Biparatopic CD73 antibodies

Abstract

To provide anti- CD73 antibodies that achieve effective inhibition of CD73 enzymatic activity, which may be useful in the treatment of CD73 mediated diseases and disorders.SOLUTION: Anti- CD73 antigen binding proteins are provided. Biparatopic anti- CD73 antigen binding proteins are provided. Also provided are methods of inhibiting CD73 activity and methods of treating CD73 mediated diseases and disorders.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 936,119, filed November 15, 2019, U.S. Provisional Application No. 63 / 023,542, filed May 12, 2020, and U.S. Provisional Application No. 63 / 086,982, filed October 2, 2020, the contents of each of which are incorporated herein by reference for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on November 5, 2020, is named 711174_SA9-282PC_ST25.txt and is 35,615 bytes in size.

[0003] This disclosure relates to compositions of bispecific antigen binding proteins and methods of making bispecific antigen binding proteins. [Background technology]

[0004] CD73 (ecto-5'-nucleotidase, NT5E) is a glycosylated 125-kDa homodimeric membrane-bound enzyme that dephosphorylates adenosine monophosphate (AMP) to adenosine (ADO) in the extracellular environment (Non-Patent Document 1). Adenosine exerts a potent immunosuppressive effect in the tumor microenvironment, and therefore CD73 has attracted widespread interest as a target for cancer therapy (Non-Patent Document 2; Non-Patent Document 3; Non-Patent Document 4; Non-Patent Document 5; Non-Patent Document 6). CD73 expression has been associated with resistance to anti-HER2 therapy (Non-Patent Document 7), poor prognosis associated with impaired anti-tumor immune responses in various tumor types (Non-Patent Document 1), and increased tumor cell proliferation, migration, and invasion in vitro (Non-Patent Document 8). Several clinical studies are underway on CD73-specific antibodies (Non-Patent Document 9) and small molecule inhibitors (Non-Patent Document 10), alone or in combination with A2a adenosine receptor antagonists and antibodies against other targets, particularly the PD-1 / PD-L1 axis (Non-Patent Document 11). MEDI9447 (oleclumab), a CD73-specific intracellular antibody with moderate inhibition of enzyme activity, has shown some clinical efficacy as monotherapy and in combination with the PD-L1 blocker durvalumab (Non-Patent Document 12). Nevertheless, there is a need in the art for CD73 antibodies with greater clinical efficacy as monotherapy and in combination with other therapeutic agents.

[0005] There are also indications that CD73 antibodies can exert their effects independently of adenosine production. One study showed that enhanced immune responses in mice were mediated through FcγRIV engagement (Non-Patent Document 13), and other studies suggested a role for CD73 internalization in suppressing metastasis (Non-Patent Document 10; Patent Document 12; Non-Patent Document 14). Nevertheless, adenosine levels in tumors can reach micromolar concentrations, and therefore, incomplete inhibition of CD73 activity may be a limiting factor for the efficacy of current CD73-targeted therapies (Non-Patent Document 15). Thus, the mechanism by which CD73 affects cancer progression may be complex, suggesting the need for very potent inhibition of enzymatic activity or a combination of mechanisms to achieve optimal efficacy.

[0006] It can be difficult to achieve potent inhibition of CD73 enzyme activity (e.g., both a high percentage of inhibition and a low EC50) with conventional monospecific CD73 antibodies (Non-Patent (Reference 16; Patent Document 1; Patent Document 2). Thus, there is a need in the art to identify anti-CD73 antibodies that achieve effective inhibition of CD73 enzymatic activity. Such anti-CD73 antibodies may be useful in the treatment of CD73-mediated diseases and disorders. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Patent Publication No. 2016055609 [Patent Document 2] International Patent Publication No. 2017118613 [Non-patent literature]

[0008] [Non-Patent Document 1] Allard et al. 2016 Immunotherapy 8:145-163 [Non-patent document 2] Allard et al. 2017 Immunological reviews 276:121-144 [Non-patent document 3] Allard et al. 2019 Immunology Letters 205:31-39 [Non-patent document 4] Kats et al. 2018 International journal of molecular sciences 156:451-457 [Non-Patent Document 5] Sek et al. 2018 Int J Mol Sci.19(12)pii:E3837 [Non-patent document 6] Yang et al. 2018 Current medicinal chemistry 25:2260~2271 [Non-Patent Document 7] Turcotte et al. 2017 Cancer research 77:5652-5663 [Non-patent document 8] Zhi et al. 2007 Clinical & experimental metastasis 24:439-448 [Non-Patent Document 9] Siu et al. 2018 Cancer research 78:CT180~CT180 [Non-Patent Document 10] Overman et al. 2018 Journal of Clinical Oncology 36(15):4123 pages [Non-Patent Document 11] Leone et al. 2018 Journal for immunotherapy of cancer 6:57 [Non-Patent Document 12] Hay et al. 2016 Oncoimmunology 5:e1208875 [Non-Patent Document 13] Vijayan et al. 2017 Oncoimmunology 6(5):e1312044 [Non-Patent Document 14] Terp et al. 2013 Journal of immunology 191:4165-4173 [Non-Patent Document 15] Blay et al. 1997 Cancer research 57:2602-2605 [Non-Patent Document 16] Geoghegan et al. 2016 mAbs. 8:454~467 Summary of the Invention [Means for solving the problem]

[0009] Disclosed herein are dual paratopic binding proteins capable of binding to two different epitopes on CD73 and achieving potent inhibition of CD73 activity. The binding proteins of the invention are particularly suitable for treating CD73-mediated diseases and disorders.

[0010] In one aspect, the disclosure provides a method for the production of antibodies having binding specificity for a CD73 epitope, including: and (b) an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVNTNY (SEQ ID NO: 4) or SGSVSTSYY (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of GTS (SEQ ID NO: 5) or STN (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQDYNLPYT (SEQ ID NO: 6) or VLFMGSGIWV (SEQ ID NO: 12).

[0011] In certain embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:15, and the VL domain comprises the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:16.

[0012] In certain embodiments, the antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 17, 18, 20, or 21, and the antibody light chain comprises the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 22.

[0013] In certain embodiments, the antigen binding protein or fragment thereof comprises a VH domain that is at least about 90% identical or at least about 95% identical to the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:15, and a VL domain that is at least about 90% identical or at least about 95% identical to the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:16.

[0014] In certain embodiments, the antigen binding protein or fragment thereof comprises an antibody heavy chain that is at least about 90% identical or at least about 95% identical to the amino acid sequence of SEQ ID NO: 17, 18, 20, or 21, and an antibody light chain that is at least about 90% identical or at least about 95% identical to the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 22.

[0015] In certain embodiments, the antigen binding protein or fragment thereof comprises: (a) a VH domain comprising a CDR-H1 sequence comprising the amino acid sequence of GGSIRNNY (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence of IYISGTT (SEQ ID NO: 2), and a CDR-H3 sequence comprising the amino acid sequence of AREHYVSGTSLDN (SEQ ID NO: 3); and (b) a VL domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVNTNY (SEQ ID NO: 4), a CDR-L2 sequence comprising the amino acid sequence of GTS (SEQ ID NO: 5), and a CDR-L3 sequence comprising the amino acid sequence of QQDYNLPYT (SEQ ID NO: 6).

[0016] In certain embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:13 and the VL domain comprises the amino acid sequence of SEQ ID NO:14.

[0017] In certain embodiments, the antibody heavy chain comprises the amino acid sequence of SEQ ID NO:17 or SEQ ID NO:18, and the antibody light chain comprises the amino acid sequence of SEQ ID NO:19.

[0018] In certain embodiments, the antigen binding protein or fragment thereof comprises: (a) a VH domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 7), a CDR-H2 sequence comprising the amino acid sequence of FWYDGSNK (SEQ ID NO: 8), and a CDR-H3 sequence comprising the amino acid sequence of ARAPNWDDAFDI (SEQ ID NO: 9); and (b) a VL domain comprising a CDR-L1 sequence comprising the amino acid sequence of SGSVSTSYY (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of STN (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of VLFMGSGIWV (SEQ ID NO: 12). hmm.

[0019] In certain embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:15 and the VL domain comprises the amino acid sequence of SEQ ID NO:16.

[0020] In certain embodiments, the antibody heavy chain comprises the amino acid sequence of SEQ ID NO:20 or SEQ ID NO:21, and the antibody light chain comprises the amino acid sequence of SEQ ID NO:22.

[0021] In certain embodiments, the antigen binding protein binds a human CD73 polypeptide comprising the amino acid sequence of SEQ ID NO:23.

[0022] In certain embodiments, the antigen binding protein binds an epitope of a human CD73 polypeptide comprising amino acids N96, G97, V98, E99, K121, P123, P156, F157, S159, N160, G162, T163, N164, L165, V166, F167, E168, R491, and D496 of SEQ ID NO: 23.

[0023] In certain embodiments, the antigen binding protein binds an epitope of a human CD73 polypeptide comprising amino acids P112, K119, A125, S126, S129, G130, L133, P134, Y135, K136, K180, L184, and N185 of SEQ ID NO:23.

[0024] In certain embodiments, the antigen binding protein is a chimeric or humanized antibody. In certain embodiments, the antigen binding protein is a human antibody.

[0025] In certain embodiments, the antigen binding protein is a monoclonal antibody.

[0026] In certain embodiments, the antigen binding protein comprises one or more full-length antibody heavy chains comprising an Fc region. In certain embodiments, the Fc region is human IgG1 The Fc region.

[0027] In certain embodiments, the human IgG1 Fc region comprises amino acid substitutions at one or more positions corresponding to positions 405 and 409 of human IgG1 according to the EU index, wherein the amino acid substitutions are F405L and K409R.

[0028] In one aspect, the present disclosure provides a pharmaceutical composition comprising an antigen-binding protein or fragment thereof listed above, and a pharmaceutically acceptable carrier.

[0029] In one aspect, the present disclosure provides an isolated nucleic acid molecule encoding the above-listed antigen binding proteins or fragments thereof.

[0030] In one aspect, the present disclosure provides an expression vector comprising the above-listed nucleic acid molecule.

[0031] In one aspect, the present disclosure provides a host cell comprising the above-listed expression vector.

[0032] In one embodiment, the disclosure provides a dual paratopic binding protein that comprises binding specificity for a first CD73 epitope and a second CD73 epitope.

[0033] In certain embodiments, the dual paratopic binding protein comprises: (b) a first VH domain having specificity for a first CD73 epitope, the first VH domain comprising a CDR-H1 sequence comprising the amino acid sequence of GGSIRNNY (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence of IYISGTT (SEQ ID NO: 2), and a CDR-H3 sequence comprising the amino acid sequence of AREHYVSGTSLDN (SEQ ID NO: 3); (b) a first VL domain having specificity for a first CD73 epitope, the first VL domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVNTNY (SEQ ID NO: 4), a CDR-L2 sequence comprising the amino acid sequence of GTS (SEQ ID NO: 5), and a CDR-L3 sequence comprising the amino acid sequence of QQDYNLPYT (SEQ ID NO: 6); (c) GFT (d) a second VH domain having specificity for a second CD73 epitope, comprising a CDR-H1 sequence comprising the amino acid sequence of FSSYG (SEQ ID NO: 7), a CDR-H2 sequence comprising the amino acid sequence of FWYDGSNK (SEQ ID NO: 8), and a CDR-H3 sequence comprising the amino acid sequence of ARAPNWDDAFDI (SEQ ID NO: 9); and (d) a second VL domain having specificity for a second CD73 epitope, comprising a CDR-L1 sequence comprising the amino acid sequence of SGSVSTSYY (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of STN (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of VLFMGSGIWV (SEQ ID NO: 12).

[0034] In certain embodiments, the first VH domain comprises the amino acid sequence of SEQ ID NO: 13; the second VH domain comprises the amino acid sequence of SEQ ID NO: 15; the first VL domain comprises the amino acid sequence of SEQ ID NO: 14; and the second VL domain comprises the amino acid sequence of SEQ ID NO: 16.

[0035] In certain embodiments, the dual paratopic binding protein comprises: (a) a first antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18; (b) a second antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21; (c) a first antibody light chain comprising the amino acid sequence of SEQ ID NO: 19; and (d) a second antibody light chain comprising the amino acid sequence of SEQ ID NO: 22.

[0036] In certain embodiments, the dual paratopic binding protein comprises: (a) a first antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 17; (b) a second antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 21; (c) a first antibody light chain comprising the amino acid sequence of SEQ ID NO: 19; and (d) a second antibody light chain comprising the amino acid sequence of SEQ ID NO: 22.

