai He 165 170 175 tea tat gat gta age aat aaa tac tac gca gac tcc gtg aag ggc ega 576

[0684] Ser Tyr Asp Vai Ser Asn Lys Tyr Tyr Ala Asp Ser Vai Lys Gly Arg

[0685] 180 185 190 ttc ace ate tcc aga gac aat tcc aag aac acg etg tat etg caa atg 624

[0686] Phe Thr l ie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin Met

[0687] 195 200 205 aac age etg aga get gag gac acg get gtg tat tac tgt geg aga tet 672

[0688] Asn Ser Leu Arg Ala Glu Asp Thr Ala Vai Tyr Tyr Cys Ala Arg Ser 210 215 220 tac tac tac tac tac ggt atg gac gtc tgg ggc caa ggg acc acg gtc 720

[0689] Tyr Tyr Tyr Tyr Tyr Gly Met Asp Vai Trp Gly Gin Gly Thr Thr Vai

[0690] 225 230 235 240 ace gtc tee tea 732

[0691] Thr Vai Ser Ser

[0692] SEQ ID No. 40 (Clone PSCWT22 seFv amino acid sequence)

[0693] Asp Vai Vai Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Vai Gly 1 5 10 15

[0694] Asp Arg Vai Thr T ie Thr Cys Arg Ala Ser Gin Ser T ie Ser Arg Tyr

[0695] 20 25 30

[0696] Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu T ie

[0697] 35 40 45

[0698] Tyr Asp Ala Ser Asn Arg Ala Thr Gly T ie Pro Vai Arg Phe Ser Gly

[0699] 50 55 60

[0700] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr T ie Ser Arg Leu Glu Pro 65 70 75 80

[0701] Glu Asp Phe Ala Vai Tyr Tyr Cys Gin Gin Phe Gly Arg Ser Pro Arg

[0702] 85 90 95

[0703] Thr Phe Gly Gin Gly Thr Arg Leu Glu T ie Lys Gly Gly Ser Ser Arg

[0704] 100 105 110

[0705] Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Vai Gin

[0706] 115 120 125

[0707] Leu Vai Glu Ser Gly Gly Gly Vai Vai Arg Pro Gly Gly Ser Leu Arg

[0708] 130 135 140

[0709] Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met His

[0710] 145 150 155 160

[0711] Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Vai Ala Vai T ie

[0712] 165 170 175

[0713] Ser Tyr Asp Vai Ser Asn Lys Tyr Tyr Ala Asp Ser Vai Lys Gly

[0714] Arg

[0715] 180 185 190

[0716] Phe Thr T ie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin Met

[0717] 195 200 205

[0718] Asn Ser Leu Arg Ala Glu Asp Thr Ala Vai Tyr Tyr Cys Ala Arg Ser

[0719] 210 215 220 Tyr Tyr Tyr Tyr Tyr Gly Met Asp Vai Trp Gly Gin Gly Thr Thr Vai

[0720] 225 230 235 240

[0721] Thr Vai Ser Ser

[0722] SEQ ID No. 41 (Clone PSC25 vH domain nucleic acid sequence) cag teg gtg gag gag tcc ggg ggt ege etg gtc acg cct ggg aca ccc 48

[0723] Gin Ser Vai Glu Glu Ser Gly Gly Arg Leu Vai Thr Pro Gly Thr Pro

[0724] 1 5 10 15 ctg aca etc acc tgc aca gtc tet gga ate gac etc agt aac aat gca 96

[0725] Leu Thr Leu Thr Cys Thr Vai Ser Gly l ie Asp Leu Ser Asn Asn Ala

[0726] 20 25 30 atg age tgg gtc ege cag get cca ggg aag ggg etg gaa tat ate gga 144

[0727] Met Ser Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Tyr He Gly 35 40 45 ate att agg agt agt ggt agt aca tat tac geg aac tgg gca aaa ggc

[0728] 192 l ie l ie Arg Ser Ser Gly Ser Thr Tyr Tyr Ala Asn Trp Ala Lys Gly 50 55 60 egg ttc acc ate tcc aaa acc teg acc acg gtg gat etg ata ate acc 240

[0729] Arg Phe Thr l ie Ser Lys Thr Ser Thr Thr Vai Asp Leu He He Thr 65 70 75 80 agt ccg aca acc gag gac acg gee acc tat ttc tgt gcc aga ggg ggt 288

[0730] Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Gly 85 90 95 ggt tet tac agt att gtc ttc tgg aac ttg tgg ggc cca ggc acc etg 336

[0731] Gly Ser Tyr Ser l ie Vai Phe Trp Asn Leu Trp Gly Pro Gly Thr Leu

[0732] 100 105 110 gtc acc gtc tcc tea 351

[0733] Vai Thr Vai Ser Ser

[0734] 115

[0735] SEQ ID No. 42 (Clone PSC25 vH domain amino acid sequence)

[0736] Gin Ser Vai Glu Glu Ser Gly Gly Arg Leu Vai Thr Pro Gly Thr Pro 1 5 10 15

[0737] Leu Thr Leu Thr Cys Thr Vai Ser Gly l ie Asp Leu Ser Asn Asn Ala 20 25 30

[0738] Met Ser Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Tyr l ie Gly 35 40 45 l ie l ie Arg Ser Ser Gly Ser Thr Tyr Tyr Ala Asn Trp Ala Lys Gly 50 55 60

[0739] Arg Phe Thr l ie Ser Lys Thr Ser Thr Thr Vai Asp Leu l ie l ie Thr 65 70 75 80

[0740] Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Gly

[0741] 85 90 95

[0742] Gly Ser Tyr Ser T ie Vai Phe Trp Asn Leu Trp Gly Pro Gly Thr Leu

[0743] 100 105 110

[0744] Vai Thr Vai Ser Ser

[0745] 115

[0746] SEQ ID No. 43 (Clone PSC25 vL domain nucleic acid sequence) gcc tat gat atg acc cag act cca gcc tct gtg gag gta get gtg gga 48

[0747] Ala Tyr Asp Met Thr Gin Thr Pro Ala Ser Vai Glu Vai Ala Vai

[0748] Gly

[0749] 1 5 10 15 ggc aca gtc acc ate aat tgc cag gcc agt gag agg att tat age aat 96

[0750] Gly Thr Vai Thr T ie Asn Cys Gin Ala Ser Glu Arg T ie Tyr Ser Asn

[0751] 20 25 30 tta gcc tgg tat cag cag aaa cca ggg cag cgt ccc aag etc etg ate 144

[0752] Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Arg Pro Lys Leu Leu T ie

[0753] 35 40 45 tat tat aca tec act etg gca tct ggg gtc tea teg egg ttc aaa ggc 192

[0754] Tyr Tyr Thr Ser Thr Leu Ala Ser Gly Vai Ser Ser Arg Phe Lys Gly

[0755] 50 55 60 agt gga tct ggg aca cag ttc act etc acc ate age ggc gtg gag tgt 240

[0756] Ser Gly Ser Gly Thr Gin Phe Thr Leu Thr T ie Ser Gly Vai Glu

[0757] Cys

[0758] 65 70 75 80 gcc gat get gcc act tac tac tgt caa cag ggt tat agt agt agt aat 288

[0759] Ala Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Gly Tyr Ser Ser Ser Asn

[0760] 85 90 95 gtt gac aat act ttc ggc gga ggg acc gag gtg gtg gtc aaa ggt 333

[0761] Vai Asp Asn Thr Phe Gly Gly Gly Thr Glu Vai Vai Vai Lys Gly 100 105 110

[0762] SEQ ID No. 44 (Clone PSC25 vL domain amino acid sequence)

[0763] Ala Tyr Asp Met Thr Gin Thr Pro Ala Ser Vai Glu Vai Ala Vai

[0764] Gly 1 5 10 15 Gly Thr Vai Thr l ie Asn Cys Gin Ala Ser Glu Arg l ie Tyr Ser Asn

[0765] 20 25 30

[0766] Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Arg Pro Lys Leu Leu l ie

[0767] 35 40 45

[0768] Tyr Tyr Thr Ser Thr Leu Ala Ser Gly Vai Ser Ser Arg Phe Lys Gly

[0769] 50 55 60

[0770] Ser Gly Ser Gly Thr Gin Phe Thr Leu Thr l ie Ser Gly Vai Glu Cys 65 70 75 80

[0771] Ala Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Gly Tyr Ser Ser Ser Asn

[0772] 85 90 95

[0773] Vai Asp Asn Thr Phe Gly Gly Gly Thr Glu Vai Vai Vai Lys Gly

[0774] 100 105 110

[0775] SEQ ID No. 45 (Humanized PSC20 vH domain nucleic acid sequence) ggc gag cag cag ctg gtg gag age ggc gga ggc etg gtg cag cct gga 48

[0776] Gly Glu Gin Gin Leu Vai Glu Ser Gly Gly Gly Leu Vai Gin Pro Gly 1 5 10 15 gga age ctg agg ctg age tgc gcc gtg tcc ggc ttc age ctg age age 96

[0777] Gly Ser Leu Arg Leu Ser Cys Ala Vai Ser Gly Phe Ser Leu Ser Ser 20 25 30 tac gcc ate age tgg gtg agg cag gcc ccc gga aag ggc ctg gag tac 144

[0778] Tyr Ala T ie Ser Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Tyr 35 40 45 ate gcc ate ate aac age tac ggc ace ace tac tac gcc age tgg gcc 192

[0779] T ie Ala T ie T ie Asn Ser Tyr Gly Thr Thr Tyr Tyr Ala Ser Trp Ala 50 55 60 aag ggc aga gtg ace ate tcc aag gat tcc tcc aag aac ace gtg tac 240

[0780] Lys Gly Arg Vai Thr T ie Ser Lys Asp Ser Ser Lys Asn Thr Vai Tyr

[0781] 65 70 75 80 ctg cag atg ggc tcc ctg aga gcc gag gat atg gcc gtg tac ttt tgc

[0782] 288

[0783] Leu Gin Met Gly Ser Leu Arg Ala Glu Asp Met Ala Vai Tyr Phe Cys 85 90 95 gcc aga ggc gat tcc tac ggc tcc ggc gtg ggc ctg ggc ctg tgg gga 336

[0784] Ala Arg Gly Asp Ser Tyr Gly Ser Gly Vai Gly Leu Gly Leu Trp Gly 100 105 110 cct gga acc ctg gtg aca gtg tcc tcc

[0785] 363

[0786] Pro Gly Thr Leu Vai Thr Vai Ser Ser

[0787] 115 120

[0788] SEQ ID No. 46 (Humanized PSC20 vH domain amino acid sequence)

[0789] Gly Glu Gin Gin Leu Vai Glu Ser Gly Gly Gly Leu Vai Gin Pro Gly 1 5 10 15

[0790] Gly Ser Leu Arg Leu Ser Cys Ala Vai Ser Gly Phe Ser Leu Ser Ser

[0791] 20 25 30

[0792] Tyr Ala l ie Ser Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Tyr

[0793] 35 40 45

[0794] l ie Ala l ie l ie Asn Ser Tyr Gly Thr Thr Tyr Tyr Ala Ser Trp Ala

[0795] 50 55 60

[0796] Lys Gly Arg Vai Thr l ie Ser Lys Asp Ser Ser Lys Asn Thr Vai Tyr 65 70 75 80

[0797] Leu Gin Met Gly Ser Leu Arg Ala Glu Asp Met Ala Vai Tyr Phe Cys

[0798] 85 90 95

[0799] Ala Arg Gly Asp Ser Tyr Gly Ser Gly Vai Gly Leu Gly Leu Trp Gly

[0800] 100 105 110

[0801] Pro Gly Thr Leu Vai Thr Vai Ser Ser

[0802] 115 120

[0803] SEQ ID No. 47 (Humanized PSC20 vL domain nucleic acid sequence) gga gac tac cag atg aca cag tcc cct age acc etg tec gcc tec gtg 48

[0804] Gly Asp Tyr Gin Met Thr Gin Ser Pro Ser Thr Leu Ser Ala Ser Vai 1 5 10 15 ggc gac aga gtg aca ate acc tgt cag gcc tcc cag aat ate tac age 96 Gly Asp Arg Vai Thr T ie Thr Cys Gin Ala Ser Gin Asn T ie Tyr Ser 20 25 30 aat etg gcc tgg tac cag cag aag cct ggc aag agg ccc aag etg etg 144

[0805] Asn Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Arg Pro Lys Leu Leu 35 40 45 ate tac aga gcc age tec etg gcc tcc ggc gtg cca tet aga ttt tcc 192

[0806] T ie Tyr Arg Ala Ser Ser Leu Ala Ser Gly Vai Pro Ser Arg Phe Ser 50 55 60 ggc tec ggc age ggc aca gag ttt acc etg aca ate age age etg cag 240 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr T ie Ser Ser Leu Gin 65 70 75 80 ccc gat gat ttc gcc acc tac tac tgt cag cag ggc ttc age age aat 288 Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Gly Phe Ser Ser Asn 85 90 95 aat gtg gac aat aca ttt ggc ggc ggc aca aag gtg gag ate aag 333

[0807] Asn Vai Asp Asn Thr Phe Gly Gly Gly Thr Lys Vai Glu l ie Lys 100 105 110

[0808] SEQ ID No. 48 (Humanized PSC20 vL domain amino acid sequence)

[0809] Gly Asp Tyr Gin Met Thr Gin Ser Pro Ser Thr Leu Ser Ala Ser Vai 1 5 10 15

[0810] Gly Asp Arg Vai Thr T ie Thr Cys Gin Ala Ser Gin Asn T ie Tyr Ser

[0811] 20 25 30

[0812] Asn Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Arg Pro Lys Leu Leu

[0813] 35 40 45 l ie Tyr Arg Ala Ser Ser Leu Ala Ser Gly Vai Pro Ser Arg Phe Ser

[0814] 50 55 60

[0815] Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr l ie Ser Ser Leu Gin 65 70 75 80

[0816] Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Gly Phe Ser Ser Asn

[0817] 85 90 95

[0818] Asn Vai Asp Asn Thr Phe Gly Gly Gly Thr Lys Vai Glu T ie Lys

[0819] 100 105 110

[0820] SEQ ID No. 49 (Humanized PSC19 vH domain nucleic acid sequence) ggc gag cag cag ctg gtg gag age ggc gga ggc etg gtg cag cct gga 48

[0821] Gly Glu Gin Gin Leu Vai Glu Ser Gly Gly Gly Leu Vai Gin Pro Gly 1 5 10 15 gga age ctg agg ctg age tgc gcc gtg tcc ggc ttt tcc ctg age aag 96

[0822] Gly Ser Leu Arg Leu Ser Cys Ala Vai Ser Gly Phe Ser Leu Ser Lys 20 25 30 age ate ate age tgg gtg agg cag gcc cct ggc aag ggc ctg gag tac 144

[0823] Ser T ie T ie Ser Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Tyr 35 40 45 ate ggc ate ate ggc age age ggc tcc ace tac tac gcc aac tgg gcc 192

[0824] T ie Gly T ie T ie Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Asn Trp Ala 50 55 60 aag ggc aga ttc aca ate tcc aag gac tcc tcc aag aat acc gtg tac 240

[0825] Lys Gly Arg Phe Thr T ie Ser Lys Asp Ser Ser Lys Asn Thr Vai Tyr

[0826] 65 70 75 80 ctg cag atg ggc tcc ctg agg gcc gag gat atg gcc gtg tac ttt tgt

