CN116847883A

CN116847883A - Dosage regimen of anti-CD 73 antibody and anti-ENTPD 2 antibody and uses thereof

Info

Publication number: CN116847883A
Application number: CN202280012128.2A
Authority: CN
Inventors: J·冈萨雷斯-玛菲; R·E·艾萨克; J·金; L·纳尔迪; J·A·奥特罗; N·帕里克; M·J·罗伊; K·K·苏布拉曼尼亚; T·翟
Original assignee: Novartis AG
Current assignee: Novartis AG
Priority date: 2021-01-29
Filing date: 2022-01-27
Publication date: 2023-10-03

Abstract

The present application relates generally to dosage regimens of an anti-cluster of differentiation 73 (CD 73) antibody and/or an anti-extracellular enzyme extracellular nucleoside triphosphate bisphosphate hydrolase 2 (ENTPD 2) antibody for use in a method of treating cancer in a subject, and dosage regimens of an anti-CD 73 antibody for use in treating cancer. The present application further relates generally to a dosage regimen of a combination of agents, e.g., a dosage regimen comprising a combination of an anti-CD 73 antibody and/or an anti-ENTPD 2 antibody and at least one or more of a PD-1 inhibitor and an adenosine A2AR antagonist.

Description

Dosage regimen of anti-CD 73 antibody and anti-ENTPD 2 antibody and uses thereof

Sequence listing

The present application contains a sequence listing that has been submitted electronically in ASCII format, and the sequence listing is hereby incorporated by reference in its entirety. The ASCII copy was created at 2021, 12, 6, under the name PAT059034_sl.txt and was 435,867 bytes in size.

Technical Field

The present application relates generally to dosage regimens of an anti-cluster of differentiation 73 (CD 73) antibody and an anti-extracellular enzyme extracellular nucleoside triphosphate bisphosphate hydrolase 2 (ENTPD 2) antibody for use in a method of treating cancer in a subject, and dosage regimens of an anti-CD 73 antibody and an anti-ENTPD 2 antibody for use in treating cancer. The present application further relates generally to a dosage regimen of a combination of agents, e.g., a dosage regimen comprising a combination of an anti-CD 73 antibody and/or an anti-ENTPD 2 antibody and at least one or more of a PD-1 inhibitor and an adenosine A2AR antagonist.

Background

Cluster of differentiation 73 (CD 73), also known as extracellular-5 '-nucleotidase (extracellular-5' NT), is a Glycosyl Phosphatidylinositol (GPI) -linked cell surface enzyme that is present in most tissues, particularly expressed in endothelial cells and hematopoietic cell subsets (Resta et al, immunol Rev [ Immunol comment ]161:95-109 (1998) and Colgan et al, prinergic Signal [ purinergic signalling ]2:351-60 (2006)). CD73 catalyzes the conversion of Adenosine Monophosphate (AMP) to adenosine. Adenosine is a signaling molecule that mediates its biological effects through several receptors, including the adenosine Al, A2A, A B and A3 receptors. The A2A receptor is of particular interest due to its broad expression on immune cells. Adenosine has pleiotropic effects in tumor microenvironments, including expansion of regulatory T cells (tregs), inhibition of Interferon (IFN) -gamma mediated effector T cell (Teff) responses, and expansion of Myeloid Derived Suppressor Cells (MDSCs). See, for example, allard B et al, curr Opin Pharmacol [ Pharmacology Studies ]29:7-16 (2016) and Allard et al, immunotherapy [ Immunotherapy ]8:145-163 (2016).

CD73 can also be expressed on Cancer cells, including colon, lung, pancreas, ovary, bladder, leukemia, glioma, glioblastoma, melanoma, thyroid, esophageal, prostate and breast cancers (Jin et al, cancer Res [ Cancer Industry ]70:2245-55 (2010) and Stagg et al, PNAS [ Proc. Natl. Acad. Sci. USA ]107:1547-52 (2010); zhang et al, cancer Res [ Cancer Industry ]70:6407-11 (2010)). High CD73 expression is reported to be associated with poor prognosis for various cancer indications (e.g., lung cancer, melanoma, triple negative breast cancer, squamous head and neck cancer, and colorectal cancer). See, e.g., allard B et al, expert Opin Ther Targets [ therapeutic target expert opinion ]18:863-881 (2014); leclerc BG et al Clin Cancer Res [ clinical Cancer research ]22:158-166 (2016); ren ZH et al, oncostarget [ tumor target ]7:61690-61702 (2016); ren ZH et al, oncol Lett [ Innovative Rev.Ind. ]12:556-562 (2016); and Turcotte M et al, cancer Res [ Cancer research ]75:4494-4503 (2015).

Cell stress and apoptosis trigger the release of ATP to the extracellular space. Increased ATP concentrations promote rapid onset of inflammation, lead to amplification of T cell signaling, inhibit regulatory T cells (tregs), and promote inflammatory body activation in dendritic cells and macrophages. Extracellular enzyme extracellular nucleoside triphosphate diphosphate hydrolase 2 (ENTPD 2) is part of the extracellular-nucleotidase family that hydrolyzes 5' -triphosphates and is an integral membrane protein involved in purinergic signalling. EnTPD2 catalyzes the conversion of Adenosine Triphosphate (ATP) to Adenosine Diphosphate (ADP) and Adenosine Monophosphate (AMP). In turn, AMP is catalyzed to adenosine by cluster of differentiation 73 (CD 73, also known as extracellular-5 '-nucleotidase (extracellular-5' NT)).

In a mouse model of hepatocellular carcinoma, it was shown that ENTPD2 converts extracellular ATP to AMP, which prevents differentiation of monocyte myelogenous suppressor cells (MDSCs) into dendritic cells, thereby promoting maintenance of MDSCs in vitro and in vivo (Chiu et al, nat Commun 8:517-28 (2017)).

In view of the continuing need for improved strategies for diseases such as cancer, new dosage regimens for modulating CD73 and/or ENTPD2 activity are highly desirable.

Disclosure of Invention

Disclosed herein are anti-CD 73 antibodies that can be used alone or in combination with other therapeutic agents, procedures, or means (e.g., in combination with one or more of a programmed death receptor 1 (PD-1) inhibitor (e.g., an anti-PD 1 antibody molecule), an adenosine A2AR antagonist, and an extracellular enzyme extracellular nucleoside triphosphatehydrolase 2 (ENTPD 2) inhibitor) to treat or prevent a disorder, such as a cancerous disorder (e.g., a solid tumor). The present invention relates generally to dosage regimens for treating cancer using the anti-CD 73 antibodies disclosed herein.

Thus, in one aspect of the invention, there is provided an antibody molecule that binds human CD73 for use in or in a method of treating cancer in a subject, wherein the antibody molecule is administered in a ascending dosing regimen such that in a primary phase the antibody molecule dose is administered in a primary dose according to a primary dosing period having a primary dosing period frequency, the primary phase being preceded by an initial phase in which the antibody molecule is administered at a dosing period frequency higher than the primary dosing period frequency and via a fractionated dose of the primary dose, wherein in the initial phase over a period equal to the primary dosing period the sum of the fractionated doses should not exceed the amount of the primary phase dose.

In one embodiment of the invention, an antibody molecule that binds human CD73 is provided for use in treating cancer in a subject, wherein the antibody molecule is administered in a ascending dosing regimen such that in a main phase the antibody molecule dose is administered in a main dose according to a main dosing phase with a main dosing phase frequency, the main phase being preceded by an initial phase in which the antibody molecule is administered at a dosing phase frequency higher than the main dosing phase frequency and via a fractionated dose of the main dose, wherein in the initial phase within a period of time equal to the main dosing phase the sum of the fractionated doses should not exceed the amount of the main phase dose, wherein the antibody molecule comprises (i) a heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (vh1) amino acid sequence of SEQ ID NO:88, the cdr2 amino acid sequence of SEQ ID NO:89 and the VHCDR3 amino acid sequence of SEQ ID NO: 37; and (ii) a light chain variable region (VL) comprising the light chain complementarity determining region 1 (VLCDR 1) amino acid sequence of SEQ ID NO. 48, the VLCDR2 amino acid sequence of SEQ ID NO. 49 and the VLCDR3 amino acid sequence of SEQ ID NO. 50.

In another embodiment of the invention, there is provided a method of treating cancer in a subject, the method comprising administering to the subject an antibody molecule that binds human CD73 in an amount effective to treat the cancer, wherein the antibody molecule is administered in a ascending dosing regimen such that in a main phase the antibody molecule dose is administered in a main dose according to a main dosing period having a main dosing period frequency, the main phase being preceded by an initial phase in which the antibody molecule is administered at a dosing period frequency higher than the main dosing period frequency and administered via divided doses of the main dose, wherein the sum of the divided doses should not exceed the amount of the main phase dose in the initial phase over a period equal to the main dosing period, wherein the antibody molecule comprises (i) a heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (VHCDR 1) amino acid sequence of SEQ ID NO:88, the VHCDR2 amino acid sequence of SEQ ID NO:89, and the cdr3 amino acid sequence of SEQ ID NO: 37; and (ii) a light chain variable region (VL) comprising the light chain complementarity determining region 1 (VLCDR 1) amino acid sequence of SEQ ID NO. 48, the VLCDR2 amino acid sequence of SEQ ID NO. 49 and the VLCDR3 amino acid sequence of SEQ ID NO. 50.

In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule binding to human CD73 comprises

(i) A VH comprising a VH CDR1 amino acid sequence of SEQ ID NO. 38, a VH CDR2 amino acid sequence of SEQ ID NO. 36 and a VH CDR3 amino acid sequence of SEQ ID NO. 37; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 48, the VLCDR2 amino acid sequence of SEQ ID NO. 49 and the VLCDR3 amino acid sequence of SEQ ID NO. 50;

(ii) A VH comprising a VH CDR1 amino acid sequence of SEQ ID NO:72, a VH CDR2 amino acid sequence of SEQ ID NO:71 and a VH CDR3 amino acid sequence of SEQ ID NO: 37; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 48, the VLCDR2 amino acid sequence of SEQ ID NO. 49 and the VLCDR3 amino acid sequence of SEQ ID NO. 50;

(iii) A VH comprising a VH CDR1 amino acid sequence of SEQ ID NO. 38, a VH CDR2 amino acid sequence of SEQ ID NO. 71 and a VH CDR3 amino acid sequence of SEQ ID NO. 37; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 48, the VLCDR2 amino acid sequence of SEQ ID NO. 49 and the VLCDR3 amino acid sequence of SEQ ID NO. 50;

(iv) A VH comprising the VH CDR1 amino acid sequence of SEQ ID NO. 137, the VH CDR2 amino acid sequence of SEQ ID NO. 136 and the VH CDR3 amino acid sequence of SEQ ID NO. 37; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 48, the VLCDR2 amino acid sequence of SEQ ID NO. 49 and the VLCDR3 amino acid sequence of SEQ ID NO. 50;

(v) A VH comprising the VH CDR1 amino acid sequence of SEQ ID NO. 137, the VH CDR2 amino acid sequence of SEQ ID NO. 146 and the VH CDR3 amino acid sequence of SEQ ID NO. 37; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 48, the VLCDR2 amino acid sequence of SEQ ID NO. 49 and the VLCDR3 amino acid sequence of SEQ ID NO. 50; or alternatively

(vi) A VH comprising a VH CDR1 amino acid sequence of SEQ ID NO. 137, a VH CDR2 amino acid sequence of SEQ ID NO. 154 and a VH CDR3 amino acid sequence of SEQ ID NO. 37; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 48, the VLCDR2 amino acid sequence of SEQ ID NO. 49 and the VLCDR3 amino acid sequence of SEQ ID NO. 50.

A VH comprising a VH CDR1 amino acid sequence of SEQ ID NO. 38, a VH CDR2 amino acid sequence of SEQ ID NO. 36 and a VH CDR3 amino acid sequence of SEQ ID NO. 37; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 48, the VLCDR2 amino acid sequence of SEQ ID NO. 49 and the VLCDR3 amino acid sequence of SEQ ID NO. 50.

In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule that binds human CD73 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO 44, 77, 84, 142, 151, or 159 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO 44, 77, 84, 142, 151, or 159.

(i) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 44 (or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto) and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 55 (or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto);

(ii) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO:77 (or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto) and a light chain variable region comprising the amino acid sequence of SEQ ID NO:55 (or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto);

(iii) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 84 (or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto) and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 55 (or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto);

(iv) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 142 (or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto) and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 55 (or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto);

(v) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 151 (or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto) and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 55 (or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto); or alternatively

(vi) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 159 (or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto) and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 55 (or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto).

In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule that binds human CD73 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 44 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID No. 44.

In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule that binds human CD73 comprises a light chain variable region comprising the amino acid sequence of SEQ ID No. 55 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID No. 55.

In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule binding to human CD73 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 44 and a light chain variable region comprising the amino acid sequence of SEQ ID No. 55.

In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule that binds human CD73 comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 46, 79, 86, 114, 116, or 117 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID No. 46, 79, 86, 114, 116, or 117.

In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule that binds human CD73 comprises:

(i) A heavy chain comprising the amino acid sequence of SEQ ID NO. 46 (or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto) and a light chain comprising the amino acid sequence of SEQ ID NO. 57 (or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto);

(ii) A heavy chain comprising the amino acid sequence of SEQ ID NO. 114 (or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto) and a light chain comprising the amino acid sequence of SEQ ID NO. 57 (or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto);

(iii) A heavy chain comprising the amino acid sequence of SEQ ID NO. 79 (or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto) and a light chain comprising the amino acid sequence of SEQ ID NO. 57 (or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto);

(iv) A heavy chain comprising the amino acid sequence of SEQ ID NO. 116 (or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto) and a light chain comprising the amino acid sequence of SEQ ID NO. 57 (or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto);

(v) A heavy chain comprising the amino acid sequence of SEQ ID NO. 86 (or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto) and a light chain comprising the amino acid sequence of SEQ ID NO. 57 (or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto); or alternatively

(vi) A heavy chain comprising the amino acid sequence of SEQ ID NO. 117 (or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto) and a light chain comprising the amino acid sequence of SEQ ID NO. 57 (or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto).