[0037] In certain embodiments, the dual paratopic binding protein comprises: (a) a first antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 18; (b) a second antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 20; (c) a first antibody light chain comprising the amino acid sequence of SEQ ID NO: 19; and (d) a second antibody light chain comprising the amino acid sequence of SEQ ID NO: 22.

[0038] In certain embodiments, the dual paratopic binding protein comprises: (a) a first VH and VL domain that binds a first epitope of a human CD73 polypeptide comprising amino acids N96, G97, V98, E99, K121, P123, P156, F157, S159, N160, G162, T163, N164, L165, V166, F167, E168, R491, and D496 of SEQ ID NO:23; and (b) a second VH and VL domain that binds a second epitope of a human CD73 polypeptide comprising amino acids P112, K119, A125, S126, S129, G130, L133, P134, Y135, K136, K180, L184, and N185 of SEQ ID NO:23.

[0039] In certain embodiments, the dual paratopic binding protein comprises greater inhibitory activity against CD73 compared to one or both of the monospecific parent antibodies.

[0040] In certain embodiments, the dual paratopic binding protein is a monospecific parent antibody. It contains higher inhibitory activity against CD73 compared to the combination of

[0041] In certain embodiments, the first VH and VL domains bind a first CD73 epitope on a first CD73 dimer molecule, and the second VH and VL domains bind a second CD73 epitope on a second CD73 dimer molecule.

[0042] In certain embodiments, the antigen binding protein is capable of cross-linking two or more CD73 dimer molecules.

[0043] In certain embodiments, dual paratopic binding proteins are generated by Fab arm exchange.

[0044] In certain embodiments, Fab arm exchange is performed according to the following steps: (a) mixing a first parent monospecific antigen-binding protein comprising an IgG1 Fc region comprising the amino acid substitution F405L according to the EU index and a second parent monospecific antigen-binding protein comprising an IgG1 Fc region comprising the amino acid substitution K409R according to the EU index to produce a mixture; (b) subjecting the mixture of step (a) to reducing conditions to produce a reduced antigen-binding protein mixture containing the dual paratopic bispecific antigen-binding protein; (c) subjecting the mixture of step (b) to oxidizing conditions to rearrange disulfide bonds between the heavy chains of the dual paratopic bispecific antigen-binding protein; and (d) isolating the dual paratopic bispecific antigen-binding protein.

[0045] In certain embodiments, the first parent monospecific antigen-binding protein and the second parent monospecific antigen-binding protein are mixed in equimolar amounts.

[0046] In certain embodiments, the reducing conditions are generated by adding a reducing agent, hi certain embodiments, the reducing agent comprises mercaptoethylamine (MEA).

[0047] In certain embodiments, the mixture of step (a) is subjected to reducing conditions at a temperature of about 18° C. to about 30° C. for about 3 hours to about 6 hours.

[0048] In another aspect, the present disclosure provides a method for treating a CD73-mediated disease or disorder in a subject, comprising administering to a subject in need thereof an antigen binding protein listed above or a fragment thereof.

[0049] In certain embodiments, the CD73-mediated disease or disorder is cancer.

[0050] In another embodiment, the present disclosure provides a method of selecting a dual paratopic antigen binding protein that comprises greater inhibitory activity against CD73 compared to one or more monospecific parent antibodies, the method comprising the steps of: a) combining the two parent antibodies under conditions to form the dual paratopic antigen binding protein; b) testing the dual paratopic antigen binding protein and one or both of the two parent antibodies in a CD73 activity assay; c) comparing the CD73 activity for the dual paratopic antigen binding protein with the CD73 activity for one or both of the two parent antibodies; and d) selecting the dual paratopic antigen binding protein if the CD73 activity is lower than the CD73 activity of one or both of the two parent antibodies.

[0051] In certain embodiments, the CD73 activity assay measures adenosine formation, hi certain embodiments, adenosine formation is quantified by liquid chromatography-mass spectrometry (LC / MS).

[0052] In certain embodiments, the CD73 activity assay is performed using COR-L23 lung cancer cells, which express human CD73.

[0053] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0054] [Figure 1] Screening of inhibitory activity against CD73 on COR-L23 cells. Percent inhibition of CD73 activity was determined using an LC / MS-based assay with heavy-isotope AMP substrate after 4 h of antibody exposure (white shading: 0-49% inhibition at 1 μg / ml; light gray: 50-69% inhibition; gray: 70-89% inhibition; and dark gray: 90-100% inhibition). Each square, except the farthest right in each row, represents a biparatopic antibody generated by combining the parent antibodies indicated on the horizontal and vertical axes. The farthest right square in each row represents a parental bivalent anti-CD73 antibody reconstituted using Fab arm exchange. The bottom row ("AS30") indicates pairing with an unrelated antibody, AS30, to generate a monovalent version of the parent antibody. [Figure 2] Figure 1 shows the relative affinity of parental and dual paratopic antibodies to CD73. Antibodies (each parental and dual paratopic antibody in monovalent form containing an irrelevant AS30 arm) were immobilized and exposed to soluble CD73 in a flow. [Figure 3]Figure 1 shows confirmation of dual paratopic antibody formation by capillary isoelectric focusing (cIEF). The duobody product and parental antibody (4 μg each) were digested with IdeZ to obtain F(ab')2 and Fc fragments, which were then separated by cIEF. For the peaks between pI 9.0 and 9.5: right peak: E3.2 (F405L) parent; left peak: H19 (K409R) parent; center peak: Fab arm exchange reaction (cFAE) product E3.2 / H19. The F(ab')2 fragment has a pI value above 8.5. The peak between pI 7.5 and 8 represents Fc fragment. The peak at 7.1 is IdeZ. [Figure 4]

[0023] Figure 1 shows the potency dose-response for 11 dual paratopic antibodies, parental antibodies, and the parental antibody mixture. COR-L23 cells were incubated with the antibodies for 3 hours, after which CD73 activity was determined by an LC / MS-based assay. Activity (percent of no antibody control) is plotted against total antibody concentration for the dual paratopic antibodies (light gray circles), the parental mixture (black), and each of the two parental antibodies (dark gray circles and squares). [Figure 5-1] Figure 5 shows epitope binning by Biolayer Interferometry (Octet). A mixture of CD73 with a molar excess of Fab was incubated with a monovalent parent antibody immobilized on a solid support. Figure 5A depicts a schematic of the assay format, showing conditions for non-overlapping epitopes and no blocking (top panel) or overlapping epitopes resulting in complete blocking of capture (bottom panel). Figure 5B depicts capture of CD73 / Fab complexes by immobilized antibodies. Capture was normalized to the signal from CD73 alone in the absence of Fab. Figure 5C depicts epitope binning based on inhibition of capture. Figure 5D depicts inhibition of CD73 activity on COR-L23 cells relative to the antibody's ability to capture CD73 / Fab complexes in vitro. Gray circles: capture of CD73 / Fab complexes of the same antibody on the support (parent pair). [Figure 5-2] Continued from Figure 5-1. [Figure 6]Figure 6A depicts the structure of TB19 with CD73. Figure 6A depicts a schematic of different conformational states of CD73. The N- and C-terminal domains of CD73 are labeled "N" and "C," respectively. The linker connecting the two domains is represented by a gray coil. The zinc cofactor is shown as a small gray sphere in the N-terminal domain. The substrate is depicted by an "S." Figure 6B depicts two TB19 Fv domains binding one CD73 dimer in an intermediate conformation from two different angles. CD73 is colored as in Figure 6A, with zinc and phosphate molecules shown as black circles. The TB19 Fab is shown as a light gray schematic. Figure 6C depicts the mapping of TB19 epitope residues on CD73. The same coloring scheme as in Figure 6B is used, with the CD73 epitope residues recognized by TB19 shown in light gray. [Figure 7A] Figure 7A depicts the spatial arrangement of the CD73 monomer with TB19 Fv. Figure 7A depicts TB19 bound to CD73 in a partially open conformation. The zinc and inorganic phosphate ions in the catalytic center are labeled "Zn" and "Pi," respectively. Interacting residues (within 4 Å) in the TB19 Fv and N-terminal domain are shown in stick form. For illustration, the substrate analog AMPCP (stick form) bound by the C-terminal domain of the closed conformer structure 4H21 is superimposed on the TB19:CD73 structure to show its position and the interacting CD73 residues Phe417 and Phe500. Note that AMPCP is not present in the TB19 structure. [Figure 7B] Figure 7B depicts the spatial arrangement of the CD73 monomer with the TB19 Fv. Figure 7B depicts the modeling of TB19 onto the closed conformation 4H21 by superimposing the CD73 N-terminal domains of the two structures. The zinc ion and β-phosphate ion of AMPCP in 4H21 occupy the same positions as the zinc and inorganic phosphate ions in the TB19-bound CD73 structure. The TB19 variable region clashes with the C-terminal domain in 4H21. [Figure 8]Figure 8 shows the structure of TB38 with CD73. The CD73 N-terminal domain is labeled "N," the CD73 C-terminal domain is labeled "C," and the grey coil is the linker. Figure 8A depicts the TB38 Fab::CD73 structure, in which CD73 is in the open conformation. The TB38 Fab is shown and labeled. Figure 8B depicts the TB38 Fv::CD73 structure, in which CD73 is in the open / closed hybrid conformation. The TB38 Fv is shown and labeled. Figure 8C depicts the mapping of TB38 epitope residues on CD73 (open / closed hybrid conformation), shown and labeled. [Figure 9] Figure 9A shows potential modes of co-association of CD73 by the TB19 / TB38 biparatopic antibody. The bispecific antibody was modeled based on the TB19:CD73, TB38:CD73, and full IgG1 (PDB 1ZHZ) structures. Figure 9A depicts a surface view of the TB19 / TB38 biparatopic antibody. The distance between the last residues in the CH1 domain is shown as a black line. The TB19 Fab, TB38 Fab, and Fc are labeled. Figure 9B depicts a model for four TB19 / TB38 biparatopic antibodies bound by CD73 in a partially open conformation. The CD73 N- and C-terminal domains are shown. The distance separating the last residues of the CH1 domain is shown as a black line. [Figure 10] FIG. 1 shows the epitopes for TB19 and TB38 mapped to one subunit of the CD73 homodimer shown in a partially open conformation as in the TB19 co-crystal structure (dark grey / light grey). [Figure 11]These figures show CD73 conformer structures related to the conceptual cartoon in Figure 6 above. Figure 11A depicts a representation of the CD73 conformation similar to that shown in Figure 6, reflecting key configurations of each conformer. Figure 11B depicts the actual structural equivalent of the diagrammatic representation in Figure 11A. Note that the N-terminal domain on the right rotates back into the plane of the page between the open, TB19, and closed conformations. Figure 11C depicts the structure of the CD73 monomer, aligning the C-terminal domain in each of the three conformations: open, TB19, and closed, to show the rotation of the N-terminal domain. The TB19 Fab is not shown for clarity. The view is perpendicular to the plane of rotation of the N-terminal domain and from below the view in Figure 11B. The C-terminal residue of the extracellular domain, the zinc atom in the closed conformer structure 4H21, and the substrate analog AMPCP in 4H21 are shown. [Figure 12] Figure 1 shows the Fo-Fc omit map for zinc and phosphate ions in the N-terminal domain structure with TB19 at 5 sigma. Ball: zinc; stick: oxygen and phosphorus of phosphate ion. [Figure 13A] Figure 13A shows fits of raw Biacore sensorgrams to kinetic response data to assess bivalent binding of dual paratopic antibodies to single CD73, as shown in Figure 2 above. The fits are based on a 1:1 Langmuir binding model. Kinetic values ​​based on these fits are shown in Table 6. Biphasic association and dissociation rates were observed for several antibodies. In those cases (indicated by an asterisk), fits were performed at two concentrations of CD73 (32 nM and 12 nM) using an iterative process to obtain abundance and rate constants for each component during association and dissociation. Figure 13A depicts the data for the monovalent parent antibody. [Figure 13B]Figure 13B depicts fits of raw Biacore sensorgrams to kinetic response data to assess bivalent binding of dual paratopic antibodies to single CD73, as shown in Figure 2 above. The fits are based on a 1:1 Langmuir binding model. Kinetic values ​​based on these fits are shown in Table 6. Biphasic association and dissociation rates were observed for multiple antibodies. In those cases (indicated by an asterisk), fits were performed at two concentrations of CD73 (32 nM and 12 nM) using an iterative process to obtain abundance and rate constants for each component during association and dissociation. Figure 13B depicts the data for the dual paratopic antibodies. [Figure 14-1] Figure 5. Epitope mapping by a premix competition approach. Capture of Fab:CD73 complexes in solution by a monovalent CD73 antibody on a Protein A biosensor chip was tracked by Octet. Title: Name of antibody loaded on chip. Sensorgram color identifies the Fab preincubated with CD73 (see legend). Blue trace: CD73 alone. The initial rise at 300 seconds reflects IgG loading on the chip. After washing, CD73 premixed with excess Fab was applied. Higher responses reflect greater mass capture. Note that preincubation with Fab for overlapping epitopes would block capture and result in no change in the sensorgram. Capture of Fab-bound CD73 for non-overlapping epitopes would yield a higher signal than CD73 alone due to the larger size of the complex. Binding shift values ​​normalized to CD73 are shown in Figure 5B. [Figure 14-2] Continued from Figure 14-1. [Figure 15-1] Figure 1 shows half-times for dissociation of CD73 from immobilized monovalent parent and biparatopic antibodies. The half-times shown are based on the first-order rate constants presented in Table 6. Where biphasic kinetics were observed, the major and minor components and their respective percentages (in italics) are shown. nd: not detectable. [Figure 15-2] Continued from Figure 15-1. DETAILED DESCRIPTION OF THE INVENTION

[0055] Anti-CD73 parent monospecific antigen-binding proteins are provided. Dual-paratopic anti-CD73 antigen-binding proteins derived from the parent antigen-binding proteins are also provided. Methods for inhibiting CD73 activity and for treating CD73-mediated diseases and disorders are also provided.