[0827] 288

[0828] Leu Gin Met Gly Ser Leu Arg Ala Glu Asp Met Ala Vai Tyr Phe Cys 85 90 95 gcc aga ggc ctg ctg tac tcc ggc aat aag tcc tgg ggc ccc ggc aca 336

[0829] Ala Arg Gly Leu Leu Tyr Ser Gly Asn Lys Ser Trp Gly Pro Gly Thr 100 105 110 ctg gtg acc gtg age tcc 354

[0830] Leu Vai Thr Vai Ser Ser

[0831] 115

[0832] SEQ ID No. 50 (Humanized PSC19 vH domain amino acid sequence)

[0833] Gly Glu Gin Gin Leu Vai Glu Ser Gly Gly Gly Leu Vai Gin Pro Gly 1 5 10 15

[0834] Gly Ser Leu Arg Leu Ser Cys Ala Vai Ser Gly Phe Ser Leu Ser Lys

[0835] 20 25 30

[0836] Ser l ie l ie Ser Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Tyr

[0837] 35 40 45 l ie Gly l ie l ie Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Asn Trp Ala

[0838] 50 55 60

[0839] Lys Gly Arg Phe Thr T ie Ser Lys Asp Ser Ser Lys Asn Thr Vai Tyr 65 70 75 80

[0840] Leu Gin Met Gly Ser Leu Arg Ala Glu Asp Met Ala Vai Tyr Phe Cys

[0841] 85 90 95

[0842] Ala Arg Gly Leu Leu Tyr Ser Gly Asn Lys Ser Trp Gly Pro Gly Thr

[0843] 100 105 110

[0844] Leu Vai Thr Vai Ser Ser

[0845] 115

[0846] SEQ ID No. 51 (Humanized PSC19 vL domain nucleic acid sequence) ggc gac ate gtg atg ace cag tec ccc gat tec ctg gcc gtg tcc ctg 48

[0847] Gly Asp He Vai Met Thr Gin Ser Pro Asp Ser Leu Ala Vai Ser Leu

[0848] 1 5 10 15 ggc gag aga gcc aca ate aat tgt cag tec tcc cag age gtg ctg ctg 96

[0849] Gly Glu Arg Ala Thr He Asn Cys Gin Ser Ser Gin Ser Vai Leu Leu

[0850] 20 25 30 aac aat cag ctg tec tgg ttc cag cag aag cct ggc cag cct ccc aag

[0851] 144

[0852] Asn Asn Gin Leu Ser Trp Phe Gin Gin Lys Pro Gly Gin Pro Pro Lys 35 40 45 ctg ctg ate tac gac gcc tec aca ctg gag tcc ggc gtg ccc gat agg 192

[0853] Leu Leu He Tyr Asp Ala Ser Thr Leu Glu Ser Gly Vai Pro Asp Arg

[0854] 50 55 60 ttc age ggc tee ggc age ggc ace gac ttt ace etg ace ate tee age 240

[0855] Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr l ie Ser Ser

[0856] 65 70 75 80 etg cag gee gag gat gtg gee gtg tac tac tgc etg ggc ggc tac age

[0857] 288

[0858] Leu Gin Ala Glu Asp Vai Ala Vai Tyr Tyr Cys Leu Gly Gly Tyr Ser 85 90 95 ggc aac etg tac gee ttt ggc ggc ggc acc aag gtg gag ate aag 333

[0859] Gly Asn Leu Tyr Ala Phe Gly Gly Gly Thr Lys Vai Glu T ie Lys

[0860] 100 105 110

[0861] SEQ ID No. 52 (Humanized PSC19 vL domain amino acid sequence)

[0862] Gly Asp l ie Vai Met Thr Gin Ser Pro Asp Ser Leu Ala Vai Ser Leu 1 5 10 15

[0863] Gly Glu Arg Ala Thr l ie Asn Cys Gin Ser Ser Gin Ser Vai Leu Leu

[0864] 20 25 30

[0865] Asn Asn Gin Leu Ser Trp Phe Gin Gin Lys Pro Gly Gin Pro Pro Lys

[0866] 35 40 45

[0867] Leu Leu l ie Tyr Asp Ala Ser Thr Leu Glu Ser Gly Vai Pro Asp Arg

[0868] 50 55 60

[0869] Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr T ie Ser Ser 65 70 75 80

[0870] Leu Gin Ala Glu Asp Vai Ala Vai Tyr Tyr Cys Leu Gly Gly Tyr Ser

[0871] 85 90 95

[0872] Gly Asn Leu Tyr Ala Phe Gly Gly Gly Thr Lys Vai Glu T ie Lys

[0873] 100 105 110

[0874] SEQ ID No. 53 (Humanized PSC21 vH domain nucleic acid sequence) ggc gag cag cag ctg gtg gag too ggc gga ggc ctg gtg cag cca gga 48

[0875] Gly Glu Gin Gin Leu Vai Glu Ser Gly Gly Gly Leu Vai Gin Pro Gly 1 5 10 15 gga age ctg agg ctg tcc tgt gcc gtg age ggc ttc tec ctg age tec 96

[0876] Gly Ser Leu Arg Leu Ser Cys Ala Vai Ser Gly Phe Ser Leu Ser Ser 20 25 30 tac gcc atg age tgg gtg agg cag gcc ccc gga aag ggc ctg gag tac 144

[0877] Tyr Ala Met Ser Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Tyr 35 40 45 ate ggc ate ate age age age ggc age aca tac tac gcc age tgg gcc 192

[0878] T ie Gly T ie T ie Ser Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala 50 55 60 aag ggc agg ttc aca ate age aag gat tcc tcc aag aat aca gtg tac 240

[0879] Lys Gly Arg Phe Thr l ie Ser Lys Asp Ser Ser Lys Asn Thr Vai Tyr 65 70 75 80 ctg cag atg ggc tcc ctg agg gcc gag gac atg gcc gtg tac ttc tgt 288

[0880] Leu Gin Met Gly Ser Leu Arg Ala Glu Asp Met Ala Vai Tyr Phe Cys 85 90 95 gcc aga gac agg gtc ate tat tcc ate ggc cct tac tac ttc aac ctg 336

[0881] Ala Arg Asp Arg Vai l ie Tyr Ser l ie Gly Pro Tyr Tyr Phe Asn Leu

[0882] 100 105 110

[0883] tgg ggc ccc ggc aca ctg gtg aca gtg tcc age

[0884] 369

[0885] Trp Gly Pro Gly Thr Leu Vai Thr Vai Ser Ser

[0886] 115 120

[0887] SEQ ID No. 54 (Humanized PSC21 vH domain amino acid sequence)

[0888] Gly Glu Gin Gin Leu Vai Glu Ser Gly Gly Gly Leu Vai Gin Pro Gly 1 5 10 15

[0889] Gly Ser Leu Arg Leu Ser Cys Ala Vai Ser Gly Phe Ser Leu Ser Ser

[0890] 20 25 30

[0891] Tyr Ala Met Ser Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Tyr

[0892] 35 40 45 l ie Gly l ie l ie Ser Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala

[0893] 50 55 60

[0894] Lys Gly Arg Phe Thr T ie Ser Lys Asp Ser Ser Lys Asn Thr Vai Tyr 65 70 75 80

[0895] Leu Gin Met Gly Ser Leu Arg Ala Glu Asp Met Ala Vai Tyr Phe Cys

[0896] 85 90 95

[0897] Ala Arg Asp Arg Vai T ie Tyr Ser T ie Gly Pro Tyr Tyr Phe Asn Leu

[0898] 100 105 110

[0899] Trp Gly Pro Gly Thr Leu Vai Thr Vai Ser Ser 115 120

[0900] SEQ ID No. 55 (Humanized PSC21 vL domain nucleic acid sequence) ggc gat tac cag atg aca cag too ccc too too ctg age gcc tcc gtg 48

[0901] Gly Asp Tyr Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Vai 1 5 10 15 gga gat agg gtg acc ate aca tgc cag gcc age gag ate ate tac age 96

[0902] Gly Asp Arg Vai Thr T ie Thr Cys Gin Ala Ser Glu T ie T ie Tyr Ser 20 25 30 aat etg gee tgg tac cag cag aag ccc ggc aag ccc ccc aag etg etg 144 Asn Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Pro Pro Lys Leu Leu 35 40 45 ate tac ggc gee tee aca etg gee age ggc gtg cct age aga ttc age

[0903] 192 l ie Tyr Gly Ala Ser Thr Leu Ala Ser Gly Vai Pro Ser Arg Phe Ser

[0904] 50 55 60 ggc age ggc tee ggc acc gat tac acc etg aca ate tee age etg cag 240 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr l ie Ser Ser Leu Gin 65 70 75 80

[0905] cct gag gat ttt gcc aca tac tac tgt cag cag tcc ttc age tcc aat 288

[0906] Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Ser Phe Ser Ser Asn 85 90 95 aac gtg ggc aac ate ttc ggc ggc ggc aca aag gtg gag ate aag 333

[0907] Asn Vai Gly Asn T ie Phe Gly Gly Gly Thr Lys Vai Glu T ie Lys 100 105 110

[0908] SEQ ID No. 56 (Humanized PSC21 vL domain amino acid sequence)

[0909] Gly Asp Tyr Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Vai

[0910] 1 5 10 15

[0911] Gly Asp Arg Vai Thr T ie Thr Cys Gin Ala Ser Glu T ie T ie Tyr Ser

[0912] 20 25 30

[0913] Asn Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Pro Pro Lys Leu Leu

[0914] 35 40 45 l ie Tyr Gly Ala Ser Thr Leu Ala Ser Gly Vai Pro Ser Arg Phe

[0915] Ser

[0916] 50 55 60

[0917] Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr T ie Ser Ser Leu Gin 65 70 75 80

[0918] Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Ser Phe Ser Ser Asn 85 90 95

[0919] Asn Vai Gly Asn T ie Phe Gly Gly Gly Thr Lys Vai Glu T ie Lys

[0920] 100 105 110

[0921] For use in human patients, it will be desirable to humanize these antibodies, replacing both the constant regions of the heavy and light chains with human constant regions, as well as replacing the framework regions of the variable regions with human antibody framework regions. In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof, is a humanized version of a rabbit antibody. In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof comprises at least one heavy chain variable is at least 60% identical to SEQ ID No. 2, 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54, and even more preferably at least 65%, 70%, 75%, 80%, 85% or even 90% identical to SEQ ID No. 2, 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54, and able to specifically bind human CD39.

[0922] In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof comprises at least one light chain variable is at least 60% identical to SEQ ID No. 4, 8, 12, 16,

[0923] 20, 24, 28, 44, 48, 52, or 56, and even more preferably at least 65%, 70%, 75%, 80%, 85% or even 90% identical to SEQ ID No. 4, 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56, and able to specifically bind human CD39.

[0924] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a nonhuman antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0925] In certain embodiments, the anti-CD39 antibody is a humanized antibody comprising a VH domain having human framework sequences associated with CDRs of a VH domain selected from SEQ ID No. 2, 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54, and the CDRs of the corresponding VE domain selected from SEQ ID No. 4, 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56. The CDRs are preferably identical, but may vary by 1, 2 or 3 amino acids across each CDR so long as the resulting antibody specifically binds human CD39.

[0926] Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, 2008, Front. Biosci. 13:1619-1633, and are further described, e.g., in Riechmann et al., 1988, Nature 332:323-329; Queen et al., 1989, Proc. Natl Acad. Sci. USA 86:10029-10033; U.S. Pat. Nos. 5, 821,337; 7,527,791; 6,982,321; and 7,087,409; Kashmiri et al., 2005, Methods 36:25-34 (describing specificity determining region (SDR) grafting); Padlan, 1991, Mol. Immunol. 28:489-498 (describing "resurfacing"); Dall'Acqua et al., 2005, Methods 36:43-60 (describing "FR shuffling"); and Osbourn et al., 2005, Methods 36:61-68 and Klimka et al., 2000, Br. J. Cancer 83:252-260 (describing the "guided selection" approach to FR shuffling).

[0927] Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims et al. 1993, J. Immunol. 151:2296); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. 1992, Proc. Natl. Acad. Sci. USA 89:4285; and Presta et al. 1993, J. Immunol. 151:2623); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, 2008, Front. Biosci. 13:1619-1633); and framework regions derived from screening FR libraries (see, e.g., Baca et al., 1997, J. Biol. Chem. 272:10678-10684 and Rosok et al., 1996, J Biol. Chem. 271:22611-22618).

[0928] In certain embodiments, an anti-CD39 antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, 2001, Curr. Opin. Pharmacol. 5:368-74 and Lonberg, 2008, Curr. Opin. Immunol. 20:450-459.

[0929] For instance, human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, 2005, Nat. Biotech. 23:1117-1125. (See also, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE technology; U.S. Pat. No. 5,770,429 describing HuMAB technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE technology; and U.S. Patent Application Publication No. US 2007 / 0061900 describing VELOCIMOUSE technology.) Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.

[0930] Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor, 1984, J. Immunol. 133:3001; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., 1991, J. Immunol. 147:86.) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., 2006, Proc. Natl. Acad. Sci USA 103:3557-3562. Additional methods include those described, for example, in U.S. Pat. No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, 2006, Xiandai Mianyixue, 26(4):265-268 (describing humanhuman hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, 2005, Histology and Histopathology 20(3):927-937 and Vollmers and Brandlein, 2005, Methods and Findings in Experimental and Clinical Pharmacology 27(3): 185- 91.

[0931] Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage, yeast or bacterial display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

[0932] To illustrate, anti-CD39 antibodies encompassed by the present invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage or yeast display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al., in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., 2001) and further described, e.g., in McCafferty et al., 1990, Nature 348:552-554; Clackson et al., 1991, Nature 352:624-628; Marks et al., 1992, J. Mol. Biol. 222:581-597; Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, N.J., 2003); Sidhu et al., 2004, J. Mol. Biol. 338(2):299-310; Lee et al., 2004, J. Mol. Biol. 340(5): 1073- 1093; Fellouse, 2004, Proc. Natl. Acad. Sci. USA 101(34): 12467- 12472; and Lee et al., 2004, J. Immunol. Methods 284(1-2): 119-132.

[0933] As an example of phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., 1994, Ann. Rev. Immunol., 12:433-455. Phage typically display antibody fragments, either as single-chain Lv (scLv) fragments or as Lab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths et al., 1993, EMBO J. 12:725-734. Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, 1992, J. Mol. Biol. 227:381-388. Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373; and US Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0934] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0935] FcyRIII binding can also be increased by methods according to the state of the art, e.g., by modifying the amino acid sequence of the Fc part or the glycosylation of the Fc part of the antibody (see e.g., EP2235061). In certain embodiments, the subject antibodies are produced by cells in which, when glycosylated, less than 50% of the oligosaccharide chains on the antibody contain oc-l,6-fucose. Typically, less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than 5% or less than 1% of the oligosaccharide chains contain oc-l,6-fucose in a "hypo-fucosylated" antibody preparation. An "afucosylated" antibody lacks oc-l,6-fucose in the carbohydrate attached to the CH2 domain of the IgG heavy chain. Mori et al., 2007, Cytotechnology 55(2-3): 109- 114 and Satoh et al., 2006, Expert Opin Biol Ther. 6:1161-1173 relate to a FUT8 (oc-l,6-fucosyltransferase) gene knockout CHO line for the generation of afucosylated antibodies.

