RU2844809C1 - Anti-cd73 biparatope antibodies - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular to a biparatope antigen-binding protein which specifically binds to a first CD73 epitope and a second CD73 epitope.

EFFECT: invention is effective for treating a CD73-mediated disease or disorder in a subject.

18 cl, 15 dwg, 7 tbl, 5 ex

Description

RELATED APPLICATIONS

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

SEQUENCE LIST

[002] This application contains a sequence listing that was filed electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy of the listing, created on November 5, 2020, is named 711174_SA9-282PC_ST25.txt and is 35,615 bytes in size.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[003] The present invention relates to compositions and methods for producing bispecific antigen-binding proteins.

BACKGROUND OF THE INVENTION

[004] CD73 (ecto-5'-nucleotidase, NT5E) is a glycosylated, homodimeric, membrane-bound enzyme of 125 kDa that dephosphorylates adenosine monophosphate (AMP) in the extracellular environment to produce adenosine (ADO) (Allard et al. 2016. Immunotherapy 8:145-163). Adenosine has strong immunosuppressive effects in the tumor microenvironment, and as a result, CD73 has attracted wide interest as a target for cancer therapy (Allard et al. 2017. Immunological reviews 276:121-144; Allard et al. 2019. Immunology letters 205:31-39; Kats et al. 2018. International journal of molecular sciences 156:451-457; Sek et al. 2018. Int J Mol Sci. 19(12) pii: E3837; Yang et al. 2018. Current medicinal chemistry. 25:2260-2271). CD73 expression is associated with resistance to HER2 antibody therapy (Turcotte et al. 2017. Cancer research. 77:5652–5663), poor prognosis with reduced antitumor immune response in a variety of tumor types (Allard 2016, above) and increased tumor cell growth, migration, and invasion in vitro (Zhi et al. 2007. Clinical & experimental metastasis. 24:439-448). A number of clinical trials are currently underway using CD73-specific antibodies (Siu et al. 2018. Cancer research. 78:CT180-CT180) and small molecule inhibitors (Overman et al. 2018. Journal of Clinical Oncology. 36(15):4123), alone or in combination with adenosine A2a receptor antagonists and antibodies to other targets, in particular those in the PD-1/PD-L1 system (Leone et al. 2018. Journal for immunotherapy of cancer. 6:57). MEDI9447 (oleclumab), a CD73-specific internalizing antibody with moderate enzyme inhibition, has demonstrated some clinical efficacy as monotherapy and in combination with the PD-L1 blocker durvalumab (Hay et al. 2016. Oncoimmunology. 5: e1208875). However, there is a need in the field for CD73 antibodies with higher clinical efficacy as monotherapy and in combination with other therapeutic agents.

[005] There are also indications that CD73 antibodies may have effects independent of adenosine production. One study showed that enhanced immune responses in mice were mediated by FcγRIV engagement (Vijayan et al. 2017. Oncoimmunology. 6(5):e1312044), and another study suggested a role for CD73 internalization in suppressing metastasis (Overman 2018, above; Hay 2016, above; Terp et al. 2013. Journal of immunology. 191:4165–4173). However, adenosine levels in tumors can reach micromolar concentrations, and therefore incomplete inhibition of CD73 activity may be a limiting factor for the efficacy of currently available CD73-targeting therapeutics (Blay et al. 1997. Cancer Research. 57:2602–2605). Thus, the mechanism by which CD73 influences cancer progression may be complex, suggesting a requirement for potent inhibition of enzymatic activity or a combination of mechanisms to achieve optimal efficacy.

[006] Achieving potent inhibition of CD73 enzymatic activity (e.g., both high percentage inhibition and low EC50) with traditional monospecific CD73 antibodies can be challenging (Geoghegan et al. 2016. mAbs. 8:454-467; WO 2016055609A1; WO 2017118613). Accordingly, there is a need in the art to identify CD73 antibodies that achieve potent inhibition of CD73 enzymatic activity. Such CD73 antibodies may be useful in the treatment of diseases and disorders mediated by CD73.

ESSENCE OF THE INVENTION

[007] Disclosed herein are biparatopic binding proteins that bind to two different epitopes on CD73 and have the ability to achieve potent inhibition of CD73 activity. The binding proteins of the present invention are particularly suitable for the treatment of diseases and disorders mediated by CD73.

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

[009] 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.

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

[011] 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.

[012] 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.

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

[014] 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.

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

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

[017] 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.

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

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

[020] In certain embodiments, the antigen-binding protein binds an epitope in 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.

[021] In certain embodiments, the antigen-binding protein binds an epitope in 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.

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

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

[024] 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 a human IgG1 Fc region.

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

[026] In one aspect, the present invention provides a pharmaceutical composition comprising an antigen-binding protein or fragment thereof as defined above and a pharmaceutically acceptable carrier.

[027] In one aspect, the present invention provides an isolated nucleic acid molecule encoding an antigen-binding protein or fragment thereof as defined above.

[028] In one aspect, the present invention provides an expression vector comprising a nucleic acid molecule as defined above.

[029] In one aspect, the present invention provides a host cell comprising the expression vector defined above.

[030] In one aspect, the present invention provides a biparatopic antigen-binding protein that provides binding specificity for a first epitope of CD73 and a second epitope of CD73.

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

[032] 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.

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

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

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

[036] In certain embodiments, a biparatopic antigen-binding protein comprises (a) first VH and VL domains that bind a first epitope in 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) second VH and VL domains that bind a second epitope in 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.

[037] In certain embodiments, the biparatopic antigen-binding protein provides greater inhibitory activity against CD73 than one or both of the monospecific parent antibodies.

[038] In certain embodiments, the biparatopic antigen-binding protein provides greater inhibitory activity against CD73 compared to a combination of monospecific parent antibodies.

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

[040] In certain embodiments, the antigen-binding protein has the ability to crosslink two or more dimeric CD73 molecules.

[041] In certain embodiments, the biparatopic antigen-binding protein is produced by exchange of Fab fragments.

[042] In certain embodiments, the exchange of Fab fragments is accomplished by performing the following steps: (a) mixing a first parent monospecific antigen-binding protein comprising an IgG1 Fc region comprising an F405L amino acid substitution according to the EU index and a second parent monospecific antigen-binding protein comprising an IgG1 Fc region comprising an K409R amino acid substitution according to the EU index to form a mixture; (b) subjecting the mixture of step (a) to reducing conditions to form a mixture of reduced antigen-binding proteins comprising a biparatopic bispecific antigen-binding protein; (c) subjecting the mixture of step (b) to oxidizing conditions to re-form disulfide bonds between the heavy chains of the biparatopic bispecific antigen-binding protein; and (d) isolating the biparatopic bispecific antigen-binding protein.

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

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

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

[046] In another aspect, the present invention provides a method of treating a CD73-mediated disease or disorder in a subject, comprising administering to a subject in need thereof the above-mentioned antigen-binding protein or fragment thereof.

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

[048] In another aspect, the present invention provides a method for selecting biparatopic antigen-binding proteins that provide superior CD73 inhibitory activity compared to one or more monospecific parent antibodies, the method comprising the steps of a) combining two parent antibodies under conditions that provide for the formation of a biparatopic antigen-binding protein; b) testing the biparatopic antigen-binding protein and one or both of the two parent antibodies in a CD73 activity assay; c) comparing the CD73 activity in the presence of the biparatopic antigen-binding protein to the CD73 activity in the presence of one or both of the two parent antibodies; and d) selecting the biparatopic antigen-binding protein if the CD73 activity is less than the CD73 activity in the presence of one or both of the two parent antibodies.

[049] In certain embodiments, the CD73 activity assay measures the level of adenosine formation. In certain embodiments, the level of adenosine formation is quantified by liquid chromatography with mass spectrometry (LC/MS).

[050] In certain embodiments, the CD73 activity assay is performed using COR-L23 lung carcinoma cells expressing human CD73.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[051] The above and other features and advantages of the present invention will be more clearly understood from the following detailed description of illustrative embodiments in conjunction with the accompanying drawings. The set of materials of the present patent or application contains at least one drawing executed in color. Copies of this patent or patent application publication with color graphic(s) material(s) will be provided by the patent office upon request and payment of the required fee.

[052] Figure 1 depicts a screen for CD73 inhibitory activity on COR-L23 cells. The inhibition activity (%) of CD73 was determined after 4 hours of exposure to antibodies using an LC/MS-based assay with a heavy isotope-labeled AMP substrate (white: 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 rightmost square in each row, represents a bivalent antibody generated by combining the parent antibodies indicated on the horizontal and vertical axes. The rightmost square in each row represents the parent bivalent anti-CD73 antibody reconstituted using Fab exchange. The bottom row ("AS30") represents combinations with the irrelevant AS30 antibody to produce monovalent versions of the parent antibodies.

[053] Figure 2 shows the relative affinities of the parent and biparatopic antibodies to CD73. The antibodies (each parent antibody in monovalent form containing an irrelevant AS30 fragment and the biparatopic antibodies) were immobilized and exposed to a soluble form of CD73 in a flow-through manner.

[054] Figure 3 shows confirmation of biparatopic antibody formation by capillary isoelectric focusing (cIEF). DuoBody products and parent antibodies (4 μg each) were digested with IdeZ to yield F(ab')2 and Fc and separated by cIEF. Peaks with pIs between 9.0 and 9.5 were as follows: the right peak corresponded to the parent antibody E3.2(F405L), the left peak corresponded to the parent antibody H19(K409R), and the middle peak corresponded to the product of the Fab exchange reaction (cFAE) E3.2/H19. F(ab')2 fragments are characterized by pI values greater than 8.5. Peaks with pIs between 7.5 and 8 correspond to Fc. The peak with a value of 7.1 corresponds to IdeZ.

[055] Figure 4 shows the effective dose-dependent responses for 11 biparatopic antibodies, parent antibodies, and parent antibody mixtures. COR-L23 cells were incubated with antibodies for 3 hours, after which CD73 activity was determined by LS/MS-based analysis. Activity (proportion relative to no-antibody control) was plotted for biparatopic antibodies (light gray circles), parent antibody mixtures (black), and each of the two parent antibodies (dark gray circles and squares) are shown relative to total antibody concentration.

[056] Figures 5A-5D depict epitope-specific sorting by biolayer interferometry (Octet). A mixture of CD73 with a molar excess of Fab was incubated with monovalent parent antibodies immobilized on a solid support. Figure 5A shows a schematic representation of the assay format showing a state of non-overlapping epitopes and no blocking (upper panel) or overlapping epitopes achieving complete blocking of capture (lower panel). Figure 5B shows capture of CD73/Fab complexes by immobilized antibodies. Capture was normalized to the signal from CD73 alone in the absence of Fab. Figure 5C shows epitope-specific sorting based on inhibition of capture. Figure 5D shows inhibition of CD73 activity on COR-L23 cells compared to the ability of antibodies to capture the CD73/Fab complex in vitro . Gray filled circles: capture of CD73/Fab complex for the same antibody on the substrate (original pair).

[057] Figures 6A-6C depict the structure of TB19 with CD73. Figure 6A depicts the various conformational states of CD73 shown in schematic form. The N-terminal domain of CD73 and the C-terminal domain of CD73 are designated "N" and "C", respectively. The linkers connecting the two domains are indicated by gray helices. The zinc cofactors are shown as small gray spheres in the N-terminal domain. The substrate is depicted by an "S". Figure 6B depicts the two TB19 Fv domains binding a single CD73 dimer in an intermediate conformation, at two different angles. CD73 is colored the same as in Figure 6A , with the zinc and phosphate molecules shown in a black circle. The TB19 Fab is shown as a light gray schematic. Figure Figure 6C shows the mapping of TB19 epitope residues to CD73. The same color scheme is used as in Figure 6B , with CD73 epitope residues recognized by TB19 shown in light gray.

[058] Figures 7A-7B depict the spatial arrangement of CD73 monomer with Fv TB19. Figure 7A depicts TB19 bound to CD73 in a partially open conformation. The zinc and inorganic phosphate in the catalytic site are designated "Zn" and "Pi", respectively. The interacting residues (within 4 Å) in Fv TB19 and the N-terminal domain are shown as rods. For illustration, the substrate analog AMPCP (in rods) bound by the C-terminal domain of the closed conformational isomer structure 4H2I is superimposed on the TB19:CD73 structure to show its position and the interacting residues Phe417 and Phe500 of CD73. It should be noted that AMPCP is not present in the TB19 structure. Figure Figure 7B shows a modeling of TB19 relative to the closed conformational structure of 4H2I by superposing the N-terminal domains of CD73 of the two structures. The zinc ions and AMPCP β-phosphonate in 4H2I occupy the same positions as the zinc and inorganic phosphate in the structure of TB19-bound CD73. There is a conflict between the variable region of TB19 and the C-terminal domain of 4H2I.