[0056] Generally, the methods described herein include cell and tissue culture, molecular biology, immunology, microbiology, and the like. The nomenclatures used in connection with biology, genetics, and protein and nucleic acid chemistry and hybridization are those well known and commonly used in the art. The methods and techniques provided herein are generally, unless otherwise indicated, performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout the specification. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with and the experimental procedures and techniques of analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0057] Unless otherwise defined herein, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In the event of any latent ambiguity, the definitions provided herein take precedence over any dictionary or extrinsic definitions. Unless otherwise required by context, singular terms shall include plurals and plural terms shall include the singular. The use of "or" means "and / or" unless otherwise specified. The use of the term "including," as well as other forms such as "includes" and "included," is not limiting.

[0058] In order that the present invention may be more readily understood, certain terms are first defined.

[0059] CD73 CD73 monomers, with N- and C-terminal domains connected through a flexible α-helical linker, are expressed on the cell surface attached to a C-terminal GPI anchor. In their physiological form, two monomers associate through extensive noncovalent contacts between the C-terminal domains to form a dimer (Heuts et al. 2012 Chembiochem: a European journal of chemical biology 13:2384-2391; Knapp et al. 2012 Structure (London, England: 1993) 20:2161-2173). The active site in each CD73 monomer consists of substrate-contacting residues in both the N- and C-terminal domains, in addition to a zinc cofactor bound by the N-terminal domain (Knapp et al. 2012). 2012, supra). After binding of the AMP substrate to the C-terminal domain, the N-terminal domain and zinc cofactor align with AMP in the "closed" CD73 conformation, where catalysis occurs to generate the adenosine product. A large lateral rotation of the N-terminal domain, which re-exposes the substrate-binding site in the "open" conformer, allows product release (Knapp 2012, supra). The limited solvent access to the active site in the closed conformer indicates that cycling between the two forms is required for substrate binding and product release, i.e., efficient enzyme activity (Knapp 2012, supra).

[0060] antigen-binding proteins As used herein, the term "antibody" or "antigen-binding protein" refers to an immunoglobulin molecule that specifically binds to or is immunologically reactive with an antigen or epitope, and includes both polyclonal and monoclonal antibodies, as well as functional antibody fragments, including, but not limited to, antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain variable fragments (scFv), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term "antibody" includes intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, meditope-enabled antibodies (meditope-enab and the like. Unless otherwise specified, the term "antibody" should be understood to include functional antibody fragments thereof.

[0061] As used herein, the term "complementarity determining region" or "CDR" refers to a non-contiguous sequence of amino acids in an antibody variable region that confers antigen specificity and binding affinity. Generally, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region, and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. "Framework region" or "FR" is known in the art to refer to the non-CDR portion of the heavy and light chain variable regions. Generally, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each heavy chain variable region, and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each light chain variable region.

[0062] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of several well-known schemes, including those described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. ("Kabat" numbering scheme); Al-Lazikani et al. (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745 ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering scheme" for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, January 2003;27(1):55-77 ("IMGT" numbering scheme), and Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool", J Mol Biol 2001 Jun 8;309(3):657-70 (AHo numbering scheme).

[0063] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering for both the Kabat scheme and the Chothia scheme is based on the most common antibody region sequence length, and in some antibodies, insertions are accommodated by insertion letters, such as "30a", and deletions appear. The two schemes place certain insertions and deletions ("indels") at different positions, resulting in different numbering. The contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme.

[0064] Thus, unless otherwise specified, the "CDRs" or "complementarity determining regions" of a given antibody or regions thereof, such as the variable region thereof, or individual identified CDRs (e.g., "CDR-H1," "CDR-H2"), are defined according to any of the known schemes. It should be understood that a given antibody or region thereof, such as a variable region thereof, includes a particular (or specified) complementarity determining region (CDR) as defined by any of the known schemes. Similarly, unless otherwise specified, the "FR" or "framework region" of a given antibody or region thereof, such as a variable region thereof, or an individually specified FR (e.g., "FR-H1," "FR-H2"), should be understood to include a particular (or specified) framework region as defined by any of the known schemes. In some cases, a scheme for identifying a particular CDR or FR is specified, such as CDRs as defined by the Kabat, Chothia, or Contact method. In other words, the specific amino acid sequence of the CDR or FR is given.

[0065] Anti-CD73 antigen-binding protein In one aspect, the present disclosure provides antigen-binding proteins that have binding specificity for CD73. As used herein, "CD73" can refer to both a CD73 monomeric protein or a CD73 homodimeric complex formed by two non-covalently associated CD73 monomeric proteins.

[0066] Exemplary anti-CD73 antigen binding protein CDRs are listed below in Table 1. Exemplary anti-CD73 antigen binding protein heavy chain variable (VH) domains and light chain variable (VL) domains are listed below in Table 2. Exemplary anti-CD73 antigen binding protein full-length heavy and light chains are listed below in Table 3.

[0067] [Table 1]

[0068] [Table 2]

[0069] [Table 3-1] [Table 3-2]

[0070] In certain embodiments, an anti-CD73 antigen binding protein of the present disclosure comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence similarity or identity to any of the sequences in Table 1, Table 2, or Table 3.

[0071] In certain embodiments, an anti-CD73 antigen binding protein of the disclosure binds a human CD73 polypeptide comprising the amino acid sequence of SEQ ID NO: 23, as shown in Table 4 below.

[0072] In certain embodiments, an anti-CD73 antigen binding protein of the disclosure comprises a human CD73 polypeptide comprising amino acids N96, G97, V98, E99, K121, P123, P156, F157, S159, N160, G162, T163, N164, L165, V166, F167, E168, R491, and D496 of SEQ ID NO: 23 as shown in Table 4 below. It binds to the epitope of the polypeptide.

[0073] In certain embodiments, an anti-CD73 antigen binding protein of the disclosure binds an epitope of a human CD73 polypeptide that includes amino acids P112, K119, A125, S126, S129, G130, L133, P134, Y135, K136, K180, L184, and N185 of SEQ ID NO: 24, as shown in Table 4 below.

[0074] [Table 4]

[0075] Dual paratopic anti-CD73 antigen-binding protein In one aspect, the present disclosure provides a dual paratopic antigen-binding protein having binding specificity for a first CD73 epitope and a second CD73 epitope. As used herein, a "dual paratopic" antigen-binding protein binds two different epitopes on the same molecular target (i.e., dual paratopic). In the present disclosure, the dual paratopic anti-CD73 antigen-binding protein is derived from two parent monospecific CD73 antigen-binding proteins. Each of the two parent antigen-binding proteins binds a different epitope on the CD73 molecule.

[0076] The dual paratopic antigen-binding proteins of the present disclosure may have advantages over monospecific antigen-binding proteins due to the potential additive or synergistic effects of combining antibody specificities. Dual paratopic antigen-binding proteins of the present disclosure may exhibit potent CD73 inhibition when combined in dual paratopic variants, provided that they bind non-overlapping epitopes on CD73. Dual paratopic antigen-binding proteins may also provide multiple mechanisms for inhibiting CD73 activity. CD73 inhibitory mechanisms include, but are not limited to, blocking the formation of catalytically active CD73 conformers, binding intermediate, partially open, inactive CD73 conformers, binding open, closed, and hybrid conformations, and cross-linking two or more CD73 dimers. A CD73 hybrid conformer is one in which one CD73 monomer is in an open conformation and the other CD73 monomer is in a closed conformation.

[0077] In certain embodiments, dual paratopic antigen binding proteins of the present disclosure comprise greater inhibitory activity against CD73 compared to one or both of the monospecific parent antibodies used to generate the respective dual paratopic antigen binding proteins. In certain embodiments, dual paratopic antigen binding proteins of the present disclosure comprise greater inhibitory activity against CD73 compared to the combination of monospecific parent antibodies used to generate the respective dual paratopic antigen binding proteins. Inhibition of CD73 activity is determined by any method known in the art. In certain embodiments, CD73 activity is determined using CD73-expressing COR-L23 cells, as described in Example 1 below and in McManus et al. 2018 SLAS discovery: advancing life sciences R & D 23, pp. 264-273.

[0078] In certain embodiments, the dual paratopic anti-CD73 antigen binding proteins of the present disclosure bind to two different CD73 epitopes on the same CD73 molecule. Dual paratopic anti-CD73 antigen binding proteins may bind to two different CD73 epitopes on the same CD73 monomer protein. Dual paratopic anti-CD73 antigen binding proteins may bind to two different CD73 epitopes on the same CD73 homodimer protein.

[0079] In certain embodiments, a dual paratopic anti-CD73 antigen binding protein of the present disclosure binds to two different CD73 epitopes on two separate CD73 molecules, hi certain embodiments, the first VH and VL domains of the dual paratopic anti-CD73 antigen binding protein bind a first CD73 epitope on a first CD73 dimer or homodimer molecule, and the second VH and VL domains bind a second CD73 epitope on a second CD73 dimer or homodimer molecule.

[0080] In certain embodiments, the biparatopic anti-CD73 antigen binding proteins of the present disclosure may be capable of cross-linking two or more CD73 dimer molecules. As used herein, "cross-linking" with an antibody refers to the binding of a first binding site on a multivalent antibody to a first target molecule. This can occur when a first epitope on a target molecule binds to a second binding site on a multivalent antibody, while simultaneously binding a second epitope on a second target molecule. Cross-linking multiple target molecules through the binding of multiple bivalent antibodies can form a higher-order structure with enhanced stability. This can result in a reduced koff rate for the cross-linked antigen-binding protein compared to a non-cross-linked antigen-binding protein. By enhancing antigen-binding protein cross-linking, antigen-binding proteins with weak antigen-binding affinity can be used. Certain antigen-binding proteins with weak binding affinity for the target antigen generally have limited utility. By combining antigen-binding proteins with weak binding affinity, the cross-linking effect of the present disclosure can enhance their efficacy through a reduced koff rate.

[0081] Methods for heterodimerization of antigen-binding proteins The dual paratopic anti-CD73 antigen binding proteins of the present disclosure are formed through heterodimerization of two parent CD73 antigen binding proteins. Any heterodimerization method known in the art can be used to form the dual paratopic anti-CD73 antigen binding proteins.

[0082] In certain exemplary embodiments, the two Fc domains of an antibody or antigen-binding fragment thereof are heterodimerized through Fab arm exchange (FAE). In certain exemplary embodiments, the human non-IgG4 CH3 sequence is modified so that it does not contain any amino acid residues involved in forming disulfide bonds or covalent or stable non-covalent inter-heavy chain bonds with other peptides containing the same amino acid sequence of its CH3 region. Such modified CH3 sequences can be IgG4-like. In certain embodiments, the antibody is an IgG1 and is modified to be IgG4-like.

[0083] An exemplary method of FAE can include the steps of: a) providing a first antigen-binding construct having a first binding specificity, wherein the first antigen-binding construct comprises an IgG4-like CH3 region; b) providing a second antigen-binding construct having a second binding specificity different from the first binding specificity, wherein the second antigen-binding construct comprises an IgG4-like CH3 region; c) incubating the first and second antigen-binding constructs together under reducing conditions that allow cysteines within the core hinge region to undergo disulfide bond isomerization; and d) obtaining a bispecific antigen-binding construct.