[0936] IV. Expression Vectors

[0937] In certain embodiments, a recombinant expression vector is used to amplify and express DNA encoding the anti-CD39 antibody described herein. For example, a recombinant expression vector can be a replicable DNA construct which has synthetic or cDNA-derived DNA fragments encoding the polypeptide chains of the anti-CD39 antibody operatively linked to suitable transcriptional and / or translational regulatory elements derived from mammalian, microbial, viral or insect genes. A transcriptional unit generally comprises an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences. Regulatory elements can include an operator sequence to control transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can additionally be incorporated. DNA regions are "operatively linked" when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operatively linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation. In some embodiments, structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell. In other embodiments, where recombinant protein is expressed without a leader or transport sequence, it can include an N- terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.

[0938] The choice of an expression control sequence and an expression vector depends upon the choice of host cell. A wide variety of expression host / vector combinations can be employed. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pCRl, pBR322, pMB9 and their derivatives, and wider host range plasmids, such as M13 and other filamentous single- stranded DNA phages.

[0939] Suitable host cells for expression of the polypeptide chains of the anti-CD39 antibody (or a protein to use as a target) include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram-negative or grampositive organisms, for example E. coli or Bacillus. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Cell-free translation systems may also be employed. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are well known by those skilled in the art.

[0940] Various mammalian cell culture systems are used to express recombinant polypeptides. Expression of recombinant proteins in mammalian cells can be preferred because such proteins are generally correctly folded, appropriately modified, and biologically functional. Examples of suitable mammalian host cell lines include COS-7 (monkey kidney-derived), L-929 (murine fibroblast-derived), C127 (murine mammary tumor-derived), 3T3 (murine fibroblast-derived), CHO (Chinese hamster ovary-derived), HeLa (human cervical cancer-derived), BHK (hamster kidney fibroblast-derived), and HEK-293 (human embryonic kidney-derived) cell lines and variants thereof. Mammalian expression vectors can comprise non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences, and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.

[0941] Expression of recombinant proteins in insect cell culture systems (e.g., baculovirus) also offers a robust method for producing correctly folded and biologically functional proteins. Baculovirus systems for production of heterologous proteins in insect cells are well-known to those of skill in the art.

[0942] In certain embodiments, the polynucleotide comprises a polynucleotide encoding an antibody light chain comprising a variable region at least 60% identical to SEQ ID No. 1, and even more preferably at least 65%, 70%, 75%, 80%, 85% or even 90% identical to SEQ ID No.

[0943] 1, and able to specifically bind human CD39.

[0944] In certain embodiments, the polynucleotide comprises a polynucleotide encoding an antibody heavy chain comprising a variable region at least 60% identical to SEQ ID No. 2, and even more preferably at least 65%, 70%, 75%, 80%, 85% or even 90% identical to SEQ ID No.

[0945] 2, and able to specifically bind human CD39.

[0946] V. Encoded anti-CD39 Antibodies for In Vivo Delivery

[0947] Therapeutic vectors for delivering the coding sequence for an anti-CD39 antibody to be expressed in the patient can be viral, non-viral, or physical. See, for example, Rosenberg et al., 1988, Science 242:1575-1578 and Wolff et al., 1989, Proc. Natl. Acad. Sci. USA 86:9011- 9014. Discussion of methods and compositions for use in gene therapy include Eck et al., 1996, in Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, Hardman et al., eds., McGraw-Hill, New York, Chapter 5, pp. 77-101; Wilson et al., 1997, Clin. Exp. Immunol. 107 (Suppl. 1):31-32; Wivel et al., 1998, Hematology / Oncology Clinics of North America, Gene Therapy, S. L. Eck, ed., 12(3):483-501; Romano et al., 2000, Stem Cells 18:19- 39; and the references cited therein. U.S. Pat. No. 6,080,728 also provides a discussion of a wide variety of gene delivery methods and compositions. The routes of delivery include, for example, systemic administration and administration in situ. Well-known viral delivery techniques include the use of adenovirus, retrovirus, lentivirus, foamy virus, herpes simplex virus, vaccinia virus and adeno-associated virus vectors. a. Viral Vectors

[0948] Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non- essential genes have been replaced with the nucleic acid construct carrying the nucleic acid sequences encoding the epitopes and targeting sequences of interest. Preferred viruses for certain embodiments encompassed by the present invention are the adenoviruses and adeno- associated (AAV) viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. In addition, preferred vectors for tolerizing do not include immune- stimulating sequences.

[0949] Adenovirus Vectors

[0950] One illustrative method for in vivo delivery of one or more nucleic acid sequences involves the use of an adenovirus expression vector. "Adenovirus expression vector" is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express a polynucleotide that has been cloned therein in a sense or antisense orientation. Of course, in the context of an antisense construct, expression does not require that the gene product be synthesized. In a specific embodiment, the delivery vector pertains to commercially available ORF of cytochrome b5 reductase 3 (CYB5R3), transcript variant 1 in adenoviral vector pAd, with C terminal Flag and His tag (Vigene Biosciences Product code AH889428). WIPO Patent Application WO / 2015 / 050364 also teaches vectors with expression constructs including a Cyb5r3 gene.

[0951] Adenoviral vectors are highly immunogenic and therefore are less preferred for administration to induce tolerance by presenting antigens, or in the case of autoimmune diseases. These vectors can be used, however to induce immunity, for example in treatment of infectious diseases and the like, include, for example, influenza, HBV, HCV and HIV.

[0952] Adeno-Associated Virus Vectors (AAV)

[0953] AAV is a good choice of delivery vehicles due to its safety, i.e., genetically engineered (recombinant) does not integrate into the host genome. Likewise, AAV is not pathogenic and not associated with any disease. The removal of viral coding sequences minimizes immune reactions to viral gene expression, and therefore, rAAV does not evoke an inflammatory response. According to a specific embodiment, an AAV vector containing an epitope sequence containing nucleic acid construct described herein is useful for transducing APCs. Typically, viral vectors containing an epitope containing nucleic acid construct are assembled from polynucleotides encoding the desired epitopes, suitable regulatory elements and elements necessary for epitope expression which mediate cell transduction. In one embodiment, adeno-associated viral (AAV) vectors are employed. In a more specific embodiment, the AAV vector is an AAV1, AAV6, or AAV8.

[0954] The AAV expression vector which harbors the DNA molecule of interest bounded by AAV ITRs, can be constructed by directly inserting the selected sequence(s) into an AAV genome which has had the major AAV open reading frames ("ORFs") excised therefrom. Examples of constitutive promoters which may be included in the AAV of this invention include, without limitation, the exemplified CMV immediate early enhancer / chicken P-actin (CBA) promoter.

[0955] For eukaryotic cells, expression control sequences typically include a promoter, an enhancer, such as one derived from an immunoglobulin gene, SV40, cytomegalovirus, etc., and a poly adenylation sequence which may include splice donor and acceptor sites. The polyadenylation sequence generally is inserted following the transgene sequences and before the 3' ITR sequence. In one embodiment, the bovine growth hormone polyA may be used.

[0956] Selection of these and other common vector and regulatory elements are conventional, and many such sequences are available. See, e.g., Sambrook et al., and references cited therein at, for example, pages 3.18-3.26 and 16.17-16.27 and Ausubel et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York). Of course, not all vectors and expression control sequences will function equally well to express all of the transgenes of this invention. However, one of skill in the art may make a selection among these expression control sequences without departing from the scope of this invention. Suitable promoter / enhancer sequences may be selected by one of skill in the art using the guidance provided by this application. Such selection is a routine matter and is not a limitation of the molecule or construct.

[0957] Retrovirus Vectors

[0958] In certain embodiments, the viral vector may be a retroviral vector. "Retroviruses" are viruses having an RNA genome. In particular embodiments, a retroviral vector contains all of the cis-acting sequences necessary for the packaging and integration of the viral genome, i.e., (a) a long terminal repeat (LTR), or portions thereof, at each end of the vector; (b) primer binding sites for negative and positive strand DNA synthesis; and (c) a packaging signal, necessary for the incorporation of genomic RNA into virions. More detail regarding retroviral vectors can be found in Boesen et al., 1994, Biotherapy 6:291-302; Clowes et al., 1994, J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; Miller et al., 1993, Meth. Enzymol. 217:581-599; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3: 110-114.

[0959] "Gammaretroviruses" refers to a genus of the retroviridae family. Exemplary gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses.

[0960] Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., 1992, J. Virol. 66:2731-2739; Johann et al., 1992, J. Virol. 66:1635-1640; Sommerfelt et al., 1990, Virol. 176:58-59; Wilson et al., 1989, J. Virol. 63:2374-2378; Miller et al., 1991, J. Virol. 65:2220-2224; and PCT / US94 / 05700).

[0961] Lentiviral vectors refer to a genus of retroviruses that are capable of infecting dividing and non-dividing cells and typically produce high viral titers. Several examples of lentiviruses include HIV (human immunodeficiency virus: including HIV type 1 and HIV type 2); equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0962] In particular embodiments, other retroviral vectors can be used. These include, e.g., vectors based on human foamy virus (HFV) or other viruses in the Spumavirus genera. Foamy viruses (FVes) are the largest retroviruses known today and are widespread among different mammals, including all non-human primate species, however are absent in humans. This complete apathogenicity qualifies FV vectors as ideal gene transfer vehicles for genetic therapies in humans and clearly distinguishes FV vectors as gene delivery system from HIV- derived and also gammaretrovirus-derived vectors.

[0963] Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are known to those of skill in the art.

[0964] The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Retroviral vectors are gene transfer plasmids wherein the heterologous nucleic acid resides between two retroviral LTRs. Retroviral vectors typically contain appropriate packaging signals that enable the retroviral vector, or RNA transcribed using the retroviral vector as a template, to be packaged into a viral virion in an appropriate packaging cell line (see, e.g., U.S. Pat. No. 4,650,764). These two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990). In order to construct a retroviral vector, a nucleic acid encoding one or more oligonucleotide or polynucleotide sequences of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. Also included are episomal or non-integrating forms of retroviral vectors based on lentiviruses (e.g., a type of retrovirus).

[0965] Lentiviral vectors are useful when stable expression is needed, but lentiviral vectors can be immunogenic, and possibly have other undesirable effects. Therefore, although lentiviral vectors are convenient for research, care should be taken when using them for human administration, particularly where it is desired to induce tolerance rather than immunity. Lentiviruses are suitable for engineering T cells or dendritic cells or other antigen presenting cells ex vivo for cancer therapy, although mRNA electroporation is more safe. However, two recent advances have made the use of lentiviruses safer and more clinically translatable. First, the coexpression of a suicide gene along with the antigens whose products become functional when a drug is administered. A typical example is Herpes simplex virus thymidine kinase (HSV-Tk). Cells that express these genes can metabolize the drug ganciclovir into a cytotoxic product that induces cell death. Thus, in case some transduced cells become malignant, they can be eradicated. About a dozen such systems exist (Duarte et al., 2012, Cancer Letters 324:160-170). Second, there are now non-integrating lentiviral vectors being developed that are therefore non-oncogenic (Nightingale et al., 2006, Mol. Ther. 13:1121-1132). These methods can be used with the invention according to the judgement of the person of skill in the art.

[0966] Suitable retroviral vectors for use herein are described, for example, in U.S. Pat. Nos. 5,399,346 and 5,252,479; and in WIPO publications WO 92 / 07573, WO 90 / 06997, WO 89 / 05345, WO 92 / 05266, and WO 92 / 14829, which provide a description of methods for efficiently introducing nucleic acids into human cells using such retroviral vectors. Other retroviral vectors include, for example, mouse mammary tumor virus vectors (e.g., Shackleford et al., 1998, Proc. Natl. Acad. Sci. USA 85:9655-9659), lentiviruses, and the like. An exemplary viral vector is plentilox-IRES-GFP.

[0967] Additional retroviral viral delivery systems that can be readily adapted for delivery of a transgene encoding an Anti-CD39 antibody Agent include, merely to illustrate Published PCT Applications WO / 2010 / 045002, WO / 2010 / 148203, WO / 2011 / 126864, WO / 2012 / 058673, WO / 2014 / 066700, WO / 2015 / 021077, WO / 2015 / 148683, WO / 2017 / 040815 - the specifications and figures of each of which are incorporated by reference herein.

[0968] In certain embodiments, the retrovirus is a recombinant replication competent retrovirus comprising: a nucleic acid sequence encoding a retroviral GAG protein; a nucleic acid sequence encoding a retroviral POL protein; a nucleic acid sequence encoding a retroviral envelope; an oncoretroviral polynucleotide sequence comprising Long-Terminal Repeat (LTR) sequences at the 5' and 3' end of the oncoretroviral polynucleotide sequence; a cassette comprising an internal ribosome entry site (IRES) operably linked to a coding sequence for an Anti-CD39 antibody Agent, wherein the cassette is positioned 5' to the U3 region of the 3' LTR and 3' to the sequence encoding the retroviral envelope; and cis-acting sequences for reverse transcription, packaging and integration in a target cell.

[0969] In certain embodiments, the retrovirus is a recombinant replication competent retrovirus comprising: a retroviral GAG protein; a retroviral POL protein; a retroviral envelope; a retroviral polynucleotide comprising Long-Terminal Repeat (LTR) sequences at the 3' end of the retroviral polynucleotide sequence, a promoter sequence at the 5' end of the retroviral polynucleotide, the promoter being suitable for expression in a mammalian cell, a gag nucleic acid domain, a pol nucleic acid domain and an env nucleic acid domain; a cassette comprising an Anti-CD39 antibody Agent coding sequence operably linked to a heterologous polynucleotide, wherein the cassette is positioned 5' to the 3' LTR and is operably linked and 3' to the env nucleic acid domain encoding the retroviral envelope; and cis-acting sequences necessary for reverse transcription, packaging and integration in a target cell.

[0970] In certain preferred embodiments of the recombinant replication competent retrovirus, the envelope is chosen from one of amphotropic, polytropic, xenotropic, 10A1, GALV, Baboon endogenous virus, RD114, rhabdovirus, alphavirus, measles or influenza virus envelopes.

[0971] In certain preferred embodiments of the recombinant replication competent retrovirus, the retroviral polynucleotide sequence is engineered from a virus selected from the group consisting of murine leukemia virus (MLV) , Moloney murine leukemia virus (MoMLV) , Feline leukemia virus (FeLV) , Baboon endogenous retrovirus (BEV) , porcine endogenous virus (PERV) , the cat derived retrovirus RD 114, squirrel monkey retrovirus, Xenotropic murine leukemia virus-related virus (XMRV) , avian reticuloendotheliosis virus (REV) , or Gibbon ape leukemia virus (GALV) .

[0972] In certain preferred embodiments of the recombinant replication competent retrovirus, retrovirus is a gammaretrovirus.

[0973] In certain preferred embodiments of the recombinant replication competent retrovirus, there is a second cassette comprising a coding sequence for a second therapeutic protein, such as another checkpoint inhibitor polypeptide, a co-stimulatory polypeptide and / or a immuno stimulatory cytokine (merely as examples), e.g., downstream of the cassette. In certain instances, the second cassette can include an internal ribosome entry site (IRES) or a minipromoter or a polIII promoter operably linked to the coding sequence for the second therapeutic protein.

[0974] In certain preferred embodiments of the recombinant replication competent retrovirus, it is a nonlytic, amphotropic retroviral replicating vector which, preferably, selectively infects and replicates in the cells of the inflammatory tissue microenvironment.