[059] Figures 8A-8C depict the structures of TB38 with CD73. The N-terminal domain of CD73 is designated by "N", the C-terminal domain of CD73 is designated by "C", and the gray helix is a linker. Figure 8A shows the structure of Fab::CD73 TB38 with CD73 in an open conformation. Fab TB38 is shown and labeled. Figure 8B depicts the structure of Fv::CD73 TB38 with CD73 in an open/closed hybrid conformation. Fv TB38 is shown and labeled. Figure 8C depicts the mapping of TB38 epitope residues on CD73 (open/closed hybrid conformation) shown and labeled.

[060] Figures 9A-9B depict potential modes of CD73 co-engagement by the TB19/TB38 bispecific antibody. The bispecific antibodies were modeled based on the structures of TB19:CD73, TB38:CD73, and full-length IgG1 (PDB 1ZHZ). Figure 9A depicts a surface representation of the TB19/TB38 bispecific antibody. The distance between the last residues in the CH1 domains is shown as a black line. TB19 Fab, TB38 Fab, and Fc are indicated. Figure 9B depicts a model for four TB19/TB38 bispecific antibodies bound to CD73 in a partially open configuration. The N-terminal domains and the C-terminal domains of CD73 are shown. The distances separating the last residues of the CH1 domains are shown as black lines.

[061] Figure 10 depicts epitopes for TB19 and TB38 mapped to one subunit of the CD73 homodimer, shown in a partially open configuration as found in a co-crystal structure with TB19 (dark gray/light gray).

[062] Figures 11A-11C depict the structures of conformational isomers of CD73 related to the conceptual drawings in Figure 6 above. Figure 11A depicts a representation of CD73 conformations similar to those shown in Figure 6, displaying key features of each conformational isomer. Figure 11B depicts the actual structural equivalents for the schematic representations 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 configurations. Figure 11C depicts the structures of CD73 monomers with the C-terminal domains aligned, showing the rotation of the N-terminal domain in each of the 3 conformations: open, TB-19, and closed. The TB19 Fab is not shown for clarity. The view is from below relative to those in Figure 11B , perpendicular to the plane of rotation of the N-terminal domain. Shown are the C-terminal residue of the extracellular domain, zinc atoms in the structure of the closed conformational isomer of 4H2I, and the AMPCP substrate analog in 4H2I.

[063] Figure 12 shows the Fo-Fc OMIT map for zinc and phosphate atoms in the N-terminal domain structure with TB19 at 5 sigma. Circles: zinc; sticks: oxygen and phosphorus of the phosphate ion.

[064] Figures 13A-13B depict the results of initial fitting of Biacore sensorgrams to kinetic reaction data to assess bivalent binding of biparatopic antibodies to CD73 alone, as shown in Figure 2 above. The fitting results are presented based on a 1:1 Langmuir binding model. The kinetic indices based on these fitting results are shown in Table 6 . Biphasic association and dissociation kinetics were observed using multiple antibodies. In these cases (indicated by asterisks), the fitting procedures were performed at two concentrations of CD73 (32 nM and 12 nM) using an iterative process to obtain abundances and rate constants for each component during association and dissociation. Figure 13A depicts data for monovalent parent antibodies. Figure 13B depicts data for biparatopic antibodies.

[065] Figure 14 shows epitope mapping using a premix competition approach. Capture of Fab:CD73 complexes in solution by monovalent anti-CD73 antibodies on Protein A-coated biosensor tips was monitored using Octet. Names: name of antibody loaded onto tips. Sensorgram colors identify Fab pre-incubated with CD73 (see legend). Blue line: CD73 only. The initial increase at 300 s reflects IgG loading onto the tips. After washing, CD73 pre-mixed with excess Fab was used. Higher response levels reflect higher mass capture. Note that pre-incubation with Fab with an overlapping epitope will block capture and result in no change in the sensorgram. Capture of CD73 bound by a Fab at a non-overlapping epitope will result in a higher signal level than CD73 alone due to the larger size of the complex. Binding shift values normalized to CD73 are shown in Figure 5B.

[066] Figure 15 shows the half-life values of CD73 from immobilized monovalent parent and biparatopic antibodies. The half-life values shown are based on the first-order rate constants presented in Table 6. When biphasic kinetics are observed, the major and minor components and the fraction attributable to each are shown (in italics). nd: not detectable.

DETAILED DESCRIPTION

[067] Parental monospecific antigen-binding proteins directed against CD73 are provided. Biparatopic antigen-binding proteins directed against CD73 derived from the parental antigen-binding proteins are also provided. Methods of inhibiting CD73 activity and methods of treating diseases and disorders mediated by CD73 are also provided.

[068] In general, the nomenclature used in the context of cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein is that well known and commonly used in the art. The methods and techniques provided herein are generally performed in accordance with conventional methods well known in the art and described in various general and more specialized literature references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification procedures are performed in accordance with the manufacturer's descriptions, as commonly performed in the art, or as described herein. The nomenclature used in the context of analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry, and the corresponding laboratory procedures and techniques described herein are that well known and commonly used in the art. Standard techniques are used in chemical synthesis procedures, chemical analysis, pharmaceutical preparation, formulation and delivery, and in patient care.

[069] Unless otherwise defined herein, scientific and technical terms used herein have meanings commonly understood by those skilled in the art. In the event of any hidden ambiguity, the definitions provided herein take precedence over any dictionary or external definition. Unless the context otherwise requires, singular terms shall include the plural form, and plural terms shall include the singular form. 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.

[070] In order that the present invention may be more readily understood, definitions of certain terms are first provided.

CD73

[071] A CD73 monomer with N- and C-terminal domains that are connected via a flexible α-helical linker is expressed on the cell surface, with a C-terminal GPI anchor attached. In the physiological form, the two monomers associate via extensive non-covalent contacts between the C-terminal domains, forming 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-terminal and C-terminal domains, in addition to zinc cofactors bound by the N-terminal domain (Knapp 2012, above). Upon binding of the AMP substrate to the C-terminal domain, the N-terminal domain and zinc cofactors align with AMP into a "closed" conformation of CD73, in which catalysis occurs to produce the adenosine product. A major lateral rotation of the N-terminal domain then re-exposes the substrate-binding site to the "open" conformational isomer, allowing product release (Knapp 2012, (above). Limited solvent access to the active site in the closed conformational isomer indicates that cycling between the two forms is required for substrate binding and product release, i.e., efficient enzymatic activity (Knapp 2012, above).

Antigen binding proteins

[072] As used herein, the term "antibody" or "antigen-binding protein" refers to an immunoglobulin molecule that specifically binds or is capable of immunologically reacting with an antigen or epitope, and includes both polyclonal and monoclonal antibodies, as well as functional fragments of an antibody, including, but not limited to, antigen-binding fragments (Fab), F(ab') ₂ fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain variable fragments (scFv), and single-domain antibody fragments (e.g., sdAb, sdFv, nanobody). The term "antibody" includes genetically engineered or otherwise modified forms of immunoglobulins such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, media-activated antibodies, heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, tandem tri-scFv), etc. Unless otherwise specified, the term "antibody" should be understood to encompass functional fragments of an antibody.

[073] As used herein, the term "complementarity determining region" or "CDR" refers to non-contiguous amino acid sequences within the variable regions of an antibody that provide specificity and binding affinity for an antigen. In general, each heavy chain variable region contains three CDRs (CDR-H1, CDR-H2, CDR-H3) and each light chain variable region contains three CDRs (CDR-L1, CDR-L2, CDR-L3). As is known in the art, "framework regions" or "FRs" refer to portions of the heavy and light chain variable regions other than the CDRs. In general, each variable region of the heavy chain contains four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) and each variable region of the light chain contains four FRs (FR-L1, FR-L2, FR-L3, and FR-L4).

[074] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (numbering scheme according to "Kabat"), Al-Lazikani et al., (1997) JMB 273, 927-948 (numbering scheme according to "Chothia"), 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 M P et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains", Dev Comp Immunol, 2003 January; 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).

[075] 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 according to both the Kabat and Chothia schemes is based on the most frequently occurring lengths of antibody sequence regions, with insertions, which are accounted for by a letter designation for the insertion, such as "30a", and deletions occurring in some antibodies. The two schemes place certain insertions and deletions ("insertions/deletions") at different positions, resulting in different numbering. The contact scheme is based on the analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.

[076] Thus, unless otherwise indicated, a "CDR" or "complementarity determining region" or individually recited CDRs (e.g., "CDR-H1," "CDR-H2") of a given antibody or a region thereof, such as a variable region thereof, should be understood to encompass any (or the recited) complementarity determining region as defined by any of the known schemes. Likewise, unless otherwise indicated, a "FR" or "framework region" or individually recited FRs (e.g., "FR-H1," "FR-H2") of a given antibody or a region thereof, such as a variable region thereof, should be understood to encompass any (or the recited) framework region as defined by any of the known schemes. In some cases, the scheme for identifying a particular CDR or FR is recited, such as a CDR as defined by the Kabat, Chothia, or contact method. In other cases, the specific amino acid sequence of the CDR or FR is given.

Antigen binding proteins directed against CD73

[077] In one aspect, the present invention provides antigen-binding proteins with binding specificity for CD73. As used herein, the term "CD73" can refer to both a monomeric CD73 protein and a homodimeric CD73 complex formed by two non-covalently associated monomeric CD73 proteins.

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

[079] Table 1. CDR sequences of the antigen-binding protein directed against CD73

SEQ ID NO: Subsequence Note 1 GGSIRNNY TB19,3 CDR-H1 2 IYISGTT TB19,3 CDR-H2 3 AREHYVSGTSLDN TB19,3_CDR-H3 4 QSVNTNY TB19,3_CDR-L1 5 GTS TB19,3_CDR-L2 6 QQDYNLPYT TB19,3_CDR-L3 7 GFTFSSYG TB38,8 CDR-H1 8 FWYDGSNK TB38,8 CDR-H2 9 ARAPNWDDAFDI TB38,8_CDR-H3 10 SGSVSTSYY TB38,8_CDR-L1 11 STN TB38,8_CDR-L2 12 VLFMGSGIWV TB38,8_CDR-L3

[080] Table 2. VH/VL sequences of the antigen-binding protein directed against CD73

SEQ ID NO: Subsequence Note 13 QEQLQESGPGLVKPSETLSLTCTVSGGSIRNNYYNWIRQPAGKGLEWIGRIYISGTTNSNPSLKSRVTMSIDTSKNQFSLKLSSVTAADTAIYYCAREHYVSGTSLDNWGQGTLVTVSS TB19,3 VH 14 EIVMTQSPTTLSLSPGERATLSCRASQSVNTNYFSWYQQKPGLTPRLLIYGTSTRATGIPARFSGSGSGTDFTLTISSLQPEDFGIYYCQQDYNLPYTFGQGTYLEIK TB19,3 VL 15 QVQLVESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKGLEWVAVFWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLSAEDTAVYYCARAPNWDDAFDIWGQGTMVTVSS TB38,8 VH 16 QTVVTQEPSFSVSPGGTVTLTCGLSSGSVSTSYYPNWYQQTPGQAPRTLIYSTNTRSSGVPDRFSGSILGNKAALTITGAQADDESDYYCVLFMGSGIWVFGGGTKLTVL TB38,8 VL

[081] Table 3. Sequences of the antigen-binding protein directed against CD73

SEQ ID NO: Subsequence Note 17 QEQLQESGPGLVKPSETLSLTCTVSGGSIRNNYYNWIRQPAGKGLEWIGRIYISGTTNSNPSLKSRVTMSIDTSKNQFSLKLSSVTAADTAIYYCAREHYVS GTSLDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS R LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG TB19.3_huIgG1_
K409R - heavy chain 18 QEQLQESGPGLVKPSETLSLTCTVSGGSIRNNYYNWIRQPAGKGLEWIGRIYISGTTNSNPSLKSRVTMSIDTSKNQFSLKLSSVTAADTAIYYCAREHYV SGTSLDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF L LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG TB19.3_huIgG1_
F405L - heavy chain 19 EIVMTQSPTTLSLSPGERATLSCRASQSVNTNYFSWYQQKPGLTPRLLIYGTSTRATGIPARFSGSGSGTDFTLTISSLQPEDFGIYYCQQDYNLPYTFGQGTYLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TB19.3_huIgG1_
- light chain 20 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVFWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLSAEDTAVYYCARAPNWDDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT
LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS R LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG TB38.8_huIgG1_
K409R - heavy chain 21 QVQLVESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKGLEWVAVFWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLSAEDTAVYYCARAPN WDDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFL L YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG TB38.8_huIgG1_
F405L - heavy chain 22 QTVVTQEPSFSVSPGGTVTLTCGLSSGSVSTSYYPNWYQQTPGQAPRTLIYSTNTRSSGVPDRFSGSILGNKAALTITGAQADDESDYYCVLFMGSGIWVFGGGTKLT VLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS TB38.8_huIgG1_
- light chain

[082] In certain embodiments, the antigen-binding proteins directed against CD73 of the present invention comprise 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 of Table 1 , Table 2 , or Table 3 .