[0084] The term "IgG4-like CH3 region" refers to a CH3 region that is identical to the CH3 of IgG4, for example, human IgG4, or a CH3 region that is functionally equivalent to the IgG4 CH3 region. In this context, functional equivalent means that a CH3 region similar to the CH3 region of IgG4 does not form stable intermolecular interactions between the half molecules. The formation of stable intermolecular interactions between half molecules by a given CH3 region can be tested, for example, by replacing the CH3 of IgG4 with that CH3 region and testing for exchange under the conditions described in U.S. Pat. No. 9,212,230, incorporated herein by reference. If exchange is observed, stable intermolecular interactions have not been formed. For example, an IgG4-like CH3 region may be a CH3 region that is as efficient at enabling half-molecule exchange as a CH3 region derived from IgG4. Thus, an IgG4-like CH3 region may be structurally similar to the CH3 region of IgG4, for example, more than 75%, e.g., more than 90%, identical to the sequence of the CH3 region of IgG4. However, an IgG4-like CH3 region in this context may additionally or alternatively be a CH3 region that is not structurally close to the CH3 region of IgG4, but has similar functional properties in that it does not contain any amino acid residues involved in the formation of stable non-covalent inter-heavy chain bonds, such as disulfide bonds or covalent or salt bridges, with other peptides containing the same amino acid sequence of the CH3 region. For example, an IgG4-like CH3 region may be involved in half-intermolecular CH3-CH3 interactions. It may be a mutant IgG1 CH3 region in which one or more of the involved amino acid residues have been changed or deleted.

[0085] Exemplary amino acid residue modifications include R238Q, D239E, K292R, K292Y, K292F, K292W, Q302E, and P328L. Additional exemplary amino acid residue modifications include a P228S hinge mutation. Further amino acid residue modifications include an F405L or K409R CH3 domain mutation. Mixing two antibodies with a reducing agent results in FAE. For example, but by no means limiting, a first parent monospecific antibody containing an F405L modification can undergo FAE with a second parent monospecific antibody containing a K409R modification. This technology is described in U.S. Patent No. 9,212,230 and Labrijn AF PNAS (2013) 110(13):5145-5150.

[0086] In certain exemplary embodiments, the two Fc domains of an antigen-binding construct are heterodimerized through knobs-into-holes pairing. This dimerization technique utilizes a "protrusion" or "knob" along with a "cavity" or "hole" engineered into the interface of the CH3 domain. When a knob or hole with appropriate position and dimensions is present at the interface of either the first or second CH3 domain, it is only necessary to engineer the corresponding hole or knob at the adjacent interface, thereby promoting and strengthening Fc domain pairing at the CH3 / CH3 domain interface. An IgG Fc domain fused to a VHH is provided with a knob, and an IgG Fc domain of a conventional antibody is provided with a hole designed to accommodate the knob, or vice versa. A "knob" refers to at least one amino acid side chain, typically a larger side chain, that protrudes from the interface of the CH3 portion of the first Fc domain. The protrusion forms a "knob" that is complementary to and accommodates a "hole" in the CH3 portion of the second Fc domain. The "hole" is at least one amino acid side chain, typically a smaller side chain, that is recessed from the interface of the CH3 portion of the second Fc domain. This technology is described in U.S. Patent No. 5,821,333; Ridgway et al., Protein Engineering (1996) 9:617-621; and Carter PJ, Immunol. Methods (2001) 248:7-15.

[0087] Exemplary amino acid residues that can serve as the knob include arginine (R), phenylalanine (F), tyrosine (Y), and / or tryptophan (W). An existing amino acid residue in the CH3 domain is replaced or substituted with the amino acid residue of the knob. Exemplary amino acids that can be substituted include any amino acid with a small side chain, such as alanine (A), asparagine (N), aspartic acid (D), glycine (G), serine (S), threonine (T), and / or valine (V).

[0088] Exemplary amino acid residues that can serve as holes include alanine (A), serine (S), threonine (T), or valine (V). An existing amino acid residue in the CH3 domain is replaced or substituted with the hole amino acid residue. Exemplary amino acids that can be substituted include any amino acid with a large side chain, such as arginine (R), phenylalanine (F), tyrosine (Y), and / or tryptophan (W).

[0089] In certain exemplary embodiments, the CH3 domain is derived from a human IgG1 antibody. Exemplary amino acid substitutions in the CH3 domain include T366Y, T366W, F405A, F405W, Y407T, Y407A, Y407V, T394S, or combinations thereof. Particularly exemplary combinations include knob mutations on the first CH3 domain. The mutations are T366Y or T366W as the primary mutations and Y407T or Y407V as the hole mutations on the second CH3 domain.

[0090] In certain exemplary embodiments, the two Fc domains of an antigen-binding construct are heterodimerized through electrostatic steering effects. This dimerization technique utilizes electrostatic steering to promote and strengthen Fc domain pairing at the CH3 / CH3 domain interface. The charge complementarity between the two CH3 domains is altered to favor heterodimerization (opposite charge pairing) over homodimerization (same charge pairing). In this method, electrostatic repulsion prevents homodimerization.

[0091] Exemplary amino acid residue substitutions include K409D, K392D, and / or K370D in the first CH3 domain, and D399K, E356K, and / or E357K in the second CH3 domain. This technology is described in U.S. Patent Publication No. 2014 / 0154254 and Gunasekaran K. JBC (2010) 285 (25): 19637-19646.

[0092] In certain exemplary embodiments, the two Fc domains of the antigen-binding construct are heterodimerized through hydrophobic interaction. This dimerization technique utilizes hydrophobic interactions instead of electrostatic interactions to promote and strengthen Fc domain pairing at the CH3 / CH3 domain interface. Exemplary amino acid residue substitutions include K409W, K360E, Q347E, Y349S, and / or S354C in the first CH3 domain, and D399V, F405T, Q347R, E357W, and / or Y349C in the second CH3 domain. Exemplary pairs of amino acid residue substitutions between the first and second CH3 domains include K409W:D399V, K409W:F405T, K360E:Q347R, Y349S:E357W, and S354C:Y349C. This technology is described in U.S. Patent Publication No. 2015 / 0307628.

[0093] Expression of antigen-binding proteins In one aspect, polynucleotides encoding the binding proteins (e.g., antigen binding proteins) disclosed herein are provided. Methods of making the binding proteins comprising expressing these polynucleotides are also provided.

[0094] Polynucleotides encoding the binding proteins disclosed herein are typically inserted into expression vectors for introduction into host cells used to produce desired quantities of the claimed antibodies or fragments thereof. Thus, in certain aspects, the present disclosure provides expression vectors comprising the polynucleotides disclosed herein, as well as host cells comprising these vectors and polynucleotides.

[0095] The term "vector" or "expression vector" is used to mean a vector used in accordance with the present invention as a vehicle for introducing into a cell and expressing a desired gene in the cell. As known to those skilled in the art, such vectors are easily selected from the group consisting of plasmids, phages, viruses, and retroviruses. Generally, vectors compatible with the present invention contain a selectable marker to facilitate cloning of the desired gene, appropriate restriction sites, and the ability to transfer and / or replicate in eukaryotic or prokaryotic cells.

[0096] Numerous expression vector systems can be used for the purposes of this invention. For example, one class of vectors is based on bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV, or MOML). Some utilize DNA elements derived from animal viruses, such as SV40, or SV41. Others involve the use of polycistronic systems containing internal ribosome binding sites. Additionally, cells that have integrated the DNA into their chromosomes are selected by introducing one or more markers that allow for selection of transfected host cells. Markers can confer prototrophy to auxotrophic hosts, resistance to biocides (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be either directly linked to the expressible DNA sequence or introduced into the same cell by cotransformation. Additional elements may also be required for optimal synthesis of mRNA. These elements include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals. In some embodiments, the cloned variable region genes are inserted into an expression vector along with heavy and light chain constant region genes (e.g., human constant region genes) synthesized as discussed above.

[0097] In other embodiments, binding polypeptides can be expressed using polycistronic constructs. In such expression systems, multiple gene products of interest, such as antibody heavy and light chains, are produced from a single polycistronic construct. These systems advantageously use internal ribosome entry sites (IRES) to provide relatively high levels of polypeptides in eukaryotic host cells. Compatible IRES sequences are disclosed in U.S. Patent No. 6,193,980, which is incorporated herein by reference in its entirety for all purposes. Those skilled in the art will understand that such expression systems can be used to effectively produce the full range of polypeptides disclosed in the present application.

[0098] More generally, once a vector or DNA sequence encoding an antibody or fragment thereof has been prepared, the expression vector is introduced into a suitable host cell; i.e., the host cell is transformed. Introduction of the plasmid into the host cell can be accomplished by a variety of techniques well known to those skilled in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion using enveloped DNA, microinjection, and infection with intact virus. Ridgway, AAG See "Mammalian Expression Vectors," Chapter 24.2, pp. 470-472, in Vectors, edited by Rodriguez and Denhardt (Butterworths, Boston, Mass. 1988). Plasmid introduction into host cells can be accomplished by electroporation. Transformed cells are grown under conditions appropriate for the production of the light and heavy chains and assayed for heavy and / or light chain protein synthesis. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence-activated cell sorter analysis (FACS), immunohistochemistry, and the like.

[0099] As used herein, the term "transformation" is used broadly to refer to the introduction of DNA into a recipient host cell, altering the genotype and resulting in a change in the recipient cell.

[0100] Similarly, a "host cell" refers to a cell that has been transformed with a vector constructed using recombinant DNA technology and encoding at least one heterologous gene. In describing processes for isolating a polypeptide from a recombinant host, the terms "cell" and "cell culture" are used interchangeably to refer to the source of the antibody, unless clearly specified otherwise. In other words, recovery of polypeptide from the "cells" can mean either from sedimented whole cells or from the cell culture containing both the medium and the suspended cells.

[0101] In one embodiment, the host cell line used for antibody expression is mammalian in origin. One of skill in the art can determine the particular host cell line best suited for the desired gene product to be expressed. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese hamster ovary lines, DHFR minus), HELA (human cervical carcinoma), CV-1 (monkey kidney line), COS (a derivative of CV-1 carrying the SV40 T antigen), R1610 (Chinese hamster fibroblasts), BALBC / 3T3 (mouse fibroblasts), HAK (hamster kidney line), SP2 / O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), 293 (human kidney). In one embodiment, the cell line provides for altered glycosylation (e.g., afucosylation) of antibodies expressed therefrom (e.g., PER.C6 (registered trademark)). (Crucell) or a FUT8 knockout CHO cell line (Potelligent® cells) (Biowa, Princeton, NJ). In one embodiment, NS0 cells are used. CHO cells are particularly useful. Host cell lines are typically available from commercial services, such as the American Tissue Culture Collection, or from published literature.

[0102] In vitro production allows for scale-up to give large amounts of the desired polypeptide. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include homogenous suspension culture, e.g., in airlift reactors or continuous stirred tank reactors, or immobilized or entrapped cell culture, e.g., on hollow fibers, in microcapsules, agarose microbeads, or ceramic cartridges. If necessary and / or desired, the solution of the polypeptide can be purified by conventional chromatographic methods, e.g., gel filtration, ion exchange chromatography, chromatography on DEAE cellulose, and / or (immuno)affinity chromatography.

[0103] Genes encoding the binding polypeptides featured in the present invention can also be expressed in non-mammalian cells, such as bacteria, yeast, or plant cells. In this regard, it will be understood that various unicellular, non-mammalian microorganisms, such as bacteria, i.e., those capable of growth in culture or fermentation, can also be transformed. Bacteria susceptible to transformation include members of the Enterobacteriaceae family, such as Escherichia coli or strains of Salmonella; Bacillaceae, such as Bacillus subtilis; Streptococcus pneumoniae; Streptococcus spp.; and Haemophilus influenzae. It will further be understood that when expressed in bacteria, the polypeptides become part of inclusion bodies. The polypeptides must be isolated, purified, and then assembled into functional molecules.

[0104] In addition to prokaryotes, eukaryotic microorganisms are also used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used eukaryotic microorganism, although several other strains are commonly available. For expression in Saccharomyces, for example, the plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)) is commonly used. This plasmid is expressed in a yeast mutant lacking the ability to grow on tryptophan, e.g., ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). The presence of the trpl disruption as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[0105] Methods of Administering Antigen Binding Proteins Methods for preparing and administering binding proteins (e.g., antigen-binding proteins disclosed herein) to a subject are well known to or readily determined by those of skill in the art. Routes of administration of the binding proteins of the present disclosure can be oral, parenteral, by inhalation, or topical. As used herein, the term parenteral includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. While all of these modes of administration are clearly considered to be within the scope of the present disclosure, the form for administration is a solution for injection, particularly intravenous or intraarterial injection or infusion. Typically, a suitable pharmaceutical composition for injection may include a buffer (e.g., acetate, phosphate, or citrate buffer), a surfactant (e.g., polysorbate), optionally a stabilizer (e.g., human albumin), and the like. However, in other methods consistent with the teachings herein, modified antibodies can be delivered directly to the site of harmful cell populations, thereby increasing the exposure of the affected tissue to the therapeutic agent.