[0975] Other Viral Vectors as Expression Constructs

[0976] Other viral vectors may be employed as expression constructs in the present invention for the delivery of oligonucleotide or polynucleotide sequences to a host cell. Vectors derived from viruses such as vaccinia virus, polioviruses and herpes viruses may be employed. They offer several attractive features for various mammalian cells. Also included are hepatitis B viruses. b. Non-Viral Vectors

[0977] Plasmid Vectors

[0978] Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al., 1989, cited above. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide epitope encoded by nucleic acid within the plasmid. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.

[0979] Thus, in one aspect, a plasmid is provided for expression of the epitope containing nucleic acid construct which includes an expression cassette; also referred to as a transcription unit. When a plasmid is placed in an environment suitable for epitope expression, the transcriptional unit will express the polynucleotide including a sequence encoding the epitopes, ETS and MHCII activator sequence, or sequence encoding the epitopes and secretion signal sequence, and anything else encoded in the construct. The transcription unit includes a transcriptional control sequence, which is transcriptionally linked with a cellular immune response element coding sequence. Transcriptional control sequence may include promoter / enhancer sequences such as cytomegalovirus (CMV) promoter / enhancer sequences. However, those skilled in the art will recognize that a variety of other promoter sequences suitable for expression in eukaryotic cells are known and can similarly be used in the constructs disclosed herein. The level of expression of the nucleic acid product will depend on the associated promoter and the presence and activation of an associated enhancer element. In certain embodiments, a sequence encoding the desired epitopes and targeting sequence can be cloned into an expression plasmid which contains the regulatory elements for transcription, translation, RNA stability and replication (i.e., including a transcriptional control sequence). Such expression plasmids are well known in the art and one of ordinary skill would be capable of designing an appropriate expression construct with a polynucleotide including a sequence encoding a cellular immune response element or fragment thereof in such a manner that the cellular immune response element is expressible. There are numerous examples of suitable expression plasmids into which a polynucleotide including a sequence could be cloned such as pCI-neo, pUMVC or pcDNA3.

[0980] Large quantities of a bacterial host harboring a plasmid for expression of cellular immune response element or fragment thereof may be fermented and the plasmid can be purified for subsequent use. Current human clinical trials using plasmids utilize this approach (Recombinant DNA Advisory Committee Data Management Report, 1994, Human Gene Therapy 6:535-548). Current DNA isolation methods known in the art include removal of lipopolysaccharides (endotoxins) that are contaminants from the bacteria used to propagate the plasmids. This step is most preferably taken for use of tolerogenic DNA vaccines as endotoxins act as strong adjuvants and can produce undesired immune stimulation.

[0981] The purpose of the plasmid is the efficient delivery of nucleic acid sequences to and expression of therapeutic epitopes in a cell or tissue. In particular, the purpose of the plasmid may be to achieve high copy number, avoid potential causes of plasmid instability and provide a means for plasmid selection. As for expression, the nucleic acid cassette contains the necessary elements for expression of the nucleic acid within the cassette. Expression includes the efficient transcription of an inserted gene, nucleic acid sequence, or nucleic acid cassette with the plasmid. Expression products may be proteins, polypeptides or RNA. The nucleic acid sequence can be contained in a nucleic acid cassette. Expression of the nucleic acid can be continuous or regulated.

[0982] Minicircle

[0983] Embodiments of nucleic acid constructs described herein may be processed in the form of minicircle DNA. Minicircle DNA pertains to small (2-4 kb) circular plasmid derivatives that have been freed from all prokaryotic vector parts. Since minicircle DNA vectors contain no bacterial DNA sequences, they are less likely to be perceived as foreign and destroyed. (Typical transgene delivery methods involve plasmids, which contain foreign DNA.) As a result, these vectors can be expressed for longer periods of time (in order of weeks or months) compared to conventional plasmids (days to weeks). The smaller size of minicircles also extends their cloning capacity and facilitates their delivery into cells. Kits for producing minicircle DNA are known in the art and are commercially available (System Biosciences, Inc., Palo Alto, Calif.). Information on minicircle DNA is provided in Dietz et al., 2013, Vector Engineering and Delivery Molecular Therapy 21(8): 1526- 1535 and Hou et al., 2015, Molecular Therapy - Methods & Clinical Development, Article number: 14062 doi:10.1038 / mtm.2014.62. More information on Minicircles is provided in Chen et al., 2003 September, Mol. Ther. 8(3):495- 500 and Minicircle DNA vectors achieve sustained expression reflected by active chromatin and transcriptional level (Gracey Maniar et al., 2013 January, Mol. Ther. 21(1): 131-8).

[0984] As an initial step in the process of ultimately obtaining expression of a product encoded by a nucleic acid, is to effect the uptake of the nucleic acid by cells. Uptake of nucleic acid by cells is dependent on a number of factors, one of which is the length of time during which a nucleic acid is in proximity to a cellular surface. For instance, after intramuscular (i.m.) administration of plasmid DNA in buffer, a marked reduction in gene expression was observed if the muscle is massaged, presumably due to DNA leakage out of the muscle either directly or via lymphatic vessels (Human Gene Therapy 4:151-159 (1993)). Accordingly, it may be desirable to formulate nucleic acids with compounds which would retard the rate at which nucleic acids diffuse or are carried away from a site at which cellular uptake of the nucleic acid is desired. Further, these compounds could be suitable for administration to an organism by means such as injection while maintaining or regaining the physical characteristics necessary to increase cellular uptake of nucleic acids.

[0985] In order to effect expression of oligonucleotide or polynucleotide sequences, the expression construct must be delivered into a cell. In certain embodiments encompassed by the present invention, an expression construct comprising one or more oligonucleotide or polynucleotide sequences may simply consist of naked recombinant DNA or plasmids.

[0986] To prime immunity, DNA vaccine vectors of any type preferably are engineered to be CpG-rich (to stimulate TER9 on immune cells) or conversely are engineered to remove CpG, and when possible, replace CpG motifs with GpG motifs (Ho et al., 2003, J. Immunol. 71(9):4920-6; Ho et al., 2005, J. Immunol. 175(9):6226-34). DNA vaccines can be engineered to contain the antigen(s) / epitope(s), and also can contain additional genes for co-expression with the antigens to act as adjuvants or immunomodulators (multiple promoter vectors. These DNA vaccines have been found to be safe clinically, for example in T1D patients (Roep et al., 2013, Sci. Transl. Med. 5(191): 191ra82).

[0987] Mechanical Delivery Systems

[0988] Additional non-viral delivery methods include but are not limited to mechanical delivery systems that can be used in vitro such as the approach described in Woffendin et al., 1994, Proc. Natl. Acad. Sci. USA 91(24): 11581 ; deposition of photopolymerized hydrogel materials or use of ionizing radiation (see, e.g., U.S. Pat. No. 5,206,152 and WO 92 / 11033); the use of a hand-held gene transfer particle gun (see, e.g., U.S. Pat. No. 5,149,655); and the use of ionizing radiation for activating transferred gene (see, e.g., U.S. Pat. No. 5,206,152 and WO 92 / 11033). Delivery devices can also be biocompatible, and may also be biodegradable. The formulation preferably provides a relatively constant level of active component release. On the other hand, a more rapid rate of release immediately upon administration may be desired. The formulation of such compositions is well within the level of ordinary skill in the art using known techniques.

[0989] Physical methods to enhance delivery include electroporation (where short pulses of high voltage carries the nucleic acid across the membrane), a gene gun (where DNA is loaded onto gold particles and forced to achieve penetration of the DNA into the cells), sonoporation, magnetofection, hydrodynamic delivery and the like, all of which are known to those of skill in the art. DNA also can be encapsulated in liposomes, preferably cationic liposomes, or polymersomes (synthetic liposomes) which can interact with the cell membrane and fuse or undergo endocytosis to effect DNA transfer into the cell. The DNA also can be formed into complexes with polymers (polyplexes) or with dendrimers which can directly release their load into the cytoplasm of a cell.

[0990] Illustrative carriers useful in this regard include microparticles of poly(lactide-co- glycolide), polyacrylate, latex, starch, cellulose, dextran and the like. Other illustrative delayed- release carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross -linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound, such as a phospholipid (see e.g., U.S. Pat. No. 5,151,254 and PCT applications WO 94 / 20078, WO / 94 / 23701 and WO 96 / 06638). The amount of active agent contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented. Biodegradable microspheres (e.g., polylactate poly glycolate) may be employed as carriers for compositions. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344; 5,407,609; and 5,942,252. Modified hepatitis B core protein carrier systems such as described in WO / 99 40934, and references cited therein, will also be useful for many applications. Another illustrative carrier / delivery system employs a carrier comprising particulate-protein complexes, such as those described in U.S. Pat. No. 5,928,647.

[0991] Biodegradable polymeric nanoparticles facilitate nonviral nucleic acid transfer to cells. Small (approximately 200 nm), positively charged (approximately 10 mV) particles are formed by the self-assembly of cationic, hydrolytically degradable poly(beta-amino esters) and plasmid DNA.

[0992] Polynucleotides may also be administered to cells by direct microinjection, temporary cell permeabilizations (e.g., co-administration of repressor and / or activator with a cell permeabilizing agent), fusion to membrane translocating peptides, and the like.

[0993] In certain particular embodiments of the present disclosure, the gene construct is introduced into target cells via electroporation. Electroporation involves the exposure of cells (or tissues) and DNA (or a DNA complex) to a high-voltage electric discharge. In vivo electroporation is a gene delivery technique that has been used successfully for efficient delivery of plasmid DNA to many different tissues. Systemic and local expression of a gene or cDNA encoded by a plasmid can be obtained with administration of in vivo electroporation. Use of in vivo electroporation enhances plasmid DNA uptake in the target inflammatory tissue, resulting in expression within the inflamed tissue, and delivers plasmids to muscle tissue, resulting in systemic expression of the anti-CD39 antibody (see, e.g., US8026223). Exemplary techniques, vectors and devices for electroporating anti-CD39 antibody transgenes into cells in vivo include PCT Publications WO / 2017 / 106795, WO / 2016 / 161201, WO / 2016 / 154473, WO / 2016 / 112359, and WO / 2014 / 066655.

[0994] U.S. Patent No. 7,245,963 describes modular electrode systems and their use for facilitating the introduction of a biomolecule into cells of a selected tissue in a body or plant. The modular electrode systems comprise a plurality of needle electrodes; a hypodermic needle; an electrical connector that provides a conductive link from a programmable constant-current pulse controller to the plurality of needle electrodes; and a power source. An operator can grasp the plurality of needle electrodes that are mounted on a support structure and firmly insert them into the selected tissue in a body or plant. The biomolecules are then delivered via the hypodermic needle into the selected tissue. The programmable constant-current pulse controller is activated and constant-current electrical pulse is applied to the plurality of needle electrodes. The applied constant-current electrical pulse facilitates the introduction of the biomolecule into the ceil between the plurality of electrodes. The entire content of U.S. Patent No. 7,245,963 is hereby incorporated by reference.

[0995] U.S. Patent Pub. 2005 / 0052630 describes an electroporation device which may be used to effectively facilitate the introduction of a biomolecule into ceils of a selected tissue in a body or plant. The electroporation device comprises an electro-kinetic device ("EKD device") whose operation is specified by software or firmware. The EKD device produces a series of programmable constant-current pulse patterns between electrodes in an array based on user control and input of the pulse parameters, and allows the storage and acquisition of current waveform data. The electroporation device also comprises a replaceable electrode disk having an array of needle electrodes, a central injection channel for an injection needle, and a removable guide disk (see, e.g., U.S. Patent Pub. 2005 / 0052630) is hereby incorporated by reference.

[0996] The electrode arrays and methods described in U.S. Patent No. 7,245,963 and U.S. Patent Pub. 2005 / 0052630 are adapted for deep penetration into not only tissues such as muscle, but also other tissues or organs. Because of the configuration of the electrode array, the injection needle (to deliver the biomolecule of choice) is also inserted completely into the target organ, and the injection is administered perpendicular to the target issue, in the area that is predelineated by the electrodes.

[0997] Typically, the electric fields needed for in vivo cell electroporation are generally similar in magnitude to the fields required for cells in vitro. In one embodiment, the magnitude of the electric field range from approximately, 10 V / cm to about 1500 V / cm, preferably from about 300 V / cm to 1500 V / cm and preferably from about 1000 V / cm to 1500 V / cm. Alternatively, lower field strengths (from about 10 V / cm to 100 V / cm, and more preferably from about 25 V / cm to 75 V / cm) the pulse length is long. For example, when the nominal electric field is about 25-75 V / cm, if is preferred that the pulse length is about 10 msec.

[0998] The pulse length can be about 10 s to about 100 ms. There can be any desired number of pulses, typically one to 100 pulses per second. The delay between pulses sets can be any desired time, such as one second. The waveform, electric field strength and pulse duration may also depend upon the type of cells and the type of molecules that are to enter the cells via electroporation.

[0999] Also encompassed are electroporation devices incorporating electrochemical impedance spectroscopy ("EIS"). Such devices provide real-time information on in vivo, in particular, inflammatory tissue electroporation efficiency, allowing for the optimization of conditions. Examples of electroporation devices incorporating EIS can be found, e.g., in W02016 / 161201, which is hereby incorporated by reference.

[1000] Uptake of the non-viral delivery vectors encompassed by the present invention may also be enhanced by plasma electroporation also termed avalanche transfection. Briefly, microsecond discharges create cavitation microbubbles at electrode surface. The mechanical force created by the collapsing microbubbles combined with the magnetic field serve to increase transport efficiency across the cell membrane as compared with the diffusion mediated transport associated with conventional electroporation. The technique of plasma electroporation is described in United States Patent Nos. 7,923,251 and 8,283,171. This technique may also be employed in vivo for the transformation of cells (Chaiberg et al., 2006, Investigative Ophthalmology & Visual Science 47:4083-4090; Chaiberg et al., United States Patent No 8, 101 169 Issued January 24, 2012).

[1001] Other alternative electroporation technologies are also contemplated. In vivo plasmid delivery can also be performed using cold plasma. Plasma is one of the four fundamental states of matter, the others being solid, liquid, and gas. Plasma is an electrically neutral medium of unbound positive and negative particles (i.e. the overall charge of a plasma is roughly zero). A plasma can be created by heating a gas or subjecting it to a strong electromagnetic field, applied with a laser or microwave generator. This decreases or increases the number of electrons, creating positive or negative charged particles called ions (Luo et al., 1998, Phys. Plasma 5:2868-2870) and is accompanied by the dissociation of molecular bonds, if present.

[1002] Cold plasmas (i.e., non-thermal plasmas) are produced by the delivery of pulsed high voltage signals to a suitable electrode. Cold plasma devices may take the form of a gas jet device or a dielectric barrier discharge (DBD) device. Cold temperature plasmas have attracted a great deal of enthusiasm and interest by virtue of their provision of plasmas at relatively low gas temperatures. The provision of plasmas at such a temperature is of interest to a variety of applications, including wound healing, anti-bacterial processes, various other medical therapies and sterilization. As noted earlier, cold plasmas (i.e., non-thermal plasmas) are produced by the delivery of pulsed high voltage signals to a suitable electrode. Cold plasma devices may take the form of a gas jet device, a dielectric barrier discharge (DBD) device or multi-frequency harmonic-rich power supply.