[083] In certain embodiments, the antigen-binding proteins directed against CD73 of the present invention bind a human CD73 polypeptide comprising the amino acid sequence of SEQ ID NO: 23, shown in Table 4 below.

[084] In certain embodiments, the antigen-binding proteins directed against CD73 of the present invention bind an epitope in 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, shown in Table 4 below.

[085] In certain embodiments, the antigen-binding proteins directed against CD73 of the present invention bind an epitope in 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: 24, shown in Table 4 below.

[086] Table 4. Human CD73 and epitopes

SEQ ID NO: Subsequence Note 23 WELTILHTNDVHSRLEQTSEDSSKCVNASRCMGGVARLFTKVQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNALRYDAMALGNHEFD NGVE GLIEPLLKEAKFPILSANIKA K G P LASQISGLYLPYKVLPVGDEVVGIVGYTSKET PF L SN P GTNLVFE DEITALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRF RECNMGNLICDAMINNNLRHTDEMFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELL R HDSG D QDINVVSTYISKMKVIYPAVEGRIKFS Human CD73
TB19.3 epitope (highlighted in bold and underlined)
Residues N96, G97, V98, E99, K121, P123, P156, F157, S159, N160, G162, T163, N164, L165, V166, F167, E168, R491, D496 24 WELTILHTNDVHSRLEQTSEDSSKCVNASRCMGGVARLFTKVQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNALRYDAMALGNHEFDNGVEGLIEPLLKEAKF P ILSANI K AKGPL AS QI SG LY LPYK VLPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDEITALQPEVD K LKT LN VNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHTDEMF WNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKFS Human CD73
Epitope TB38.8 (highlighted in bold and underlined)
Residues P112, K119, A125, S126, S129, G130, L133, P134, Y135, K136, K180, L184, N185

Biparatopic antigen-binding proteins directed against CD73

[087] In one aspect, the present invention provides biparatopic antigen-binding proteins with binding specificity for a first epitope of CD73 and a second epitope of CD73. As used herein, a "biparatopic" antigen-binding protein binds two different epitopes on the same molecular target (i.e., biparatopic). In the present invention, a biparatopic antigen-binding protein directed against CD73 is derived from two parent monospecific antigen-binding proteins specific for CD73. Each of the two parent antigen-binding proteins binds a different epitope on the CD73 molecule.

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

[089] In certain embodiments, the biparatopic antigen-binding proteins of the present invention provide higher CD73 inhibitory activity than one or both of the monospecific parent antibodies used to produce each biparatopic antigen-binding protein. In certain embodiments, the biparatopic antigen-binding proteins of the present invention provide higher CD73 inhibitory activity than a combination of the monospecific parent antibodies used to produce each biparatopic antigen-binding protein. Inhibition of CD73 activity can be determined by any method known in the art. In certain embodiments, CD73 activity is determined using COR-L23 cells expressing CD73, as described below in Example 1 and in McManus et al. 2018. SLAS discovery: advancing life sciences R & D 23, 264-273.

[090] In certain embodiments, the biparatopic antigen-binding proteins directed against CD73 of the present invention bind to two different CD73 epitopes on the same CD73 molecule. The biparatopic antigen-binding proteins directed against CD73 can bind to two different CD73 epitopes on the same monomeric CD73 protein. The biparatopic antigen-binding proteins directed against CD73 can bind to two different CD73 epitopes on the same homodimeric CD73 protein.

[091] In certain embodiments, the biparatopic antigen-binding proteins directed against CD73 of the present invention bind to two different epitopes of CD73 on two separate CD73 molecules. In certain embodiments, the first VH and VL domains of the biparatopic antigen-binding protein directed against CD73 bind a first epitope of CD73 on a first dimeric or homodimeric CD73 molecule, and the second VH and VL domains bind a second epitope of CD73 on a second dimeric or homodimeric CD73 molecule.

[092] In certain embodiments, the biparatopic antigen-binding proteins directed against CD73 of the present invention may have the ability to cross-link two or more dimeric CD73 molecules. As used herein, "cross-linking" by antibodies can occur when a first binding site on a multivalent antibody binds a first epitope on a first target molecule while a second binding site on the multivalent antibody binds a second epitope on a second target molecule. Cross-linking of multiple target molecules through the binding of multiple bivalent antibodies can result in the formation of higher order structures with increased stability. This can result in a decrease in the koff of the cross-linked antigen-binding proteins relative to the uncross-linked antigen-binding proteins. By enhancing the cross-linking of antigen-binding proteins, antigen-binding proteins with weak antigen-binding affinity can be used. Certain antigen-binding proteins that have weak binding affinity for their target antigen generally have limited utility. By combining antigen-binding proteins with weak binding affinity, the cross-linking effect of the present invention can increase their efficiency by reducing the koff value.

Methods of heterodimerization of antigen-binding proteins

[093] The biparatopic CD73 antigen-binding proteins of the present invention can be formed by heterodimerization of the parent CD73 antigen-binding proteins. Any heterodimerization method known in the art can be used to form the biparatopic CD73 antigen-binding proteins.

[094] In certain exemplary embodiments, two Fc domains of an antibody or antigen-binding fragment thereof heterodimerize via Fab exchange (FAE). In certain exemplary embodiments, a human CH3 sequence other than that of IgG4 is modified such that it does not contain any amino acid residues that participate in the formation of disulfide bonds or covalent or stable non-covalent bonds between heavy chains with other peptides comprising the identical amino acid sequence of the CH3 region. Such a modified CH3 sequence may be similar to the CH3 of IgG4. In certain embodiments, the antibody is IgG1 and is modified such that it is IgG4-like.

[095] An exemplary FAE method may comprise the steps of a) providing a first antigen-binding construct characterized by a first binding specificity, wherein said first antigen-binding construct comprises a CH3 region similar to the CH3 of IgG4; b) providing a second antigen-binding construct characterized by a second binding specificity that differs from said first binding specificity, wherein said second antigen-binding construct comprises a CH3 region similar to the CH3 of IgG4; c) co-incubating said first and second antigen-binding constructs under reducing conditions that allow cysteine residues in the core hinge region to undergo disulfide bond isomerization; and d) producing a bispecific antigen-binding construct.

[096] The term "CH3 region similar to CH3 of IgG4" refers to a CH3 region that is identical to the CH3 of IgG4, such as human IgG4, or a CH3 region that is functionally equivalent to a CH3 region of IgG4. The term "functionally equivalent" in this context means that the CH3 region, like the CH3 region of IgG4, does not form stable interactions between half molecules. The formation of stable interacting half molecules by a given CH3 region can, for example, be tested by replacing the CH3 of IgG4 with the CH3 region and testing for exchange under the conditions described in U.S. Patent No. 9,212,230, incorporated herein by reference. If exchange is observed, then the formation of stable interactions between half molecules is not observed. For example, a CH3 region similar to CH3 of IgG4 may be a CH3 region that is equally effective in allowing half-molecule exchange compared to a CH3 region of IgG4. Accordingly, a CH3 region similar to CH3 of IgG4 may be structurally similar to a CH3 region of IgG4, such as being more than 75%, such as more than 90%, identical to a CH3 region of IgG4. However, the CH3 region similar to CH3 of IgG4 in the context of the present invention may additionally or alternatively be a CH3 region which is not structurally related to the CH3 region of IgG4, but has similar functional characteristics, which is due to the fact that it does not contain any amino acid residues that are involved in the formation of disulfide bonds or covalent or stable non-covalent bonds between the heavy chains, such as salt bridges, with other peptides containing an identical amino acid sequence of the CH3 region. For example, the CH3 region similar to CH3 of IgG4 may be a mutant CH3 region of IgG1, in which one or more amino acid residues that are involved in CH3-CH3 interactions between the half molecules have been changed or deleted.

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

[098] In certain exemplary embodiments, two Fc domains of an antigen-binding construct undergo heterodimerization via knob-into-hole pairing. This dimerization technique utilizes "bumps" or "knobs" with "cavities" or "holes" engineered onto the surface of the CH3 domains. In the event that there is a knob or hole of suitable position and size on the surface of either the first or second CH3 domain, it is only necessary to engineer a corresponding hole or knob, respectively, on the adjacent surface, thereby facilitating the Fc domain pairing that occurs on the surface of the CH3/CH3 domain and also reinforcing it. An IgG Fc domain that is fused to a VHH is provided with a knob, and an IgG Fc domain from a conventional antibody is provided with a hole designed to accommodate the knob, or vice versa. "Knob" refers to at least one amino acid side chain, typically a larger side chain, that projects from the surface of the CH3 portion of a first Fc domain. The protrusion creates a "knob" that is complementary to and fits into a "hole" in the CH3 portion of a second Fc domain. The "hole" is at least one amino acid side chain, typically a smaller side chain, that projects from the surface 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 P. J. Immunol. Methods (2001) 248: 7-15.

[099] Exemplary amino acid residues that can act as a protrusion include arginine (R), phenylalanine (F), tyrosine (Y), and/or tryptophan (W). An existing amino acid residue in the CH3 domain can be replaced or substituted with an amino acid residue that provides for the formation of a protrusion. Exemplary amino acids for substitution can 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).

[0100] Exemplary amino acid residues that can act as a hole include alanine (A), serine (S), threonine (T), or valine (V). An existing amino acid residue in the CH3 domain can be replaced or substituted with an amino acid residue that provides for the formation of a hole. Exemplary amino acids for substitution can include amino acids with a large side chain, such as arginine (R), phenylalanine (F), tyrosine (Y), and/or tryptophan (W).

[0101] In certain exemplary embodiments, the CH3 domain is derived from a human IgG1 antibody. Exemplary amino acid substitutions for the CH3 domain include T366Y, T366W, F405A, F405W, Y407T, Y407A, Y407V, T394S, or combinations thereof. A particular exemplary combination is T366Y or T366W for a knob mutation on the first CH3 domain and Y407T or Y407V for a hole mutation on the second CH3 domain.

[0102] In certain exemplary embodiments, two Fc domains of an antigen-binding construct undergo heterodimerization via electrostatic coordination effects. This dimerization technique utilizes electrostatic coordination to promote and enhance pairing of the Fc domain on the surface of the CH3/CH3 domain. The charge complementarity between the two CH3 domains is altered to promote heterodimerization (pairing of opposite charges) over homodimerization (pairing of like charges). In this method, electrostatic repulsive forces prevent homodimerization.

[0103] An exemplary amino acid residue substitution may 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 A1 and Gunasekaran K. JBC (2010) 285(25):19637-19646.

[0104] In certain exemplary embodiments, two Fc domains of an antigen-binding construct undergo heterodimerization via hydrophobic interaction effects. This dimerization technique utilizes hydrophobic interactions instead of electrostatic interactions to promote and enhance Fc domain pairing at the surface of the CH3/CH3 domains. An exemplary amino acid residue substitution can 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. Illustrative pairs of amino acid residue substitutions between the first CH3 domain and the second CH3 domain include K409W:D399V, K409W:F405T, K360E:Q347R, Y349S:E357W, and S354C:Y349C. This technology is described in U.S. Patent Publication No. 2015/0307628 A1.

Expression of antigen-binding proteins

[0105] In one aspect, polynucleotides encoding the binding proteins (e.g., antigen-binding proteins) disclosed herein are provided. Methods of producing the binding proteins comprising expressing these polynucleotides are also provided.