[0106] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. 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. In the compositions and methods of the present disclosure, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1M or 0.05M phosphate buffer, or 0.8% saline. Other common parenteral vehicles include sodium phosphate solution, Ringer's 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 antimicrobials, antioxidants, chelating agents, and inert gases and the like. More particularly, pharmaceutical compositions suitable for injection include sterile aqueous solutions (if water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and also preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerin, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0107] Prevention of microbial activity can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Isotonic agents, such as sugars, polyalcohols such as mannitol and sorbitol, or sodium chloride may also be included in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[0108] In any case, sterile injectable solutions can be prepared by incorporating the active compound (e.g., modified hydroxybenzoates, alone or in combination with other active agents) in an appropriate solvent with one or a combination of ingredients enumerated herein. The dispersions can be prepared by incorporating the modified binding polypeptide) in the required amount, followed by filtered sterilization, as required. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, preparation methods typically include vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution thereof. Preparations for injection are processed and filled into containers such as ampoules, bags, bottles, syringes, or vials, and sealed under aseptic conditions by methods known in the art. Furthermore, the preparations may be packaged and sold in the form of a kit, such as those described in co-pending U.S. patent application Ser. No. 09 / 259,337 and U.S. patent application Ser. No. 09 / 259,338 (each of which is incorporated herein by reference). Such articles of manufacture may include a label or package insert indicating that the associated composition is useful for treating a subject suffering from or predisposed to an autoimmune or neoplastic disorder.

[0109] The effective amount of the composition of the present disclosure for treating the above conditions varies depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, other medications administered, and whether the treatment is prophylactic or therapeutic. Usually, the patient is human, but non-human mammals, including transgenic mammals, can also be treated. Treatment dosages can be determined using routine methods known to those skilled in the art to optimize safety and efficacy.

[0110] For passive immunization with a binding polypeptide, dosages can range, for example, from about 0.0001 mg / kg to 100 mg / kg of host body weight, more usually 0.01-5 mg / kg (e.g., 0.02 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 0.75 mg / kg, 1 mg / kg, 2 mg / kg, etc.). For example, dosages can be 1 mg / kg or 10 mg / kg body weight or within the range of 1-10 mg / kg, e.g., at least 1 mg / kg. Doses intermediate in the above ranges are also intended to be within the scope of the present disclosure. Subjects are administered such doses daily, every other day, weekly, or according to any other schedule determined by empirical analysis. Exemplary treatments entail administration in multiple dosages over an extended period of time, for example, for at least six months. Additional exemplary treatment regimens entail administration once every two weeks, once a month, or once every three to six months. Exemplary dosing schedules include 1-10 mg / kg or 15 mg / kg daily, 30 mg / kg every other day, or 60 mg / kg weekly. In some methods, two or more binding proteins with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated.

[0111] The binding proteins described herein can be administered on multiple occasions. The interval between single doses can be weekly, monthly, or yearly. The intervals can also be irregular, as indicated by measuring the patient's blood levels of the modified binding polypeptide or antigen. In some methods, the dosage is adjusted to achieve a plasma modified binding polypeptide concentration of 1-1000 μg / ml, and in some methods, 25-300 μg / ml. Alternatively, the binding polypeptide can be administered as a sustained-release formulation, requiring less frequent administration. For antibodies, dosage and frequency vary depending on the half-life of the antibody in the patient. Generally, humanized antibodies exhibit the longest half-life, followed by chimeric antibodies and nonhuman antibodies.

[0112] The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, compositions containing the present antibodies or a cocktail thereof are administered to patients not already in the disease state. In this application, the antibody is administered to enhance the patient's resistance. Such an amount is defined as a "prophylactically effective amount." In this use, the exact amount again depends on the patient's health and general immune status, but generally ranges from 0.1 mg to 25 mg per dose, particularly 0.5-2.5 mg per dose. Relatively low dosages are administered at relatively infrequent intervals over an extended period of time. Some patients continue to receive treatment until death. In therapeutic applications, relatively high dosages at relatively short intervals (e.g., about 1 mg / kg to 400 mg / kg of antibody per dose, with dosages of 5 mg to 25 mg for radioimmunoconjugates and higher doses more commonly used for cytotoxic drug-modified antibodies) may be required until disease progression slows or terminates, or the patient shows partial or complete remission of disease symptoms. The patient can then undergo a prophylactic regimen.

[0113] The binding polypeptides described herein can optionally be administered in combination with other agents that are effective in treating the disorder or condition in need of treatment (e.g., prophylactic or therapeutic). 90 Effective single treatment dosages (ie, therapeutically effective amounts) of Y-labeled modified antibodies range from between about 5 mCi and about 75 mCi, such as between about 10 mCi and about 40 mCi. 131 Effective single treatment non-marrow ablative doses of I-modified antibodies range from between about 5 mCi and about 70 mCi, such as between about 5 mCi and about 40 mCi. 131 Effective single-treatment ablative doses of I-labeled antibodies (i.e., which may require autologous bone marrow transplantation) range from about 30 mCi to about 600 mCi, such as from about 50 mCi to less than about 500 mCi. With chimeric antibodies, due to their longer circulating half-lives compared to murine antibodies, 131 An effective single treatment non-marrow ablative dose of I-labeled chimeric antibody ranges between about 5 mCi and about 40 mCi, e.g., less than about 30 mCi. 111 For In labels, imaging criteria are typically less than about 5 mCi.

[0114] While the binding polypeptides are administered as described immediately above, it should be emphasized that in other embodiments, the binding polypeptides are administered as a first-line treatment to otherwise healthy patients. In such embodiments, the binding polypeptides are administered to patients with normal or average red bone marrow reserve and / or to patients who have not received and will not receive one or more other therapies. As used herein, administration of a modified antibody or fragment thereof in conjunction with or in combination with an adjunctive therapy refers to sequential, simultaneous, coextensive, concurrent, concomitant, or contemporaneous administration or application of the disclosed antibody with that therapy. Those skilled in the art will understand that the timing of administration or application of the various components of a combined therapeutic regimen will be determined to enhance the overall effectiveness of the treatment. A skilled practitioner (e.g., an experienced oncologist) will readily be able to identify an effective combined therapeutic regimen based on the selected adjunctive therapy and the teachings herein without undue experimentation.

[0115] As previously discussed, the disclosed binding polypeptides, immunoreactive fragments, or recombinant forms thereof are administered in pharmaceutically effective amounts for the in vivo treatment of mammalian disorders. In this regard, the disclosed binding polypeptides are formulated to facilitate administration and promote stability of the active agent.

[0116] Pharmaceutical compositions according to the present disclosure typically comprise a pharmaceutically acceptable, non-toxic, sterile carrier, such as saline, non-toxic buffers, preservatives, and the like. For purposes of this application, a pharmaceutically effective amount of a modified binding polypeptide, immunoreactive fragment, or recombinant thereof, conjugated or unconjugated to a therapeutic agent, is an amount sufficient to achieve effective binding with an antigen and obtain a benefit, e.g., ameliorate the symptoms of a disease or disorder, or detect a substance or cell. In the case of tumor cells, the modified binding polypeptides are typically capable of interacting with selected immunoreactive antigens on neoplastic or immunoreactive cells and conferring increased killing of those cells. Of course, the pharmaceutical compositions of the present disclosure are administered in single or multiple doses to provide a pharmaceutically effective amount of the modified binding protein.

[0117] In accordance with the scope of the present disclosure, the binding proteins of the present disclosure are administered to humans or other animals in an amount sufficient to produce a therapeutic or prophylactic effect, according to the treatment methods described above. The binding polypeptides of the present disclosure can be administered to such humans or other animals in conventional dosage forms prepared by combining the antibodies of the present disclosure with conventional pharmaceutically acceptable carriers or diluents by known techniques. Those skilled in the art will recognize that the form and characteristics of the pharmaceutically acceptable carrier or diluent will be determined by the amount of active ingredient with which it is to be combined, the route of administration, and other well-known variables. Those skilled in the art will further appreciate that cocktails comprising one or more species of binding polypeptides described in the present disclosure may prove particularly effective.

[0118] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made, using appropriate equivalents, without departing from the scope of the embodiments disclosed herein. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting. [Example]

[0119] Experimental procedure Generation of dual paratopic antibodies CD73-specific monoclonal antibodies were isolated using a general mouse immunization and phage display approach with soluble human CD73 as the antigen (data not shown). Twelve sequence-unrelated parent antibodies with IC50s ranging from 1 to 25 nM and at least 50% inhibition of CD73 in a cell-based assay at saturating antibody concentrations were selected for this study.

[0120] Bispecific variants were generated using a modified version of the published Duobody procedure (Gramer et al., 2013 mAbs 5, 962-973), except for the use of microdialysis for product purification. Equimolar amounts of the F405L and K409R Fc variants of each parent huIgG1 (25-50 μg each) were combined in a total volume of 90 μL of PBS, to which 10 μL of 7.5 M mercaptoethylamine (MEA) pH 7.4 was added. The mixture was incubated at 30°C in a forced-air incubator for 4 hours, then transferred to individual cassettes removed from a 96-well dialysis plate strip (Pierce) and subjected to three rounds of dialysis (1 hour, 1.5 hours, and overnight) at room temperature. For samples with more than six samples, the reactions were transferred to a dialysis cassette strip mounted on a carrier plate. The plate was suspended over a reservoir and transferred between reservoirs containing fresh PBS after each round of dialysis. After the second dialysis, the total free thiols in the retentate were below the limit of detection with DTNB. The final product was stored at 4°C. Product formation was determined by cIEF. The parental antibody for analysis was reconstituted by hybridizing the F405L and K409R parents in the same format as the test duobody.

[0121] Characterization of dual paratopic antibodies The formation of the duobody product of the Fab arm exchange reaction (cFAE) was analyzed by capillary isoelectric focusing (cIEF) (Maurice, Protein Simple, San Jose, CA). This approach was chosen because the pI of the bispecific daughter molecule is expected to be between the pIs of its two parents. To increase the relative contribution of charge differences in the peaks, cIEF was performed on the F(ab')2 fragments obtained by IdeZ digestion of the cFAE product. The cFAE product (4 μL, 1 mg / mL) was mixed with 4 μL of 1 U / μL IdeZ (Fabricator Z, Genovis) in water and mixed by trituration. The tube was incubated at 37°C for 4 hours in an air incubator. Subsequently, 36 μL of a 1.1x Pharmalyte methylcellulose / ampholine mixture was added, mixed, and centrifuged at 13 kG for 4 minutes. The supernatant (30 μL) was transferred to a 96-well plate for analysis. The sample was loaded into a cIEF cassette for 55 seconds and focused at 1.5 kV for 1.5 minutes, followed by 3 kV for 6 minutes. Separated products were detected by fluorescence. Formation of the desired duobody product was assessed by the absence of the F405L parent Fc peak at approximately pI 7.6, along with the disappearance of the parent antibody F(ab')2 peak and the formation of an F(ab')2 peak with a pI close to the average of the two parent F(ab')2 peaks. The duobody Fc fragment with both mutations (F405L:K409R) was not resolved from the K409R parent, likely due to limited changes in the pKa of the arginine in the environment surrounding this residue. IdeZ focused at pI 7.14 and below. Example results are shown in Figure 3.