[1003] Dielectric barrier discharge device relies on a different process to generate the cold plasma. A dielectric barrier discharge (DBD) device contains at least one conductive electrode covered by a dielectric layer. The electrical return path is formed by the ground that can be provided by the target substrate undergoing the cold plasma treatment or by providing an inbuilt ground for the electrode. Energy for the dielectric barrier discharge device can be provided by a high voltage power supply, such as that mentioned above. More generally, energy is input to the dielectric barrier discharge device in the form of pulsed DC electrical voltage to form the plasma discharge. By virtue of the dielectric layer, the discharge is separated from the conductive electrode and electrode etching and gas heating is reduced. The pulsed DC electrical voltage can be varied in amplitude and frequency to achieve varying regimes of operation. Any device incorporating such a principle of cold plasma generation (e.g., a DBD electrode device) falls within the scope of various embodiments encompassed by the present invention.

[1004] In certain illustrative embodiments, the transgene construct encoding the anti-CD39 antibody agent encompassed by the present invention is delivered using an electroporation device comprising: an applicator; a plurality of electrodes extending from the applicator, the electrodes being associated with a cover area; a power supply in electrical communication with the electrodes, the power supply configured to generate one or more electroporating signals to cells within the cover area; and a guide member coupled to the electrodes, wherein the guide member is configured to adjust the cover area of the electrodes. At least a portion of the electrodes can be positioned within the applicator in a conical arrangement. The one or more electroporating signals may be each associated with an electric field. The device may further comprise a potentiometer coupled to the power supply and electrodes. The potentiometer may be configured to maintain the electric field substantially within a predetermined range.

[1005] The one or more electroporating signals may be each associated with an electric field. The device may further comprise a potentiometer coupled to the power supply and the electrodes. The potentiometer may be configured to maintain the electric field within a predetermined range so as to substantially prevent permanent damage in the cells within the cover area and / or substantially minimize pain. For instance, potentiometer may be configured to maintain the electric field to about 1300 V / cm. The power supply may provide a first electrical signal to a first electrode and a second electrical signal to a second electrode. The first and second electrical signals may combine to produce a wave having a beat frequency. The first and second electrical signals may each have at least one of a unipolar waveform and a bipolar waveform. The first electrical signal may have a first frequency and a first amplitude. The second electrical signal may have a second frequency and a second amplitude. The first frequency may be different from or the same as the second frequency. The first amplitude may be different from or the same as the second amplitude.

[1006] In certain embodiments, the present invention provides a method for treating a subject having an abnormal scar formation or suffering from scarring, the method comprising: injecting the scarring tissue (or tissue proximate to it) with an effective dose of plasmid coding for an anti-CD39 antibody; and administering electroporation therapy to the target tissue. In certain embodiments, the electroporation therapy further comprises the administration of at least one voltage pulse of about 200 V / cm to about 1500 V / cm over a pulse width of about 100 microseconds to about 20 milliseconds.

[1007] In certain embodiments, the plasmid (or a second electroporated plasmid) further encodes at least one or more additional immunosuppressive biologic(s), such as adalimumab, certolizumab, etanercept, golimumab, infliximab, risankizumab, and ustekinumab.

[1008] Lipids and Polycationic Molecules for Delivering Anti-CD39 antibody Encoding Nucleic Constructs

[1009] Lipid-mediated nucleic acid delivery and expression of foreign nucleic acids, including mRNA, in vitro and in vivo has been very successful. Lipid based non-viral formulations provide an alternative to adenoviral gene therapies. Current in vivo lipid delivery methods use subcutaneous, intradermal, pulmonary, gastrointestinal, submucosal, intrasynovial, intrathecal or intracranial injection. Advances in lipid formulations have improved the efficiency of gene transfer in vivo (see PCT Application WO 98 / 07408). For instance, a lipid formulation composed of an equimolar ratio of l,2-bis(oleoyloxy)-3-(trimethyl ammonio)propane (DOTAP) and cholesterol can significantly enhances systemic in vivo gene transfer. The DOTAP:cholesterol lipid formulation forms unique structure termed a "sandwich liposome". This formulation is reported to "sandwich" DNA between an invaginated bi-layer or "vase" structure. Beneficial characteristics of these lipid structures include a positive p, colloidal stabilization by cholesterol, two-dimensional nucleic acid packing and increased serum stability.

[1010] Cationic liposome technology is based on the ability of amphipathic lipids, possessing a positively charged head group and a hydrophobic lipid tail, to bind to negatively charged DNA or RNA and form particles that generally enter cells by endocytosis. Some cationic liposomes also contain a neutral co-lipid, thought to enhance liposome uptake by mammalian cells. Similarly, other polycations, such as poly-l-lysine and polyethylene-imine, complex with nucleic acids via charge interaction and aid in the condensation of DNA or RNA into nanoparticles, which are then substrates for endosome-mediated uptake. Several of these cationic-nucleic acid complex technologies have been developed as potential clinical products, including complexes with plasmid DNA (pDNA), oligodeoxynucleotides, and various forms of synthetic RNA.

[1011] The nucleic acid constructs disclosed herein may be associated with polycationic molecules that serve to enhance uptake into cells. Complexing the nucleic acid construct with polycationic molecules also helps in packaging the construct such their size is reduced, which is believed to assist with cellular uptake. Once in the endosome, the complex dissociates due to the lower pH, and the polycationic molecules can disrupt the endosome's membrane to facilitate DNA escape into the cytoplasm before it can be degraded. Preliminary data shows that the nucleic acid construct embodiments had enhanced uptake into SCs over DCs when complexed with the polycationic molecules polylysine or polyethyleneimine.

[1012] One example of polycationic molecules useful for complexing with nucleic acid constructs includes cell penetrating peptides (CPP), examples include polylysine (described above), polyarginine and Tat peptides. Cell penetrating peptides (CPP) are small peptides which can bind to DNA and, once released, penetrate cell membranes to facilitate escape of the DNA from the endosome to the cytoplasm. Another example of a CPP pertains to a 27-residue chimeric peptide, termed MPG, was shown some time ago to bind ss- and ds-oligonucleotides in a stable manner, resulting in a non-covalent complex that protected the nucleic acids from degradation by DNase and effectively delivered oligonucleotides to cells in vitro (Mahapatro et al., 2011, J Nanobio technol 9:55). The complex formed small particles of approximately 150 nm to 1 um when different peptide:DNA ratios were examined, and the 10:1 and 5:1 ratios (150 nm and 1 um respectively). Another CPP pertains to a modified tetrapeptide (tetralysine containing guanidinocarbonylpyrrole (GCP) groups (TL-GCP)), which was reported to bind with high affinity to a 6.2 kb plasmid DNA resulting in a positive charged aggregate of 700- 900 nm (Li et al., 2015, Agnew Chem Int Ed Enl, 54(10):2941-4). RNA can also be complexed by such polycationic molecules for in vivo delivery.

[1013] Other examples of polycationic molecules that may be complexed with the nucleic acid constructs described herein include polycationic polymers commercially available as JETPRIME® and In Vivo JET (Polypus-transfection, S.A., Illkirch, France).

[1014] VI. Methods of Use and Pharmaceutical Compositions

[1015] The anti-CD39 antibodies encompassed by the present invention are useful in a variety of applications including, but not limited to, therapeutic and cosmetic treatment methods, such as therapy for scarring disorders or wound healing applications marked by infiltration of CD39highinflammation / granulation cells. In certain embodiments, an anti-CD39 antibody described herein is useful for inactivating or otherwise decreasing wound healing inflammation / granulation cells mediated aspects of scar formation.

[1016] The present invention provides compositions comprising a CD39-targeted wound healing inflammation / granulation cell-depleting agent, such as an anti-CD39 antibody described herein. The present invention also provides pharmaceutical compositions comprising an anti-CD39 antibody described herein and a pharmaceutically acceptable vehicle. In some embodiments, the pharmaceutical compositions find use in treating a human patient.

[1017] Formulations are prepared for storage and use by combining a purified agent encompassed by the present invention with a pharmaceutically acceptable vehicle (e.g., a carrier or excipient). Those of skill in the art generally consider pharmaceutically acceptable carriers, excipients, and / or stabilizers to be inactive ingredients of a formulation or pharmaceutical composition.

[1018] The pharmaceutical compositions encompassed by the present invention can be administered in any number of ways for either local or systemic treatment. Administration can be topical by epidermal or transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary by inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal, and intranasal; oral; or parenteral including intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion), or intracranial (e.g., intrathecal or intraventricular) and intrasynovial.

[1019] In certain embodiments, the CD39-targeted wound healing inflammation / granulation cell-depleting agent (such as an ADCC+ anti-CD39 antibody) is administered. For instance, local administration can be achieved by injection (e.g., subcutaneous injection, intramuscular injection). In certain embodiments, the injection is an intradermal injection.

[1020] Certain embodiments herein provide a kit for treating or preventing scar formation, the kit comprising a CD39-targeted wound healing inflammation / granulation cell-depleting agent (such as an ADCC+ anti-CD39 antibody), a delivery device (e.g., for topical administration), and instructions for use. In some embodiments, the delivery device comprises a device for injection.

[1021] In some embodiments, local administration is achieved by topical administration (e.g., by irrigation, by application of ointments, salves, powders, or the like which include the CD39- targeted wound healing inflammation / granulation cell-depleting agent).

[1022] In still other embodiments, the local administration is achieved by application of a bandage or other drug eluting wound dressing or suture for preventing or reducing the severity of epithelial scar formation, where the bandage or dressing includes a CD39-targeted wound healing inflammation / granulation cell-depleting agent (such as an ADCC+ anti-CD39 antibody) to the subject in an amount sufficient to reduce the number of CD39highinflammation / granulation cells at the site of scar formation.

[1023] In some embodiments, administration occurs proximal to the wound site. In some embodiments, administration occurs within the wound site. The methods are not limited by the nature of the wound. Wounds may occur via trauma, bums, surgical or other medical procedures, or as a result of a physiological condition (e.g., pathological conditions, pressure sores, ulcers).

[1024] The anti-CD39 antibody can be administered one time or over a series of treatments lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient and will vary depending on the relative potency of an individual agent. The administering physician can determine optimum dosages, dosing methodologies, and repetition rates. In certain embodiments, dosage is from 0.01 pg to 100 mg / kg of body weight, from 0.01 pg to 10 mg / kg of body weight, from 0.1 pg to 100 mg / kg of body weight, from 0.1 pg to 10 mg / kg of body weight, from 1 pg to 100 mg / kg of body weight, from 1 pg to 10 mg / kg of body weight, from 0.01 mg to 100 mg / kg of body weight, from 0.01 mg to 50 mg / kg of body weight, from 0.01 mg to 25 mg / kg of body weight, from 0.01 mg to 10 mg / kg of body weight, from 0.01 mg to 5 mg / kg of body weight, from 0.1 mg to 100 mg / kg of body weight, from 0.1 mg to 50 mg / kg of body weight, from 0.1 mg to 25 mg / kg of body weight, from 0.1 mg to 10 mg / kg of body weight, from 0.1 mg to 5 mg / kg of body weight, from 1 mg to 100 mg / kg of body weight, 1 mg to 50 mg / kg of body weight, from 1 mg to 25 mg / kg of body weight, from 1 mg to 10 mg / kg of body weight, or from 1 mg to 5 mg / kg of body weight. In certain embodiments, the dosage of the anti-CD39 antibody is from about 0.01 mg to about 10 mg / kg of body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 0.01 mg / kg of body weight.

[1025] In some embodiments, the dosage of the anti-CD39 antibody is about 0.025 mg / kg of body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 0.05 mg / kg of body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 0.1 mg / kg of body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 0.25 mg / kg of body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 0.5 mg / kg of body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 1 mg / kg of body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 1.5 mg / kg of body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 2 mg / kg of body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 2.5 mg / kg of body weight. In some embodiments, the dosage of the anti- CD39 antibody is about 5 mg / kg of body weight. In some embodiments, the dosage of the anti- CD39 antibody is about 7.5 mg / kg of body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 10 mg / kg of body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 25 mg / kg of body weight. In some embodiments, the dosage is a range bounded by dosages described herein, such as 0.025 mg / kg to 25 mg / kg of body weight, or any range in between, such as 2-25 mg / kg body weight, 5-10 mg / kg, and the like. In certain embodiments, the dosage can be given once or more daily, trice a week, twice a week, weekly, monthly, or yearly. In certain embodiments, the anti-CD39 antibody is given once every week, once every two weeks, once every three weeks, or once every four weeks.

[1026] In some embodiments, an anti-CD39 antibody may be administered at an initial higher "loading" dose, followed by one or more lower doses. In some embodiments, the frequency of administration may also change. In some embodiments, a dosing regimen may comprise administering an initial dose, followed by additional doses (or "maintenance" doses) once a week, once every two weeks, once every three weeks, or once every month. For example, a dosing regimen may comprise administering an initial loading dose, followed by a weekly maintenance dose of, for example, one-half of the initial dose. In some embodiments, a dosing regimen may comprise administering an initial loading dose, followed by maintenance doses of, for example one-half of the initial dose every other week. In some embodiments, a dosing regimen may comprise administering three initial doses for 3 weeks, followed by maintenance doses of, for example, the same amount every other week.

[1027] In some embodiments, the dosing schedule may be limited to a specific number of administrations or "cycles". In some embodiments, the anti-CD39 antibody is administered for 2, 3, 4, 5, 6, 7, 8, or more cycles. For example, the anti-CD39 antibody is administered every 2 weeks for 6 cycles, the anti-CD39 antibody is administered every 3 weeks for 6 cycles, the anti-CD39 antibody is administered every 2 weeks for 4 cycles, the anti-CD39 antibody is administered every 3 weeks for 4 cycles, etc. Dosing schedules can be decided upon and subsequently modified by those skilled in the art.

[1028] Thus, the present invention provides methods of administering to a subject the anti- CD39 antibody described herein comprising using an intermittent dosing strategy for administering one or more agents, which may reduce side effects and / or toxicities associated with administration of an anti-CD39 antibody, anti-inflammatory agent, etc. In some embodiments, a method for treating a disease or condition associated with unwanted wound healing inflammation / granulation cell activity in a human subject comprises administering to the subject a therapeutically effective dose of an anti-CD39 antibody in combination with a therapeutically effective dose of an anti-inflammatory agent, wherein one or both of the agents are administered according to an intermittent dosing strategy. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of an anti-CD39 antibody to the subject, and administering subsequent doses of the anti-CD39 antibody about once every 2 weeks. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of an anti-CD39 antibody to the subject, and administering subsequent doses of the anti-CD39 antibody about once every 3 weeks. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of an anti-CD39 antibody to the subject, and administering subsequent doses of the anti-CD39 antibody about once every 4 weeks. In some embodiments, the anti-CD39 antibody is administered using an intermittent dosing strategy and the anti-inflammatory agent is administered weekly. VII. Anti-CD39 Antibody Conjugates

[1029] The anti-CD39 antibodies disclosed herein may also be conjugated to a cytotoxic moiety. In some embodiments, bispecific anti-CD39 antibodies disclosed herein are conjugated to a cytotoxic moiety to further improve the specificity.