[0106] Polynucleotides encoding the binding proteins disclosed herein are typically inserted into an expression vector for introduction into host cells that can be used to produce a desired amount of the claimed antibodies or fragments thereof. Accordingly, in certain aspects, the present invention provides expression vectors comprising the polynucleotides disclosed herein and host cells comprising the vectors and polynucleotides.

[0107] As used herein, the term "vector" or "expression vector" refers to vectors used in accordance with the present invention as a carrier medium for introducing a desired gene into a cell and expressing it therein. As is known to those skilled in the art, such vectors can be readily selected from the group consisting of plasmids, phages, viruses, and retroviruses. In general, vectors compatible with the present invention will contain a selection marker, suitable restriction sites to allow cloning of the desired gene, and the ability to enter and/or replicate in eukaryotic or prokaryotic cells.

[0108] Numerous expression vector systems can be used for the purposes of the present invention. For example, one class of vectors uses DNA elements derived from animal viruses such as bovine papillomavirus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV, or MOMLV), or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites. In addition, cells with integrated DNA in their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. The marker can provide prototrophy to an auxotrophic host, resistance to biocides (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be directly linked to the DNA sequences to be expressed or introduced into the same cell by co-transformation. Additional elements may also be necessary for optimal mRNA synthesis. These elements may include signal sequences, splicing 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 the heavy and light chain constant region genes (e.g., human constant region genes) by synthesis, as discussed above.

[0109] In other embodiments, the binding polypeptides can be expressed using polycistronic constructs. In such expression systems, multiple gene products of interest, such as the heavy and light chains of antibodies, can be produced from a single polycistronic construct. These systems advantageously utilize an internal ribosome entry site (IRES) to produce 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 appreciate that such expression systems can be used to efficiently produce the full range of polypeptides disclosed herein.

[0110] More generally, once a vector or DNA sequence encoding an antibody or fragment thereof has been prepared, the expression vector can be introduced into a suitable host cell. That is, the host cells can be transformed. Introduction of a plasmid into a 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 with enveloped DNA, microinjection, and infection with intact virus. See Ridgway, A. A. G. "Mammalian Expression Vectors" Chapter 24.2, pp. 470-472 Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988). Introduction of a plasmid into a host cell can be accomplished by electroporation. The transformed cells are grown under conditions suitable for the production of light chains and heavy chains and analyzed for heavy and/or light chain protein synthesis. Exemplary assays include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or fluorescence-activated cell sorter (FACS) analysis, immunohistochemistry, etc.

[0111] As used herein, the term "transformation" shall be used in a broad sense to refer to the introduction of DNA into a recipient host cell that alters the genotype and subsequently results in a change in the recipient cell.

[0112] Additionally, the term "host cells" refers to cells that have been transformed with vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene. In descriptions of processes for isolating polypeptides from recombinant hosts, the terms "cell" and "cell culture" are used interchangeably to indicate the source of the antibody, unless explicitly stated otherwise. In other words, recovery of a polypeptide from "cells" may mean either recovery from whole cells pelleted by centrifugation or recovery from a cell culture containing both medium and suspended cells.

[0113] In one embodiment, the host cell line used to express the antibody is mammalian. Those skilled in the art can determine particular host cell lines that are best suited to express the desired gene product. Illustrative host cell lines include, but are not limited to, DG44 and DUXB11 (DHFR-negative Chinese hamster ovary cells), HELA (human cervical carcinoma cells), CV-1 (monkey kidney cell line), COS (SV40 T-antigen derivative of CV-1), R1610 (Chinese hamster fibroblasts), BALBC/3T3 (mouse fibroblasts), HAK (hamster kidney cell line), SP2/O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), and 293 (human kidney cells). In one embodiment, the cell line provides altered glycosylation, such as afucosylation, of the antibody expressed therein (e.g., PER.C6® (Crucell) or FUT8 knockout CHO (Potelligent® cells) (Biowa, Princeton, NJ) cell lines). In one embodiment, NS0 cells can be used. CHO cells are particularly useful. Host cell lines are generally available from commercial services, such as the American Tissue Culture Collection, or from published literature.

[0114] In vitro production allows for scale-up to produce large quantities of the desired polypeptides. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include culturing in a homogeneous suspension, such as in an airlift reactor or in a continuously stirred reactor, or culturing immobilized or captured cells, such as in hollow fibers, microcapsules, on agarose microbeads, or in ceramic cartridges. If necessary and/or desired, polypeptide solutions can be purified using conventional chromatographic techniques, such as gel filtration, ion exchange chromatography, DEAE-cellulose chromatography, and/or (immuno-)affinity chromatography.

[0115] The genes encoding the binding polypeptides of the present invention can also be expressed in cells other than mammalian cells, such as bacteria or yeast or plant cells. In this regard, it will be appreciated that various non-mammalian unicellular microorganisms, such as bacteria, can also be transformed, i.e., those that can be grown in cultures or fermentations. Bacteria susceptible to transformation include members of the Enterobacteriaceae, such as strains of Escherichia coli or Salmonella ; Bacillaceae , such as Bacillus subtilis ; Pneumococcus ; Streptococcus and Haemophilus influenzae . It will further be appreciated that when expressed in bacteria, the polypeptides can become part of inclusion bodies. The polypeptides should be isolated, purified and then assembled into functional molecules.

[0116] In addition to prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae , or common baker's yeast, is the most commonly used eukaryotic microorganism, although a number of other strains are widely available. For expression in Saccharomyces , for example, the YRp7 plasmid is widely used (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)). This plasmid already contains the TRP1 gene, which is a selection marker for a mutant yeast strain that lacks the ability to grow on tryptophan, such as ATCC #44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). The presence of a TRP1 lesion as a characteristic of the yeast host cell genome in this case provides an effective medium for detecting transformation by growth in the absence of tryptophan.

Methods of administration of antigen-binding proteins

[0117] Methods for producing and administering binding proteins (e.g., antigen-binding proteins disclosed herein) to a subject are well known to those skilled in the art or can be readily determined by them. The route of administration of the binding proteins of the present invention can be oral, parenteral, inhalation, or topical. As used herein, the term "parenteral" includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. Although all of these forms of administration are clearly considered to be within the scope of the present invention, the form of administration can be an injection solution, particularly for intravenous or intraarterial injection or drip administration. A suitable pharmaceutical composition for injection may typically contain a buffer (e.g., acetate, phosphate, or citrate buffer), a surfactant (e.g., polysorbate), optionally a stabilizer (e.g., human albumin), etc. However, in other methods consistent with the teachings herein, the modified antibodies may be delivered directly to the site of the unwanted cell population, thereby increasing the exposure of the therapeutic agent to the affected tissue.

[0118] Parenteral preparations include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include 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 invention, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1 M or 0.05 M phosphate buffer or 0.8% saline. Other common parenteral carrier media include sodium phosphate solutions, Ringer's dextrose solution, dextrose and sodium chloride solution, lactated Ringer's solution, or fixed oils. Carrier media for intravenous administration include solutions with added fluid and nutrients, solutions with added electrolytes, such as based on Ringer's dextrose solution, etc. Preservatives and other additives such as, for example, antimicrobial agents, antioxidants, chelating agents and inert gases, etc. may also be present. More specifically, pharmaceutical compositions suitable for injection use include sterile aqueous solutions (in the case of water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injection solutions or dispersions. In such cases, the composition must be sterile and must be fluid to such an extent that it is easy to administer by syringe. It must be stable under the conditions of manufacture and storage, and must also be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion and by using surfactants.

[0119] Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. Isotonic agents, such as sugars, polyhydric alcohols such as mannitol, sorbitol, or sodium chloride, can also be included in the composition. Prolonged absorption of injection compositions can be ensured by including in the composition an agent that slows down absorption, such as aluminum monostearate and gelatin.

[0120] In any case, sterile injectable solutions can be prepared by incorporating the active compound (e.g., a modified binding polypeptide, alone or in combination with other active ingredients) in the required amount in an appropriate solvent together with one or a combination of ingredients listed herein, optionally followed by filtered sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile carrier medium that contains a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation typically include vacuum drying and freeze-drying, which yield a powder form of the active ingredient with any additional required ingredient from a previously filtered sterilization solution thereof. Injectable preparations are processed, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art. Additionally, the preparations may be packaged and sold as a kit, such as those described in co-pending U.S.S.N. 09/259,337 and U.S.S.N. 09/259,338, each of which is incorporated herein by reference. Such articles may include labels or package inserts indicating that the compositions are useful for treating a subject suffering from or predisposed to autoimmune or neoplastic disorders.

[0121] Effective doses of the compositions of the present invention for treating the conditions described above vary depending on a variety of factors, including the route of administration, the target site, the physiological state of the patient, whether the patient is a human or an animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but it is also possible to treat non-human mammals, including transgenic mammals. Treatment doses can be adjusted using standard techniques known to those skilled in the art to optimize safety and efficacy.

[0122] For passive immunization using a binding polypeptide, the dosage can be in the range of, for example, about 0.0001 mg/kg to 100 mg/kg, and more typically 0.01 mg/kg to 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.), based on the body weight of the host. For example, dosages can be 1 mg/kg body weight or 10 mg/kg body weight, or within the range of 1-10 mg/kg, such as at least 1 mg/kg. Intermediate dosages in the above ranges are also intended to be within the scope of the present invention. Subjects can be administered such dosages daily, every other day, weekly, or any other schedule determined by empirical analysis. Exemplary treatment includes administration in multiple doses over an extended period, such as at least six months. Additional exemplary treatment regimens include once every two weeks, or once monthly, or once every 3-6 months. Exemplary dosing regimens include administration of 1-10 mg/kg, or 15 mg/kg on consecutive days, 30 mg/kg on alternate days, or 60 mg/kg weekly. In some methods, two or more binding proteins with different binding specificities are administered simultaneously, in which case the dose of each antibody administered is within the ranges indicated.

[0123] The binding proteins described herein can be administered multiple times. The intervals between individual dose administrations can be weekly, monthly, or yearly. The intervals can also be irregular and determined by measuring the levels of the modified binding polypeptide or antigen in the patient's blood. In some methods, the dose is adjusted to achieve a plasma concentration of the modified binding polypeptide of 1-1000 μg/mL, and in some methods 25-300 μg/mL. Alternatively, the binding polypeptides can be administered as a sustained release formulation, in which case less frequent administration is required. For antibodies, the dose and frequency vary depending on the half-life of the antibody in the patient. Generally, humanized antibodies have the longest half-life, followed by chimeric antibodies and non-human antibodies.

[0124] The dose and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic use, compositions containing the antibodies of the present invention or a cocktail thereof are administered to a patient who is not yet in a state of disease, to enhance the patient's resistance. Such an amount is defined as a "prophylactically effective dose". In such use, the exact amounts again depend on the health condition and general immunity of the patient, but are typically in the range of 0.1 to 25 mg per dose, in particular 0.5 to 2.5 mg per dose. A relatively low dose is administered at relatively long intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dose (e.g., about 1 to 400 mg/kg antibody per dose, with doses of 5 to 25 mg more commonly used for radioimmunoconjugates and higher doses used for antibodies modified with a cytotoxic drug) is sometimes required at relatively short intervals until disease progression is reduced or stopped or until the patient exhibits partial or complete amelioration of disease symptoms. The patient can then be administered prophylactically.

[0125] 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., prophylactically or therapeutically). Effective single therapeutic doses (i.e., therapeutically effective amounts) of ^{the 90} Y-labeled modified antibodies of the present invention are in the range of about 5 to about 75 mCi, such as about 10 to about 40 mCi. Effective single therapeutic doses of ¹³¹ I-modified antibodies that are not intended for bone marrow ablation are in the range of about 5 mCi to about 70 mCi, such as about 5 mCi to about 40 mCi. Effective single therapeutic doses of ¹³¹ I-labeled antibodies intended for ablation (i.e., which may require autologous bone marrow transplantation) are in the range of about 30 mCi to about 600 mCi, such as about 50 mCi to less than about 500 mCi. In combination with a chimeric antibody, due to its longer circulating half-life compared to murine antibodies, an effective single therapeutic dose of ¹³¹ I-labeled chimeric antibodies not intended for bone marrow ablation is in the range of about 5 mCi to about 40 mCi, such as less than about 30 mCi. The criteria for ensuring imaging in the case of, for example, ¹¹¹ In labeling typically provide for less than about 5 mCi.