In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule that binds human CD73 comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 46 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID No. 46. In an embodiment, X in SEQ ID NO:46 is K.

In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule that binds human CD73 comprises a light chain comprising the amino acid sequence of SEQ ID No. 57 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID No. 57.

In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule binding to human CD73 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 46 and a light chain comprising the amino acid sequence of SEQ ID NO. 57. In an embodiment, X in SEQ ID NO:46 is K.

In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule that binds human CD73 is antibody 373.A.

In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule that binds to human CD73 is a human antibody, a full length antibody, a bispecific antibody, fab, F (ab') 2, fv, or single chain Fv fragment (scFv).

In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule that binds human CD73 comprises a heavy chain constant region selected from the group consisting of IgG1, igG2, igG3, and IgG4, and a light chain constant region selected from the group consisting of kappa and lambda light chain constant regions.

In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule that binds human CD73 comprises a heavy chain constant region of IgG4 and a light chain constant region of κ.

In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule binding to human CD73 comprises a heavy chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 92-103, 119, and 120 and/or a light chain constant region comprising the amino acid sequence of SEQ ID NO: 104.

i) A human IgG4 heavy chain constant region having a mutation at position 228 according to EU numbering, or

ii) a human IgG4 heavy chain constant region with serine to proline mutation at position 228 according to EU numbering, or

iii) A heavy chain constant region comprising the amino acid sequence of SEQ ID NO. 92 or 93.

In embodiments of the antibodies used according to the invention or the methods according to the invention, the antibody molecules that bind human CD73 comprise one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more, e.g., all) of the following properties:

(i) For example, when the antibody molecule is tested as a bivalent antibody molecule using Octet, it binds to human CD73 with a dissociation constant (KD) of less than about 1x 10 "8M;

(ii) Binds to soluble human CD73 and/or membrane bound human CD73;

(iii) For example, by using Octet, does not bind murine CD73;

(iv) Inhibiting or reducing the enzymatic activity of CD73 (e.g., soluble human CD73 or membrane bound human CD 73), such as human CD 73-mediated conversion of Adenosine Monophosphate (AMP) to adenosine, as measured, for example, by a Malachite Green (MG) phosphate assay or a modified Cell Titer Glo (CTG) assay;

(v) For example, when the antibody molecule is tested as a bivalent antibody molecule using a modified Cell Titer Glo (CTG) assay, the membrane-bound human CD73 is inhibited by at least about 60%, 70%, 80% or 90% of the enzymatic activity;

(vi) Increasing proliferation of anti-CD 3/anti-CD 28 stimulated T cells (e.g., cd4+ T cells) in the presence of Adenosine Monophosphate (AMP), as measured, for example, by a CellTrace Violet (CTV) cell proliferation assay;

(vii) An N-terminal domain that binds human CD 73;

(viii) For example, when the antibody molecule is tested as a bivalent antibody molecule using hydrogen deuterium exchange mass spectrometry, hydrogen deuterium exchange is reduced for one or more regions selected from the group consisting of residues 158-172, residues 206-215, residues 368-387 and residues 87-104 of SEQ ID No. 105 when bound to one or more regions of a protein comprising the amino acid sequence of residues 27-547 of SEQ ID No. 105 (e.g., a protein consisting of the amino acid sequence of SEQ ID No. 171);

(ix) When bound to a protein comprising the amino acid sequence of residues 27-547 of SEQ ID NO. 105 (e.g., a protein consisting of the amino acid sequence of SEQ ID NO. 171), a conformational change of residues 368-387 of SEQ ID NO. 105 is induced;

(x) For example with at least one, two, three or four residues within residues 158-172 of SEQ ID NO. 105;

(xi) For example with at least one, two, three, four or five residues within residues 206-215 of SEQ ID NO. 105;

(xii) For example with at least one, two, three, four or five residues within residues 368-387 of SEQ ID NO. 105 or 106;

(xiii) For example with at least one, two, three, four or five residues within residues 87-104 of SEQ ID NO. 105;

(xiv) Binding a human CD73 dimer, said dimer consisting of a first CD73 monomer and a second CD73 monomer, wherein when the antibody molecule comprises a first antigen binding domain and a second antigen binding domain, the first antigen binding domain binds the first CD73 monomer and the second antigen binding domain binds the second CD73 monomer, e.g., when tested using size exclusion chromatography;

(xv) Binding to catalytically active, closed conformation human CD73 with a lower affinity, e.g., 50%, 60%, 70%, 80%, 90%, 95%, or 99% lower affinity, than when the antibody molecule binds to catalytically inactive, open conformation human CD73;

(xvi) Locking human CD73 into a catalytically inactive open conformation; or alternatively

(xvii) Preventing or reducing the conversion of human CD73 from a catalytically inactive open conformation to a catalytically active closed conformation, e.g., reducing the conversion by at least 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold as compared to the conversion in the absence of the antibody molecule.

In an embodiment of the antibody used according to the invention or of the method according to the invention, the sum of the divided doses added in an initial phase within a period of time equal to the main dosing period is equal to the main dose.

In an embodiment of the antibody for use according to the invention or the method according to the invention, the initial stage is used to prevent or reduce the incidence or severity of headache and/or migraine. In embodiments, the initial stage is used to prevent the incidence and/or severity of headache or migraine. In an embodiment, the initial stage is used to prevent the incidence of headache or migraine. In an embodiment, the initial stage is used to prevent the severity of headache or migraine. In embodiments, the initial stage is used to reduce the incidence and/or severity of headache or migraine. In an embodiment, the initial stage is used to reduce the incidence of headache or migraine. In an embodiment, the initial stage is used to reduce the severity of headache or migraine. The antibodies or methods used in such embodiments involving headache and/or migraine are anti-CD 73 antibodies.

In an embodiment of the antibody used according to the invention or the method according to the invention, the main dose of antibody molecules binding to human CD73 is 600mg. In an embodiment, the primary dose frequency is Q2W.

In an embodiment of the antibody used according to the invention or the method according to the invention, in the initial phase, the antibody molecule binding to human CD73 is administered at the frequency of QW.

In an embodiment of the antibody used according to the invention or the method according to the invention, the initial phase of administration of the anti-CD 73 antibody is two weeks.

In an embodiment of the antibody used according to the invention or the method according to the invention, in this initial phase the sum of the divided doses administered over a period equal to the main dosing period is equal to the main dose. In an embodiment, these initial stage doses are administered first in a lower amount than the main stage dose, and then in an intermediate amount that alters the main stage dose. The lower amount is lower than the main stage dose and the intermediate dose is a value between these two amounts. In an embodiment, in the initial phase, the divided doses are about 200mg and about 400mg, and the divided doses are administered within two weeks. In an alternative embodiment, in the initial phase, the divided doses are about 100mg and about 500mg, and the divided doses are administered within two weeks. In another alternative embodiment, in the initial phase, the sum of the divided doses administered over a period equal to the main dosing period is equal to the main dose, and wherein the divided doses are equal amounts, e.g., about 300mg and about 300mg. In these embodiments, these amounts refer to the amount of anti-CD 73 antibody.

In an embodiment, the anti-CD 73 antibody is administered on a ascending dosing regimen such that the anti-CD 73 antibody is administered as follows: in the initial phase of two weeks, about 200mg was administered once in the first week, about 400mg was administered once in the second week, and then about 600mg q2w was administered in the main phase.

In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule binding to human CD73 is administered as follows: in the initial phase, about 200mg was administered on day 1, about 400mg was administered on day 8, then about 600mg was administered at the beginning of the main phase on day 15, after which about 600mg was administered with Q2W continued.

In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule binding to human CD73 is administered by IV infusion within 1 to 2 hours, the frequency of administration being dependent on the frequency of dosing periods of each stage.

In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule that binds human CD73 is administered in combination with one or more therapeutic agents or procedures.

In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule that binds human CD73 is administered in combination with one or more therapeutic agents or procedures selected from one or more of the following: chemotherapy, targeted anti-cancer therapy, oncolytic drugs, cytotoxic agents, immune-based therapies, cytokines, surgery, radiological procedures, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, vaccines, or cell therapies. In embodiments, the one or more therapeutic agents are selected from one or more of the following: 1) Protein Kinase C (PKC) inhibitors; 2) Heat shock protein 90 (HSP 90) inhibitors; 3) Inhibitors of phosphoinositide 3-kinase (PI 3K) and/or rapamycin target protein (mTOR); 4) Inhibitors of cytochrome P450 (e.g., CYP17 inhibitors or 17α -hydroxylase/C17-20 lyase inhibitors); 5) An iron chelator; 6) An aromatase inhibitor; 7) Inhibitors of p53, e.g., inhibitors of the p53/Mdm2 interaction; 8) Apoptosis inducers; 9) An angiogenesis inhibitor; 10 Aldosterone synthase inhibitor; 11 A Smooth (SMO) receptor inhibitor; 12 A prolactin receptor (PRLR) inhibitor; 13 Wnt signaling inhibitors; 14 CDK4/6 inhibitors; 15 Fibroblast growth factor receptor 2 (FGFR 2)/fibroblast growth factor receptor 4 (FGFR 4) inhibitor; 16 Inhibitors of macrophage colony-stimulating factor (M-CSF); 17 Inhibitors of one or more of c-KIT, histamine release, flt3 (e.g., FLK2/STK 1), or PKC; 18 Inhibitors of one or more of VEGFR-2 (e.g., FLK-1/KDR), PDGFRβ, C-KIT or Raf kinase C; 19 A somatostatin agonist and/or a somatostatin release inhibitor; 20 Anaplastic Lymphoma Kinase (ALK) inhibitors; 21 Insulin-like growth factor 1 receptor (IGF-1R) inhibitors; 22 P-glycoprotein 1 inhibitors; 23 Vascular Endothelial Growth Factor Receptor (VEGFR) inhibitors; 24 BCR-ABL kinase inhibitors; 25 FGFR inhibitors; 26 Inhibitors of CYP11B 2; 27 An inhibitor of HDM2, such as an inhibitor of HDM2-p53 interaction; 28 Inhibitors of tyrosine kinase; 29 Inhibitors of c-MET; 30 Inhibitors of JAK; 31 DAC inhibitors; 32 11 beta-hydroxylase inhibitor; 33 An inhibitor of IAP; 34 Inhibitors of PIM kinase; 35 Inhibitors of bockipine (Porcupine); 36 Inhibitors of BRAF (e.g., BRAF V600E or wild-type BRAF); 37 An inhibitor of HER 3; 38 Inhibitors of MEK; or 39) inhibitors of lipid kinases.

In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule binding to human CD73 is administered in combination with triptan. In embodiments, the triptan is administered prior to one or more doses of the antibody molecule that binds human CD 73. In an embodiment, the triptan is selected from almotriptan, eletriptan, frovatriptan, naratriptan, rizatriptan, sumatriptan, zolmitriptan, lamiditan, optionally in combination with another agent, such as sumatriptan in combination with naproxen sodium. In embodiments, the triptan is sumatriptan or zolmitriptan.

In an embodiment of the antibody for use according to the invention or the method according to the invention, the antibody molecule that binds human CD73 is administered in combination with a PD-1 inhibitor. In embodiments, the PD-1 inhibitor is selected from the group consisting of: stadalimumab, nivolumab, pembrolizumab, pidazumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224. In embodiments, the PD-1 inhibitor is selected from the group consisting of: tirilizumab, stbadizumab, nivolumab, pembrolizumab, pierizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224. In embodiments, the PD-1 inhibitor is swabber. In embodiments, the PD-1 inhibitor is tirelimumab. In embodiments, the PD-1 inhibitor is an anti-PD-1 antibody molecule, wherein the anti-PD-1 antibody molecule is administered at a dose Q3W of about 300mg or at a dose Q4W of about 400 mg. In embodiments, the PD-1 inhibitor is an anti-PD-1 antibody molecule, wherein the anti-PD-1 antibody molecule is administered at a dose of about 400mg Q4W. In embodiments, the PD-1 inhibitor is swabber and is administered at a dose of about 400mg Q4W. In embodiments, the PD-1 inhibitor is tirelimumab and is administered at a dose of about 300mg Q4W.

In an embodiment of the antibody for use according to the invention or the method according to the invention, the antibody molecule that binds human CD73 is administered in combination with an adenosine A2AR antagonist. In the case of an embodiment of the present invention,

(i) The adenosine A2AR antagonist is selected from the group consisting of: PBF509, CPI444, AZD4635, vipadnan, GBV-2034 and AB928; or alternatively

(ii) The adenosine A2AR antagonist is selected from the group consisting of: 5-bromo-2, 6-di- (1H-pyrazol-1-yl) pyrimidin-4-amine; (S) -7- (5-methylfuran-2-yl) -3- ((6- (((tetrahydrofuran-3-yl) oxy) methyl) pyridin-2-yl) methyl) -3H- [1,2,3] triazolo [4,5-d ] pyrimidin-5-amine; (R) -7- (5-methylfuran-2-yl) -3- ((6- (((tetrahydrofuran-3-yl) oxy) methyl) pyridin-2-yl) methyl) -3H- [1,2,3] triazolo [4,5-d ] pyrimidin-5-amine, or a racemate thereof; 7- (5-methylfuran-2-yl) -3- ((6- (((tetrahydrofuran-3-yl) oxy) methyl) pyridin-2-yl) methyl) -3H- [1,2,3] triazolo [4,5-d ] pyrimidin-5-amine; 6- (2-chloro-6-methylpyridin-4-yl) -5- (4-fluorophenyl) -1,2, 4-triazin-3-amine.

In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule binding to human CD73 is administered in combination with the adenosine A2AR antagonist PBF 509. The PBF509 is also referred to as NIR178.

In an embodiment of the antibody used according to the invention or the method according to the invention, the adenosine A2AR antagonist is administered at a dose of about 80mg, about 160mg, 240mg or about 320 mg. In an embodiment of the antibody used according to the invention or the method according to the invention, the adenosine A2AR antagonist is administered twice daily (BID) at a dose of about 160mg or twice daily (BID) at a dose of about 240 mg. In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule binding to human CD73 is administered in combination with the adenosine A2AR antagonist PBF509 at a dose of about 240mg twice daily (BID).