[1030] In certain embodiments, the anti-CD39 antibody (e.g., the bispecific anti-CD39 antibody described herein) is capable of inducing cytotoxicity in a CD39-expressing cell (e.g., wound healing inflammation / granulation cells) by internalization of the antibody conjugated to or associated with a cytotoxic moiety. The cytotoxic moiety may, for example, be selected from the group consisting of taxol; cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; tenoposide; vincristine; vinblastine; colchicin; doxorubicin; daunorubicin; dihydroxy anthracin dione; a tubulin-inhibitor such as maytansine or an analog or derivative thereof; an antimitotic agent such as monomethyl auristatin E or F or an analog or derivative thereof; dolastatin 10 or 15 or an analogue thereof; irinotecan or an analogue thereof; mitoxantrone; mithramycin; actinomycin D; 1-dehydrotestosterone; a glucocorticoid; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin or an analog or derivative thereof; an antimetabolite such as methotrexate, 6 mercaptopurine, 6 thioguanine, cytarabine, fludarabin, 5 fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, or cladribine; an alkylating agent such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C; a platinum derivative such as cisplatin or carboplatin; duocarmycin A, duocarmycin SA, rachelmycin (CC-1065), or an analog or derivative thereof; an antibiotic such as dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)); pyrrolo [2, 1-c] [ 1,4] -benzodiazepines (PDB); diphtheria toxin and related molecules such as diphtheria A chain and active fragments thereof and hybrid molecules, ricin toxin such as ricin A or a deglycosylated ricin A chain toxin, cholera toxin, a Shiga-like toxin such as SLT I, SLT II, SLT IIV, LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin, gelanin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins such as PAPI, PAPII, and PAP-S, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin toxins; ribonuclease (RNase); DNase I, Staphylococcal enterotoxin A; pokeweed antiviral protein; diphtherin toxin; and Pseudomonas endotoxin. In one embodiment, the anti-CD39 antibody (e.g.. the bispecific anti-CD39 antibody described herein) is conjugated to an auristatin or a peptide analog, derivative or prodrug thereof. Auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis and nuclear and cellular division (Woyke et al., 2001, Antimicrob. Agents and Chemother. 45(12): 3580-3584) and have anti-cancer (US5663149) and anti-fungal activity (Pettit et al., 1998, Antimicrob. Agents and Chemother. 42:2961-2965). For example, auristatin E can be reacted with para- acetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatin derivatives include AFP, MMAF (monomethyl auristatin F), and MMAE (monomethyl auristatin E). Suitable auristatins and auristatin analogs, derivatives and prodrugs, as well as suitable linkers for conjugation of auristatins to Abs, are described in, e.g., U.S. Pat. Nos. 5,635,483, 5,780,588 and 6,214,345 and in International patent application publications W002088172, W02004010957, W02005081711, W02005084390, W02006132670, WO03026577, W0200700860, W0207011968, and W0205082023.

[1031] In another embodiment, the anti-CD39 antibody (e.g., the bispecific anti-CD39 antibody described herein) is conjugated to pyrrolo[2,l-c][ 1,4] -benzodiazepine (PDB) or an analog, derivative or prodrug thereof. Suitable PDBs and PDB derivatives, and related technologies are described in, e.g., Sagnou et al., 2000, Bioorg Med Chem Lett 10(18):2083- 2086; Antonow et al., 2008, Cancer J 14(3): 154-169; Howard et al., 2009, Bioorg Med Chem Lett 19:6463-6466; and Hartley et al., 2010, Cancer Res 70(17):6849-6858.

[1032] In another embodiment, the anti-CD39 antibody (e.g., the bispecific anti-CD39 antibody described herein) is conjugated to a cytotoxic moiety selected from the group consisting of an anthracycline, maytansine, calicheamicin, duocarmycin, rachelmycin (CC- 1065), dolastatin 10, dolastatin 15, irinotecan, monomethyl auristatin E, monomethyl auristatin F, a PDB, or an analog, derivative, or prodrug of any thereof.

[1033] In a particular embodiment, the anti-CD39 antibody (e.g., the bispecific anti-CD39 antibody described herein) is conjugated to an anthracycline or an analog, derivative or prodrug thereof. In another particular embodiment, the anti-CD39 antibody (e.g., the bispecific anti- CD39 antibody described herein) is conjugated to maytansine or an analog, derivative or prodrug thereof. In another particular embodiment, the anti-CD39 antibody (e.g., the bispecific anti-CD39 antibody described herein) is conjugated to calicheamicin or an analog, derivative or prodrug thereof. In another particular embodiment, the anti-CD39 antibody (e.g., the bispecific anti-CD39 antibody described herein) is conjugated to duocarmycin or an analog, derivative or prodrug thereof. In another particular embodiment, the anti-CD39 antibody (e.g., the bispecific anti-CD39 antibody described herein) is conjugated to rachelmycin (CC-1065) or an analog, derivative or prodrug thereof. In another particular embodiment, the anti-CD39 antibody (e.g., the bispecific anti-CD39 antibody described herein) is conjugated to dolastatin 10 or an analog, derivative or prodrug thereof. In another particular embodiment, the anti-CD39 antibody (e.g., the bispecific anti-CD39 antibody described herein) is conjugated to dolastatin 15 or an analog, derivative or prodrug thereof. In another particular embodiment, the anti-CD39 antibody (e.g., the bispecific anti-CD39 antibody described herein) is conjugated to monomethyl auristatin E or an analog, derivative or prodrug thereof. In another particular embodiment, the anti-CD39 antibody (e.g., the bispecific anti-CD39 antibody described herein) is conjugated to monomethyl auristatin F or an analog, derivative or prodrug thereof. In another particular embodiment, the anti-CD39 antibody (e.g., the bispecific anti-CD39 antibody described herein) is conjugated to pyrrolo [2, 1-c] [ 1,4] -benzodiazepine or an analog, derivative or prodrug thereof. In another particular embodiment, the anti-CD39 antibody (e.g., the bispecific anti-CD39 antibody described herein) is conjugated to irinotecan or an analog, derivative or prodrug thereof.

[1034] VIII. Pharmaceutical Compositions

[1035] Anti-CD39 antibodies, antibody fragments, nucleic acids, or vectors encompassed by the present invention can be formulated in compositions, especially pharmaceutical compositions. Such compositions comprise a therapeutically or prophylactically effective amount of an anti-CD39 antibody, antibody fragment, nucleic acid, or vector encompassed by the present invention in admixture with a suitable carrier, e.g., a pharmaceutically acceptable agent. Typically, anti-CD39 antibodies, antibody fragments, nucleic acids, or vectors encompassed by the present invention are sufficiently purified for administration to an animal before formulation in a pharmaceutical composition.

[1036] Pharmaceutically acceptable agents for use in the present pharmaceutical compositions include carriers, excipients, diluents, antioxidants, preservatives, coloring, flavoring and diluting agents, emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents, wetting agents, complexing agents, buffering agents, antimicrobials, and surfactants.

[1037] Neutral buffered saline or saline mixed with serum albumin are exemplary appropriate carriers. The pharmaceutical compositions can include antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween, pluronics, or polyethylene glycol (PEG). Also by way of example, suitable tonicity enhancing agents include alkali metal halides (preferably sodium or potassium chloride), mannitol, sorbitol, and the like. Suitable preservatives include benzalkonium chloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid and the like. Hydrogen peroxide also can be used as preservative. Suitable cosolvents include glycerin, propylene glycol, and PEG. Suitable complexing agents include caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxy-propyl-beta-cyclodextrin. Suitable surfactants or wetting agents include sorbitan esters, polysorbates such as polysorbate 80, tromethamine, lecithin, cholesterol, tyloxapal, and the like. The buffers can be conventional buffers such as acetate, borate, citrate, phosphate, bicarbonate, or Tris-HCl. Acetate buffer may be about pH 4-5.5, and Tris buffer can be about pH 7-8.5. Additional pharmaceutical agents are set forth in Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company, 1990.

[1038] The composition can be in liquid form or in a lyophilized or freeze-dried form and may include one or more lyoprotectants, excipients, surfactants, high molecular weight structural additives and / or bulking agents (see for example U.S. Pat. Nos. 6,685,940; 6,566,329; and 6,372,716). In one embodiment, a lyoprotectant is included, which is a non-reducing sugar such as sucrose, lactose or trehalose. The amount of lyoprotectant generally included is such that, upon reconstitution, the resulting formulation will be isotonic, although hypertonic or slightly hypotonic formulations also may be suitable. In addition, the amount of lyoprotectant should be sufficient to prevent an unacceptable amount of degradation and / or aggregation of the protein upon lyophilization. Exemplary lyoprotectant concentrations for sugars (e.g., sucrose, lactose, trehalose) in the pre-lyophilized formulation are from about 10 mM to about 400 mM. In another embodiment, a surfactant is included, such as for example, nonionic surfactants and ionic surfactants such as polysorbates (e.g. polysorbate 20, polysorbate 80); poloxamers (e.g. poloxamer 188); poly (ethylene glycol) phenyl ethers (e.g. Triton); sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl- sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl- betaine; lauroamidopropyl-, cocamidopropyl-, Hnoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g. lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl ofeyl-taurate; and the MONAQUAT™. series (Mona Industries, Inc., Paterson, NJ.), polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g. Pluronics, PF68 etc). Exemplary amounts of surfactant that may be present in the prelyophilized formulation are from about 0.001-0.5%. High molecular weight structural additives (e.g. fillers, binders) may include for example, acacia, albumin, alginic acid, calcium phosphate (dibasic), cellulose, carboxymethylcellulose, carboxymethylcellulose sodium, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, dextran, dextrin, dextrates, sucrose, tylose, pregelatinized starch, calcium sulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose, disodium hydrogen phosphate, disodium phosphate, disodium pyrosulfite, polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose, compressible sugar, magnesium aluminum silicate, maltodextrin, polyethylene oxide, polymethacrylates, povidone, sodium alginate, tragacanth microcrystalline cellulose, starch, and zein. Exemplary concentrations of high molecular weight structural additives are from 0.1% to 10% by weight. In other embodiments, a bulking agent (e.g., mannitol, glycine) may be included.

[1039] Compositions can be suitable for parenteral administration. Exemplary compositions are suitable for injection or infusion into an animal by any route available to the skilled worker, such as intraarticular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, or intralesional routes. A parenteral formulation typically will be a sterile, pyrogen-free, isotonic aqueous solution, optionally containing pharmaceutically acceptable preservatives.

[1040] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringers' dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, anti-microbials, anti-oxidants, chelating agents, inert gases and the like. See generally, Remington's Pharmaceutical Science, 16th Ed., Mack Eds., 1980, which is incorporated herein by reference.

[1041] Pharmaceutical compositions described herein can be formulated for controlled or sustained delivery in a manner that provides local concentration of the product (e.g., bolus, depot effect) and / or increased stability or half-life in a particular local environment. The compositions can include the formulation of anti-CD39 antibodies, antibody fragments, nucleic acids, or vectors encompassed by the present invention with particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc., as well as agents such as a biodegradable matrix, injectable microspheres, microcapsular particles, microcapsules, bioerodible particles beads, liposomes, and implantable delivery devices that provide for the controlled or sustained release of the active agent which then can be delivered as a depot injection. Techniques for formulating such sustained- or controlled- delivery means are known and a variety of polymers have been developed and used for the controlled release and delivery of drugs. Such polymers are typically biodegradable and biocompatible. Polymer hydrogels, including those formed by complexation of enantiomeric polymer or polypeptide segments, and hydrogels with temperature or pH sensitive properties, may be desirable for providing drug depot effect because of the mild and aqueous conditions involved in trapping bioactive protein agents (e.g., antibodies). See, for example, the description of controlled release porous polymeric microparticles for the delivery of pharmaceutical compositions in PCT Application Publication WO 93 / 15722.

[1042] Suitable materials for this purpose include polylactides (see, e.g., U.S. Patent 3,773,919), polymers of poly-(a-hydroxycarboxylic acids), such as poly-D-(-)-3- hydroxybutyric acid (EP 133,988A), copolymers of L-glutamic acid and gamma ethyl-L- glutamate (Sidman et al., 1983, Biopolymers 22:547-556), poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15:167-277 and Langer et al., 1982, Chem. Tech. 12:98-105), ethylene vinyl acetate, or poly-D(~)~3-hydroxybutyric acid. Other biodegradable polymers include poly(lactones), poly(acetals), poly(orthoesters), and poly(orthocarbonates). Sustained-release compositions also may include liposomes, which can be prepared by any of several methods known in the art (see, e.g., Eppstein et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-92). The carrier itself, or its degradation products, should be nontoxic in the target tissue and should not further aggravate the condition. This can be determined by routine screening in animal models of the target disorder or, if such models are unavailable, in normal animals. Microencapsulation of recombinant proteins for sustained release has been performed successfully with human growth hormone (rhGH), interferon (rhIFN), interleukin-2, and MNrgpl20 (Johnson et al., 1996, Nat. Med. 2:795-799; Yasuda et al., 1993, Biomed. Ther. 27:1221-1223; Hora et al., 1990, Bio / Technologv. 8:755-758; Cleland et al., "Design and Production of Single Immunization Vaccines Using Polylactide Polyglycolide Microsphere Systems," in Vaccine Design: The Subunit and Adjuvant Approach, Powell and Newman, eds, (Plenum Press: New York, 1995), pp. 439-462; WO 97 / 03692, WO 96 / 40072, WO 96 / 07399; and U.S. Pat. No. 5,654,010). The sustained-release formulations of these proteins were developed using poly-lactic-coglycolic acid (PLGA) polymer due to its biocompatibility and wide range of biodegradable properties. The degradation products of PLGA, lactic and glycolic acids can be cleared quickly within the human body. Moreover, the degradability of this polymer can be depending on its molecular weight and composition. Lewis et al., "Controlled release of bioactive agents from lactide / glycolide polymer," in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New York, 1990), pp. 1- 41. Additional examples of sustained release compositions include, for example, EP 58,48 IA, U.S. Patent No. 3,887,699, EP 158,277A, Canadian Patent No. 1176565; Sidman et al., 1983, Biopolymers 22:547; Langer et al., 1982, Chem. Tech. 12:98; Sinha et al., 2003, J. Control. Release 90:261; Zhu et al., 2000, Nat. Biotechnol. 18:24; and Dai et al., 2005, Colloids Surf B Biointerfaces 41:117.

[1043] Bioadhesive polymers are also contemplated for use in or with compositions encompassed by the present invention. Bioadhesives are synthetic and naturally occurring materials able to adhere to biological substrates for extended time periods. For example, Carbopol and polycarbophil are both synthetic cross-linked derivatives of poly (acrylic acid). Bioadhesive delivery systems based on naturally occurring substances include for example hyaluronic acid, also known as hyaluronan. Hyaluronic acid is a naturally occurring mucopolysaccharide consisting of residues of D-glucuronic and N-acetyl-D-glucosamine. Hyaluronic acid is found in the extracellular tissue matrix of vertebrates, including in connective tissues, as well as in synovial fluid and in the vitreous and aqueous humour of the eye. Esterified derivatives of hyaluronic acid have been used to produce microspheres for use in delivery that are biocompatible and biodegradable (see for example, Cortivo et al., 1991, Biomaterials 12:727-730; European Publication No. 517,565; International Publication No. WO 96 / 29998; Ilium et al., 1994, J. Controlled Rel. 29:133-141). Exemplary hyaluronic acid containing compositions encompassed by the present invention comprise a hyaluronic acid ester polymer in an amount of approximately 0.1% to about 40% (w / w) of an IL- 1 / 3 binding antibody or fragment to hyaluronic acid polymer. Both biodegradable and non-biodegradable polymeric matrices can be used to deliver compositions encompassed by the present invention, and such polymeric matrices may comprise natural or synthetic polymers. Biodegradable matrices are preferred. The period of time over which release occurs is based on selection of the polymer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable. Exemplary synthetic polymers which can be used to form the biodegradable delivery system include: polymers of lactic acid and glycolic acid, polyamides, polycarbonates, poly alkylenes, poly alkylene glycols, poly alkylene oxides, poly alkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyanhydrides, polyurethanes and copolymers thereof, poly(butic acid), poly(valeric acid), alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly (hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, poly(ethylene glycol), polyethylene oxide), polyethylene terephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinyl chloride, polystyrene and polyvinylpyrrolidone. Exemplary natural polymers include alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion. The polymer optionally is in the form of a hydrogel (see for example WO 04 / 009664; WO 05 / 087201; Sawhney et al., 1993, Macromolecules 26:581-587) that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multivalent ions or other polymers.