[0126] While the binding polypeptides can be administered as directly described above, it should be emphasized that in other embodiments, the binding polypeptides can be administered to otherwise healthy patients as a first-line therapy. In such embodiments, the binding polypeptides can be administered to patients who have normal or moderate red marrow reserves and/or to patients who have not undergone and are not undergoing one or more other therapies. As used herein, administration of the modified antibodies or fragments thereof in conjunction or in combination with an adjuvant therapy means sequential, simultaneous, coeval, parallel, concomitant, or coinciding administration or use of the therapy and the disclosed antibodies. Those skilled in the art will appreciate that the administration or use of the various components of a combination therapy regimen can be timed to enhance the overall effectiveness of the treatment. One skilled in the art (e.g., an experienced oncologist) will be able to readily discern effective combination therapy regimens without undue experimentation based on the selected adjuvant therapy and the teachings of the present invention.

[0127] As discussed previously, the binding polypeptides of the present invention, immunoreactive fragments thereof, or recombinant variants thereof can be administered in a pharmaceutically effective amount to treat disorders in mammals in vivo. In this regard, it will be appreciated that the disclosed binding polypeptides will be formulated to facilitate administration and ensure stability of the active agent.

[0128] The pharmaceutical compositions of the present invention typically include a pharmaceutically acceptable, non-toxic sterile solvent such as saline, non-toxic buffers, preservatives, and the like. For the purposes of the present application, a pharmaceutically effective amount of a modified binding polypeptide, immunoreactive fragment or recombinant variant thereof, conjugated or unconjugated to a therapeutic agent, is to be understood to mean an amount sufficient to achieve effective binding to an antigen and achieve a beneficial effect, such as ameliorating the symptoms of a disease or disorder, or detecting a substance or cell. In the case of tumor cells, the modified binding polypeptide will typically be capable of interacting with selected immunoreactive antigens on neoplastic or immunoreactive cells and providing an increase in the level of cell death of these cells. Of course, the pharmaceutical compositions of the present invention can be administered in single or multiple doses to provide a pharmaceutically effective amount of the modified binding proteins.

[0129] In accordance with the scope of the present invention, the binding proteins of the present invention can be administered to a human or other animal according to the above-mentioned methods of treatment in an amount sufficient to obtain a therapeutic or prophylactic effect. The binding polypeptides of the present invention can be administered to such a human or other animal in a conventional dosage form prepared by combining an antibody of the present invention with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. One skilled in the art will understand that the form and type of pharmaceutically acceptable carrier or diluent is determined by the amount of active ingredient with which they are to be combined, the route of administration, and other well-known variables. One skilled in the art will also understand that a cocktail containing one or more types of binding polypeptides described in the present invention can be particularly effective.

[0130] It will be apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein can be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. Having described certain embodiments in detail, they will be better understood with reference to the following examples, which are included for illustrative purposes only and are not intended to be limiting.

EXAMPLES

Example 1. Experimental procedures

Obtaining biparatopic antibodies

[0131] CD73-specific monoclonal antibodies were isolated using widely used mouse immunization and phage display approaches using a soluble form of human CD73 as the antigen (data not shown). Twelve sequence-unrelated parent antibodies with IC50s in the range of 1-25 nM and at least 50% inhibition of CD73 demonstrated in cell-based assays at saturating antibody concentrations were selected for testing.

[0132] Bispecific variants were generated using a modification of the published Duobody procedure (Gramer et al. 2013. mAbs 5, 962-973), except for the use of microdialysis to purify the product. Equimolar amounts of F405L and K409R Fc variants from each huIgG1 parent (25-50 μg each) were combined in a total volume of 90 μL of PBS supplemented with 10 μL of 7.5 M mercaptoethylamine (MEA), pH 7.4. The mixture was incubated for 4 h at 30°C in a forced air incubator, transferred to individual cassettes taken from 96-well dialysis plate strips (Pierce), and dialyzed for three cycles (1 h, 1.5 h, and overnight) at room temperature. For more than 6 samples, reactions were transferred to cassette dialysis strips mounted on a carrier plate. The plate was suspended over the reservoir and transferred between reservoirs containing fresh PBS after each dialysis cycle. After the second dialysis, the total free thiol level in the retentate was below the detection limit using DTNB. The final products were stored at 4°C. Product formation was determined by cIEF. The parent antibodies for the assay were re-engineered by cross-hybridizing the parent antibodies to F405L and K409R in the same manner as for the Duobody test products.

Characterization of biparatopic antibodies

[0133] The formation of Duobody products from the Fab fragment exchange reaction (cFAE) was determined using capillary isoelectric focusing (cIEF) (Maurice, Protein Simple, San Jose, CA). This approach was chosen because the expected pI for the bispecific progeny molecules was in the range between that of each of the two parent antibodies. To enhance the relative impact of charge differences in the CDR and framework regions, cIEF was performed on the F(ab')2 fragments generated by IdeZ digestion of the cFAE products. The cFAE product (4 μL, 1 mg/mL) was mixed with 4 μL of IdeZ at a concentration of 1 U/μL (Fabricator Z, Genovis) in water and mixed by trituration. The tubes were incubated for 4 hours at 37°C in a ventilated incubator followed by the addition of 36 µl of 1.1x methylcellulose/ampholine Pharmalyte, mixed and centrifuged for 4 minutes at 13 kG. The supernatant (30 µl) was transferred to a 96-well assay plate. Samples were loaded onto a cIEF cassette for 55 seconds and focused for 1.5 minutes at 1.5 kV followed by 6 minutes at 3 kV. Separated products were detected by fluorescence. Formation of the desired Duobody product was assessed by the disappearance of the parental antibody F(ab')2 peaks and the formation of an F(ab')2 peak with a pI close to the average of the two parental F(ab')2, along with the absence of the parental antibody Fc peak with F405L at ~pI 7.6. The Fc fragment of the Duobody product with both mutations (F405L:K409R) was not separated from the parent antibody with K409R, probably due to the limited pKa change of arginine in the environment surrounding this residue. IdeZ focusing was performed at pI 7.14 and below. An illustrative result is shown in Fig. 3 .

Biparatopic binding assay

[0134] The ability of biparatopic antibodies to interact bivalently with CD73 (e.g., with two epitopes) was determined by comparing the affinity of biparatopic antibodies and monovalent antibodies using surface plasmon resonance (SPR). Monovalent antibodies were used to prevent bivalent interactions with CD73. SPR was performed on a Biacore T200 device (GE Healthcare) at 25°C using HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% (v/v) P20 surfactant, pH 7.4) as the running buffer and S-series Protein A sensor chips (GE Healthcare). To minimize avidity effects due to CD73 binding by individual monovalent antibodies on the chip, binding was detected at a very low response level (less than 10 RU). Antibodies were threshold-cut diluted to 5-30 RU during a 30-second injection at 10 μl/min. CD73 at several concentrations (32 nM, 12 nM, and 3 nM) was then passed over the captured antibodies for 5 minutes at 30 μl/min. Dissociation was measured over 30 minutes. The sensor surface was then regenerated with 10 mM glycine-HCl pH 1.5 for 30 seconds at 20 μl/min. Kinetic constants were calculated using a 1:1 Langmuir binding model using Biacore T200 evaluation software (GE Healthcare). The 1:1 Langmuir binding model was not applied for cases where the fitting results for the bivalents, performed using the BiaEvaluation software, showed lower apparent residue levels, raising the possibility of biphasic binding. In these cases, kd values for each component during dissociation were determined starting with the fitting of the component with the longer half-life relative to the first order degradation determined by the kinetics at 1000 seconds. An exponential fit to the residue for this component in the range 100-200 seconds was used to calculate the amount and kd for the rapidly dissociating component(s). The criterion used was that the interval used to fit the more slowly dissociating component began after a time period at least four times longer than the t1/2 for the rapidly dissociating component. The components for association were obtained separately by an initial fit of the saturation approach (RUmax) within a range of 100 seconds to a point of at least 0.2 RU from RUmax as a first-order response for a range of expected RUmax values. The best-fit parameters and RUmax were then used as a starting point for further refinement. The presence of a positive residual between the observed RU and the results of this fit, extended to earlier periods, was taken as an independent pseudo-first-order response reflecting a fast-binding component. An iterative process with varying rate constants and the proportion of each component at the binding level (RU) at 300 seconds was used to obtain fit results within 0.2 of the observed RU and measure a near-zero slope for the net residual over the 300-second period. Testing of variance showed that the values represented true minimum ^R2 for the overall fit. RUmax had little effect on the rate constants or proportions of each component. Fitting procedures were performed using Excel.

Cellular CD73 Inhibition (Activity) Assay

[0135] Biparatopic antibody activity was determined using a modification of a previously reported method (McManus et al. 2018. SLAS discovery: advancing life sciences R & D 23, 264-273). CD73-expressing COR-L23 cells (4 × 103/well) were grown overnight to ~50% confluency in 40 μl of 1640 medium with L-glutamine and 10% heat-inactivated FBS in a 384-well clear-bottom plate (Greiner Bio One). Antibodies diluted in 1640 medium (10 μl) were added and the plates were incubated for 3 h at 37°C. Antibody dilution and addition procedures were performed using an Agilent Bravo liquid handler. AMPCP (100 μM) was substituted for the antibodies as a zero activity control (23, 25). Substrate (5 μl of 200 μM ¹⁵ N ₅ -AMP, Silantes GmbH, Munich, Germany) was added using a GNF Dispenser II (GNF Systems, San Diego, CA) and the plates were incubated at 37°C for 1 h. Reactions were then quenched with 5 μl of 12% formic acid in 1640 medium and a portion of the quenched reactions (40 μl) were filtered by centrifugation for 30 min at 3.5 kG through a 10 kDa MWCO ultrafiltration plate (Pall). The filtrates were stored at -80°C. The adenosine product was determined by LC/MS/MS analysis as described previously (23). Data were analyzed using nonlinear least squares data fitting (GraphPad Prism). Activity is shown relative to no-antibody controls in the same plate sector and normalized to the least-squares-fitted maximum activity (%CNTL). Results from the activity determination procedures ( Table 5 ) were expressed as the predicted maximum % inhibition at saturating antibody concentrations. In the initial screening, three concentrations (0.25 μg/mL, 0.5 μg/mL, and 1 μg/mL) were tested in a four-fold dilution series, and the mean % inhibition shown ( Fig. 1 ) is based on the residual activity at one concentration (1 μg/mL).

Epitope-specific sorting

[0136] Epitope-specific antibody subset sorting was performed using a premix format and biolayer interferometry (BLI) using a modification of a previously described method (Abdiche et al. 2009. Analytical biochemistry 386, 172-180). In this format, binding of antigen premixed with a molar excess of Fab is compared to binding of antigen alone. The assay was performed in 16-channel mode using an Octet QK384 (Pall Life Sciences). Antibodies were bound to the Protein A biosensors for 5 min, baselined for 1 min, then transferred to 100 nM CD73 or 100 nM CD73 with a 4-fold molar excess of Fab for 3 min, followed by transfer to dissociation buffer for 3 min. 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 assays were performed at 30°C. Data were analyzed using ForteBio Data Analysis software version 7.1 (Pall Life Sciences) by acquiring acquisition points at the end of the association phase. Normalized uptake values were calculated using the signal (nm) divided by the signal from CD73 alone, multiplied by the relative mass of CD73 compared to the CD73::(Fab)2 complex (0.56).

Procedures for determining structure

[0137] Recombinant TB19 and TB38 Fabs were expressed in Expi293F cells, purified using CaptureSelect CH1-XL Affinity Matrix (ThermoFisher), and buffer exchanged with 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 with PBS, deglycosylated overnight using PNGaseF, and further purified using size exclusion chromatography. The molar mass of the product was determined by SEC using 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 software version 6.1. 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. The fractions corresponding to the peak of the eluted complex were pooled and concentrated to 9 mg/mL for crystallization studies. Fab::CD73 TB19 was crystallized in 0.1 M sodium potassium phosphate pH 6.2, 35% 5-methyl-2,4-pentanediol, and 2.5% pentaerythritol ethoxylate at 4°C. The crystals were cryoprotected in 20% ethylene glycol and stock solution. X-ray diffraction data were collected on an EMBL Hamburg P14 using an Eiger 16M detector. 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 assay assemblies: separated N- and C-terminal domains of CD73 (PDB: 4H2I) and a model Fv TB19.3 generated by MOE (Molecular Operating Environment (MOE) 2013.8 Ed., Chemical Computing Group). Fab::CD73 TB38 formed crystals at 4°C in 1.6 M sodium phosphate monobasic monohydrate, 0.4 M potassium phosphate dibasic, and 0.1 M sodium citrate-phosphate, pH 5.3. Crystals were flash frozen in liquid nitrogen using 20% glycerol in the stock solution as a cryoprotectant. X-ray diffraction data were collected using the European Synchrotron Radiation Facility Beamline ID-30b with a Pilatus 3 6M detector. 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 MOE-derived Fab model of TB38 were used as exploratory models for MOE. For the second round, the previously discovered CD73 monomer was separated into its N- and C-terminal domains and analyzed along with the isolated Fv domain of TB38. For both structures, the model was rebuilt in Coot (Emsley et al. 2010. Acta crystallographica. Section D, Biological crystallography 66, 486–501) and refined using Phenix (Adams et al. 2010. Acta crystallographica. Section D, Biological crystallography 66, 213–221). Data collection and refinement statistics are provided ( Table 7 ). The software used in this project was accessed through the SBGrid consortium (Morin et al. 2013. eLife 2, e01456).