In an embodiment of the antibody for use according to the invention or the method according to the invention, the antibody molecule that binds human CD73 is administered in combination with an anti-human ENTPD2 antibody. In an embodiment, an anti-human ENTPD2 antibody is administered in combination with an anti-human ENTPD2 antibody having the sequence given in table 9. In embodiments, the anti-human ENTPD2 antibody comprises a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO. 401, the VH CDR2 amino acid sequence of SEQ ID NO. 402 and the VH CDR3 amino acid sequence of SEQ ID NO. 403; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 414, the VLCDR2 amino acid sequence of SEQ ID NO. 415 and the VLCDR3 amino acid sequence of SEQ ID NO. 416. In an embodiment, the anti-human ENTPD2 antibody comprises a VH having the sequence of SEQ ID NO. 410 and a VL having the sequence of SEQ ID NO. 421. In an embodiment, the anti-human ENTPD2 antibody comprises a heavy chain having the sequence of SEQ ID NO:412 and a light chain having the sequence of SEQ ID NO: 423.

In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule binding to human CD73 is administered as follows: in the initial phase, about 200mg QW was administered, then about 400mg QW was administered, followed by about 600mg Q2W in the main phase. In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule binding to human CD73 is administered in combination with swabber administered at about 400mg q4w and PBF509 administered at about 240mg BID according to the following: in the initial phase, about 200mg QW was administered, then about 400mg QW was administered, followed by about 600mg Q2W in the main phase.

In an embodiment of the antibody for use according to the invention or the method according to the invention, the cancer is selected from lung cancer (e.g. non-small cell lung cancer), pancreatic cancer (e.g. pancreatic ductal adenocarcinoma), breast cancer (e.g. triple negative breast cancer), melanoma, head and neck cancer (e.g. squamous head and neck cancer), colorectal cancer (e.g. microsatellite stabilized (MSS) colorectal cancer), ovarian cancer, metastatic castration-resistant prostate cancer or renal cancer (e.g. renal cell carcinoma). In an embodiment of the antibody for use according to the invention or the method according to the invention, the cancer is selected from non-small cell lung cancer, pancreatic ductal adenocarcinoma, triple negative breast cancer, microsatellite stabilized (MSS) colorectal cancer, metastatic castration-resistant prostate cancer, ovarian cancer or renal cell carcinoma.

In an embodiment of the antibody for use according to the invention or the method according to the invention, the antibody molecule that binds human CD73 is in the form of a pharmaceutical composition comprising an anti-CD 73 antibody molecule as defined herein and a pharmaceutically acceptable carrier, excipient or stabilizer.

Disclosed herein are anti-ENTPD 2 antibodies, which can be used alone or in combination with other therapeutic agents, procedures, or means (e.g., in combination with one or more of a programmed death receptor 1 (PD-1) inhibitor (e.g., an anti-PD 1 antibody molecule), an adenosine A2AR antagonist, and a CD73 inhibitor (e.g., an anti-CD 73 antibody molecule) to treat or prevent a disorder, such as a cancerous disorder (e.g., a solid tumor). The present invention relates generally to dosage regimens for treating cancer using the anti-ENTPD 2 antibodies disclosed herein.

In one embodiment of the invention, an antibody molecule that binds to human ENTPD2 is provided for use in treating cancer in a subject, wherein the antibody molecule is administered in a ascending dosing regimen such that in a main phase the antibody molecule dose is administered in a main dose according to a main dosing phase with a main dosing phase frequency, the main phase being preceded by an initial phase in which the antibody molecule is administered at a dosing phase frequency equal to or higher than the main dosing phase frequency and via a fractionated dose of the main dose, wherein in the initial phase within a period of time equal to the main dosing phase the sum of the fractionated doses should not exceed the amount of the main phase dose.

In another embodiment of the invention, there is provided a method of treating cancer in a subject, the method comprising administering to the subject an antibody molecule that binds human ENTPD2 in an amount effective to treat the cancer, wherein the antibody molecule is administered in a ascending dosing regimen such that in a primary phase the antibody molecule dose is administered in a primary dose according to a primary dosing period having a primary dosing period frequency, the primary phase being preceded by an initial phase in which the antibody molecule is administered at a dosing period frequency equal to or higher than the primary dosing period frequency, and via a fractionated dose of the primary dose, wherein the sum of the fractionated doses should not exceed the amount of the primary phase dose in the initial phase over a period of time equal to the primary dosing period.

In an embodiment of the anti-ent pd2 antibody for use according to the invention or of the method of treatment according to the invention involving an anti-ent pd2 antibody, the antibody molecule comprises:

(i) A heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (VHCDR 1) amino acid sequence of SEQ ID NO. 401, the VHCDR2 amino acid sequence of SEQ ID NO. 402 and the VHCDR3 amino acid sequence of SEQ ID NO. 403; and

(ii) A light chain variable region (VL) comprising the light chain complementarity determining region 1 (VLCDR 1) amino acid sequence of SEQ ID NO:414, the VLCDR2 amino acid sequence of SEQ ID NO:415 and the VLCDR3 amino acid sequence of SEQ ID NO: 416.

In an embodiment of the anti-ent pd2 antibody for use according to the invention or of the method of treatment according to the invention involving an anti-ent pd2 antibody, the antibody molecule comprises: a heavy chain variable region (VH) comprising SEQ ID NO 410 or a sequence having at least about 95% or more identity thereto and a light chain variable region (VL) comprising SEQ ID NO 421 or a sequence having at least about 95% or more identity thereto.

In an embodiment of the anti-ent pd2 antibody for use according to the invention or of the method of treatment according to the invention involving an anti-ent pd2 antibody, the antibody molecule comprises: a heavy chain comprising SEQ ID NO. 412 or a sequence having at least about 95% or more identity thereto and a light chain comprising SEQ ID NO. 423 or a sequence having at least about 95% or more identity thereto.

In an embodiment of the antibody for use according to the invention or the method according to the invention, the initial stage is for preventing or reducing the incidence or severity of Cytokine Release Syndrome (CRS). The antibody or method used in such an embodiment is an anti-ENTPD 2 antibody.

The following examples relate to dosage regimens for anti-ENTPD 2 antibodies. In an embodiment of the antibody used according to the invention or the method according to the invention, the primary dose is 300mg, 600mg, 1200mg, or 2400mg and/or the primary dose frequency is Q2W. In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule is administered at the frequency of QW or Q2W in the initial phase. In an embodiment of the antibody used according to the invention or the method according to the invention, the split dose in the initial phase is 100mg. In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule is administered once or twice within two weeks in the initial phase. In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule is administered as follows: in the initial phase, about 100mg was administered on day 1, then about 300mg was administered at the beginning of the main phase on day 15, after which Q2W administration was continued for about 300mg. In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule is administered as follows: in the initial phase, about 100mg was administered on day 1, about 100mg was administered on day 8, then about 300mg was administered at the beginning of the main phase on day 15, after which about 300mg was administered with Q2W continued. In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule is administered intravenously to the subject in a 1 hour (up to 2 hours if clinically indicated) infusion.

In an embodiment of the antibody used according to the invention or the method according to the invention, the initial phase of administration of the anti-ent pd2 antibody is two weeks.

In an embodiment, the primary dose frequency of the anti-ent pd2 antibody is Q2W.

In an embodiment of the antibody for use according to the invention or the method according to the invention, the antibody molecule is administered in combination with one or more therapeutic agents or procedures.

The following examples relate to dosage regimens of anti-ent pd2 antibodies and combination agents administered with anti-ent pd2 antibodies. In an embodiment of the antibody for use according to the invention or the method according to the invention, the antibody molecule is administered in combination with a PD-1 inhibitor. In one embodiment, the PD-1 inhibitor is selected from the group consisting of: tirilizumab, stbadizumab, nivolumab, pembrolizumab, pierizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224. In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody molecule, wherein the anti-PD-1 antibody molecule is administered at a dose of about 400mg Q4W.

The following examples relate to dosage regimens of anti-ent pd2 antibodies and combination agents administered with anti-ent pd2 antibodies. In an embodiment of the antibody for use according to the invention or the method according to the invention, the antibody molecule is administered in combination with an adenosine A2AR antagonist. In one embodiment, (i) the adenosine A2AR antagonist is selected from the group consisting of: PBF509, CPI444, AZD4635, vipadnan, GBV-2034 and AB928; or (ii) an adenosine A2AR antagonist is selected from the group consisting of: 5-bromo-2, 6-di- (1H-pyrazol-1-yl) pyrimidin-4-amine; (S) -7- (5-methylfuran-2-yl) -3- ((6- (((tetrahydrofuran-3-yl) oxy) methyl) pyridin-2-yl) methyl) -3H- [1,2,3] triazolo [4,5-d ] pyrimidin-5-amine; (R) -7- (5-methylfuran-2-yl) -3- ((6- (((tetrahydrofuran-3-yl) oxy) methyl) pyridin-2-yl) methyl) -3H- [1,2,3] triazolo [4,5-d ] pyrimidin-5-amine, or a racemate thereof; 7- (5-methylfuran-2-yl) -3- ((6- (((tetrahydrofuran-3-yl) oxy) methyl) pyridin-2-yl) methyl) -3H- [1,2,3] triazolo [4,5-d ] pyrimidin-5-amine; 6- (2-chloro-6-methylpyridin-4-yl) -5- (4-fluorophenyl) -1,2, 4-triazin-3-amine; preferably, wherein the adenosine A2AR antagonist is PBF509. In one embodiment, the adenosine A2AR antagonist is administered at a dose of about 80mg, about 160mg, 240mg, or about 320mg, preferably wherein the adenosine A2AR antagonist is administered at a dose of about 160mg twice daily (BID).

The following examples relate to dosage regimens of anti-ent pd2 antibodies and combination agents administered with anti-ent pd2 antibodies. In an embodiment of the antibody for use according to the invention or the method according to the invention, the antibody molecule is administered in combination with an anti-human CD73 antibody. In one embodiment, the anti-human CD73 antibody comprises: (i) A heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (VHCDR 1) amino acid sequence of SEQ ID NO. 88, the VHCDR2 amino acid sequence of SEQ ID NO. 89 and the VHCDR3 amino acid sequence of SEQ ID NO. 37; and (ii) a light chain variable region (VL) comprising the light chain complementarity determining region 1 (VLCDR 1) amino acid sequence of SEQ ID NO. 48, the VLCDR2 amino acid sequence of SEQ ID NO. 49 and the VLCDR3 amino acid sequence of SEQ ID NO. 50. In one embodiment, the anti-human CD73 antibody comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 44 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO. 44 and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO. 55 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO. 55. In one embodiment, the anti-human CD73 antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO. 46 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO. 46, wherein X in SEQ ID NO. 46 is K, and/or a light chain comprising the amino acid sequence of SEQ ID NO. 57 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO. 57.

In an embodiment of the antibody for use according to the invention or the method according to the invention, the cancer is MSS colorectal cancer (CRC), cholangiocarcinoma (intrahepatic or extrahepatic), pancreatic cancer, esophageal-gastric junction (EGJ) cancer or gastric cancer.

In an embodiment of the antibody for use according to the invention or the method according to the invention, the antibody molecule that binds to human ENTPD2 is in the form of a pharmaceutical composition comprising the antibody molecule of claim 33 and a pharmaceutically acceptable carrier, excipient or stabilizer.

In an embodiment of the antibody for use according to the invention or the method according to the invention, the antibody molecule that binds to human ENTPD2 is in the form of a pharmaceutical composition comprising an anti-ENTPD 2 antibody as disclosed herein and a pharmaceutically acceptable carrier, excipient or stabilizer.

In one aspect of the invention, both the anti-CD 73 antibody and the anti-ENTPD 2 antibody are administered in ascending dosing regimens as described herein. In embodiments, both the anti-CD 73 antibody and the anti-ENTPD 2 antibody are administered in ascending dosing regimens, such that

a) In a primary phase of administration of an anti-CD 73 antibody molecule, the dose is administered at a primary dose of the anti-CD 73 antibody according to a primary administration phase and a primary administration phase frequency, the primary phase being preceded by an initial phase in which the anti-CD 73 antibody molecule is administered at a higher administration phase frequency than the primary administration phase frequency of the anti-CD 73 antibody and via a fractionated dose of the primary dose of the anti-CD 73 antibody, wherein the sum of the fractionated doses of the anti-CD 73 antibody should not exceed the amount of the primary phase dose of the anti-CD 73 antibody in the initial phase within a period of time equal to the primary administration phase of the anti-CD 73 antibody;

And is also provided with

b) In a main phase of administration of the anti-ENTPD 2 antibody molecule, the dose is administered at a main dose of the anti-ENTPD 2 antibody according to a main administration phase and a main administration phase frequency of the anti-ENTPD 2 antibody, the main phase being preceded by an initial phase in which the anti-ENTPD 2 antibody molecule is administered at a dosing phase frequency higher than the main administration phase frequency of the anti-ENTPD 2 antibody and via divided doses of the main dose of the anti-ENTPD 2 antibody, wherein the sum of the divided doses of the anti-ENTPD 2 antibody should not exceed the amount of the main phase dose of the anti-ENTPD 2 antibody in the initial phase within a period equal to the main administration phase of the anti-ENTPD 2 antibody.

In embodiments, both the anti-CD 73 antibody and the anti-ENTPD 2 antibody are administered in ascending dosing regimens, such that

a) The anti-CD 73 antibody was administered as follows: in the initial phase of two weeks, about 200mg once in the first week, about 400mg once in the second week, then the main phase, thereafter about 600mg q2w, and

b) The anti-ENTPD 2 antibody was administered as follows: in the initial phase of two weeks i) at about 100mg once, or ii) at 100mg in the first week, at about 100mg once in the second week, and then at about 300mg q2w in the main phase.

Description of the Table

The amino acid and nucleotide sequences of exemplary anti-CD 73 antibodies are provided in table 1.

Table 2 provides the consensus CDR sequences of exemplary anti-CD 73 antibodies.

The amino acid sequences of human IgG heavy and human kappa light chains are provided in table 3.

Table 4 provides exemplary sequences for CD 73.

Tables 5 and 6 provide amino acid and/or nucleotide sequences of exemplary anti-PD-1 antibody molecules.