[0122] Analysis of double paratopic binding The ability of dual paratopic antibodies to associate with CD73 bivalently (e.g., at two epitopes) was determined using surface plasmon resonance (SPR) to compare their ability with monovalent antibodies. Monovalent antibodies were used to block bivalent interactions with CD73. SPR was performed at 25°C on a Biacore T200 instrument (GE Healthcare) using HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% (v / v) surfactant P20, pH 7.4) as the running buffer and a Protein A Series S sensor chip (GE Healthcare). Binding was measured at very low responses (<10 RU) to minimize avidity effects from binding of CD73 by separate monovalent antibodies on the chip. Antibodies were diluted to limit capture to between 5 RU and 30 RU during a 30-second injection at 10 μL / min. CD73 at multiple concentrations (32 nM, 12 nM, and 3 nM) was then passed over the captured antibody at 30 μL / min for 5 min. Dissociation was measured for 30 min. The sensor surface was then regenerated with 10 mM glycine-HCl pH 1.5 at 20 μL / min for 30 s. Rate constants were measured using a Biacore T200. Calculations were performed using a 1:1 Langmuir binding model using BiaEvaluation software (GE Healthcare). In cases where a bivalent fit using BiaEvaluation software yielded lower apparent residuals, raising the possibility of biphasic binding, the 1:1 Langmuir binding model was not used. In these cases, the kd value of each component during dissociation was determined by fitting the longer half-life component to a first-order decay defined by the rate equation after 1000 s. Abundance and kd for the rapidly dissociating component were calculated using an exponential fit to the residuals for that component over a period of 100–200 s. The criterion applied was that the interval used to fit the slower component begin at least four times the t1 / 2 of the rapidly dissociating component. The association components were extracted separately by first fitting the approach to saturation (RUmax) within a window from 100 s to a point at least 0.2 RU from RUmax as a first-order reaction, for various assumed RUmax values. The best fit parameters and RUmax were then used as the starting point for further refinement. Positive residuals between the observed RU and this fit extended to earlier time points were treated as independent pseudo-first-order reactions reflecting rapidly binding components. Using an iterative process of varying the rate constant and rate of binding for each component with a level (RU) of binding after 300 seconds, fits within 0.2 of the observed RU and net residuals over the 300 second measurement were obtained. The variation test showed that the value was the true R for the overall fit. 2 The effect of each component of RUmax on the rate constant or rate was negligible. Fits were performed in Excel.

[0123] Cell-based assay of CD73 inhibition (potency) The efficacy of the dual paratopic antibodies was assessed using a previously described method (McManus et al. 2018). The results were determined using a modified version of the SLAS discovery: advancing life sciences R & D 23, pp. 264-273. CD73-expressing COR-L23 cells (4 × 10 3 Cells (1 / well) were grown overnight in 40 μL of 1640 medium containing L-glutamine and 10% heat-inactivated FBS in 384-well clear-bottom plates (Greiner Bio One) to approximately 50% confluence. Antibodies diluted in 1640 medium (10 μL) were added, and plates were incubated at 37°C for 3 h. Antibody dilutions and additions were performed with an Agilent Bravo liquid handler. AMPCP (100 μM) was used in place of antibody as a zero activity control (23, 25). Substrate (200 μM 15 N5-AMP (5 μL, Silantes GmbH Munich Germany) was dispensed into a GNF dispenser II (GNF Systems, San Antibodies were added using a 500-milliliter (1000-milliliter) ELISA kit (Diamond City, CA) and the plate was incubated at 37°C for 1 hour. The reaction was then quenched with 5 μL of 12% formic acid in 1640 medium, and a portion of the quenched reaction (40 μL) was filtered by centrifugation at 3.5 kG for 30 minutes through a 10 kDa MWCO ultrafiltration plate (Pall). The filtrate was stored at -80°C. Adenosine production was determined by LC / MS / MS analysis as previously described (23). Data were analyzed by nonlinear least-squares fit (GraphPad Prism). Activity (%CNTL) normalized to a no-antibody control in the same plate sector and to the least-squares fit maximum activity is shown. Potency determination results (Table 5) are expressed as estimated maximum % inhibition at saturating antibody concentrations. In the initial screen, three concentrations (0.25 μg / mL, 0.5 μg / mL, and 1 μg / mL) were tested in a 4-fold dilution series, and the average % inhibition shown (Figure 1) is based on residual activity at a single concentration (1 μg / mL).

[0124] Epitope binning Epitope binning of antibody subsets was performed as previously described (Abdiche et al. Assays were performed using a premix format and biolayer interferometry (BLI) using a modified version of the method described in J. Biol. Chem. 2009, 386, pp. 172-180. In this format, binding of premixed antigen with a molar excess of Fab is compared to binding of antigen alone. Analysis was performed in 16-channel mode on an Octet QK384 (Pall Life Sciences). Antibodies were bound by the Protein A biosensor for 5 minutes, a baseline was established for 1 minute, and then transferred to 100 nM CD73 or 100 nM CD73 with a 4-fold molar excess of Fab for 3 minutes, followed by transfer to buffer and dissociation monitoring for 3 minutes. All samples were diluted in PBS pH 7.4 containing 0.1% (w / v) bovine serum albumin and 0.01% (v / v) Tween 20, and the assay was performed at 30°C. Data were analyzed using ForteBio Data Analysis 7.1 software (Pall Life Sciences) by taking the report point at the end of the association phase. Normalized capture values ​​were calculated by dividing the signal (nm) by the signal from CD73 alone and multiplying by the relative mass of CD73 compared to the CD73::(Fab)2 complex (0.56).

[0125] Structure determination Recombinant TB19 and TB38 Fabs were expressed in Expi293F cells and bound to CaptureSelect CH1-XL affinity matrix (ThermoF Purification was performed using a chromatograph (Diamond-type chromatograph) and buffer-exchanged into PBS. Human CD73 27-549 was cloned with a C-terminal His6 tag and expressed in ExpiHEK293 cells. CD73 was purified using a nickel column, buffer-exchanged into PBS, deglycosylated with PNGaseF overnight, and further purified using size-exclusion chromatography. The molar mass of the product was determined by SEC on a Superdex 200 column in 150 mM NaCl, 20 mM HEPES pH 7.0 using multi-angle light scattering (Wyatt miniDAWN® Treos and Wyatt Optilab® T-rEX online refractometer). Data were evaluated using Wyatt ASTRA 6.1 software. Each respective Fab was then incubated with CD73 on ice for 1 hour and loaded onto a Superdex 200 10 / 300 GL column (GE Healthcare) pre-equilibrated with 20 mM HEPES pH 7.0, 150 mM NaCl. Fractions corresponding to the eluted complex peak were pooled and concentrated to 9 mg / ml for crystallization trials. TB19 Fab::CD73 was crystallized at 4°C in 0.1 M disodium hydrogen phosphate / potassium dihydrogen phosphate pH 6.2, 35% 5-methyl-2,4-pentanediol, and 2.5% pentaerythritol ethoxylate. These crystals were cryoprotected in 20% ethylene glycol and mother liquor. X-ray diffraction data were collected using an Eiger 16M detector at EMBL Hamburg P14. Data were indexed / integrated using XDS and scaled using Aimless (Evans et al. 2013 Acta crystallographica Section D, Biological crystallography 69, 1204-1214; Kabsch et al. 2010 Acta crystallographica Section D, Biological crystallography 66, 125-132).Molecular replacement was performed using Phaser (McCoy et al. 2007 Journal of applied crystallography 40, 658-674) and three search ensembles: isolated CD73 N- and C-terminal domains (PDB: 4H2I) and MOE (Molecular Operating Environment (MOE)). The analysis was performed using the TB19.3 Fv model generated by the Chemical Computing Group (August 2013). TB38 Fab::CD73 crystallized at 4°C in 1.6 M monobasic sodium phosphate monohydrate, 0.4 M dibasic potassium phosphate, and 0.1 M sodium citrate phosphate solution (pH 5.3). Crystals were flash-frozen in liquid nitrogen using 20% ​​glycerol in the mother liquor as cryoprotectant. X-ray diffraction data were collected using a Pilatus 3 6M detector at the European Synchrotron Radiation Facility Beamline ID-30b. Data were indexed / integrated using XDS and scaled using Aimless (Evans, supra; Kabsch, supra). Molecular replacement was performed iteratively using Phaser (McCoy, supra). For the first round of molecular replacement, the CD73 monomer (PDB:4H2F) and the TB38 Fab MOE-generated model were used as search models for MOE. For the second round, the previously found CD73 monomer was separated into its N- and C-terminal domains and searched alongside the Fv domain of TB38 alone. For both structures, model reconstructions were performed using the same method as Coot (Emsley et al. 2010 Acta crystallographica Section D, Biological crystallography 66, 486-501), and refinement was completed using Phenix (Adams et al. 2010 Acta crystallographica Section D, Biological crystallography 66, 213-221). Data collection and refinement statistics are tabulated (Table 7). Software used for this project was accessed through the SBGrid consortium (Morin et al. 2013 eLife 2, e01456). [Example]

[0126] Generation of dual paratopic antibodies A panel of dual paratopic antibodies against CD73, representing pairwise combinations of 11 sequence-unrelated parent antibodies previously shown to inhibit CD73 activity by >50% in cell-based assays, was generated using Fab arm exchange (cFAE). Each Fab was expressed as a fusion with a human IgG1 Fc containing either the F405L or K409R mutation, which destabilizes the parent Fc and stabilizes the Fc of the dual paratopic duobody product (Gramer et al. 2013 mAbs 5, 962-973; Labrijn et al. 2013 PNAS 110, 5145-5150; Labrijn et al. 2014 Nat. Protoc. 9, 2450-2463). The parent antibodies were expressed in small-scale cultures, purified using Protein A, and recombined by cFAE. Production of the desired product was verified by cIEF (Figure 3). Of the 121 (11 x 11) possible combinations, 88 dual paratopic variants were generated, covering all possible combinations in at least one orientation. Eleven monospecific parent antibodies were also reconstructed as comparators by combining the parent F405L and K409R Fc variants to control for potential effects of Fc mutations on antibody structure and function. In addition, 21 pairings were generated in both Fc orientations to control for possible positional effects of mutations. [Example]

[0127] Efficacy of parental and dual paratopic antibodies in inhibiting cellular CD73 enzymatic activity Purified parental and dual paratopic antibodies were tested for potency at 1 μg / mL on COR-L23 lung cancer cells expressing human CD73, and the product adenosine was quantified by an LC / MS-based assay (McManus et al. 2018 SLAS discovery: advancing life sciences R & D 23, 264–273). The percentage of inhibition of CD73 enzymatic activity by dual paratopic antibodies at 1 nM is shown in Figure 1. While the degree of inhibition varied widely, the majority of dual paratopic combinations, in the form of duobodies, demonstrated greater potency than either parental antibody. Some parental antibodies, when combined with more than one other antibody, generated highly potent daughter dual paratopic variants that exhibited ≥90% inhibition. Of these, TB19 and E3.2 formed the largest number of variants with ≥90% inhibition, and several TB19 pairs, including TB19 with E3.2, H19, TB38, or TC29, achieved ≥95% inhibition. TB19 and E3.2 antibodies also achieved ≥80% inhibition in combination with several other antibodies. While both of these antibodies exhibited this promiscuous pairing ability, they were distinguished from each other by the complementarity in their pairing patterns. No significant differences in the degree of inhibition were observed between the dual paratopic variants tested in both Fc orientations (16 in total), indicating that the position of the duobody mutation in the Fc did not significantly affect the results (data not shown).

[0128] To assess whether both parental Fabs are required for potency, the parental antibodies were also hybridized with an unrelated antibody (AS30) to generate monovalent variant IgGs with only a single Fab capable of interacting with CD73. All of these antibodies showed negligible potency, demonstrating that Fabs from the two cognate parental antibodies must be involved (Figure 1). To determine whether this was due to lower affinity, the affinity of the most potent biparatopic antibody monovalent molecules was compared to that of the biparatopic variants of which they were part. First, the antibodies were conjugated to a solid support, and binding of soluble CD73 dimers in solution was followed by SPR (experimental procedure described above). As can be seen in Figure 2, in most cases, the affinity (KD) of the biparatopic variants was similar to that of the more affine parental antibodies, indicating that their affinity can be attributed to binding of the Fab alone. In two cases ( Only in the two cases (H19 / TB19 and CL25 / TB19) did the dual paratopic variant exhibit significantly higher affinity than either parental antibody (approximately 15-fold lower KD in both cases), suggesting synergy that may be due to bivalent binding to the CD73 dimer or conformational effects promoting binding. However, neither of the parental antibodies in these two cases produced a similar enhancement when combined with the other antibody, suggesting that conformational effects are less likely. The affinity of most monovalent antibodies for individual CD73 dimers was not increased by the addition of a second cognate Fab, despite being required for potency, suggesting that dual paratopic IgG interactions with more than a single CD73 are required for potent inhibition.

[0129] To further evaluate the benefit of combining these antibodies in a dual paratopic format, the EC50 and maximum inhibition at saturating antibody concentrations were determined for the most active dual paratopic antibodies, along with their parental mAbs, either alone or in mixtures, on COR-L23 cells (Table 5, Figure 4). Consistent with the results in Figure 1, each dual paratopic antibody was more potent than either of its two parental antibodies, which showed only partial inhibition up to 10 nM. EC50 values ​​for all of the dual paratopic antibodies ranged from 0.2 to 0.8 nM. In most cases, the mixture of parental antibodies produced similar maximum inhibition as the dual paratopic antibodies, but in half of the combinations tested, the dual paratopic variants also showed lower EC50s. In the most striking case (TB19 / TC29), the dual paratopic antibody exhibited an EC50 50-fold lower than that of the antibody mixture, despite nearly identical affinity of the dual paratopic antibody and the more affine TC29 monovalent parent antibody for CD73 (Figure 2). In only one case (CL25 / TA10) was the mixture more potent (approximately 4-fold), indicating that the interaction with CD73 conferred by the mixture could not be reproduced by the dual paratopic antibody.