Example 2. Obtaining biparatopic antibodies

[0138] A panel of biparatopic antibodies directed against CD73 was generated using Fab fragment exchange (cFAE) and consisted of paired combinations of 11 sequence-unrelated parental antibodies that had previously demonstrated greater than 50% inhibition of CD73 activity in cell-based assays. Each Fab was expressed as a fusion to a human IgG1 Fc containing either the F405L or K409R mutation that destabilizes the parental Fc and stabilizes the Fc of the biparatopic 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. The generation of the desired products was verified by cIEF ( Fig. 3 ). Of the 121 (11 × 11) possible combinations, 88 biparatopic variants were generated, covering all possible combinations in at least one orientation. Eleven monospecific parent antibodies were also recombined as reference molecules by combining the parental Fc variants F405L and K409R to control for the potential effect of Fc mutations on antibody structure and function. In addition, 21 pairs were generated in both Fc orientations to control for the potential effects of mutation position.

Example 3. Activity of parent and biparatopic antibodies in inhibiting CD73 cellular enzymatic activity

[0139] Purified parent and biparatopic antibodies were tested for activity at 1 μg/mL in COR-L23 lung carcinoma cells expressing human CD73, and the adenosine product was quantified using an LC/MS-based assay (McManus et al. 2018. SLAS discovery: advancing life sciences R & D 23, 264-273). The percentage inhibition of CD73 enzymatic activity by the biparatopic antibodies at 1 nM is shown in Fig. 1 . Although the level of inhibition varied widely, most biparatopic combinations demonstrated higher activity than any parent antibody in the Duobody product form. A number of parent antibodies generated highly active progeny biparatopic variants, demonstrating inhibition levels of 90% or greater when combined with more than one other antibody. Of these, TB19 and E3.2 generated the largest number of variants with inhibition levels of 90% or greater, and several pairs with TB19, including those with E3.2, H19, TB38, or TC29, achieved inhibition levels of 95% or greater. TB19 and E3.2 antibodies were also combined with several other antibodies, achieving inhibition levels of 80% or greater. Although both of these antibodies exhibited this universal pairing ability, they differed in the complementarity of their pairing patterns. Among the biparatopic variants tested in both Fc orientations (a total of 16), no significant differences in inhibition were observed, indicating that the position of the mutations in the Fc of the Duobody product did not significantly influence the outcome (data not shown).

[0140] To assess whether both parental Fabs were required for activity, the parental antibodies were also cross-hybridized with an irrelevant antibody (AS30) to generate monovalent IgG variants with only one Fab capable of interacting with CD73. All of these antibodies showed little activity, demonstrating that Fabs from two cognate parental antibodies must be involved ( Fig. 1 ). To determine whether this was due to lower affinity, the affinity of the monovalent molecules of the most active biparatopic antibodies was compared to that of the biparatopic variants of which they were a part. The antibodies were first bound to a solid support and, after binding of the soluble form of the CD73 dimer in solution, SPR was performed (experimental procedures described above). As shown in Figure 2, in most cases the affinity of the biparatopic variant (KD) was similar to that of the higher affinity parent antibody, indicating that its affinity was due to binding of this single Fab. Only in two cases (H19/TB19 and CL25/TB19) did the biparatopic variant show significantly higher affinity than either parent antibody (~15-fold lower KD in both cases), suggesting synergistic effects potentially due to bivalent binding to the CD73 dimer or conformational effects promoting binding. However, neither parent antibody in these two cases resulted in similar enhancement when combined with the other antibodies, suggesting that a conformational effect was less likely. Since the affinity of most monovalent antibodies for individual CD73 dimers was not increased by the addition of a second cognate Fab, despite it being required for activity, this suggests that interaction of biparatopic IgG with more than one CD73 is required for significant inhibition.

[0141] To further evaluate the utility of combining these antibodies in a biparatopic format, the EC50 and maximal inhibition at saturating antibody concentrations were determined for the most potent biparatopic antibodies along with their parent mAbs, presented either alone or in a mixture, on COR-L23 cells ( Table 5, Fig. 4 ). As shown in Fig. 1, each biparatopic antibody was more potent than either of its two parent antibodies, which showed only partial inhibition down to 10 nM. The EC50 values for all of the biparatopic antibodies were in the range of 0.2-0.8 nM. In most cases, mixtures of parent antibodies resulted in maximal inhibition similar to that of the biparatopic antibodies, however, in half of their combinations tested, the biparatopic variant also showed a lower EC50. In the most pronounced case (TB19/TC29), the biparatopic antibody showed an EC50 50-fold lower than the antibody mixture, despite the nearly identical affinity of the higher affinity monovalent parent antibody TC29 and the biparatopic antibody for CD73 ( Fig. 2 ). Only in one case (CL25/TA10) was the mixture more potent (~4-fold), indicating that the interactions with CD73 mediated by this mixture could not be reproduced using the biparatopic antibody.

[0142] Table 5. Activity of biparatopic antibodies and parent antibody mixtures against CD73 on COR-L23 cells. EC50 and maximum inhibition rates are based on nonlinear regression analysis

[0143] The affinity of the biparatopic antibodies for CD73 was compared with that of the parent antibodies in monovalent form. To avoid potential avidity effects due to binding of CD73 in solution by individual antibodies on the chip surface, the parent antibodies were loaded onto the chip at the lowest level sufficient to reliably estimate the kinetic constants. The data are grouped as shown in Figure 2. It should be noted that kinetic parameters for parent antibodies that are common to several biparatopic antibodies are shown in each case to facilitate comparison. Values represent curve fits obtained using 3 nM, 12 nM, and 32 nM CD73 in flow. Association rate constants for biphasic kinetics are shown with their amounts after the 300 s binding phase in parentheses. The amounts for the dissociation rate constant are based on the T0 intercept of the fitted values for each component. Table 6 below shows the binding data used to generate Fig. 2 .

[0144] Table 6. Representative SPR kinetic data for assessing bivalent engagement of single CD73

[0145] Nine of the eleven biparatopic antibodies exhibited biphasic dissociation kinetics ( Figs. 13A-13B ), although primarily as a result of a minor fraction (15% or less) of the more rapidly dissociating component. In one case (H19/C16), the amount of this component was similar to that of the monovalent parent C16/AS30 (31% versus 38%), suggesting heterogeneity of the monoclonal C16 used to generate both. TA9/AS30 exhibited similar heterogeneity (29% lower stability), which was not reflected in the biparatopic daughter TA9/H7. Half-life values compared to the monovalent parent antibodies are shown in Fig. 15 . In eight of eleven cases, the kd of the principal dissociation component was within 2.2-fold of the most stable monovalent parent antibody. In contrast, the difference between the kd values for the monovalent parent antibodies averaged 15-fold (range 1.5- to 73-fold, median 6.2), suggesting that in these cases CD73 is bound by a single parent Fab fragment on the immobilized antibody. However, in three cases (E3.2/TB19, CL25/TB19, and H19/TB19), the interaction with the biparatopic antibody was significantly more stable than with any of the monovalent parent antibodies (5.4-, 8.8-, and 26-fold, respectively), suggesting the presence of additional contacts with the biparatopic antibody.

[0146] The presence of bivalent association kinetics was also evident from the rapid increase in RU immediately after administration, followed by a significant decrease in rate at 100 seconds. Prediction of the expected early RU value based on the kinetic values at 100 seconds, implying a pseudo first-order kinetic reaction, revealed the presence of a significant residue corresponding to a rapidly binding component showing a first-order kinetic reaction, accounting for a significant fraction of RU at 300 seconds (30-49%). Fitting both components through an iterative process resulted in a combined value within ±0.2 of the observed RU value over 90% of the binding cycle ( Figures 13A-13B ). Similar to the dissociation case, the calculated ka values for each of the two components were within 3-fold of the values for the monovalent parent antibody (2.04 ± 1.4-fold difference, range 1.02–2.71) compared to an average ~6-fold difference between them (5.9 ± 2.1, Table 6), suggesting that they reflect independent binding to CD73 of each parent Fab fragment.

[0147] Because each kinetic component for association cannot be uniquely assigned to a specific component for dissociation, the relative affinities of the biparatopic antibodies and monovalent parent antibodies for CD73 were compared using KD values based on the kd value for the principal dissociation component and a ka value based on a 1:1 Langmuir binding model. The latter was within 30% of the average ka value for the two components for biphasic binding ( Table 6 ). Consistent with the pattern observed for dissociation kinetics, the apparent affinity of the biparatopic variants ( _KD ) was similar to that of the higher affinity monovalent parent antibodies, indicating that the interaction of the biparatopic antibodies may be primarily due to binding of a single Fab fragment. However, in two cases (CL25/TB19 and TB19/H19) the biparatopic variant showed a significant enhancement compared to either monovalent parent antibody (26- and 69-fold, respectively). This enhancement was specific to these combinations, as the parent antibodies (TB19, H19, CL25) did not produce similar enhancements with the other partners. Since this enhancement required two cognate fragments, it was concluded that it reflected the interaction of both fragments of these biparatopic variants with CD73. However, in most cases, affinity for CD73 was not increased by the addition of a second cognate Fab fragment, despite it being required for activity, suggesting that interaction of the biparatopic antibody with additional CD73 is required for significant inhibition in cells.

Example 4. Epitope-specific sorting

[0148] Epitopes of parent antibodies with the highest number of highly active combinations (TB19, E3.2, TB38, H19, and E3.2) were sorted using biolayer interferometry ( Fig. 5A ). Monovalent IgG antibodies were used to coat a solid support to capture CD73 from a mixture with competing Fabs.

[0149] The result of the parent antibody subset assay is shown in Fig. 5B . Higher values indicate capture of CD73 bound by the competing Fab and no/low competition for binding (i.e., the Fab binds to an epitope of CD73 that does not overlap with that of the coating antibody), while lower values reflect blocking of the epitope by the bound Fab from capture by the immobilized antibody. The distribution of antibodies into different epitope groups based on these results is shown in Fig. 5C . One of the groups contained TB38, H19, and the mostly overlapping TC29, all of which showed susceptibility to each of the Fabs except TB19. However, these three proteins also showed differences in susceptibility to competition from the different Fabs. For example, CD73 capture by monovalent IgG TB38 was more susceptible to competition from Fab H19 than capture by either TC29 or H19, while TC29 differed from the other two by being partially resistant to competition from Fab F1.2, which was unique among all antibodies. While the groups were clearly delineated in most cases, intermediate levels of inhibition were observed in a few cases (H19+H19, TC29+H19, TC29+F1.2, TB19+H19, F1.2+H19, and F1.2+TB19), possibly reflecting partially overlapping epitopes (Abdiche et al. 2017. PloS one 12, e0169535) and/or significant differences in affinity. E3.2 could not be assigned to any group due to its aspecific interaction with the solid support.

[0150] Capture of the CD73::Fab complex by the antibody in this sorting experiment, reflecting the lack of competition between the parent antibodies, showed a high level of correlation with the inhibition of cellular CD73 enzymatic activity by the corresponding biparatopic antibodies ( Fig. 5D ). Antibody pairs that showed greater than 35% Fab capture consistently resulted in inhibition of 85% or greater at 1 μg/ml as the biparatopic variant, and, in contrast, combinations with less than 35% capture achieved less than 70% inhibition as the biparatopic variant. These data indicate that to achieve high activity, both antibodies comprising a biparatopic variant must bind non-overlapping epitopes on CD73.