Tables 7 and 8 provide amino acid and/or nucleotide sequences of exemplary anti-PD-L1 antibody molecules.

Table 9 provides amino acid and nucleotide sequences of exemplary anti-ENTPD 2 antibodies.

Table 10 provides nomenclature for two lineages of anti-CD 73 antibodies.

Table 11 provides the affinities of anti-CD 73 antibodies.

Table 12 provides the affinities of anti-CD 73 Fab.

Table 13 provides temporary dose levels of anti-CD 73 antibody 373. A.

Table 14 provides temporary dose levels of anti-CD 73 antibody 373.A in combination with PBF 509.

Table 15 provides temporary dose levels of anti-CD 73 antibody 373.A in combination with swabber.

Table 16 provides temporary dose levels of PBF509 in combination with anti-CD 73 antibody 373.A and swabber.

Table 17 provides the corresponding germline sequences for anti-CD 73 antibodies.

Table 18 provides a list of study drugs.

Table 19 describes the initial and temporary dose levels for anti-ENTPD 2 mAb1 schedule 1.

Table 20 describes temporary dose levels for anti-ent pd2 mAb1, schedules 2 and 3.

Table 21 depicts temporary dose levels of anti-ENTPD 2 mAb1 combined with Stdazumab.

Table 22 describes temporary dose levels of anti-ent pd2 mAb1 in combination with NIR 178.

Table 23 depicts temporary dose levels of anti-ENTPD 2 mAb1 combined with anti-CD 73 antibodies.

Detailed Description

As used herein, the term "CD73" refers to "cluster of differentiation 73", also known as 5' -nucleotidase (5 ' -NT) or extracellular 5' -nucleotidase. The term "CD73" includes mutants, fragments, variants, isoforms and homologs of full length wild-type CD73. In one embodiment, the CD73 protein is encoded by the NT5E gene. Exemplary CD73 sequences are available in Uniprot databases under accession numbers Q6NZX and P21589. An exemplary immature CD73 amino acid sequence is provided as SEQ ID NO. 105-107."CD73 monomer" refers to a polypeptide comprising the extracellular domain of CD73. In one embodiment, the CD73 monomer is full length CD73."CD73 dimer" refers to two polypeptides (e.g., two non-covalently associated polypeptides) that consist of two CD73 monomers (e.g., two identical CD73 monomers) that interact with each other to form a stable dimer (e.g., a dimer formed by protein-protein interactions between the C-terminal domains of CD73 monomers). In one embodiment, the CD73 dimer is a naturally occurring CD73 dimer.

Without wishing to be bound by theory, human CD73 has two domains. The conserved N-terminal domain (corresponding approximately to residues 29-310 of SEQ ID NO: 105) and the conserved C-terminal domain (corresponding approximately to residues 343-513 of SEQ ID NO: 105) are linked by a single alpha-helix (corresponding approximately to residues 318-336 of SEQ ID NO: 105). The active site is detected predominantly in the closed conformation and is formed between the C-and N-terminal domains. For enzyme catalysis, a domain movement of about 100 ° relative to the C-terminal domain of the N-terminal domain may enable substrate binding and release, which occurs in an open (catalytically inactive) conformation. Human CD73 forms dimers through protein-protein interactions between C-terminal domains. The buried area between the active and inactive conformations of the enzyme and the molecular interactions at the dimer interface are significantly different. See, e.g., knapp K et al, structure 20:2161-73 (2012), which is incorporated herein by reference in its entirety.

Disclosed herein are antibody molecules that bind CD73 with high affinity and specificity. In one embodiment, disclosed herein are human antibodies that bind CD 73. In one embodiment, disclosed herein are antibody molecules capable of inhibiting or reducing the enzymatic activity of CD73 (e.g., human CD73, e.g., soluble human CD73 or membrane-bound human CD 73). In one embodiment, disclosed herein are antibody molecules that are capable of inhibiting or reducing CD 73-mediated conversion of Adenosine Monophosphate (AMP) to adenosine. The anti-CD 73 antibody molecules disclosed herein are useful for the treatment, prevention and/or diagnosis of cancerous or malignant disorders, such as solid tumors and liquid tumors, for example, lung cancer (e.g., non-small cell lung cancer), pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), breast cancer (e.g., triple negative breast cancer), melanoma, head and neck cancer (e.g., squamous head and neck cancer), colorectal cancer (e.g., microsatellite stabilized (MSS) colorectal cancer), ovarian cancer, metastatic castration-resistant prostate cancer, or renal cancer (e.g., renal cell carcinoma). In embodiments of the invention, the cancer to be treated is non-small cell lung cancer, pancreatic ductal adenocarcinoma, triple negative breast cancer, microsatellite stabilized (MSS) colorectal cancer, metastatic castration-resistant prostate cancer, ovarian cancer, or renal cell carcinoma.

As used herein, extracellular nucleoside diphosphate hydrolase 2 (ENTPD 2) (also known as CD39 antigen-like 1, CD39L1, extracellular-ATP diphosphate hydrolase 2, extracellular-atpase 2, NTPD enzyme-2, NTPD enzyme 2) refers to an enzyme of type 2 of the extracellular-nucleoside diphosphate hydrolase family (E-NTPD enzyme), which is the extracellular-nucleoside enzyme family that hydrolyzes 5' -triphosphates. The ENTPD2 enzyme is encoded by the gene ENTPD 2. The human ENTPD2 gene maps to chromosomal location 9q34.3, and the genomic sequence of the human ENTPD2 gene can be found in GenBank at nc_ 000009.12. mRNA and protein sequences for variants of the ENTPD2 human transcript can be found in GenBank under the following accession numbers:

isoform 1: NM-203468.2 (mRNA) →NP-982293.1 (protein with 495 aa);

isoform 2: NM_001246.3 (mRNA) → NP_001237.1 (protein having 472 aa);

extracellular nucleoside diphosphate hydrolase 2 isoform 1[ Chile, NP-982293.1 ]

Chile extracellular nucleoside diphosphate hydrolase 2 (ENTPD 2), transcript variant 1, mRNA [ NM-203468.2 ]

/>

Extracellular nucleoside diphosphate hydrolase 2 isoform 2[ Chile, NP-001237.1 ]

Chile extracellular nucleoside diphosphate hydrolase 2 (ENTPD 2), transcript variant 2, mRNA [ NM-001246.3 ]

/>

As used herein, human ENTPD2 protein also encompasses proteins having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over their entire length to any of the ENTPD2 isoforms. The sequence of murine, cynomolgus and other animal ENTPD2 proteins is known in the art.

Additional terms are defined below and throughout the application.

As used herein, the articles "a" and "an" refer to one or to more than one (e.g., to at least one) of the grammatical object of the article. As used herein, "plurality" refers to two or more.

The term "or" means and is used interchangeably with the term "and/or" herein unless the context clearly indicates otherwise.

"about" and "approximately" generally mean an acceptable degree of error in the measured quantity given the nature or accuracy of the measurement. Exemplary degrees of error are within 20%, typically within 10%, and more typically within 5% of a given value or range of values.

The compositions and methods disclosed herein encompass polypeptides and nucleic acids having a specified sequence or a sequence that is substantially identical or similar thereto, e.g., a sequence having at least about 85%, 90%, 95%, 97%, or 99% sequence identity to the specified sequence. In the context of amino acid sequences, the term "substantially identical" is used herein to refer to such first amino acid: it contains i) the same as the aligned amino acid residues in the second amino acid sequence, or ii) a sufficient or minimum number of amino acid residues that are conservative substitutions of aligned amino acid residues in the second amino acid sequence, such that the first amino acid sequence and the second amino acid sequence may have a common domain and/or a common functional activity. Such as an amino acid sequence that contains a common domain that is at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence (e.g., a sequence provided herein).

In the context of nucleotide sequences, the term "substantially identical" is used herein to refer to such first nucleic acid sequences: it contains a sufficient or minimal number of nucleotides that are identical to the aligned nucleotides in the second nucleic acid sequence such that the first nucleotide sequence and the second nucleotide sequence encode a polypeptide having a common functional activity, or encode a common structural polypeptide domain or have a common functional polypeptide activity. Such as a nucleotide sequence having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence (e.g., a sequence provided herein).

The term "functional variant" refers to a polypeptide that has, or is encoded by, a substantially identical amino acid sequence to a naturally occurring sequence, and is capable of having one or more activities of the naturally occurring sequence.

The calculation of homology or sequence identity between sequences (these terms are used interchangeably herein) is performed as follows.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps are introduced in one or both of the first amino acid and the second amino acid or the first nucleic acid sequence and the second nucleic acid sequence for optimal alignment, and non-homologous sequences are negligible for comparison purposes). In preferred embodiments, the length of the reference sequences aligned for comparison purposes is at least 30%, such as at least 40%, 50%, 60%, such as at least 70%, 80%, 90%, 100% of the length of the reference sequences. The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in a first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in a second sequence, then the molecules are identical at that position.

Taking into account the number of gaps and the length of each gap, the percent identity between two sequences is a function of the number of identical positions shared by the sequences, which gaps need to be introduced for optimal alignment of the two sequences.

Sequence comparison and percent identity determination between two sequences can be accomplished using mathematical algorithms. In some embodiments, percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) j.mol.biol. [ journal of molecular biology ] 48:444-453) algorithm, which has been incorporated into the GAP program in the GCG software package (available in http:// www.gcg.com), using either the Blossum 62 matrix or PAM250 matrix, the GAP weights of 16, 14, 12, 10, 8, 6, or 4, and the length weights of 1, 2, 3, 4, 5, or 6. In certain embodiments, the percentage identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available from http:// www.gcg.com), using the NWS gapdna.CMP matrix, and a GAP weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. One suitable set of parameters (and parameters that should be used unless otherwise indicated) is the Blossum 62 scoring matrix, with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

The percent identity between two amino acid sequences or nucleotide sequences can be determined using the algorithm of E.Meyers and W.Miller ((1989) computer application in CABIOS [ biosciences ]4:11-17, which has been incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, the gap length penalty of 12, and the gap penalty of 4.

The nucleic acid sequences and protein sequences described herein may be used as "query sequences" to search public databases, for example, to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs of Altschul et al (1990) J.mol.biol. [ journal of molecular biology ]215:403-10 (version 2.0). BLAST nucleotide searches can be performed using the NBLAST program (score=100, word length=12) to obtain nucleotide sequences homologous to the nucleic acids described herein. BLAST protein searches can be performed using the XBLAST program (score=50, word length=3) to obtain amino acid sequences homologous to protein molecules described herein. To obtain a gap alignment for comparison purposes, gap BLAST (Gapped BLAST) may be used as described in Altschul et al, (1997) Nucleic Acids Res [ nucleic acids Ind 25:3389-3402. When using BLAST and empty BLAST programs, default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http:// www.ncbi.nlm.nih.gov.

As used herein, the term "hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions" describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology [ guidelines for molecular biology experiments ], john Wiley & Sons, N.Y [ John Weili father-son publishing company ], new York state (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and non-aqueous methods are described in this reference and either may be used. Specific hybridization conditions referred to herein are as follows: 1) Low stringency hybridization conditions: hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45℃followed by washing twice in 0.2 XSSC, 0.1% SDS at least 50℃for low stringency conditions (the temperature of washing can be increased to 55 ℃); 2) Moderately stringent hybridization conditions: hybridization in 6 XSSC at about 45℃followed by one or more washes in 0.2 XSSC, 0.1% SDS at 60 ℃; 3) High stringency hybridization conditions: hybridization in 6 XSSC at about 45℃followed by one or more washes in 0.2 XSSC, 0.1% SDS at 65 ℃; and preferably, 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS,65℃followed by one or more washes in 0.2 XSSC, 1% SDS,65 ℃. Unless otherwise specified, very high stringency conditions (4) are suitable conditions, and these conditions should be used.

It will be appreciated that the molecules used in the dosage regimen of the invention may have additional conservative or non-essential amino acid substitutions that have no substantial effect on the function of the molecule.

The term "amino acid" is intended to include all molecules, whether natural or synthetic, that include both amino and acid functionalities and can be included in polymers of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; their analogs, derivatives and congeners; amino acid analogs having variant side chains; and all stereoisomers of any of the foregoing. As used herein, the term "amino acid" includes D-optical isomers or L-optical isomers and peptide mimetics.

A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with the following side chains: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

The terms "polypeptide", "peptide" and "protein" (if single-chain) are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation to a labeling component. The polypeptide may be isolated from a natural source, may be produced from a eukaryotic or prokaryotic host by recombinant techniques, or may be the product of a synthetic procedure.

The terms "nucleic acid", "nucleic acid sequence", "nucleotide sequence" or "polynucleotide sequence" and "polynucleotide" are used interchangeably. They refer to nucleotides of any length in polymeric form, i.e., deoxyribonucleotides or ribonucleotides, or analogs thereof. The polynucleotide may be single-stranded or double-stranded, and if single-stranded, the polynucleotide may be the coding strand or the non-coding (antisense) strand. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The nucleic acid may be a recombinant polynucleotide, or a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin, which does not exist in nature or is linked to another polynucleotide in a non-natural arrangement.

As used herein, the term "isolated" refers to a material that is removed from its original or natural environment (e.g., its naturally occurring natural environment). For example, instead of isolating naturally occurring polynucleotides or polypeptides present in a living animal, the same polynucleotides or polypeptides are isolated from some or all of the coexisting materials in the natural system by human intervention. Such polynucleotides may be part of a vector and/or such polynucleotides or polypeptides may be part of a composition and still be isolated in that such vector or composition is not part of its naturally occurring environment.

Antibody molecules

In one embodiment of a dosage regimen according to the invention, the antibody molecule binds to mammalian (e.g., human) CD73. For example, the antibody molecule binds an epitope on CD73, such as a linear or conformational epitope (e.g., an epitope as described herein).

In another embodiment of a dosage regimen according to the invention, the antibody molecule binds to mammalian (e.g., human) ENTPD2. For example, the antibody molecule binds an epitope on ENTPD2, such as a linear or conformational epitope (e.g., an epitope as described herein).