[0130] [Table 5]

[0131] The affinity of the dual paratopic antibodies for CD73 was compared to that of the parent antibodies in monovalent form. To avoid potential avidity effects from binding of CD73 in solution by separate antibodies on the chip surface, the parent antibodies were bound to the chip at the lowest level sufficient to reliably assess rate constants. The data are grouped as shown in Figure 2. Note that the kinetic parameters for the parent antibody shared among the dual paratopic antibodies are shown in each case for ease of comparison. Values ​​represent fits to curves obtained for 3 nM, 12 nM, and 32 nM CD73 in flow. Association rate constants in the biphasic kinetic case are shown along with their abundance ratios after a 300-second association phase in parentheses. Dissociation rate constants and abundance ratios are based on the TO intercept of the fits to each component. Table 6 below shows the binding data used to generate Figure 2.

[0132] [Table 6]

[0133] Nine of eleven dual-paratopic antibodies exhibited biphasic dissociation kinetics (Figures 13A-13B), largely as a result of a small proportion (≤15%) of a faster dissociating component. In one case (H19 / C16), the abundance of this component was similar to that of the monovalent parent C16 / AS30 (31% vs. 38%), making both constructs more efficient. This suggests heterogeneity of the C16 monoclonal used for this study. TA9 / AS30 showed similar heterogeneity (~29% stability), which was not reflected in the biparatopic daughter TA9 / H7. The half-times of dissociation compared to the monovalent parental antibodies are shown in Figure 15. In 8 of 11 cases, the kd of the major dissociation component was within 2.2-fold of the most stable monovalent parent. In contrast, the difference between the kd values ​​for the monovalent parental antibodies averaged 15-fold (range 1.5-73-fold, median 6.2), suggesting that in these cases, CD73 is bound by a single parental Fab arm on the immobilized antibody. However, in three cases (E3.2 / TB19, CL25 / TB19, and H19 / TB19), the interaction with the dual paratopic antibody was significantly more stable than with either monovalent parent antibody (5.4-, 8.8-, and 26-fold, respectively), suggesting the presence of additional contacts with the dual paratopic antibody.

[0134] Bivalent kinetics of association was also evident from the rapid increase in RU immediately after injection, followed by a significant drop in kinetics after 100 seconds. Estimation of the expected RU at early times from the 100-second kinetics assuming pseudo-first-order kinetics showed significant residual binding consistent with the rapidly binding component exhibiting first-order kinetics contributing a significant proportion of the RU after 300 seconds (30-49%). Fitting both components by an iterative process yielded a sum within ±0.2 of the observed RU over 90% of the association course (Figures 13A-13B). Similar to the dissociation case, the calculated k values ​​for each of the two components were within 3-fold of the monovalent parent (2.04 ± 1.4-fold, range 1.02-2.71), in contrast to the average approximately 6-fold difference between them (5.9 ± 2.1, Table 6), suggesting that they reflect independent binding to CD73 by each parental Fab arm.

[0135] Because each kinetic component of association cannot be unambiguously assigned to a specific one of dissociation, the relative affinities of the dual paratopic and monovalent parent antibodies for CD73 were compared by KD values ​​based on the kd values ​​of the major dissociation components and the ka values ​​based on the Langmuir 1:1 binding model. The latter was within 30% of the average of the two ka components in the case of biphasic binding (Table 6). Consistent with the pattern seen for the dissociation kinetics, the apparent affinities (K D ) were similar to those of the more affine monovalent parent antibodies, indicating that the interaction of dual paratopic antibodies can be largely attributed to the binding of a single Fab arm. However, in two cases (CL25 / TB19 and TB19 / H19), the dual paratopic variants showed significant increases over either monovalent parent antibody (26-fold and 69-fold, respectively). This increase was specific to the parent (TB19, H19, CL25) because similar enhancements did not occur with other partners. Because these increases required two cognate arms, it was speculated that this reflected the interaction of both arms of these dual paratopic variants with CD73. However, in most cases, the affinity for CD73 was not increased by the addition of a second cognate Fab arm, despite its necessity for potency, suggesting that the additional CD73 interaction of dual paratopic antibodies is required for potent inhibition on cells. [Example]

[0136] Epitope binning The epitopes of the parent antibodies (TB19, E3.2, TB38, H19, and E3.2) that yielded the greatest number of highly potent combinations were binned using biolayer interferometry (Figure 5A). For capture of CD73 from a mixture with competitor Fab, a monovalent IgG antibody was used for coating on a solid support.

[0137] The results of examining a subset of parental antibodies are shown in Figure 5B. Higher values ​​indicate no / low competition for capture and binding of CD73 bound by exposed Fab (i.e. A higher value indicates that the Fab binds to a CD73 epitope that does not overlap with that of the coated antibody, while a lower value reflects epitope blocking by the bound Fab due to capture by the immobilized antibody. Based on these results, the assignment of antibodies to different epitope bins is shown in Figure 5C. One bin contained TB38, H19, and the largely overlapping TC29, all of which showed sensitivity to each of the Fabs except for TB19. However, these three also showed differences in sensitivity to competition by different Fabs. For example, capture of CD73 by monovalent TB38 Ig was more sensitive to competition by H19 Fab than capture by either TC29 or H19, while TC29 was distinguished from the other two by its partial resistance to competition by F1.2 Fab, which was unique among all of the antibodies. Although bins were clearly delineated in most cases, intermediate levels of inhibition were observed in some cases (H19+H19, TC29+H19, TC29+F1.2, TB19+H19, F1.2+H19, and F1.2+TB19), likely reflecting overlapping epitopes (Abdiche et al. 2017 PloS One 12, e0169535) and / or significant differences in affinity. E3.2 could not be binned due to nonspecific interactions with the solid support.

[0138] The capture of CD73::Fab complexes by antibodies in this binning experiment, reflecting the lack of competition between the parent antibodies, correlated highly with the inhibition of cellular CD73 enzymatic activity by the corresponding dual paratopic antibodies (Figure 5D). Antibody pairings in which greater than 35% capture of the Fab was consistently detected produced ≥85% inhibition at 1 µg / mL as dual paratopic antibodies. Conversely, combinations with less than 35% capture achieved less than 70% inhibition as dual paratopic antibodies. These data demonstrate that to achieve high potency, both antibodies constituting a dual paratopic antibody must bind non-overlapping epitopes on CD73. [Example]

[0139] Structure of TB19 and TB38 Fab in complex with CD73 Because the TB19 antibody successfully paired with several other antibodies, including TB38 in the most potent dual-paratopic variant, it was important to understand its mechanism of action by examining its interaction with CD73 through structural analysis. Prior to preparing complexes with TB19 and TB38 recombinant Fabs, the extracellular domain of human CD73 (residues 27–549) was deglycosylated with PNGaseF. The PNGaseF-treated product exhibited a molecular weight (MW) of 118 kDa by SEC-MALS, which was slightly larger than the MW of the polypeptide (116 kDa). This may be due to the glycan observed in the structure at position Asn311, which was not susceptible to PNGaseF cleavage. Crystallization parameters are shown in Table 7 below.

[0140] [Table 7]

[0141] The structure of CD73 complexed with TB19 Fab is shown in Figures 6A-6B and 7A-7B. In the crystal asymmetric unit, one TB19 binds to one CD73 monomer, and only the Fv of the Fab could be assembled due to the weak electron density in the CH1 / CL domain. The biological assembly of the dimeric CD73 complex was obtained through two-fold crystallographic symmetry operations. In the resulting structure, CD73 dimerizes through the interface between the C-terminal domains (Figure 6B), which closely resembles that of published structures (Heuts et al. 2012 Chembiochem: a European journal of chemical biology 13, 2384-2391; Knapp et al. 2012 Structure (London, England: 1993) 20, 2161-2173).

[0142] Within CD73 in complex with TB19, a well-defined positive density is observed in the active site of the N-terminal domain. Accordingly, as the TB19 complex was crystallized in the presence of phosphate ions, two zinc ions and one phosphate ion were constructed and coordinated by residues Asp36, His38, Asp85, Asn117, His118, His220, and His243 in the catalytic center. These zinc ions and phosphate ions are in the same positions as the two zinc ions and β-phosphate ion of the substrate analog AMPCP in the closed conformer of CD73 (PDB 4H2I) (Figures 7A-7B). The conserved dimerization interface and the positions of the zinc and phosphate ions indicate that the structure of the CD73 dimer in complex with TB19 is biologically relevant.

[0143] CD73 has previously been reported in either an open or closed conformation, depending on the absence or presence, respectively, of a substrate in the active site (Knapp, supra) (Figure 6A). However, when bound by TB19, CD73 exhibits a cytosolic structure consisting of the N- and The C-terminal domain assumes a conformation intermediate between those previously reported for the open and closed conformers (Figures 6A-6B and 7A-7B). When the previous structure and the C-terminal domain of CD73 bound to TB19 are superimposed, the position of the zinc-coordinating residue H220 in the N-terminal domain is approximately 22 Å away from its position in the closed conformer (PDB 4H2I) and approximately 27 Å away from its position in the open conformer (PDB 4H2F).

[0144] Although all of the TB19 CDR loops, except for CDRL2, contact portions of the N-terminal domain adjacent to the zinc and phosphate binding sites (Figures 7A-7B), none of the antibody residues directly interact with any of the catalytic center-forming residues. In addition, TB19 CDRH2 residue Ser62 and CDRL1 residue Ser26 (Figures 6B-6C) are spatially close to the C-terminal domain but are 20 Å away from substrate-binding residues, including Arg354, Asn390, Arg395, Phe417, Phe500, and Asp506. In the presence of TB19, these substrate-binding residues are far from the zinc in the catalytic center and N-terminal domain. For example, residues Phe417 and Phe500, which bind the adenine ring, are 11-13 Å away from their positions in the closed conformer (PDB 4H2I) with the substrate.

[0145] Due to the orientation of TB19 and its epitope location, when the N-terminal domains of CD73 and the closed conformer of CD73 in our structure are superimposed, a clash between the C-terminal domain and TB19 is observed (Figures 7A-7B). Thus, bound TB19 blocks the alignment of the N- and C-terminal domains in CD73, preventing the formation of the closed conformer. As a result, TB19 binding separates the zinc ion and catalytic residues of the N-terminal domain from the substrate phosphate anhydride bond, thereby blocking enzymatic activity.

[0146] In contrast to TB19, TB38 Fab and CD73 yielded structures in which each asymmetric unit contained two CD73 dimers in different conformations, with all monomers bound by one Fab (Figures 8A-8C). In the first structure (Figure 8A), the electron density for the CH1 / CL domain was clearly defined, allowing the complete Fab structure to be constructed. In the second structure (Figure 8B), weak density for the constant domains was observed; therefore, only the Fv domain was constructed. Notably, the conformations of CD73 in the two structures are different. In the first, CD73 is in a symmetric open conformation that can be superimposed onto the reference open conformer in PDB 4H2F with a root-mean-square deviation of 1 Å. However, in the second structure, the CD73 dimer is in a previously unreported asymmetric conformation, with the monomers in different conformations. In this hybrid structure, one monomer is in the open conformation (PDB 4H2F) previously observed in crystals with bound adenosine, while the other is in the closed conformation (PDB 4H2I) seen in the presence of the substrate analog AMPCP (Knapp, supra). In both complexes, TB38 Fab contacts exclusively with residues in the N-terminal domain (including Lys145, Ser152, Ser155, Gly156, Leu159-Lys162, Glu203, Lys206, Leu210, and Asn211), with all six CDRs involved in the interaction. Mapping of the epitope residues of TB19 and TB38 on the partially open conformation of CD73 (Figure 10) and sequence alignment show that the epitopes are closely spaced but non-overlapping, consistent with the binning results.

[0147] To assess the possible association of a CD73 dimer with the bispecific TB19 / TB38 antibody, we modeled the IgG by replacing the Fv of the complete IgG antibody structure (PDB 1HZH) with those of TB19 and TB38 (Figures 9A-9B). The distance between the CH1 domains of TB19 and TB38 in this model (Figure 9A) is approximately 40 Å. (Measured between the Cα of Ala225 in the CH1 domain). Although modeling bivalent binding of this dual paratopic antibody to CD73 in a partially open conformation by binding two epitopes on either the same or opposing monomers was not possible, each CD73 monomer could be monovalently bound by two antibodies, as illustrated in Figure 9B. For a single antibody to bivalently bind a CD73 dimer, the C-terminal residues of the Fab CH1 domains would need to be approximately 120 Å and 140 Å apart to bind epitopes on either the same or opposing monomers, respectively, which is much farther than can be achieved with IgG. It was concluded that the dual paratopic TB19 / TB38 antibody was likely incapable of binding a single CD73 dimer in a bivalent manner.