Example 5. Structures of Fab TB19 and TB38 in complex with CD73

[0151] Since the TB19 antibody successfully coupled with a number of other antibodies, including TB38, in the most active biparatopic variants, it was important to understand the mechanism of action by studying their interactions with CD73 through structural analysis. Prior to complexing with recombinant TB19 and TB38 Fabs, the extracellular domain of human CD73 (residues 27-549) was deglycosylated with PNGaseF. The PNGaseF-digested product exhibited a molecular weight (MW) of 118 kDa by SEC-MALS, which was slightly higher than the MW of the polypeptide (116 kDa). This could be due to the glycan observed in the structures at position Asn311, which was not susceptible to cleavage by PNGaseF. Crystallization parameters are shown in Table 7 below.

[0152] Table 7. Crystallographic parameters

[0153] The structure of CD73 in complex with the Fab TB19 is shown in Figs. 6A-6B and Figs. 7A-7B . In the asymmetric crystal unit, one TB19 is bound to one CD73 monomer and only Fv of the Fab can be built due to weak electron densities in the CH1/CL domains. Biological assembly of the dimeric CD73 complex was mediated by a 2-fold crystallographic symmetry operation. In the resulting structure, CD73 dimerizes via the interface between the C-terminal domains ( Fig. 6B ), which has close similarity to 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).

[0154] Within CD73 in complex with TB19, well-defined positive ion densities are observed in the active site in the N-terminal domain. Two zinc ions and one phosphate were incorporated, respectively, and coordinated by residues Asp36, His38, Asp85, Asn117, His118, His220, and His243 in the catalytic center as the TB19 complex was crystallized in the presence of phosphate. These zinc ions and the phosphate are in the same position as the two zinc ions and the β-phosphonate of the AMPCP substrate analog in the closed conformational isomer of CD73 (PDB 4H2I) ( Figure 7A-7B ). The conserved dimerization surface and the positions of the zinc and phosphate atoms indicate that the structure of the CD73 dimer in complex with TB19 is biologically relevant.

[0155] CD73 has previously been described in either an open or closed conformation depending on the presence or absence of a substrate in the active site, respectively (Knapp, supra) ( Fig. 6A ). However, upon binding TB19, CD73 adopts a conformation where the N- and C-terminal domains are in an intermediate position, located between those previously described as the open and closed conformational isomers ( Figs. 6A-6B and Figs. 7A-7B ). When the C-terminal domains of the earlier structures and TB19-bound CD73 are superposed, the position of the zinc ion coordinating residue H220 in the N-terminal domain is approximately 22 Å further from its position in the closed conformational isomer (PDB 4H2I) and 27 Å further from its position in the open conformational isomer (PDB 4H2F).

[0156] All of the TB19 CDR loops, except CDRL2, contact a portion of the N-terminal domain adjacent to the zinc-phosphate binding site ( Figures 7A-7B ), although none of the antibody residues interact directly with any of the residues that form the catalytic site. In addition, residue Ser62 of CDRH2 and residue Ser26 of CDRL1 of TB19 ( Figure 6B, Figure 6C ) are spatially located close to the C-terminal domain, but 20 Å away from the substrate-binding residues including Arg354, Asn390, Arg395, Phe417, Phe500, and Asp506. In the presence of TB19, these substrate-binding residues are located far from the catalytic site and the zinc atoms in the N-terminal domain. For example, residues Phe417 and Phe500, which bind the adenine ring, are 11–13Å further from their positions in the closed conformational isomer with the substrate (PDB 4H2I).

[0157] Due to the orientation of TB19 and the location of its epitope, there are conflicts between the C-terminal domain and TB19 when aligning the N-terminal domains of CD73 in our structure and in the closed conformational isomer of CD73 ( Figures 7A-7B ). Thus, bound TB19 will block the alignment of the N- and C-terminal domains in CD73 and prevent the formation of the closed conformational isomer. Binding of TB19 will dissociate zinc ions and catalytic residues of the N-terminal domain from the phosphoanhydride bonds of the substrate, thereby blocking enzymatic activity.

[0158] In contrast to TB19, TB38 and CD73 Fabs yielded structures where each asymmetric unit contained two CD73 dimers in different conformations, with all monomers linked by a single Fab ( Figures 8A-8C ). In the first structure ( Figure 8A ), the electron densities for the CH1/CL domains were well defined and the full Fab structure could be reconstructed. In the second structure ( Figure 8B ), little density was observed for the constant domains, so only the Fv domains were reconstructed. Surprisingly, the conformation of CD73 in the two structures was different. In the first structure, CD73 is in a symmetric open conformation that can be aligned with the canonical open conformational isomer in PDB 4H2F with a standard deviation of 1 Å. However, the CD73 dimer in the second structure is in an asymmetric conformation that has not been described previously, where the monomers are in different conformations. In this hybrid structure, one monomer is in the open conformation previously observed in the crystal with bound adenosine (PDB 4H2F), while the other is in the closed conformation observed in the presence of the AMPCP substrate analogue (PDB 4H2I) (Knapp, supra). In both complexes, the TB38 Fab contacts residues exclusively in the N-terminal domain (including Lys145, Ser152, Ser155, Gly156, Leu159-Lys162, Glu203, Lys206, Leu210, and Asn211) and all 6 CDRs are involved in the interactions. Mapping of epitope residues for TB19 and TB38 on the partially exposed structure of CD73 ( Fig. 10 ) and sequence alignment show that the epitopes are non-overlapping, although in close proximity according to the sorting results.

[0159] To assess the possible engagement of the CD73 dimer by the TB19/TB38 bispecific antibody, IgG was modeled by replacing the Fv from the complete IgG antibody structure (PDB 1HZH) with those of TB19 and TB38 ( Fig. 9A-9B ). The distance between the CH1 domains of TB19 and TB38 in this model ( Fig. 9A ) is approximately 40 Å (measured between the Cα Ala225 of the CH1 domain). Modeling of bivalent binding to CD73 in a partially open conformation was not possible with this biparatopic variant, whether binding to two epitopes on the same or on opposite monomers, although each CD73 monomer could be bound by the two antibodies in a monovalent manner, as depicted in Fig. 9B . For a single antibody to bivalently bind a CD73 dimer, the C-terminal residues of the Fab CH1 domains must be separated by a distance of ~120 Å and ~140 Å to bind epitopes on either the same or opposite monomers, respectively, which is much greater than can be achieved with IgG. It was concluded that the biparatopic TB19/TB38 antibody would likely not be able to bivalently bind a single CD73 dimer.

--->

SEQUENCE LIST

<110> GENZIM CORPORATION

<120> BIPARATOPIC ANTIBODIES TO CD73

<130> SA9-282PC: 711174

<150> 62/936,119

<151> 2019-11-15

<150> 63/023,542

<151> 2020-05-12

<150> 63/086,982

<151> 2020-10-02

<160> 24

<170> PatentIn version 3.5

<210> 1

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence

<400> 1

Gly Gly Ser Ile Arg Asn Asn Tyr

1 5

<210> 2

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence

<400> 2

Ile Tyr Ile Ser Gly Thr Thr

1 5

<210> 3

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence

<400> 3

Ala Arg Glu His Tyr Val Ser Gly Thr Ser Leu Asp Asn

1 5 10

<210> 4

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence

<400> 4

Gln Ser Val Asn Thr Asn Tyr

1 5

<210> 5

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence

<400> 5

Gly Thr Ser

1

<210> 6

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence

<400> 6

Gln Gln Asp Tyr Asn Leu Pro Tyr Thr

1 5

<210> 7

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence

<400> 7

Gly Phe Thr Phe Ser Ser Tyr Gly

1 5

<210> 8

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence

<400> 8

Phe Trp Tyr Asp Gly Ser Asn Lys

1 5

<210> 9

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence

<400> 9

Ala Arg Ala Pro Asn Trp Asp Asp Ala Phe Asp Ile

1 5 10

<210> 10

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence

<400> 10

Ser Gly Ser Val Ser Thr Ser Tyr Tyr

1 5

<210> 11

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence

<400> 11

Ser Thr Asn

1

<210> 12

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence

<400> 12

Val Leu Phe Met Gly Ser Gly Ile Trp Val

1 5 10

<210> 13

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence

<400> 13

Gln Glu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Asn Asn

20 25 30

Tyr Tyr Asn Trp Ile Arg Gln Pro Ala Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Tyr Ile Ser Gly Thr Thr Asn Ser Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Met Ser Ile Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Glu His Tyr Val Ser Gly Thr Ser Leu Asp Asn Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 14

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence

<400> 14

Glu Ile Val Met Thr Gln Ser Pro Thr Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asn Thr Asn

20 25 30

Tyr Phe Ser Trp Tyr Gln Gln Lys Pro Gly Leu Thr Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Thr Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Phe Gly Ile Tyr Tyr Cys Gln Gln Asp Tyr Asn Leu Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Tyr Leu Glu Ile Lys

100 105

<210> 15

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence

<400> 15

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Phe Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Ser Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Pro Asn Trp Asp Asp Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser

115

<210> 16

<211> 110

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence

<400> 16

Gln Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser

20 25 30

Tyr Tyr Pro Asn Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Thr

35 40 45

Leu Ile Tyr Ser Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Val Leu Phe Met Gly Ser

85 90 95

Gly Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 17

<211> 448

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence

<400> 17

Gln Glu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Asn Asn

20 25 30

Tyr Tyr Asn Trp Ile Arg Gln Pro Ala Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Tyr Ile Ser Gly Thr Thr Asn Ser Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Met Ser Ile Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Glu His Tyr Val Ser Gly Thr Ser Leu Asp Asn Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 18

<211> 448

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence

<400> 18

Gln Glu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Asn Asn

20 25 30

Tyr Tyr Asn Trp Ile Arg Gln Pro Ala Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Tyr Ile Ser Gly Thr Thr Asn Ser Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Met Ser Ile Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Glu His Tyr Val Ser Gly Thr Ser Leu Asp Asn Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Leu Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 19

<211> 215

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence

<400> 19

Glu Ile Val Met Thr Gln Ser Pro Thr Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asn Thr Asn

20 25 30

Tyr Phe Ser Trp Tyr Gln Gln Lys Pro Gly Leu Thr Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Thr Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Phe Gly Ile Tyr Tyr Cys Gln Gln Asp Tyr Asn Leu Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Tyr Leu Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 20

<211> 448

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence

<400> 20

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Phe Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Ser Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Pro Asn Trp Asp Asp Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 21

<211> 448

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence

<400> 21

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Phe Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Ser Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Pro Asn Trp Asp Asp Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Leu Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 22

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence

<400> 22

Gln Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser

20 25 30

Tyr Tyr Pro Asn Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Thr

35 40 45

Leu Ile Tyr Ser Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Val Leu Phe Met Gly Ser