[1044] Delivery systems also include non-polymer systems that are lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri- glycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which the product is contained in a form within a matrix such as those described in U.S. Pat. Nos. 4,452,775; 4,675,189; and 5,736,152; and (b) diffusional systems in which a product permeates at a controlled rate from a polymer such as described in U.S. Pat. Nos. 3,854,480; 5,133,974; and 5,407,686. Liposomes containing the product may be prepared by methods known methods, such as for example (DE 3,218,121; Epstein et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688- 3692; Hwang et al., 1980, Proc. Natl. Acad. Sci. USA 77:4030-4034; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patent application 83-118008; U.S. Patent Nos. 4,485,045 and 4,544,545; and EP 102,324).

[1045] Alternatively or additionally, the compositions can be administered locally via implantation into the affected area of a membrane, sponge, or other appropriate material on to which an anti-CD39 antibody, antibody fragment, nucleic acid, or vector encompassed by the present invention has been absorbed or encapsulated. Where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of an anti-CD39 antibody, antibody fragment, nucleic acid, or vector encompassed by the present invention can be directly through the device via bolus, or via continuous administration, or via catheter using continuous infusion.

[1046] A pharmaceutical composition comprising an anti-CD39 antibody, antibody fragment, nucleic acid, or vector encompassed by the present invention can be formulated for inhalation, such as for example, as a dry powder. Inhalation solutions also can be formulated in a liquefied propellant for aerosol delivery. In yet another formulation, solutions may be nebulized. Additional pharmaceutical composition for pulmonary administration includes, those described, for example, in PCT Application Publication WO 94 / 20069, which discloses pulmonary delivery of chemically modified proteins. For pulmonary delivery, the particle size should be suitable for delivery to the distal lung. For example, the particle size can be from 1 pm to 5 pm; however, larger particles may be used, for example, if each particle is fairly porous.

[1047] Certain formulations containing anti-CD39 antibody, antibody fragments, nucleic acids, or vectors encompassed by the present invention can be administered orally. Formulations administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. For example, a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents can be included to facilitate absorption of a selective binding agent. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders also can be employed.

[1048] Another preparation can involve an effective quantity of an anti-CD39 antibody, antibody fragment, nucleic acid, or vector encompassed by the present invention in a mixture with non-toxic excipients which are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or another appropriate vehicle, solutions can be prepared in unit dose form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.

[1049] IX. Exemplary Materials and Methods Reagents

[1050] All chemical reagents were purchased from Sigma- Aldrich (St. Louis, MO), cell culture media from Life Technologies (Carlsbad, CA), cell culture consumables from CELLTREAT® Scientific Products (Shirley, MA), and commercial antibodies from BioLegend (San Diego, CA), unless otherwise stated. Secondary antibody Alexa Fluor® 488-conjugated AffiniPure Donkey anti-human IgG (Fc specific) (#709-545-098) was obtained from Jackson ImmunoResearch (West Grove, PA), Bio-Gio™ (#G7941) from Promega (Madison, WI), 2- Deoxy-2-fluoro-L-fucose (#MD06089) from BIOSYNTH Carbosynth (Gardner, MA), Buprenorphine HCL Injection (#42023-0179-05) from PAR Pharmaceutical (Woodcliff Lake, NJ), Tissue’s Digestion Buffer (#130-096-730) from Miltenyi Biotech, and 0.9% Sodium Chloride Injection (#401694H) from Fresenius Kabi USA LLC (Lake Zurich, IL). Human Recombinant IL-2 (#78036.1) was purchased from STEMCELL Technologies (Cambridge, MA), 10% Buffered Formalin Phosphate (#SF100-4) from Fisher Scientific (Waltham, MA), Gill's Hematoxylin Solution (#26030-24) from Electron Microscopy Sciences (Hatfield, PA), Eosin Y Stain (#STE0150) from American MasterTech Scientific (St Lodi, CA), Masson's Trichrome Stain Kit (#HT15) from Sigma-Aldrich, and Surgical Polypropylene Veterinary Hernia Mesh (#ZMS1515) from Med-Vet International (Mettawa, IL).

[1051] The anti-human CD39 reference antibody (hCD39 Ref) was produced by transient transfection using the ExpiCHO™ Expression System Kit (#A29133; Thermo Fisher Scientific, Waltham, MA) and antibody sequences were obtained as published (Perrot et al., Cell Reports 27:2411-2425 (2019)). Both hCD39 Ref antibody and our fully human anti-CD39 monoclonal antibody (PSCWT22) contain the same ADCC-competent human IgGl Fc fraction. The hCD39 Ref antibody shares the antigen binding sites with an antibody in the art. However, that prior art antibody, unlike the Ref antibody used in the current examples, was generated with an Fc portion specifically designed to have an abrogated ADCC function (i.e., was taught to have been generated specifically to bind CD39 and inhibit NTPase activity without invoking CD39- dependent ADCC cell killing).

[1052] Cell culture

[1053] Epstein-Barr virus (EBV)-transformed human B lymphoblastoid HCC1739BL cells (ATCC #CRL-2334), Raji cells (Raji-hCD39neg), and human CD39 stably transfected Raji cells (Raji-hCD39hi expressing high level of hCD39 and Raji-hCD391o expressing low level of hCD39) were cultured in RPMI-1640 supplemented with 10% FBS, 1% penicillinstreptomycin. Human melanoma cells (SK-MEL-28, ATCC #HTB-72) were grown in EMEM plus 10% FBS, 1% penicillin- streptomycin. Human natural killer cells (NK-92-CD16 V / V) (ATCC #PTA-6967) were cultured in MEM- Alpha medium with IL-2 (10 ng / ml). Human umbilical vein endothelial cells (HUVEC), Single Donor, EGM™-2 (Lonza #C2517A, Basel, Switzerland) were grown in EGM™ Endothelial Cell Growth Medium BulletKit (Lonza #CC3124). All cell lines were maintained in culture flasks at 37°C in a 5% CO2atmosphere at 100% humidity, except for Jurkat cells / NFAT-luc+FcyRIIIA (Promega #G7011), which were thawed in water bath at 37°C prior to use for experiments.

[1054] Production, afucosylation, and optimization of our fully human anti-CD39 antibodies

[1055] Fully human anti-CD39 antibodies were produced by transient transfection using FreeStyle™ 293-F cells (Thermo Fisher Scientific #R79007) either in the absence (PSCWT22, the parent fully fucosylated version of anti-CD39 antibody) or presence of fucosylation inhibitor 2-Deoxy-2-fluoro-L-fucose (PSCAF22, the afucosylated version of anti-CD39 antibody). Antibody ADCC activity ranking is: PSCAF22 > PSCWT22.

[1056] Optimized counterparts PSCNP22 and PSC22 (afucosylated versions of anti-CD39 antibody) were produced by CHO cells stably transfected with the parental PSCWT22 plasmid using chemically modified cell culture media for ADCC activity enhancement. Antibody ADCC activity ranking is: PSC22 > PSCNP22 > PSCWT22.

[1057] No genetic modifications were employed during either afucosylation process.

[1058] Binding Affinity to hCD39KI mouse scar tissue-derived EC and MAC

[1059] Mouse scar was reproduced by an open excisional wound healing by secondary intention model as described below. Six days after the surgical procedure, single-cell suspensions were prepared and incubated with Fc Blocker (anti-CD16 / CD32 antibody; BD Biosciences, #553142) for 10 minutes at 4°C. Afterwards, cells were incubated with different concentrations of PSC22 or isotype control (For EC: top dose starting from 11 pg / mL with a 3-fold dilution; For MAC: top dose starting from 1.2 pg / mL with a 3-fold dilution) for 15 minutes at 4°C, followed by two washes and staining with several cell surface markers antibody mixture (Table 1) as well as anti-human IgGl secondary antibody (Alexa Fluor® 488 AffiniPure Donkey Anti-Human IgG, Fey fragment specific) for 15 minutes at 4°C. At the end, cells were washed and stained with 7-ADD for 5 minutes at room temperature. Viable EC and MAC were analyzed by flow cytometry (FACS) and gated as: CD45'CD31+cells and CD45+CDl lb+Grl" Ly6C'F4 / 80hlghcells, respectively. The anti-human IgGl secondary antibody mean fluorescence intensity (MFI) in each cell subpopulation surface was determined by using FCS Express 7 software (De Novo Software, Pasadena, CA) and GraphPad Prism software (San Diego, CA) was used to perform a four-parameter fitting curve to obtain the EC50.

[1060] NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC: NK cytotoxicity assay)

[1061] HCC1739BL target cells were pre-labeled with CFSE (0.025 pM) for 5 minutes at 37°C in water bath. After two washes with IX DPBS, cells were incubated in MEM- Alpha medium (Thermo Fisher Scientific #32561037) containing 4% ultra- low IgG FBS (Thermo Fisher Scientific #A3381901) with or without monoclonal antibodies for 30 minutes at 37°C in 5% CO2. Target cells were then co-cultured with NK-92-CD16 V / V effector cells at E:T=1:8 ratio for 6 hours at 37°C in 5% CO2. After incubation, cells were stained with Propidium Iodide (P / I) (200 ng / mL) for 10 minutes at room temperature and target cell death was analyzed by a Cytek™ Aurora flow cytometer (Cytek Biosciences). Results were expressed as % of cytotoxicity which represents the % of CFSE+P / I+cells. For NK cell killing assay on granulation tissue cells from hCD39KI mice: Mouse scar was reproduced by an open excisional wound healing by secondary intention model as described below. Six days after the surgical procedure, single-cell suspensions (containing the target cells EC and MAC) were prepared and resuspended in ADCC buffer (RPMI1640 + 4% ultra-low IgG FBS). Target cells (2X105cells) were then incubated with Fc Blocker (anti- CD16 / CD32 antibody; BD Biosciences, #553142) for 10 minutes at RT, followed by incubation with different concentrations of PSC22 or isotype control (top dose starting from 10 pg / mL with a 10-fold dilution) for 30 minutes at 37°C in 5% CO2. Next, effector cells NK- 92-CD16 V / V (1X105cells) were added to target cells (E:T=1:2) and incubated for 6 hours at 37°C in 5% CO2. After that, fluorophore-conjugated primary antibodies against several cell surface markers (Table 1) were added to the co-culture for 20 minutes at 4°C. At the end, cells were washed and stained with 7- ADD for 5 minutes on ice. EC and MAC were analyzed by FACS and gated as: CD45’CD31+cells and CD45+CDl lb+Grl-Ly6CF4 / 80highcells, respectively. The percentage of 7-ADD+EC and MAC (% of cytotoxicity) were determined by using FCS Express 7 software (De Novo Software, Pasadena, CA) and GraphPad Prism software (San Diego, CA) was used to perform a four-parameter fitting curve to obtain the EC50.

[1062] NFAT luciferase reporter Jurkat system (Luc-reporter ADCC assay)

[1063] Attached target cells (SK-MEL-28 human melanoma cells endogenously expressing CD39 at an intermediate level or HUVEC cells endogenously expressing low level of CD39) were seeded in a 96-well plate (8X103cells / 100 pL / well) (BRANDplates #781965) and grown for 24 hours, while suspension target cells (HCC1739BL) were seeded right before the experiment (5X105cells / mL). Cells were then washed twice with ADCC assay buffer (DMEM or RPMI-1640 medium supplemented with 4% ultra-low IgG FBS) and incubated with serially diluted monoclonal antibodies for 30 minutes at 37°C. Effector cells (Jurkat cells / NFAT- luc+FcyRIIIA; 3X106cells / mL) were then added to the wells and the mixture (E:T=6:1) was incubated for 6 hours at 37°C. Bio-Gio™ was finally added into wells and luminescence values were read at 5, 15, and 30 minutes using a Synergy™ Neo2 Multi-Mode Reader (BioTeK Instruments Inc.). ADCC activity was indicated by an increase of luciferase activity over background. Epitope competition assay

[1064] Unconjugated anti-human CD39 monoclonal antibodies (Human / Rabbit chimeric clones; 2 pg / mE) were incubated with HCC1739BL cells (1X105cells) for 30 minutes at 4°C. Cells were then washed twice with cell staining buffer and stained with PE-conjugated mouse anti-human CD39 monoclonal antibody Clone Al (0.25 pg / mE, Biolegend #328208) for 30 minutes at 4°C. Cells were then washed twice and analyzed by a Cytek™ Aurora flow cytometer. PE median fluorescence intensity (MFI) was detected, and data was analyzed by FCS 30 Express 7 software (De Novo Software). Cells incubated with media instead of the chimeric antibody were used as control.

[1065] Stable immune complex assay

[1066] HCC1739BL cells (5X105cells / mL) were incubated with anti-human CD39 monoclonal antibodies (2 pg / mL) or left untreated for 24 hours at 37 °C in 5% CO2. The following day, untreated cells were exposed to the same panel of monoclonal antibodies (2 pg / mL) but for 20 minutes at 4°C to obtain the basal level of CD39 expression. Cells were then washed twice with cell staining buffer and stained with anti-human IgG (Fc specific) Alexa Fluor® 488 (1:2000) for 30 minutes at 4°C, followed by two additional washes and fixation with paraformaldehyde (PFA, 2%) for 10 minutes at room temperature. Eastly, cells were washed twice and analyzed by a Cytek™ Aurora flow cytometer (Cytek Biosciences). Alexa Fluor® 488 (AF488) MFI was detected, and data was analyzed by FCS Express 7 software (De Novo Software). The percentage of human CD39 loss on cell membrane at 24 hours was calculated as: [(20 min MFI - 24 h MFI / 20 min MFI)] X100.

[1067] Conformational epitope mapping

[1068] This was done using a proprietary CEIPS technology by Pepscan (Eelystad, The Netherlands).

[1069] Animals

[1070] C57BE6 human CD39 knock-in (hCD39KI) mice were licensed from Beth Israel Deaconess Medical Center and bred and housed in a specific pathogen-free Vivarium at Purinomia Biotech, Inc. All mice were kept in a temperature-controlled room with alternating 12-hour dark light cycles. Open excisional wound healing by secondary intention model

[1071] 8-12 weeks h(uman) CD39KI female or male mice were subjected to anesthesia with isoflurane gas, followed by dorsum’s hair shaving and skin disinfection. Then, a single 1.5 cm full-thickness excisional round wound was created with sterilized round head tongue forceps and scissors. At the end of the surgical procedure, animals received 100 pL of Buprenorphine HCL (0.5 mg / mL) subcutaneously for pain management. The open wound was allowed to heal naturally (healing by secondary intention).