As used herein, the term "antibody molecule" refers to a protein, such as an immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence. The term "antibody molecule" includes, for example, monoclonal antibodies (including full length antibodies having an immunoglobulin Fc region). In embodiments, the antibody molecule comprises a full length antibody or a full length immunoglobulin chain. In embodiments, the antibody molecule comprises an antigen binding or functional fragment of a full length antibody or full length immunoglobulin chain.

As used herein, an antibody molecule "binds" to an antigen, as such binding is understood by those skilled in the art. In one embodiment, the antibody is at about 1x 10 ^-3 M or less, 1x 10 ^-4 M or less or 1x 10 ^-5 Dissociation constant (K) of M or less _D ) Binding to the antigen.

In embodiments, the antibody molecule is a monospecific antibody molecule and binds a single epitope, e.g., a monospecific antibody molecule having multiple immunoglobulin variable domain sequences, each immunoglobulin variable domain sequence binding the same epitope.

In embodiments, the antibody molecule is a multi-specific antibody molecule, e.g., comprising a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence in the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence in the plurality has binding specificity for a second epitope. In embodiments, the first epitope and the second epitope are on the same antigen (e.g., the same protein (or subunit of a multimeric protein)). In embodiments, the first epitope and the second epitope overlap or substantially overlap. In embodiments, the first epitope and the second epitope do not overlap or substantially do not overlap. In embodiments, the first epitope and the second epitope are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In embodiments, the multispecific antibody molecule comprises a third, fourth, or fifth immunoglobulin variable domain. In embodiments, the multispecific antibody molecule is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

In embodiments, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibodies are specific for no more than two antigens. Bispecific antibody molecules are characterized by a first immunoglobulin variable domain sequence having binding specificity for a first epitope and a second immunoglobulin variable domain sequence having binding specificity for a second epitope. In embodiments, the first epitope and the second epitope are on the same antigen (e.g., the same protein (or subunit of a multimeric protein)). In embodiments, the first epitope and the second epitope overlap or substantially overlap. In embodiments, the first epitope and the second epitope do not overlap or substantially do not overlap. In embodiments, the first epitope and the second epitope are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In embodiments, the bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a second epitope. In an embodiment, the bispecific antibody molecule comprises a half-antibody having binding specificity for a first epitope and a half-antibody having binding specificity for a second epitope. In embodiments, the bispecific antibody molecule comprises a half-antibody or fragment thereof having binding specificity for a first epitope and a half-antibody or fragment thereof having binding specificity for a second epitope. In an embodiment, the bispecific antibody molecule comprises an scFv or fragment thereof having binding specificity for a first epitope and an scFv or fragment thereof having binding specificity for a second epitope.

In embodiments, antibody molecules include diabodies, and single chain molecules, as well as antigen binding fragments of antibodies (e.g., fab, F (ab') ₂ And Fv). For example, an antibody molecule may include a heavy (H) chain variable domain sequence (abbreviated herein as VH), and a light (L) chain variable domain sequence (abbreviated herein as VL). In embodiments, the antibody molecule comprises or consists of heavy and light chains (referred to herein as half antibodies). In another example, an antibody molecule, such as Fab, fab ', F (ab'), comprises two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequences, thereby forming two antigen binding sites ₂ Fc, fd', fv, single chain antibodies (e.g., scFv), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be produced by modification of an intact antibody or may be synthesized de novo using recombinant DNA techniques. These functional antibody fragments retain the ability to selectively bind to their respective antigens or receptors. Antibodies and antibody fragments may be from any class of antibodies, including but not limited to IgG, igA, igM, igD and IgE, as well as from any subclass of antibodies (e.g., igG1, igG2, igG3, and IgG 4). The preparation of antibody molecules may be monoclonal or polyclonal. Antibody molecules may also be Human, humanized, CDR-grafted or in vitro generated antibodies. The antibody may have a heavy chain constant region selected from, for example, igG1, igG2, igG3, or IgG 4. Antibodies may also have a light chain selected from, for example, kappa or lambda. The term "immunoglobulin" (Ig) is used interchangeably herein with the term "antibody".

Examples of antigen binding fragments of antibody molecules include: (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) A F (ab') 2 fragment, which is a bivalent fragment comprising two Fab fragments linked at the hinge region by a disulfide bridge; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of a single arm of an antibody, (v) diabody antibody (dAb) fragments consisting of VH domains; (vi) Camelidae (camelid) or camelized (camelized) variable domains; (vii) Single chain Fv (scFv) (see, e.g., bird et al (1988) Science [ Science ]242:423-426; and Huston et al (1988) Proc.Natl. Acad. Sci. USA [ Proc. Natl. Acad. Sci. USA ] 85:5879-5883); (viii) single domain antibodies. These antibody fragments may be obtained using any suitable method, including conventional techniques known to those skilled in the art, and can be screened for efficacy in the same manner as whole antibodies.

The term "antibody" includes intact molecules as well as functional fragments thereof. The constant region of an antibody can be altered (e.g., mutated) to modify the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, or complement function).

The antibodies disclosed herein may also be single domain antibodies. Single domain antibodies may include antibodies whose complementarity determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies that naturally lack a light chain, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. The single domain antibody may be any antibody of the art, or any future single domain antibody. The single domain antibody may be derived from any species including, but not limited to, mouse, human, camel, llama, fish, shark, goat, rabbit, and cow. In embodiments, the single domain antibody is a naturally occurring single domain antibody, referred to as a heavy chain antibody lacking a light chain. Such single domain antibodies are disclosed, for example, in WO 9404678. For clarity reasons, such variable domains derived from heavy chain antibodies that naturally lack light chains are referred to herein as VHHs or nanobodies to distinguish them from conventional VH's of four-chain immunoglobulins. Such VHH molecules may be derived from camelidae species, such as camels, llamas, dromedaries, alpacas and antibodies raised in dromedaries.

VH and VL regions can be subdivided into regions of higher variability termed "complementarity determining regions" (CDRs) with more conserved regions termed "framework regions" (FR or FW) interposed therebetween.

The framework regions and CDR ranges have been precisely defined by a number of methods (see Kabat, E.A. et al (1991) Sequences of Proteins of Immunological Interest [ protein sequences with immunological significance ], 5 th edition, U.S. device of Health and Human Services [ U.S. health and public service ], NIH publication No. 91-3242; chothia, C. et al (1987) J.mol. Biol. [ journal of Molecular biology ]196:901-917; and AbM definitions used by Oxford Molecular AbM antibody modeling software (Oxford Molecular's AbM antibody modelling software). See, e.g., protein Sequence and Structure Analysis of Antibody Variable Domains [ protein sequence and structural analysis of antibody variable domains ] the literature is described in: antibody Engineering Lab Manual [ handbook of antibody engineering laboratories ] (editions: duebel, S. And Kontermann, R., springer-Verlag, sea Derburg).

As used herein, the terms "complementarity determining regions" and "CDRs" refer to amino acid sequences within antibody variable regions that confer antigen specificity and binding affinity. In some embodiments, there are three CDRs (HCDR 1, HCDR2, HCDR 3) in each heavy chain variable region, and three CDRs (LCDR 1, LCDR2, LCDR 3) in each light chain variable region.

The exact amino acid sequence boundaries for a given CDR may be determined using any of the well-known schemes, including those described below: kabat et Al (1991), "Sequences of Proteins of Immunological Interest [ immunologically significant protein sequences ]", 5 th edition, public Health Service [ U.S. public health service center ], national Institutes of Health [ U.S. national institutes of health ], bethesda, MD [ Bezistada, mallotus ] ("kappa" numbering scheme), al-Lazikani et Al, (1997) JMB 273,927-948 ("Qiao Xiya" numbering scheme). As used herein, CDRs defined according to the "Qiao Xiya" numbering scheme are sometimes also referred to as "hypervariable loops".

For example, according to cabazite, CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR 1), 50-65 (HCDR 2) and 95-102 (HCDR 3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR 1), 50-56 (LCDR 2) and 89-97 (LCDR 3). According to Qiao Xiya, the CDR amino acids in the VH are numbered 26-32 (HCDR 1), 52-56 (HCDR 2) and 95-102 (HCDR 3); and amino acid residues in VL are numbered 26-32 (LCDR 1), 50-52 (LCDR 2) and 91-96 (LCDR 3). By definition with CDRs binding to cabazite and Qiao Xiya, the CDRs consist of amino acid residues 26-35 (HCDR 1), 50-65 (HCDR 2) and 95-102 (HCDR 3) in human VH and amino acid residues 24-34 (LCDR 1), 50-56 (LCDR 2) and 89-97 (LCDR 3) in human VL.

Each VH and VL typically comprises three CDRs and four FRs, according to all definitions, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

Generally, unless specifically indicated, anti-CD 73 antibody molecules and anti-ENTPD 2 antibody molecules may include, for example, any combination of one or more of the carboplatin CDRs, qiao Xiya CDRs, combinations of carboplatin and Qiao Xiya CDRs, IMGT CDRs, and/or alternative definitions as set forth in tables 1 and 9.

As used herein, "immunoglobulin variable domain sequence" refers to an amino acid sequence that can form the structure of an immunoglobulin variable domain. For example, the sequence may include all or part of the amino acid sequence of a naturally occurring variable domain. For example, the sequence may or may not include one, two or more N-or C-terminal amino acids, or may include other changes that are compatible with the formation of protein structures.

The term "antigen binding site" refers to a portion of an antibody molecule that comprises determinants forming an interface that binds to a CD73 polypeptide or an ENTPD2 polypeptide or an epitope thereof. With respect to proteins (or protein mimics), an antigen binding site typically includes one or more loops (e.g., having at least four amino acids or amino acid mimics) that form an interface with a CD73 polypeptide or an ENTPD2 polypeptide. Typically, the antigen binding site of an antibody molecule comprises at least one or two CDRs and/or hypervariable loops, or more typically at least three, four, five or six CDRs and/or hypervariable loops.

As used herein, the term "Eu numbering" refers to the Eu numbering convention for antibody constant regions, as described in Edelman, G.M. et al, proc.Natl.Acad.USA [ Proc. Natl.Acad.Sci.USA ],63,78-85 (1969) and Kabat et al, "Sequences of Proteins of Immunological Interest [ protein sequence of immunological significance ], U.S. Dept.health and Human Services [ U.S. department of health and public service ], 5 th edition, 1991.

The terms "compete" or "cross-compete" are used interchangeably herein to refer to the ability of an antibody molecule to interfere with the binding of an anti-CD 73 antibody molecule or an anti-ENTPD 2 antibody molecule (e.g., an anti-CD 73 antibody molecule or an anti-ENTPD 2 antibody molecule provided herein) to a target (e.g., human CD73 or human ENTPD 2). The interference with binding may be direct or indirect (e.g., through allosteric modulation of the antibody molecule or target). The extent to which an antibody molecule can interfere with the binding of another antibody molecule to a target and thus whether it can be referred to as competition can be determined using competition binding assays (e.g., flow cytometry assays, ELISA or BIACORE assays). In some embodiments, the competitive binding assay is a quantitative competitive assay. In some embodiments, in a competitive binding assay (e.g., the competitive assay described herein), a first anti-CD 73 antibody molecule or anti-ENTPD 2 antibody molecule is said to compete for binding to a target with a second anti-CD 73 antibody molecule or anti-ENTPD 2 antibody molecule when the binding of the first antibody molecule to the target is reduced by 10% or more, e.g., 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more.

As used herein, the term "epitope" refers to the portion of an antigen (e.g., human CD73 or human ENTPD 2) that specifically interacts with an antibody molecule. Such moieties, also referred to herein as epitope determinants, typically comprise or are part of elements such as amino acid side chains or sugar side chains. Epitope determinants may be defined by methods known in the art or disclosed herein (e.g., by crystallography or by hydrogen deuterium exchange). At least one or some portion of the antibody molecule that specifically interacts with an epitope determinant is typically located in one or more CDRs. Typically, epitopes have specific three-dimensional structural features. Typically, an epitope has a specific charge characteristic. Some epitopes are linear epitopes while others are conformational epitopes.

In embodiments, an epitope determinant is a moiety (e.g., an amino acid side chain or a sugar side chain) on an antigen or a fraction thereof, which epitope determinant is within a predetermined distance, e.g., within 5 angstroms of the moiety on an antibody molecule, when the antigen and antibody molecule co-crystallize, referred to herein as a "crystallographic epitope determinant". The crystallographic epitope determinants of an epitope are collectively referred to as "crystallographic epitopes".

For example, when interactions are measured in the same manner for a first antibody and a second antibody or reference antibody, a first antibody molecule binds to the same epitope as a second antibody molecule (e.g., a reference antibody molecule, such as the antibody molecules disclosed herein) if the antibodies interact with the same epitope determinants of the second antibody or reference antibody on the antigen. Overlapping epitopes share at least one epitope determinant. When two antibody molecules interact with a common epitope determinant, a first antibody molecule binds to an overlapping epitope with a second antibody molecule (e.g., a reference antibody molecule, such as an antibody disclosed herein). If at least half of the epitope determinants of the second antibody or reference antibody are found to be epitope determinants in the epitope of the first antibody molecule, the first antibody molecule and the second antibody molecule (e.g., reference antibody molecule, e.g., an antibody molecule disclosed herein) bind to substantially overlapping epitopes. If the first antibody molecule binds to at least half of the core epitope determinant of an epitope of the second antibody or reference antibody, then the first antibody molecule and the second antibody molecule (e.g., reference antibody molecules, such as those disclosed herein) bind to substantially the same epitope, wherein the core epitope determinant is defined by, for example, crystallography or hydrogen deuterium exchange.

As used herein, an antibody molecule "reduces hydrogen deuterium exchange in an antigen fragment" when the hydrogen deuterium exchange in the antigen fragment in the presence of the antibody molecule is lower than the hydrogen deuterium exchange in the antigen fragment in the absence of the antibody molecule, as measured in a hydrogen deuterium exchange assay.

As used herein, the reduction in "average deuterium exchange" depends on the normalized level of deuterium exchange (Da/residue) in the antigen fragment in the absence of the antibody minus the normalized level of deuterium exchange (Da/residue) in the antigen fragment in the presence of the antibody.

As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules having a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope. Monoclonal antibodies can be prepared by hybridoma technology or methods that do not use hybridoma technology (e.g., recombinant methods).