85 90 95

Gly Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 23

<211> 523

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence

<400> 23

Trp Glu Leu Thr Ile Leu His Thr Asn Asp Val His Ser Arg Leu Glu

1 5 10 15

Gln Thr Ser Glu Asp Ser Ser Lys Cys Val Asn Ala Ser Arg Cys Met

20 25 30

Gly Gly Val Ala Arg Leu Phe Thr Lys Val Gln Gln Ile Arg Arg Ala

35 40 45

Glu Pro Asn Val Leu Leu Leu Asp Ala Gly Asp Gln Tyr Gln Gly Thr

50 55 60

Ile Trp Phe Thr Val Tyr Lys Gly Ala Glu Val Ala His Phe Met Asn

65 70 75 80

Ala Leu Arg Tyr Asp Ala Met Ala Leu Gly Asn His Glu Phe Asp Asn

85 90 95

Gly Val Glu Gly Leu Ile Glu Pro Leu Leu Lys Glu Ala Lys Phe Pro

100 105 110

Ile Leu Ser Ala Asn Ile Lys Ala Lys Gly Pro Leu Ala Ser Gln Ile

115 120 125

Ser Gly Leu Tyr Leu Pro Tyr Lys Val Leu Pro Val Gly Asp Glu Val

130 135 140

Val Gly Ile Val Gly Tyr Thr Ser Lys Glu Thr Pro Phe Leu Ser Asn

145 150 155 160

Pro Gly Thr Asn Leu Val Phe Glu Asp Glu Ile Thr Ala Leu Gln Pro

165 170 175

Glu Val Asp Lys Leu Lys Thr Leu Asn Val Asn Lys Ile Ile Ala Leu

180 185 190

Gly His Ser Gly Phe Glu Met Asp Lys Leu Ile Ala Gln Lys Val Arg

195 200 205

Gly Val Asp Val Val Val Gly Gly His Ser Asn Thr Phe Leu Tyr Thr

210 215 220

Gly Asn Pro Pro Ser Lys Glu Val Pro Ala Gly Lys Tyr Pro Phe Ile

225 230 235 240

Val Thr Ser Asp Asp Gly Arg Lys Val Pro Val Val Gln Ala Tyr Ala

245 250 255

Phe Gly Lys Tyr Leu Gly Tyr Leu Lys Ile Glu Phe Asp Glu Arg Gly

260 265 270

Asn Val Ile Ser Ser His Gly Asn Pro Ile Leu Leu Asn Ser Ser Ile

275 280 285

Pro Glu Asp Pro Ser Ile Lys Ala Asp Ile Asn Lys Trp Arg Ile Lys

290 295 300

Leu Asp Asn Tyr Ser Thr Gln Glu Leu Gly Lys Thr Ile Val Tyr Leu

305 310 315 320

Asp Gly Ser Ser Gln Ser Cys Arg Phe Arg Glu Cys Asn Met Gly Asn

325 330 335

Leu Ile Cys Asp Ala Met Ile Asn Asn Asn Leu Arg His Thr Asp Glu

340 345 350

Met Phe Trp Asn His Val Ser Met Cys Ile Leu Asn Gly Gly Gly Ile

355 360 365

Arg Ser Pro Ile Asp Glu Arg Asn Asn Gly Thr Ile Thr Trp Glu Asn

370 375 380

Leu Ala Ala Val Leu Pro Phe Gly Gly Thr Phe Asp Leu Val Gln Leu

385 390 395 400

Lys Gly Ser Thr Leu Lys Lys Ala Phe Glu His Ser Val His Arg Tyr

405 410 415

Gly Gln Ser Thr Gly Glu Phe Leu Gln Val Gly Gly Ile His Val Val

420 425 430

Tyr Asp Leu Ser Arg Lys Pro Gly Asp Arg Val Val Lys Leu Asp Val

435 440 445

Leu Cys Thr Lys Cys Arg Val Pro Ser Tyr Asp Pro Leu Lys Met Asp

450 455 460

Glu Val Tyr Lys Val Ile Leu Pro Asn Phe Leu Ala Asn Gly Gly Asp

465 470 475 480

Gly Phe Gln Met Ile Lys Asp Glu Leu Leu Arg His Asp Ser Gly Asp

485 490 495

Gln Asp Ile Asn Val Val Ser Thr Tyr Ile Ser Lys Met Lys Val Ile

500 505 510

Tyr Pro Ala Val Glu Gly Arg Ile Lys Phe Ser

515 520

<210> 24

<211> 523

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence

<400> 24

Trp Glu Leu Thr Ile Leu His Thr Asn Asp Val His Ser Arg Leu Glu

1 5 10 15

Gln Thr Ser Glu Asp Ser Ser Lys Cys Val Asn Ala Ser Arg Cys Met

20 25 30

Gly Gly Val Ala Arg Leu Phe Thr Lys Val Gln Gln Ile Arg Arg Ala

35 40 45

Glu Pro Asn Val Leu Leu Leu Asp Ala Gly Asp Gln Tyr Gln Gly Thr

50 55 60

Ile Trp Phe Thr Val Tyr Lys Gly Ala Glu Val Ala His Phe Met Asn

65 70 75 80

Ala Leu Arg Tyr Asp Ala Met Ala Leu Gly Asn His Glu Phe Asp Asn

85 90 95

Gly Val Glu Gly Leu Ile Glu Pro Leu Leu Lys Glu Ala Lys Phe Pro

100 105 110

Ile Leu Ser Ala Asn Ile Lys Ala Lys Gly Pro Leu Ala Ser Gln Ile

115 120 125

Ser Gly Leu Tyr Leu Pro Tyr Lys Val Leu Pro Val Gly Asp Glu Val

130 135 140

Val Gly Ile Val Gly Tyr Thr Ser Lys Glu Thr Pro Phe Leu Ser Asn

145 150 155 160

Pro Gly Thr Asn Leu Val Phe Glu Asp Glu Ile Thr Ala Leu Gln Pro

165 170 175

Glu Val Asp Lys Leu Lys Thr Leu Asn Val Asn Lys Ile Ile Ala Leu

180 185 190

Gly His Ser Gly Phe Glu Met Asp Lys Leu Ile Ala Gln Lys Val Arg

195 200 205

Gly Val Asp Val Val Val Gly Gly His Ser Asn Thr Phe Leu Tyr Thr

210 215 220

Gly Asn Pro Pro Ser Lys Glu Val Pro Ala Gly Lys Tyr Pro Phe Ile

225 230 235 240

Val Thr Ser Asp Asp Gly Arg Lys Val Pro Val Val Gln Ala Tyr Ala

245 250 255

Phe Gly Lys Tyr Leu Gly Tyr Leu Lys Ile Glu Phe Asp Glu Arg Gly

260 265 270

Asn Val Ile Ser Ser His Gly Asn Pro Ile Leu Leu Asn Ser Ser Ile

275 280 285

Pro Glu Asp Pro Ser Ile Lys Ala Asp Ile Asn Lys Trp Arg Ile Lys

290 295 300

Leu Asp Asn Tyr Ser Thr Gln Glu Leu Gly Lys Thr Ile Val Tyr Leu

305 310 315 320

Asp Gly Ser Ser Gln Ser Cys Arg Phe Arg Glu Cys Asn Met Gly Asn

325 330 335

Leu Ile Cys Asp Ala Met Ile Asn Asn Asn Leu Arg His Thr Asp Glu

340 345 350

Met Phe Trp Asn His Val Ser Met Cys Ile Leu Asn Gly Gly Gly Ile

355 360 365

Arg Ser Pro Ile Asp Glu Arg Asn Asn Gly Thr Ile Thr Trp Glu Asn

370 375 380

Leu Ala Ala Val Leu Pro Phe Gly Gly Thr Phe Asp Leu Val Gln Leu

385 390 395 400

Lys Gly Ser Thr Leu Lys Lys Ala Phe Glu His Ser Val His Arg Tyr

405 410 415

Gly Gln Ser Thr Gly Glu Phe Leu Gln Val Gly Gly Ile His Val Val

420 425 430

Tyr Asp Leu Ser Arg Lys Pro Gly Asp Arg Val Val Lys Leu Asp Val

435 440 445

Leu Cys Thr Lys Cys Arg Val Pro Ser Tyr Asp Pro Leu Lys Met Asp

450 455 460

Glu Val Tyr Lys Val Ile Leu Pro Asn Phe Leu Ala Asn Gly Gly Asp

465 470 475 480

Gly Phe Gln Met Ile Lys Asp Glu Leu Leu Arg His Asp Ser Gly Asp

485 490 495

Gln Asp Ile Asn Val Val Ser Thr Tyr Ile Ser Lys Met Lys Val Ile

500 505 510

Tyr Pro Ala Val Glu Gly Arg Ile Lys Phe Ser

515 520

<---

Claims (45)

1. A biparatopic antigen-binding protein that specifically binds to a first epitope of CD73 and a second epitope of CD73, comprising: (a) a first VH domain with specificity for a first epitope of CD73, comprising a CDR-H1 sequence comprising the amino acid sequence GGSIRNNY (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence IYISGTT (SEQ ID NO: 2), and a CDR-H3 sequence comprising the amino acid sequence AREHYVSGTSLDN (SEQ ID NO: 3); (b) a first VL domain with specificity for a first epitope of CD73, comprising a CDR-L1 sequence comprising the amino acid sequence QSVNTNY (SEQ ID NO: 4), a CDR-L2 sequence comprising the amino acid sequence GTS (SEQ ID NO: 5), and a CDR-L3 sequence comprising the amino acid sequence QQDYNLPYT (SEQ ID NO: 6); (c) a second VH domain with specificity for a second epitope of CD73 that comprises a CDR-H1 sequence comprising the amino acid sequence GFTFSSYG (SEQ ID NO: 7), a CDR-H2 sequence comprising the amino acid sequence FWYDGSNK (SEQ ID NO: 8), and a CDR-H3 sequence comprising the amino acid sequence ARAPNWDDAFDI (SEQ ID NO: 9); and (d) a second VL domain with specificity for a second epitope of CD73, which comprises a CDR-L1 sequence comprising the amino acid sequence SGSVSTSYY (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence STN (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence VLFMGSGIWV (SEQ ID NO: 12). 2. The biparatopic antigen-binding protein according to claim 1, wherein 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. 3. The biparatopic antigen-binding protein according to claim 1, wherein (a) the first heavy chain of the antibody comprises the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18; (b) the second heavy chain of the antibody comprises the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21; (c) the first light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 19; and (d) the second light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 22. 4. The biparatopic antigen-binding protein according to paragraphs 1-3, wherein (a) the first VH and VL domains bind a first epitope in 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 domains bind a second epitope in 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. 5. A biparatopic antigen-binding protein according to claims 1-4, which provides for a higher inhibitory activity against CD73 compared to one or both of the monospecific parent antibodies. 6. The biparatopic antigen-binding protein of claim 5, wherein the inhibitory activity of CD73 is determined using COR-L23 cells expressing CD73. 7. The biparatopic antigen-binding protein of claims 1-6, wherein the first VH and VL domains bind a first epitope of CD73 on a first dimeric CD73 molecule, and the second VH and VL domains bind a second epitope of CD73 on a second dimeric CD73 molecule. 8. The biparatopic antigen-binding protein of claims 1-7, wherein the antigen-binding protein has the ability to crosslink two or more dimeric CD73 molecules. 9. A biparatopic antigen-binding protein according to claim 1, obtained by exchanging Fab fragments. 10. The biparatopic antigen-binding protein according to claim 9, wherein the exchange of Fab fragments is carried out by performing the following steps: (a) mixing a first parent monospecific antigen-binding protein comprising an IgG1 Fc region comprising an F405L amino acid substitution according to the EU index and a second parent monospecific antigen-binding protein comprising an IgG1 Fc region comprising an K409R amino acid substitution according to the EU index, to obtain a mixture; (b) subjecting the mixture of step (a) to reducing conditions to obtain a mixture of reduced antigen-binding proteins comprising a biparatopic bispecific antigen-binding protein; (c) subjecting the mixture from step (b) to oxidative conditions to re-form disulfide bonds between the heavy chains of the biparatopic bispecific antigen-binding protein and (d) isolation of a biparatopic bispecific antigen-binding protein. 11. The biparatopic antigen-binding protein of claim 10, wherein the first initial monospecific antigen-binding protein and the second initial monospecific antigen-binding protein are mixed in equimolar amounts. 12. The biparatopic antigen-binding protein of claim 10 or 11, wherein the reducing conditions are obtained by adding a reducing agent. 13. The biparatopic antigen-binding protein of any one of claims 10-12, wherein the mixture of step (a) is placed under reducing conditions for a period of from about 3 hours to about 6 hours at a temperature of from about 18°C to about 30°C. 14. The biparatopic antigen-binding protein of any one of the preceding claims for treating a CD73-mediated disease or disorder in a subject, comprising administering to a subject in need thereof the biparatopic antigen-binding protein. 15. The biparatopic antigen-binding protein according to claim 1, wherein: (a) the heavy chain of the first antibody comprises the amino acid sequence of SEQ ID NO: 17; (b) the heavy chain of the second antibody comprises the amino acid sequence of SEQ ID NO: 21; (c) the light chain of the first antibody comprises the amino acid sequence of SEQ ID NO: 19; and (d) the light chain of the second antibody comprises the amino acid sequence of SEQ ID NO: 22; or (a) the heavy chain of the first antibody comprises the amino acid sequence of SEQ ID NO: 18; (b) the heavy chain of the second antibody comprises the amino acid sequence of SEQ ID NO: 20; (c) the light chain of the first antibody comprises the amino acid sequence of SEQ ID NO: 19; and (d) the second light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 22. 16. A biparatopic antigen-binding protein according to any one of claims 1-4, comprising: higher inhibitory activity of CD73 compared to the combination of monospecific parental antibodies. 17. The biparatopic antigen-binding protein of claim 10 or 11, wherein the reducing conditions are created by adding a reducing agent, wherein the reducing agent comprises mercaptoethylamine (MEA). 18. The use of a biparatopic antigen-binding protein according to any one of claims 1-17 for the treatment of a CD73-mediated disease or disorder in a subject, comprising administering to a subject in need thereof a biparatopic antigen-binding protein, wherein the CD73-mediated disease or disorder is cancer.