[1072] Open excisional wound healing by primary intention model

[1073] 8-12 weeks h(uman) CD39KI female or male mice were subjected to anesthesia with isoflurane gas, followed by dorsum’s hair shaving and skin disinfection. Then, a 10 mm linear full-thickness wound was created as midline laparotomy incision by surgical blade. The incision was then closed by standard 6.0 absorbable suture to closely re- approximate the wound edges (healing by primary intention). At the end of the surgical procedure, animals received 100 pL of Buprenorphine HCL (0.5 mg / mL) subcutaneously for pain management.

[1074] Mesh implantation model

[1075] H(uman) CD39KI mice were first subjected to anesthesia with isoflurane gas, followed by dorsum’s hair shaving and skin disinfection. Then, a 0.8 cm linear incision was created, and a subcutaneous tunnel was made by separating connective tissue from the underlying muscular fascia with forceps. Afterwards, a sterile polypropylene mesh (0.5 cm2) was inserted into the tunnel on the top of the fascia and the incision was closed with surgical staples. At the end of the surgical procedure, animals received 100 pL of Buprenorphine HCL (Bup-HCL) (0.5 mg / mL), subcutaneously, for pain management.

[1076] PSC22 treatment schedule

[1077] In all studies, animals were randomly enrolled into two study groups to receive either saline (10 pL / g) or PSC22 (3 mg / kg), intraperitonially, every two days. The studies were terminated one day after the last dosing.

[1078] For Figure 13, hCD39KI female mice received treatments on days 5 and 7 after the surgical procedure. For Figures 14, 15 and 16, hCD39KI male mice received treatments on days 10 and 12 after the surgical procedure.

[1079] For Figures 17, 18 and 19, hCD39KI male mice received treatments on days 7, 9, 11, and 13 after the surgical procedure.

[1080] For Figure 20, hCD39KI male mice received treatments on days 10, 12, 14, and 16 after the surgical procedure.

[1081] Gross pictures of scar tissue healing

[1082] Gross pictures of scar tissue were taken before the first dose treatment (on day 7), then every two days (on days 9, 11 and 13) using a microscope digital camera (AmScope, #MU900).

[1083] Images and weight of implanted meshes

[1084] Meshes with underlying granular / connective tissues were harvested from each mouse, weighed with an analytical balance (Sartorious, # Quintixl24-lS) and photographed with a digital camera (AmScope, # MU900) at the end of the study (on day 17).

[1085] Assessments of wound area and percentage of wound closure over time

[1086] Wound size was measured before the first dose treatment (on day 7), then every two days (on days 9, 11 and 13), in two dimensions using a digital caliper (Clockwise Tools, # DCLA-0605). Wound areas (A) were calculated and expressed in mm2, using the formula: A = (L / 2) x (W / 2) x 7t, where L and W were the long and short dimensions of the wound, respectively. Based on the wound area’s values, the percentage of wound closure was calculated as [(wound area on day 7-wound area on day 13) / wound area on day 7] x 100.

[1087] Tissue pathology

[1088] Scar tissues were harvested from euthanized animals, fixed into formalin-fixed paraffin embedded (FFPE) blocks, and sent to an outsourcing partner for Hematoxylin & Eosin (H&E) and Masson's Trichrome staining.

[1089] Underlying connective tissue analysis

[1090] After anesthesia, animals’ scar tissue was surgically opened, and the wound scab was removed to expose the granular tissue and surrounding subcutaneous scar fiber or fibrogenic tissue. The incision borders were held by forceps while pictures of the tissue structure were taken with a microscope digital camera (AmScope, #MU900). Tissue harvesting and single-cell suspension preparation

[1091] Scar tissues were harvested and dissociated into single-cell suspensions by enzymatic digestion using Tissue’s Digestion Buffer in GentleMACS Dissociator (Program 37C_m_TDK_l for 40 minutes, followed by m-impTumor_04_01 for 30 seconds). Red blood cells were then removed with lysis buffer (Life Technologies, #A10492-01) and the remaining cell suspension was washed and aliquoted for further downstream analysis.

[1092] Analysis of granulation / scar tissue cells by flow cytometry (FACS)

[1093] Single-cell suspensions of granulation / scar tissue were incubated with Fc Blocker (anti- CD16 / CD32 antibody; BD Biosciences, #553142) for 10 minutes at 4°C. Afterwards, cells were stained with several cell surface markers for fibroblasts, endothelial cells, and macrophages (Table 1) for 15 minutes at 4°C. At the end, cells were washed and stained with 7-ADD for 5 minutes at room temperature. Viable cells were analyzed by flow cytometry and gated as fibroblasts (FB): Linl'CD45'CDl lb'CD31'CD202b'CD90.2+cells; endothelial cells (EC): CD45'CD31+cells; and macrophages (MAC): CD45+CDl lb+Grl-Ly6CF4 / 80highcells. Human CD39 (hCD39) expression levels on each subpopulation were also calculated and expressed as mean fluorescence intensity (MFI).

[1094] Statistical analyses

[1095] Results are presented as mean (± SEM) and statistical analyses were performed using GraphPad Prism 9 (GraphPad Software, San Diego, CA). P<0.05 was considered statistically significant.

[1096] Table 1. Detection Antibodies for Granulation / Scar Tissue Cell Analysis

[1097] Equivalents and Scope

[1098] The details of one or more embodiments encompassed by the present invention are set forth in the description above. Although the preferred materials and methods have been described above, any materials and methods similar or equivalent to those described herein may be used in the practice or testing of embodiments encompassed by the present invention. Other features, objects and advantages related to the present invention are apparent from the description. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the case of conflict, the present description provided above will control.

[1099] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments encompassed by the present invention described herein. The scope encompassed by the present invention is not intended to be limited to the description provided herein and such equivalents are intended to be encompassed by the appended claims.

[1100] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article unless indicated to the contrary or otherwise evident from the context. By way of example, "an element" means one element or more than one element. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The present invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The present invention also includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.

[1101] It is also noted that the term "comprising" is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term "comprising" is used herein, the term "consisting of" is thus also encompassed and disclosed.

[1102] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges may assume any specific value or subrange within the stated ranges in different embodiments encompassed by the present invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[1103] In addition, it is to be understood that any particular embodiment encompassed by the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions encompassed by the present invention (e.g., any antibiotic, therapeutic or active ingredient; any method of production; any method of use; etc.) may be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

[1104] It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the purview of the appended claims without departing from the true scope and spirit encompassed by the present invention in its broader aspects.

[1105] While the present invention has been described at some length and with some particularity with respect to several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be construed with references to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope encompassed by the present invention.

Claims

What is claimed is:

1. A method for preventing or reducing the severity of epithelial scar formation in a subject comprising administering a CD39-targeted wound healing inflammation / granulation cell-depleting agent to the subject in an amount sufficient to reduce the number of CD39highinflammation / granulation cells at the site of scar formation.

2. The method of claim 1, wherein the CD39-targeted wound healing inflammation / granulation cell-depleting agent is an anti-CD39 antibody, or antigenbinding fragment thereof to the subject, wherein the anti-CD39 antibody, or antigen-binding fragment thereof, comprises: i. at least one antigen binding domain that binds ectonucleoside triphosphate diphosphohydrolase- 1 (CD39) at a site such that the anti- CD39 antibody forms a stable immune complex, and ii. an FcyRIIIa binding moiety that binds FcyRIIIa receptor and confers a) antibody-dependent cellular cytotoxicity (ADCC) activity and / or b) antibody-dependent cellular phagocytosis (ADCP) activity against CD39+ cells to the anti-CD39 antibody.

3. The method of claim 2, wherein the CD39-targeted wound healing inflammation / granulation cell-depleting agent is an ADCC competent anti-CD39 antibody.

4. The method of claim 3, wherein the anti-CD39 antibody is characterized by one or more of the following features: i. stable immune complex formation when incubated with HCC1739BL cells as characterized by loss of less than 30% of the immune complex after 24 hours, optionally wherein the immune complex formation is detected by fluorescence intensity using a fluorescently labeled secondary antibody; ii. binding to a CD39 epitope having a sequence selected from the group of CD39 amino acid epitope sequences listed in Figure 21; and / or iii. binding to CD39 in a manner that is non-competitive or only partially competitive with monoclonal antibody Clone Al binding to CD39.

5. The method of claim 1, wherein the CD39-targeted wound healing inflammation / granulation cell-depleting agent is an anti-CD39 antibody, or antigenbinding fragment thereof to the subject, which is conjugated to a toxin, which CD39-targeted wound healing inflammation / granulation cell-depleting agent when taken up by CD39highinflammation / granulation cells, e.g., endothelial cells, monocytes / macrophages and fibroblasts etc., and is toxic to the CD39highinflammation / granulation cells at the site of scar formation.

6. The method of any one of claims 1-5, wherein the CD39-targeted wound healing inflammation / granulation cell-depleting agent is an anti-CD39 antibody, or antigenbinding fragment thereof, has a VH domain with an amino acid sequence that can be encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid of SEQ ID NO. 1 and a VL domain with an amino acid sequence that can be encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid of SEQ ID NO. 3.

7. The method of any one of claims 1-5, wherein the CD39-targeted wound healing inflammation / granulation cell-depleting agent is an anti-CD39 antibody, or antigenbinding fragment thereof, comprises a heavy chain having CDRs at least 60% identical to the CDRs of SEQ ID NO. 2, 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54, and a light chain having CDRs at least 60% identical to the CDRs of SEQ ID NO. 4, 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56.

8. The method of any one of claims 1-5, wherein the CD39-targeted wound healing inflammation / granulation cell-depleting agent is an anti-CD39 antibody, or antigenbinding fragment thereof, comprises a variable heavy (VH) chain at least 60% identical to SEQ ID NO. 2, 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54, and a variable light (VL) chain at least 60% identical to SEQ ID NO. 4, 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56.

9. The method of any one of claims 1-5, wherein the CD39-targeted wound healing inflammation / granulation cell-depleting agent is an anti-CD39 antibody, or antigenbinding fragment thereof, comprising: i. a heavy chain variable domain comprising a complementarity determining region (CDR) Hl having an amino acid sequence at least 80% identical to SEQ ID NO. 29, a CDRH2 having an amino acidsequence at least 80% identical to SEQ ID NO. 30, and a CDRH3 having an amino acid sequence at least 80% identical to SEQ ID NO. 31; and ii. a light chain variable domain comprising a CDRL1 having an amino acid sequence at least 80% identical to SEQ ID NO. 32, a CDRL2 having an amino acid sequence at least 80% identical to SEQ ID NO. 33, and a CDRL3 having an amino acid sequence at least 80% identical to SEQ ID NO. 34.

10. The method of claim 9, wherein the anti-CD39 antibody, or antigen-binding fragment thereof, comprises i. a heavy chain variable domain comprising a CDRH1 having the sequence of SEQ ID NO: 29, a CDRH2 having the amino acid sequence of SEQ ID NO: 30, and a CDRH3 having the sequence of SEQ ID NO: 31; and ii. a light chain variable domain comprising a CDRL1 having the sequence of SEQ ID NO: 32, a CDRL2 having the sequence of SEQ ID NO: 33, and a CDRL3 having the sequence of SEQ ID NO: 34.

11. The method of claim 10, wherein the anti-CD39 antibody, or antigen-binding fragment thereof, comprises: i. a heavy chain variable domain comprising the sequence of SEQ ID NO: 2; and ii. a light chain variable domain comprising the sequence of SEQ ID NO: 4.

12. The method of claim 11, wherein the anti-CD39 antibody, or antigen-binding fragment thereof, comprises: i. a heavy chain comprising the sequence of SEQ ID NO: 36; and ii. a light chain comprising the sequence of SEQ ID NO: 38.

13. The method of any one of claims 1-12, wherein the CD39-targeted wound healing inflammation / granulation cell-depleting agent is an anti-CD39 antibody, or antigenbinding fragment thereof, comprising a heavy chain having CDRs selected from the group consisting of CDRs of SEQ ID NO. 6, 10, 14, 18, 22, 26, 42, 46, 50, and 54,and a light chain having CDRs selected from the group consisting of CDRs of SEQ ID NO. 8, 12, 16, 20, 24, 28, 44, 48, 52, and 56, and human framework sequences to form humanized heavy and light chains with an antigen binding site able to specifically bind human CD39.

14. The method of any one of claims 1-13, the CD39-targeted wound healing inflammation / granulation cell-depleting agent is an anti-CD39 antibody having an Fc domain of an IgGl or IgG3 isotype.

15. The method of any one of claims 1-14, the CD39-targeted wound healing inflammation / granulation cell-depleting agent is an anti-CD39 antibody which is hypo-fucosylated or afucosylated.

16. The method of any of claims 1-15, for preventing or reducing the severity of epithelial scar formation after surgery.

17. The method of any of claims 1- 15, for preventing or reducing the severity of keloid formation.

18. The method of any of claims 1-15, for preventing or reducing the severity of hypertrophic scars, atrophic scars or sclerodermas.

19. The method of claim 18, for preventing or reducing the severity of scarring caused by acne.

20. The method of any of claims 1-15, for preventing or reducing the severity of scarring in the cornea and / or conjunctiva.

21. The method of claim 20, for preventing or reducing the severity of scarring in the cornea or conjunctiva resulting from alkali bum (e.g., alkali burn to the cornea), post-cataract surgery, excess scarring in the tissue around the extraocular muscles in the strabismus surgery, and tractional retinal detachment in association with contraction of the tissue in diabetic retinopathy.

22. The method of any of claims 1-15, for preventing or reducing the severity of scarring as part of a treatment to promote periodontal wound healing.

23. The method of any of claims 1-15, for preventing or reducing the severity of scarring as part of a treatment to treat a skin wound.

24. The method of claim 23, wherein treatment with the CD39-targeted wound healing inflammation / granulation cell-depleting agent results in at least a 10% reduction in dermal thickness at the site of the wound relative to a wound in the absence of treatment with the CD39-targeted wound healing inflammation / granulation celldepleting agent.

25. The method of claim 23, wherein treatment with the CD39-targeted wound healing inflammation / granulation cell-depleting agent results in at least a 5% reduction in density of collagen I or dermal thickness at the site of the wound relative to a wound in the absence of treatment with the CD39-targeted wound healing inflammation / granulation cell-depleting agent.

26. The method of any of claims 1-25, wherein the CD39-targeted wound healing inflammation / granulation cell-depleting agent is administered intravenously.

27. The method of any of claims 1-25, wherein the CD39-targeted wound healing inflammation / granulation cell-depleting agent is administered sub-cutaneously.

28. The method of any of claims 1-25, wherein the CD39-targeted wound healing inflammation / granulation cell-depleting agent is administered locally at the site of scar formation.

29. The method of any of claims 1-25, wherein the CD39-targeted wound healing inflammation / granulation cell-depleting agent is administered topically.

30. A topical formulation for preventing or reducing the severity of epithelial scar formation in a subject comprising a CD39-targeted wound healing inflammation / granulation cell-depleting agent to the subject in an amount sufficient to reduce the number of CD39highinflammation / granulation cells at the site of scar formation.

31. The topical formulation of claim 30, wherein the formulation is a sustained release formulation.

32. The topical formulation of claim 30, wherein the sustained release formulation is a hydrogel.

33. A bandage or drug eluting wound dressing or suture for preventing or reducing the severity of epithelial scar formation in a subject comprising a CD39-targeted wound healing inflammation / granulation cell-depleting agent to the subject in an amountsufficient to reduce the number of CD39highinflammation / granulation cells at the site of scar formation.

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