An "effective human" protein is one that does not elicit a neutralizing antibody response, such as a human anti-murine antibody (HAMA) response. In many cases, for example, HAMA can be problematic if antibody molecules are repeatedly administered, for example in the treatment of chronic or recurrent disease conditions. The HAMA response can potentially negate repeated antibody administration due to increased antibody clearance in serum (see, e.g., saleh et al, cancer immunol. Immunother. [ Cancer immunology and immunotherapy ],32:180-190 (1990)), and also due to potential allergic reactions (see, e.g., loBuglio et al, hybrid mia [ hybridomas ],5:5117-5123 (1986)).

The antibody molecule may be a polyclonal antibody or a monoclonal antibody. In other embodiments, the antibodies may be recombinantly produced, such as by yeast display, phage display, or by combinatorial methods. Alternatively, such antibodies may be selected from synthetic yeast-based antibody presentation systems, for example as described in Y.xu et al Addressing polyspecificity of antibodies selected from an in vitro yeast presentation system:a FACS-based, high-throughput selection and analytical tool [ solving the problem of multi-specificity of antibodies selected from in vitro yeast presentation systems: FACS-based, high throughput screening and analysis tools ] PEDS26.10,663-70 (2013); WO 2009036379; WO 2010105256; and those described in WO 2012009568, which are incorporated herein by reference in their entirety.

In one embodiment, the antibody is a fully human antibody (e.g., an antibody produced by yeast display, an antibody produced by phage display, or an antibody prepared in a mouse that has been genetically engineered to produce antibodies from human immunoglobulin sequences), or a non-human antibody, such as a rodent (mouse or rat), goat, primate (e.g., monkey), or camel antibody. Methods for producing rodent antibodies are known in the art.

Transgenic mice carrying human immunoglobulin genes can be used to produce human monoclonal antibodies rather than the mouse system. Spleen cells from these transgenic mice immunized with the antigen of interest are used to generate hybridomas secreting human mAbs that have specific affinity for epitopes from human proteins (see, e.g., wood et al, international application WO 91/00906; kucherapati et al, PCT publication WO 91/10741; lonberg et al, international application WO 92/03918; kay et al, international application 92/03917; lonberg, N et al, 1994Nature [ Nature ]368:856-859; green, L.L. et al, 1994Nature Genet [ Nature Genet ] [ Nature genetics ]7:13-21; morrison, S.L. et al, 1994Proc. Natl. Acad. Sci. USA [ Proc. Natl. Acad. Sci. USA ]81:6851-6855; bruggeman et al, 1993Year Immunol. Ind. Sci. Emi ]7:33-40; tuaillon et al, 1993PNAS [ Proc. Natl. Acad. Sci. U.S. 90:3720-3724; bruggeman et al, 1991Eur J Immunol [ European Immunol ] 21:1323-1326).

The antibody may be an antibody that produces a variable region or a portion thereof (e.g., CDR) in a non-human organism (e.g., rat or mouse). Chimeric antibodies, CDR-grafted antibodies and humanized antibodies belong to the antibodies that can be used in the dosage regimen of the invention. Antibodies that are produced in a non-human organism (e.g., a rat or mouse) and then modified in, for example, a variable framework or constant region to reduce antigenicity in humans are among the antibodies that can be used in the dosage regimen of the present invention.

Antibodies may be produced by any suitable recombinant DNA technique known in the art (see Robinson et al, international patent publication PCT/US86/02269; akira et al, european patent application 184,187; taniguchi, M.; european patent application 171,496; morrison et al, european patent application 173,494; neuberger et al, international application WO 86/01533; cabilly et al, U.S. Pat. No. 4,816,567; cabilly et al, european patent application 125,023; better et al (1988 Science 240:1041-1043); liu et al (1987) PNAS [ Proc. Natl. Acad. Sci. USA ]84:3439-3443; liu et al, 1987, J.Immunol. [ J.Immunol. 139:3521-3526; sun et al (1987) PNAS [ Proc. Natl. Acad. Sci. USA ]84:214-218; nishimura et al, 1987, canc. Res. [ cancer research ]47:999-1005; wood et al (1985) Nature [ Nature ]314:446-449; shaw et al, 1988,J.Natl Cancer Inst. [ J. Natl. Cancer research U.S. 80:1553-1559).

At least one or two but typically all three recipient CDRs (of the heavy and/or light immunoglobulin chains) of the humanized antibody or CDR-grafted antibody are replaced by donor CDRs. An antibody may be replaced with at least a portion of a non-human CDR, or only some CDRs may be replaced with non-human CDRs. Only the number of CDRs required for binding of the humanized antibody to CD73 or ENTPD2 need be replaced. In some embodiments, the donor is a rodent antibody, e.g., a rat or mouse antibody, and the recipient is a human framework or a human consensus framework. Typically, an immunoglobulin providing CDRs is referred to as a "donor" and an immunoglobulin providing framework is referred to as an "acceptor". In one embodiment, the donor immunoglobulin is non-human (e.g., rodent). The acceptor framework is a naturally occurring (e.g., human) framework or a consensus framework, or a sequence having about 85% or more, e.g., 90%, 95%, 99% or more identity thereto.

As used herein, the term "consensus sequence" refers to a sequence formed by the most frequently occurring amino acids (or nucleotides) in the related sequence family (see, e.g., winnaker, from Genes to Clones [ from gene to clone ] (german Wei Yinhai m press (Verlagsgesellschaft, weinheim, germany)) 1987). In a family of proteins, each position in the consensus sequence is occupied by the amino acid most frequently present at that position in the family. If two amino acids are also commonly present, either may be included in the consensus sequence. "consensus framework" refers to a framework region in a consensus immunoglobulin sequence.

Antibodies can be humanized by methods known in the art (see, e.g., morrison, S.L.,1985, science [ science ]229:1202-1207, oi et al, 1986, bioTechniques [ biotechnology ]4:214, and Queen et al, U.S. Pat. No. 5,585,089, U.S. Pat. No. 5,693,761, and U.S. Pat. No. 5,693,762, the contents of all of which are incorporated herein by reference).

Humanized antibodies or CDR-grafted antibodies may be produced by CDR grafting or CDR substitution, wherein one, two or all of the CDRs of an immunoglobulin chain may be replaced. See, for example, U.S. Pat. nos. 5,225,539; jones et al 1986Nature 321:552-525; verhoeye et al 1988Science [ Science ]239:1534; beidler et al 1988J.Immunol. [ J.Immunol. ]141:4053-4060; winter US 5,225,539, the content of all documents is expressly incorporated by reference herein.

Humanized antibodies are also within the scope of antibodies useful in the dosage regimen of the invention, wherein specific amino acids have been substituted, deleted or added. Criteria for selection of amino acids from donors are described in U.S. Pat. No. 5,585,089, e.g. in columns 12-16 of U.S. Pat. No. 5,585,089, the contents of which are hereby incorporated by reference. Other techniques for humanizing antibodies are described in Padlan et al, EP 519596A1, published at 12/23 1992.

The antibody molecule may be a single chain antibody. Single chain antibodies (scFV) can be engineered (see, e.g., colcher, D.et al (1999) Ann N Y Acad Sci [ New York academy of sciences annual. 880:263-80; and Reiter, Y. (1996) Clin Cancer Res [ clinical Cancer research ] 2:245-52). Single chain antibodies can be dimerized or multimerized to produce multivalent antibodies specific for different epitopes of the same target protein.

In still other embodiments, the antibody molecule has a heavy chain constant region selected from the group consisting of, for example, the heavy chain constant regions of IgG1, igG2, igG3, igG4, igM, igA1, igA2, igD, and IgE; in particular, the (e.g. human) heavy chain constant region is selected from e.g. IgG1, igG2, igG3 and IgG 4. In another embodiment, the antibody molecule has a light chain constant region selected from (e.g., human) light chain constant regions such as kappa or lambda. The constant region can be altered (e.g., mutated) to modify a property of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, and/or complement function). In some embodiments, the antibody has effector function and can fix complement. In other embodiments, the antibody does not recruit effector cells or fix complement. In certain embodiments, the antibody has a reduced or no ability to bind to Fc receptors. For example, it may be an isoform or subtype, fragment or other mutant that does not support binding to Fc receptors, e.g., having a mutagenized or deleted Fc receptor binding region.

Methods for altering the constant regions of antibodies are known in the art. Antibodies with altered function (altered affinity for effector ligands (e.g., fcrs on cells) or C1 components of complement) can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see, e.g., EP 388,151A1, U.S. Pat. No. 5,624,821, and U.S. Pat. No. 5,648,260, the contents of all of which are hereby incorporated by reference). Amino acid mutations that stabilize the antibody structure, such as S228P (Eu numbering) in human IgG4, are also contemplated. Similar types of changes can be described that would reduce or eliminate these functions if applied to immunoglobulins in a murine or other species.

The antibody molecule may be derivatized or linked to another functional molecule (e.g., another peptide or protein). As used herein, a "derivatized" antibody molecule is an antibody molecule that has been modified. Derivatization methods include, but are not limited to, addition of fluorescent moieties, radionucleotides, toxins, enzymes or affinity ligands such as biotin. Thus, antibody molecules for use in the dosage regimen of the invention are intended to include derivatized forms and other modified forms of the antibodies described herein, including immunoadhesion molecules. For example, an antibody molecule may be functionally linked (by chemical coupling, genetic fusion, non-covalent association, or other means) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody antibody), a detectable agent, a cytotoxic agent, an agent, and/or a protein or peptide that may mediate the association of an antibody or antibody portion with another molecule (e.g., a streptavidin core region or a polyhistidine tag).

One type of derivatized antibody molecule is produced by cross-linking two or more antibodies (of the same type or different types, e.g., to produce bispecific antibodies). Suitable cross-linking agents include those that are heterobifunctional (having two distinct reactive groups separated by a suitable spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester)) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from pierce chemical company (Pierce Chemical Company, rockford, ill) of rocford, il.

Useful detectable agents that can derivatize (or label) antibody molecules include fluorescent compounds, various enzymes, prosthetic groups, luminescent materials, bioluminescent materials, fluorescent emitting metal atoms (e.g., europium (Eu) and other lanthanides), and radioactive materials (described below). Exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and the like. Antibodies may also be derivatized with detectable enzymes (e.g., alkaline phosphatase, horseradish peroxidase, beta-galactosidase, acetylcholinesterase, glucose oxidase, etc.). When the antibody is derivatized with a detectable enzyme, the antibody is detected by the addition of an additional reagent, which is used by the enzyme to produce a detectable reaction product. For example, when the detectable agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine results in a detectable colored reaction product. Antibody molecules may also be derivatized with prosthetic groups (e.g., streptavidin/biotin and avidin/biotin). For example, antibodies can be derivatized with biotin and detected by indirect measurement of avidin or streptavidin binding. Examples of suitable fluorescent materials include umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent materials include luminol; and examples of bioluminescent materials include luciferase, luciferin and aequorin.

The labeled antibody molecules may be used in a variety of contexts, for example in diagnostics and/or experiments, including (i) isolation of a predetermined antigen by standard techniques such as affinity chromatography or immunoprecipitation; (ii) Detecting a predetermined antigen (e.g., in a cell lysate or cell supernatant) to assess the abundance and expression pattern of the protein; (iii) As part of a clinical test procedure, protein levels in tissue are monitored, for example, to determine the efficacy of a given treatment regimen.

The antibody molecule may be conjugated to another molecular entity (typically a label or therapeutic agent (e.g., an immunomodulator, immunostimulant, cytotoxic agent or cytostatic agent) or moiety). Radioisotopes may be used in diagnostic or therapeutic applications. Radioisotopes that may be conjugated to an anti-CD 73 antibody or an anti-ENTPD 2 antibody include, but are not limited to, alpha-, beta-, or gamma-emitters, or beta-and gamma-emitters. Such radioisotopes include, but are not limited to, iodine @, and ¹³¹ i or ¹²⁵ I) Yttrium [ ] ⁹⁰ Y, lutetium ] ¹⁷⁷ Lu), actinium ²²⁵ Ac), praseodymium, astatine ²¹¹ At, re% ¹⁸⁶ Re, bi% ²¹² Bi or Bi ²¹³ Bi, in ] ¹¹¹ In, technetium ] ⁹⁹ mTc, phosphorus% ³² P and rhodium ¹⁸⁸ Rh and sulfur ³⁵ S, C ¹⁴ C) The tritium is ³ H) The chromium is ⁵¹ Cr and chlorine% ³⁶ Cl, co% ⁵⁷ Co or ⁵⁸ Co, fe ⁵⁹ Fe and Se ⁷⁵ Se), or Ga ⁶⁷ Ga). The radioisotope useful as a therapeutic agent comprises yttrium ⁹⁰ Y, lutetium ] ¹⁷⁷ Lu), actinium ²²⁵ Ac), praseodymium, astatine ²¹¹ At, re% ¹⁸⁶ Re, bi% ²¹² Bi or Bi ²¹³ Bi) and rhodium% ¹⁸⁸ Rh). Radioisotopes useful as markers (e.g., for diagnosis) include iodine @, and ¹³¹ i or ¹²⁵ I) The indium is ¹¹¹ In, technetium ] ⁹⁹ mTc, phosphorus% ³² P, C ¹⁴ C) And tritium% ³ H) Or one or more of the therapeutic isotopes listed above.

As described above, the antibody molecule may be conjugated to a therapeutic agent. Therapeutically active radioisotopes have been mentioned. Examples of other therapeutic agents include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids such as maytansinol (see U.S. Pat. No. 5,208,020), CC-1065 (see U.S. Pat. No. 5,475,092, 5,585,499, 5,846,545), and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil amamide), alkylating agents (e.g., nitrogen mustard, thiotepa, chlorambucil, CC-1065, melphalan, carmustine (BSNU) and lomustine (CCNU)), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cisplatin-dichlorodiamplatinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunorubicin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin and Aflatoxin (AMC)), and antimitotics (e.g., vincristine, vinblastine, paclitaxel and maytansinoid).