Claims

1. An antigen binding protein or fragment thereof having binding specificity for a CD73 epitope, (a) an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GGSIRNNY (SEQ ID NO: 1) or GFTFSSYG (SEQ ID NO: 7), a CDR-H2 sequence comprising the amino acid sequence of IYISGTT (SEQ ID NO: 2) or FWYDGSNK (SEQ ID NO: 8), and a CDR-H3 sequence comprising the amino acid sequence of AREHYVSGTSLDN (SEQ ID NO: 3) or ARAPNWDDAFDI (SEQ ID NO: 9); and (b) an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVNTNY (SEQ ID NO: 4) or SGSVSTSYY (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of GTS (SEQ ID NO: 5) or STN (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQDYNLPYT (SEQ ID NO: 6) or VLFMGSGIWV (SEQ ID NO: 12); The antigen-binding protein or fragment thereof, comprising:

2. 2. The antigen-binding protein or fragment thereof of claim 1, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15, and the VL domain comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO:

16.

3. The antigen-binding protein or fragment thereof of claim 1, wherein the antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 17, 18, 20, or 21, and the antibody light chain comprises the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO:

22.

4. 3. The antigen-binding protein or fragment thereof of claim 2, comprising a VH domain that is at least about 90% identical or at least about 95% identical to the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15, and a VL domain that is at least about 90% identical or at least about 95% identical to the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO:

16.

5. 4. The antigen-binding protein or fragment thereof of claim 3, comprising an antibody heavy chain that is at least about 90% identical or at least about 95% identical to the amino acid sequence of SEQ ID NO: 17, 18, 20, or 21, and an antibody light chain that is at least about 90% identical or at least about 95% identical to the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO:

22.

6. (a) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GGSIRNNY (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence of IYISGTT (SEQ ID NO: 2), and a CDR-H3 sequence comprising the amino acid sequence of AREHYVSGTSLDN (SEQ ID NO: 3); and (b) the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVNTNY (SEQ ID NO: 4), a CDR-L2 sequence comprising the amino acid sequence of GTS (SEQ ID NO: 5), and a CDR-L3 sequence comprising the amino acid sequence of QQDYNLPYT (SEQ ID NO: 6); The antigen-binding protein or fragment thereof according to any one of claims 1 to 5.

7. 7. The antigen-binding protein or fragment thereof of claim 6, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 13 and the VL domain comprises the amino acid sequence of SEQ ID NO:

14.

8. 7. The antigen-binding protein or fragment thereof of claim 6, wherein the antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18, and the antibody light chain comprises the amino acid sequence of SEQ ID NO:

19.

9. (a) the VH domain has a CDR comprising the amino acid sequence GFTFSSYG (SEQ ID NO: 7); - comprising an H1 sequence, a CDR-H2 sequence comprising the amino acid sequence of FWYDGSNK (SEQ ID NO: 8), and a CDR-H3 sequence comprising the amino acid sequence of ARAPNWDDAFDI (SEQ ID NO: 9); and (b) the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of SGSVSTSYY (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of STN (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of VLFMGSGIWV (SEQ ID NO: 12), or a fragment thereof according to any one of claims 1 to 5.

10. 10. The antigen-binding protein or fragment thereof of claim 9, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 15 and the VL domain comprises the amino acid sequence of SEQ ID NO:

16.

11. 10. The antigen-binding protein or fragment thereof of claim 9, wherein the antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, and the antibody light chain comprises the amino acid sequence of SEQ ID NO:

22.

12. 12. The antigen-binding protein or fragment thereof of any one of claims 1 to 11, which binds a human CD73 polypeptide comprising the amino acid sequence of SEQ ID NO:

23.

13. 13. The antigen-binding protein or fragment thereof of any one of claims 1 to 8 and 12, which binds an epitope of human CD73 polypeptide comprising amino acids N96, G97, V98, E99, K121, P123, P156, F157, S159, N160, G162, T163, N164, L165, V166, F167, E168, R491, and D496 of SEQ ID NO:

23.

14. 13. The antigen-binding protein or fragment thereof of any one of claims 1 to 5 and 9 to 12, which binds an epitope of human CD73 polypeptide comprising amino acids P112, K119, A125, S126, S129, G130, L133, P134, Y135, K136, K180, L184, and N185 of SEQ ID NO:

23.

15. The antigen-binding protein or fragment thereof according to any one of claims 1 to 14, which is a chimeric or humanized antibody.

16. The antigen-binding protein or fragment thereof according to any one of claims 1 to 14, which is a human antibody.

17. The antigen-binding protein or fragment thereof according to any one of claims 1 to 16, which is a monoclonal antibody.

18. 18. The antigen-binding protein or fragment thereof of any one of claims 1 to 17, comprising one or more full-length antibody heavy chains including an Fc region.

19. 19. The antigen-binding protein or fragment thereof of claim 18, wherein the Fc region is a human IgG1 Fc region.

20. 20. The antigen-binding protein or fragment thereof of claim 19, wherein the human IgG1 Fc region comprises amino acid substitutions at one or more positions corresponding to positions 405 and 409 of human IgG1 according to the EU index, the amino acid substitutions being F405L and K409R.

21. A pharmaceutical composition comprising the antigen-binding protein or fragment thereof according to any one of claims 1 to 20 and a pharmaceutically acceptable carrier.

22. An isolated nucleic acid molecule encoding the antigen-binding protein or fragment thereof of any one of claims 1 to 20.

23. 23. An expression vector comprising the nucleic acid molecule of claim 22.

24. A host cell comprising the expression vector of claim 23.

25. A dual paratopic antigen-binding protein, the dual paratopic antigen-binding protein comprising binding specificity for a first CD73 epitope and a second CD73 epitope.

26. (a) a first VH domain having specificity for a first CD73 epitope, comprising a CDR-H1 sequence comprising the amino acid sequence of GGSIRNNY (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence of IYISGTT (SEQ ID NO: 2), and a CDR-H3 sequence comprising the amino acid sequence of AREHYVSGTSLDN (SEQ ID NO: 3); (b) a first VL domain having specificity for a first CD73 epitope, comprising a CDR-L1 sequence comprising the amino acid sequence of QSVNTNY (SEQ ID NO: 4), a CDR-L2 sequence comprising the amino acid sequence of GTS (SEQ ID NO: 5), and a CDR-L3 sequence comprising the amino acid sequence of QQDYNLPYT (SEQ ID NO: 6); (c) a second VH domain having specificity for a second CD73 epitope, comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 7), a CDR-H2 sequence comprising the amino acid sequence of FWYDGSNK (SEQ ID NO: 8), and a CDR-H3 sequence comprising the amino acid sequence of ARAPNWDDAFDI (SEQ ID NO: 9); and (d) a second VL domain having specificity for a second CD73 epitope, comprising a CDR-L1 sequence comprising the amino acid sequence of SGSVSTSYY (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of STN (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of VLFMGSGIWV (SEQ ID NO: 12).

26. The dual paratopic antigen binding protein of claim 25, comprising:

27. the first VH domain comprises the amino acid sequence of SEQ ID NO: 13; the second VH domain comprises the amino acid sequence of SEQ ID NO: 15; the first VL domain comprises the amino acid sequence of SEQ ID NO: 14; and The second VL domain comprises the amino acid sequence of SEQ ID NO: 16; 26. The dual paratopic antigen-binding protein of claim 25.

28. (a) the first antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18; (b) the second antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21; (c) the first antibody light chain comprises the amino acid sequence of SEQ ID NO: 19; and (d) the second antibody light chain comprises the amino acid sequence of SEQ ID NO: 22; 26. The dual paratopic antigen-binding protein of claim 25.

29. (a) the first antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 17; (b) the second antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 21; (c) the first antibody light chain comprises the amino acid sequence of SEQ ID NO: 19; and (d) the second antibody light chain comprises the amino acid sequence of SEQ ID NO: 22; 29. The dual paratopic antigen-binding protein of claim 28.

30. (a) the first antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 18; (b) the second antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 20; (c) the first antibody light chain comprises the amino acid sequence of SEQ ID NO: 19; and (d) the second antibody light chain comprises the amino acid sequence of SEQ ID NO: 22; 29. The dual paratopic antigen-binding protein of claim 28.

31. (a) the first VH and VL bind a first epitope of a human CD73 polypeptide comprising amino acids N96, G97, V98, E99, K121, P123, P156, F157, S159, N160, G162, T163, N164, L165, V166, F167, E168, R491, and D496 of SEQ ID NO: 23; and (b) the second VH and VL bind a second epitope of a human CD73 polypeptide comprising amino acids P112, K119, A125, S126, S129, G130, L133, P134, Y135, K136, K180, L184, and N185 of SEQ ID NO: 23; The dual paratopic antigen-binding protein of any one of claims 25 to 30.

32. 32. The dual paratopic antigen-binding protein of any one of claims 25 to 31, comprising a higher inhibitory activity against CD73 compared to one or both of the monospecific parent antibodies.

33. 32. The dual paratopic antigen-binding protein of any one of claims 25 to 31, comprising a higher inhibitory activity against CD73 compared to the combination of the monospecific parent antibodies.

34. 34. The dual paratopic antigen-binding protein of any one of claims 25 to 33, wherein the first VH and VL domains bind a first CD73 epitope on a first CD73 dimer molecule, and the second VH and VL domains bind a second CD73 epitope on a second CD73 dimer molecule.

35. 34. The dual paratopic antigen-binding protein of any one of claims 25 to 33, which is capable of cross-linking two or more CD73 dimer molecules.

36. 26. The dual paratopic antigen-binding protein of claim 25, generated by Fab arm exchange.

37. Fab arm exchange involves the following steps: (a) mixing a first parent monospecific antigen binding protein comprising an IgG1 Fc region comprising the amino acid substitution F405L according to the EU index, and a second parent monospecific antigen binding protein comprising an IgG1 Fc region comprising the amino acid substitution K409R according to the EU index, to produce a mixture; (b) subjecting the mixture of step (a) to reducing conditions to produce a reduced antigen-binding protein mixture containing dual-paratopic bispecific antigen-binding proteins; (c) subjecting the mixture of step (b) to oxidizing conditions to rearrange disulfide bonds between the heavy chains of the dual paratopic bispecific antigen-binding protein; and (d) isolating the dual paratopic bispecific antigen-binding protein.

37. The dual paratopic antigen-binding protein of claim 36, wherein the dual paratopic antigen-binding protein is

38. 37. The dual paratopic antigen-binding protein of claim 36, wherein the first parent monospecific antigen-binding protein and the second parent monospecific antigen-binding protein are mixed in equimolar amounts.

39. 37. The dual paratopic antigen binding protein of claim 36, wherein the reducing conditions are generated by adding a reducing agent.

40. 40. The dual paratopic antigen binding protein of claim 39, wherein the reducing agent comprises mercaptoethylamine (MEA).

41. 37. The dual-paratopic antigen-binding protein of claim 36, wherein the mixture of step (a) is subjected to reducing conditions at a temperature of about 18°C ​​to about 30°C for about 3 hours to about 6 hours.

42. 42. A method for treating a CD73-mediated disease or disorder in a subject, comprising administering to a subject in need thereof an antigen-binding protein or fragment thereof of any one of claims 1-20 and claims 25-41.

43. 43. The method of claim 42, wherein the CD73-mediated disease or disorder is cancer.

44. 1. A method for selecting a dual paratopic antigen binding protein that contains greater inhibitory activity against CD73 compared to one or more monospecific parent antibodies, comprising the steps of: a) combining two parent antibodies under conditions to form the dual paratopic antigen binding protein; b) testing the dual paratopic antigen-binding protein and one or both of the two parent antibodies in a CD73 activity assay; c) comparing the CD73 activity for the dual paratopic antigen-binding protein with the CD73 activity for one or both of the two parent antibodies; and d) selecting the dual paratopic antigen-binding protein if its CD73 activity is lower than the CD73 activity of one or both of the two parent antibodies. The method comprising:

45. 45. The method of claim 44, wherein the CD73 activity assay measures adenosine formation.

46. 46. ​​The method of claim 45, wherein adenosine formation is quantified by liquid chromatography-mass spectrometry (LC / MS).

47. 45. The method of claim 44, wherein the CD73 activity assay is performed using COR-L23 lung cancer cells that express human CD73.

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