In certain embodiments, the antibody molecule is a multi-specific (e.g., bispecific or trispecific) antibody molecule. Protocols for the production of bispecific or heterodimeric antibody molecules are known in the art; these schemes include, but are not limited to: for example, a "knob in a hole" method, for example, as described in US 5731168; electrostatically steered Fc pairing as described for example in WO 09/089004, WO 06/106905 and WO 2010/129304; chain exchange engineering domain (SEED) heterodimer formation as described, for example, in WO 07/110205; fab arm exchange as described for example in WO 08/119353, WO 2011/131746 and WO 2013/060867; diabody conjugates, for example using heterobifunctional reagents having amine-reactive groups and thiol-reactive groups, are cross-linked by antibodies to produce bispecific structures as described, for example, in US 4433059; bispecific antibody determinants produced by recombination of half antibodies (heavy-light chain pairs or Fab) from different antibodies by cycles of reduction and oxidation of disulfide bonds between the two heavy chains, as described for example in US 4444878; trifunctional antibodies, for example three Fab' fragments crosslinked by thiol-reactive groups, as described for example in US 5273743; biosynthesis of binding proteins, e.g. scFv pairs crosslinked by C-terminal tail, preferably by disulfide or amine reactive chemical crosslinking, as described e.g. in US 5534254; bifunctional antibodies, e.g. Fab fragments with different binding specificities, dimerized by leucine zippers (e.g. c-fos and c-jun) that have replaced constant domains, as described e.g. in US 5582996; bispecific and oligospecific monovalent and oligovalent receptors, for example the VH-CH1 regions of two antibodies (two Fab fragments), which VH-CH1 regions are linked by a polypeptide spacer between the CH1 region of one antibody and the VH region of the other antibody (typically with an associated light chain), as described for example in US 5591828; bispecific DNA-antibody conjugates, e.g. cross-linked antibodies or Fab fragments by double stranded DNA fragments, as described for example in US 5635602; bispecific fusion proteins, for example expression constructs comprising two scFv (with a hydrophilic helical peptide linker between them) and a fully constant region, as described for example in US 5637481; multivalent and multispecific binding proteins, such as polypeptide dimers having a first domain of an Ig heavy chain variable region binding region and a second domain of an Ig light chain variable region binding region, are commonly referred to as diabodies (also disclosing higher order structures, resulting in bispecific, trispecific or tetraspecific molecules) as described, for example, in US 5837242; miniantibody constructs having linked VL and VH chains (which are further linked to antibody hinge and CH3 regions with peptide spacers) which can dimerise to form bispecific/multivalent molecules as described, for example, in US 5837821; VH and VL domains linked with a short peptide linker (e.g., 5 or 10 amino acids) or not linked at all at any orientation, which can form dimers to form bispecific diabodies; trimers and tetramers as described, for example, in US 5844094; a string of VH domains (or VL domains in family members) linked by peptide bonds to C-terminal crosslinkable groups which are further associated with the VL domains to form a series of FV (or scFv) as described, for example, in US 5864019; and single chain binding polypeptides having both VH and VL domains linked via peptide linkers are combined into multivalent structures by non-covalent or chemical cross-linking to form, for example, homobivalent, heterobivalent, trivalent and tetravalent structures using scFV or diabody type formats, as described, for example, in US 5869620. Additional exemplary multispecific and bispecific molecules and methods for their preparation are found, for example, in US, US US, US A1 US, US US, US A1 US A1, US A1 US A1, US A1. The contents of the above-referenced applications are incorporated herein by reference in their entirety.

In other embodiments, an anti-CD 73 antibody molecule or an anti-ENTPD 2 antibody molecule (e.g., a monospecific, bispecific, or multispecific antibody molecule) is covalently linked (e.g., fused) to another partner (e.g., a protein, e.g., as a fusion molecule, such as one, two, or more cytokines of a fusion protein).

"fusion protein" and "fusion polypeptide" refer to polypeptides having at least two moieties covalently linked together, wherein each moiety is a polypeptide having different properties. The property may be a biological property, such as in vitro or in vivo activity. The property may also be a simple chemical or physical property, such as binding to a target molecule, catalysis of a reaction, etc. The two moieties may be directly linked by a single peptide bond or by a peptide linker, but in frame with each other.

Isolated nucleic acid molecules encoding the above antibody molecules, vectors and host cells thereof are disclosed herein. Nucleic acid molecules include, but are not limited to, RNA, genomic DNA, and cDNA.

Exemplary anti-CD 73 antibody molecules

In one embodiment of the dosage regimen of the invention, the anti-CD 73 antibody is an anti-CD 73 antibody molecule, as described in WO 2018237157 (incorporated by reference in its entirety) published on 2018, month 12, 27, entitled "Antibody Molecules to CD and Uses therapy of [ antibody molecules to CD73 and Uses Thereof ]".

In some embodiments, an anti-CD 73 antibody molecule comprises at least one antigen binding region, e.g., a variable region, or antigen binding fragment thereof, from an antibody described herein, e.g., an antibody selected from 918, 350, 356, 358, 930, 373, 374, 376, 377, 379, 363, 366, 407, 893, 939, 430, or 398; or as described in table 1; or encoded by the nucleotide sequence in table 1; or a sequence that is substantially identical (e.g., has at least about 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) to any of the preceding sequences.

In some embodiments, an anti-CD 73 antibody molecule comprises at least one or two heavy chain variable regions from an antibody described herein, e.g., an antibody selected from 918, 350, 356, 358, 930, 373, 374, 376, 377, 379, 363, 366, 407, 893, 939, 430, or 398; or as described in table 1; or encoded by the nucleotide sequence in table 1; or a sequence that is substantially identical (e.g., has at least about 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) to any of the preceding sequences.

In certain embodiments, an anti-CD 73 antibody molecule comprises at least one or two light chain variable regions from an antibody described herein, e.g., an antibody selected from 918, 350, 356, 358, 930, 373, 374, 376, 377, 379, 363, 366, 407, 893, 939, 430, or 398; or as described in table 1; or encoded by the nucleotide sequence in table 1; or a sequence that is substantially identical (e.g., has at least about 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) to any of the preceding sequences.

In one embodiment, the anti-CD 73 antibody molecule comprises a heavy chain constant region of IgG4 (e.g., human IgG 4). In another embodiment, human IgG4 comprises a substitution (e.g., ser to Pro substitution) at position 228 according to Eu numbering. In yet another embodiment, the anti-CD 73 antibody molecule comprises a heavy chain constant region of IgG1 (e.g., human IgG 1). In one embodiment, human IgG1 includes a substitution at position 297 (e.g., an Asn-Ala substitution) according to Eu numbering. In one embodiment, human IgG1 includes a substitution at position 265 (e.g., asp to Ala substitution) according to Eu numbering, a substitution at position 329 (e.g., pro to Ala substitution) according to Eu numbering, or both. In one embodiment, human IgG1 includes a substitution at position 234 according to Eu numbering (e.g., leu to Ala substitution), a substitution at position 235 according to Eu numbering (e.g., leu to Ala substitution), or both. In one embodiment, the heavy chain constant region comprises or is substantially identical (e.g., has at least about 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) to an amino acid sequence set forth in table 3.

In yet another embodiment, the anti-CD 73 antibody molecule comprises a kappa light chain constant region, e.g., a human kappa light chain constant region. In one embodiment, the light chain constant region comprises or is substantially identical (e.g., has at least about 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) to an amino acid sequence set forth in table 3.

In another embodiment, the anti-CD 73 antibody molecule comprises a heavy chain constant region of IgG4 (e.g., human IgG 4), and a kappa light chain constant region (e.g., human kappa light chain constant region), such as heavy and light chain constant regions comprising or substantially identical (e.g., having at least about 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) to the amino acid sequences listed in table 3. In yet another embodiment, the anti-CD 73 antibody molecule comprises a heavy chain constant region of IgG1 (e.g., human IgG 1), and a kappa light chain constant region (e.g., human kappa light chain constant region), such as heavy and light chain constant regions comprising the amino acid sequences listed in table 3, or sequences substantially identical thereto (e.g., having at least about 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity). In one embodiment, human IgG1 includes a substitution at position 297 (e.g., an Asn-Ala substitution) according to Eu numbering. In one embodiment, human IgG1 includes a substitution at position 265 according to Eu numbering, a substitution at position 329 according to Eu numbering, or both (e.g., asp to Ala substitution at position 265 and/or Pro to Ala substitution at position 329). In one embodiment, human IgG1 includes a substitution at position 234 according to Eu numbering, a substitution at position 235 according to Eu numbering, or both (e.g., a Leu-to-Ala substitution at position 234 and/or a Leu-to-Ala substitution at position 235).

In another embodiment, the anti-CD 73 antibody molecule comprises a heavy chain variable region and a constant region, a light chain variable region and a constant region, or both, and the antibody is an antibody comprising an amino acid sequence of 918, 350, 356, 358, 930, 373, 374, 376, 377, 379, 363, 366, 407, 893, 939, 430, or 398; or as described in table 1; or encoded by the nucleotide sequence in table 1; or a sequence that is substantially identical (e.g., has at least about 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) to any of the preceding sequences.

In some embodiments, an anti-CD 73 antibody molecule comprises at least one, two, or three Complementarity Determining Regions (CDRs) from a heavy chain variable region of an antibody described herein, e.g., an antibody selected from 918, 350, 356, 358, 930, 373, 374, 376, 377, 379, 363, 366, 407, 893, 939, 430, or 398; or as described in table 1, or encoded by the nucleotide sequence in table 1; or a sequence that is substantially identical (e.g., has at least about 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) to any of the preceding sequences.

In some embodiments, an anti-CD 73 antibody molecule comprises at least one, two, or three Complementarity Determining Regions (CDRs) from a light chain variable region of an antibody described herein, e.g., an antibody selected from 918, 350, 356, 358, 930, 373, 374, 376, 377, 379, 363, 366, 407, 893, 939, 430, or 398; or as described in table 1, or encoded by the nucleotide sequence in table 1; or a sequence that is substantially identical (e.g., has at least about 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) to any of the preceding sequences.

In certain embodiments, an anti-CD 73 antibody molecule comprises all six CDRs from an antibody described herein, e.g., an antibody selected from 918, 350, 356, 358, 930, 373, 374, 376, 377, 379, 363, 366, 407, 893, 939, 430, or 398; or as described in table 1; or encoded by the nucleotide sequences in table 1, or include closely related CDRs, e.g., CDRs that are identical or have at least one amino acid change, but no more than two, three, or four changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions). In certain embodiments, an anti-CD 73 antibody molecule may include any of the CDRs described herein. In certain embodiments, the anti-CD 73 antibody molecule comprises a substitution in a CDR of a heavy chain, e.g., one or more substitutions in CDR1, CDR2, and/or CDR3 of the heavy chain.

In some embodiments, the anti-CD 73 antibody molecule comprises all six CDRs according to Kabat et al (e.g., all six CDRs according to the cabat definition set forth in table 1) from the heavy and light chain variable regions of an antibody described herein, which antibody is, for example, an antibody selected from 918, 350, 356, 358, 930, 373, 374, 376, 377, 379, 363, 366, 407, 893, 939, 430, or 398; or as described in table 1; or encoded by the nucleotide sequence in table 1; or a sequence that is substantially identical (e.g., has at least about 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) to any of the preceding sequences; or CDRs with at least one amino acid change but no more than two, three or four changes (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to all six CDRs according to Kabat et al as shown in table 1. In one embodiment, the anti-CD 73 antibody molecule may include any of the CDRs described herein.

In some embodiments, the anti-CD 73 antibody molecule comprises all six hypervariable loops (e.g., all six hypervariable loops defined according to Qiao Xiya listed in table 1) of an antibody described herein (e.g., an antibody selected from 918, 350, 356, 358, 930, 373, 374, 376, 377, 379, 363, 366, 407, 893, 939, 430, or 398); or closely related hypervariable loops, such as hypervariable loops that are identical or have at least one amino acid change, but no more than two, three, or four changes (e.g., substitutions, deletions, or insertions, such as conservative substitutions); or hypervariable loops having at least one amino acid change but no more than two, three or four changes (e.g., substitutions, deletions or insertions, such as conservative substitutions) relative to all six hypervariable loops according to Chothia et al, as shown in table 1. In one embodiment, the anti-CD 73 antibody molecule may include any hypervariable loop described herein.

In certain embodiments, the anti-CD 73 antibody molecule comprises a combination of CDRs or hypervariable loops as defined by Kabat et al and Chothia et al.

In some embodiments, an anti-CD 73 antibody molecule comprises all six CDRs defined according to IMGT from the heavy and light chain variable regions of an antibody described herein (e.g., all six CDRs defined according to IMGT listed in table 1), which antibody is, for example, an antibody selected from 918, 350, 356, 358, 930, 373, 374, 376, 377, 379, 363, 366, 407, 893, 939, 430, or 398; or as described in table 1; or encoded by the nucleotide sequence in table 1; or a sequence that is substantially identical (e.g., has at least about 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) to any of the preceding sequences; or CDRs with at least one amino acid change but no more than two, three or four changes (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to all six CDRs defined according to IMGT as shown in table 1. In one embodiment, the anti-CD 73 antibody molecule may include any of the CDRs described herein.

In some embodiments, the heavy or light chain variable domain or both of an anti-CD 73 antibody molecule comprises an amino acid sequence that is substantially identical to an amino acid disclosed herein, e.g., has at least about 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity to a variable domain of an antibody described herein, e.g., an antibody selected from 918, 350, 356, 358, 930, 373, 374, 376, 377, 379, 363, 366, 407, 893, 939, 430, or 398; or as described in table 1; or encoded by the nucleotide sequence in table 1; or at least 1 or 5 residues but less than 40, 30, 20, or 10 residues from the variable regions of the antibodies described herein.

In certain embodiments, the heavy or light chain variable region of an anti-CD 73 antibody molecule, or both, comprises an amino acid sequence encoded by a nucleic acid sequence described herein or a nucleic acid that hybridizes to a nucleic acid sequence described herein (e.g., a nucleic acid sequence shown in table 1) or its complement, e.g., under low stringency, medium stringency, or high stringency, or other hybridization conditions described herein.

In some embodiments, the antibody molecule has a variable region that is identical in sequence to a variable region described herein (e.g., an FR region disclosed herein), or differs by 1, 2, 3, or 4 amino acids.

In one embodiment, the anti-CD 73 antibody molecule comprises a heavy chain variable region (VH) comprising the VHCDR3 amino acid sequence of GGLYGSGSYLSDFDL (SEQ ID NO: 37). In one embodiment, the anti-CD 73 antibody molecule comprises a heavy chain variable region (VH) comprising the VHCDR3 amino acid sequence of ESQESPYNNWFDP (SEQ ID NO: 3).

In one embodiment, the anti-CD 73 antibody molecule comprises a heavy chain variable region (VH) comprising the VH CDR1 amino acid sequence of SEQ ID NO. 88, the VH CDR2 amino acid sequence of SEQ ID NO. 89 and the VH CDR3 amino acid sequence of SEQ ID NO. 37; and a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO:48, the VLCDR2 amino acid sequence of SEQ ID NO:49 and the VLCDR3 amino acid sequence of SEQ ID NO:50, each as disclosed in Table 2. In one embodiment, the anti-CD 73 antibody molecule comprises a heavy chain variable region (VH) comprising the VH CDR1 amino acid sequence of SEQ ID NO. 90, the VH CDR2 amino acid sequence of SEQ ID NO. 91, and the VH CDR3 amino acid sequence of SEQ ID NO. 3; and a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO. 14, the VLCDR2 amino acid sequence of SEQ ID NO. 15 and the VLCDR3 amino acid sequence of SEQ ID NO. 16, each as disclosed in Table 2.

In one embodiment, the anti-CD 73 antibody molecule comprises a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO. 38, the VH CDR2 amino acid sequence of SEQ ID NO. 36 and the VH CDR3 amino acid sequence of SEQ ID NO. 37; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 48, the VLCDR2 amino acid sequence of SEQ ID NO. 49 and the VLCDR3 amino acid sequence of SEQ ID NO. 50. In one embodiment, the anti-CD 73 antibody molecule comprises a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO:72, the VH CDR2 amino acid sequence of SEQ ID NO:71 and the VH CDR3 amino acid sequence of SEQ ID NO: 37; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 48, the VLCDR2 amino acid sequence of SEQ ID NO. 49 and the VLCDR3 amino acid sequence of SEQ ID NO. 50. In one embodiment, the anti-CD 73 antibody molecule comprises a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO. 38, the VH CDR2 amino acid sequence of SEQ ID NO. 71 and the VH CDR3 amino acid sequence of SEQ ID NO. 37; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 48, the VLCDR2 amino acid sequence of SEQ ID NO. 49 and the VLCDR3 amino acid sequence of SEQ ID NO. 50. In one embodiment, the anti-CD 73 antibody molecule comprises a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO. 137, the VH CDR2 amino acid sequence of SEQ ID NO. 136 and the VH CDR3 amino acid sequence of SEQ ID NO. 37; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 48, the VLCDR2 amino acid sequence of SEQ ID NO. 49 and the VLCDR3 amino acid sequence of SEQ ID NO. 50. In one embodiment, the anti-CD 73 antibody molecule comprises a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO. 137, the VH CDR2 amino acid sequence of SEQ ID NO. 146 and the VH CDR3 amino acid sequence of SEQ ID NO. 37; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 48, the VLCDR2 amino acid sequence of SEQ ID NO. 49 and the VLCDR3 amino acid sequence of SEQ ID NO. 50. In one embodiment, the anti-CD 73 antibody molecule comprises a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO:137, the VH CDR2 amino acid sequence of SEQ ID NO:154 and the VH CDR3 amino acid sequence of SEQ ID NO: 37; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 48, the VLCDR2 amino acid sequence of SEQ ID NO. 49 and the VLCDR3 amino acid sequence of SEQ ID NO. 50. In one embodiment, the anti-CD 73 antibody molecule comprises a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO. 61, the VH CDR2 amino acid sequence of SEQ ID NO. 60 and the VH CDR3 amino acid sequence of SEQ ID NO. 3; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 14, the VLCDR2 amino acid sequence of SEQ ID NO. 15 and the VLCDR3 amino acid sequence of SEQ ID NO. 16. In one embodiment, the anti-CD 73 antibody molecule comprises a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO. 4, the VH CDR2 amino acid sequence of SEQ ID NO. 26 and the VH CDR3 amino acid sequence of SEQ ID NO. 3; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 14, the VLCDR2 amino acid sequence of SEQ ID NO. 15 and the VLCDR3 amino acid sequence of SEQ ID NO. 16. In one embodiment, the anti-CD 73 antibody molecule comprises a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO. 4, the VH CDR2 amino acid sequence of SEQ ID NO. 2 and the VH CDR3 amino acid sequence of SEQ ID NO. 3; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 14, the VLCDR2 amino acid sequence of SEQ ID NO. 15 and the VLCDR3 amino acid sequence of SEQ ID NO. 16. In one embodiment, the anti-CD 73 antibody molecule comprises a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO. 163, the VH CDR2 amino acid sequence of SEQ ID NO. 162 and the VH CDR3 amino acid sequence of SEQ ID NO. 3; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 14, the VLCDR2 amino acid sequence of SEQ ID NO. 15 and the VLCDR3 amino acid sequence of SEQ ID NO. 16.

In one embodiment, the anti-CD 73 antibody molecule comprises a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO 39, the VH CDR2 amino acid sequence of SEQ ID NO 40 and the VH CDR3 amino acid sequence of SEQ ID NO 37; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 51, the VLCDR2 amino acid sequence of SEQ ID NO. 52 and the VLCDR3 amino acid sequence of SEQ ID NO. 53. In one embodiment, the anti-CD 73 antibody molecule comprises a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO. 73, the VH CDR2 amino acid sequence of SEQ ID NO. 74 and the VH CDR3 amino acid sequence of SEQ ID NO. 37; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 51, the VLCDR2 amino acid sequence of SEQ ID NO. 52 and the VLCDR3 amino acid sequence of SEQ ID NO. 53. In one embodiment, the anti-CD 73 antibody molecule comprises a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO. 82, the VH CDR2 amino acid sequence of SEQ ID NO. 74 and the VH CDR3 amino acid sequence of SEQ ID NO. 37; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 51, the VLCDR2 amino acid sequence of SEQ ID NO. 52 and the VLCDR3 amino acid sequence of SEQ ID NO. 53. In one embodiment, the anti-CD 73 antibody molecule comprises a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO. 138, the VH CDR2 amino acid sequence of SEQ ID NO. 139 and the VH CDR3 amino acid sequence of SEQ ID NO. 37; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 51, the VLCDR2 amino acid sequence of SEQ ID NO. 52 and the VLCDR3 amino acid sequence of SEQ ID NO. 53. In one embodiment, the anti-CD 73 antibody molecule comprises a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO:147, the VH CDR2 amino acid sequence of SEQ ID NO:148 and the VH CDR3 amino acid sequence of SEQ ID NO: 37; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 51, the VLCDR2 amino acid sequence of SEQ ID NO. 52 and the VLCDR3 amino acid sequence of SEQ ID NO. 53. In one embodiment, the anti-CD 73 antibody molecule comprises a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO:155, the VH CDR2 amino acid sequence of SEQ ID NO:156 and the VH CDR3 amino acid sequence of SEQ ID NO: 37; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 51, the VLCDR2 amino acid sequence of SEQ ID NO. 52 and the VLCDR3 amino acid sequence of SEQ ID NO. 53. In one embodiment, the anti-CD 73 antibody molecule comprises a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO. 62, the VH CDR2 amino acid sequence of SEQ ID NO. 63 and the VH CDR3 amino acid sequence of SEQ ID NO. 3; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 17, the VLCDR2 amino acid sequence of SEQ ID NO. 18 and the VLCDR3 amino acid sequence of SEQ ID NO. 19. In one embodiment, the anti-CD 73 antibody molecule comprises a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO. 27, the VH CDR2 amino acid sequence of SEQ ID NO. 28 and the VH CDR3 amino acid sequence of SEQ ID NO. 3; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 17, the VLCDR2 amino acid sequence of SEQ ID NO. 18 and the VLCDR3 amino acid sequence of SEQ ID NO. 19. In one embodiment, the anti-CD 73 antibody molecule comprises a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO. 5, the VH CDR2 amino acid sequence of SEQ ID NO. 6 and the VH CDR3 amino acid sequence of SEQ ID NO. 3; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 17, the VLCDR2 amino acid sequence of SEQ ID NO. 18 and the VLCDR3 amino acid sequence of SEQ ID NO. 19. In one embodiment, the anti-CD 73 antibody molecule comprises a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO. 164, the VH CDR2 amino acid sequence of SEQ ID NO. 165 and the VH CDR3 amino acid sequence of SEQ ID NO. 3; and a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 17, the VLCDR2 amino acid sequence of SEQ ID NO. 18 and the VLCDR3 amino acid sequence of SEQ ID NO. 19.

In other embodiments, the foregoing antibodies comprise a heavy chain variable region comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs 44, 77, 84, 142, 151, or 159. In other embodiments, the foregoing antibodies comprise a heavy chain variable region comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to any of SEQ ID NOs 66, 31, 10, or 168.

In other embodiments, the foregoing antibodies comprise a light chain variable region comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOS: 55 or 21.

In other embodiments, the foregoing antibodies comprise a heavy chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to any of SEQ ID NOs 46, 79, 86, 114, 116, or 117. In other embodiments, the foregoing antibodies comprise a heavy chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to any of SEQ ID NOs 68, 33, 12, 115, 113, or 112.

In other embodiments, the foregoing antibodies comprise a light chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to either of SEQ ID NOS: 57 or 23.

In other embodiments, the antibody molecule comprises a heavy chain variable region comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 44; and a light chain variable region comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 55. In other embodiments, the antibody molecule comprises a heavy chain variable region comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 77; and a light chain variable region comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 55. In other embodiments, the antibody molecule comprises a heavy chain variable region comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 84; and a light chain variable region comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 55. In other embodiments, the antibody molecule comprises a heavy chain variable region comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 142; and a light chain variable region comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 55. In other embodiments, the antibody molecule comprises a heavy chain variable region comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 151; and a light chain variable region comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 55. In other embodiments, the antibody molecule comprises a heavy chain variable region comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO 159; and a light chain variable region comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 55. In other embodiments, the antibody molecule comprises a heavy chain variable region comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 66; and a light chain variable region comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 21. In other embodiments, the antibody molecule comprises a heavy chain variable region comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 31; and a light chain variable region comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 21. In other embodiments, the antibody molecule comprises a heavy chain variable region comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 10; and a light chain variable region comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 21. In other embodiments, the antibody molecule comprises a heavy chain variable region comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 168; and a light chain variable region comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 21.

In other embodiments, the antibody molecule comprises a heavy chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 46; and a light chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 57. In other embodiments, the antibody molecule comprises a heavy chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 79; and a light chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 57. In other embodiments, the antibody molecule comprises a heavy chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 86; and a light chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 57. In other embodiments, the antibody molecule comprises a heavy chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 114; and a light chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 57. In other embodiments, the antibody molecule comprises a heavy chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 116; and a light chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 57. In other embodiments, the antibody molecule comprises a heavy chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 117; and a light chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 57. In other embodiments, the antibody molecule comprises a heavy chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 68; and a light chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 23. In other embodiments, the antibody molecule comprises a heavy chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 33; and a light chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 23. In other embodiments, the antibody molecule comprises a heavy chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 12; and a light chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 23. In other embodiments, the antibody molecule comprises a heavy chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 115; and a light chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 23. In other embodiments, the antibody molecule comprises a heavy chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 113; and a light chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 23. In other embodiments, the antibody molecule comprises a heavy chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 112; and a light chain comprising an amino acid sequence having at least about 85% (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID No. 23.

In other embodiments, the foregoing antibody molecule is selected from a complete antibody, a bispecific antibody, a Fab, a F (ab') 2, an Fv, or a single chain Fv fragment (scFv).

In other embodiments, the foregoing antibody molecules comprise a heavy chain constant region selected from the group consisting of IgG1, igG2, igG3, and IgG 4.

In other embodiments, the foregoing antibody molecules comprise a light chain constant region selected from the group consisting of kappa or lambda light chain constant regions.

In some embodiments, an anti-CD 73 antibody molecule comprises a heavy chain variable region, a light chain variable region, a heavy chain constant region, and/or a light chain constant region as disclosed in table 1. In some embodiments, the anti-CD 73 antibody molecule comprises the heavy chain constant regions and/or light chain constant regions disclosed in table 3. In some embodiments, the anti-CD 73 antibody molecule comprises a heavy chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS 92-103, 119 and 120. In some embodiments, the anti-CD 73 antibody molecule comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO. 104.

Exemplary sequences of anti-CD 73 antibodies are described in tables 1 and 2 below.

TABLE 1 amino acid and nucleotide sequences of exemplary anti-CD 73 antibodies

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TABLE 2 consensus CDR sequences of exemplary anti-CD 73 antibodies

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TABLE 17 corresponding germline sequences of anti-CD 73 antibodies

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TABLE 3 constant region amino acid sequences of human IgG heavy and human kappa light chains

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Table 4 exemplary sequences of cd73

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In other embodiments, the foregoing antibody molecules can be present in an amount of less than about 1x 10 ^-4 M、1x 10 ^-5 M、1x 10 ^-6 M、1x 10 ^-7 M、1x 10 ^-8 M、1x 10 ^-9 Dissociation constant of M (K _D ) Binding to human CD73, for example, as measured by Biacore, octet, flow cytometry or ELISA.

In some embodiments, the antibody molecule binds to mammalian (e.g., human or cynomolgus monkey) CD73. For example, the antibody molecule binds an epitope on CD73, such as a linear or conformational epitope (e.g., an epitope as described herein). In certain aspects, it is advantageous to identify antibodies that bind with high affinity to human and cynomolgus monkey homologs of the protein of interest. This ideal cross-reactivity can be tested for the same antibody (or two antibodies with the same CDRs or variable regions) in an animal model and then administered as a therapeutic to a human patient.

In some embodiments, disclosed herein are isolated antibody molecules that compete with the aforementioned anti-CD 73 antibody molecules for binding to human CD73.

In some embodiments, disclosed herein are isolated antibody molecules that bind to, substantially the same epitope as, overlap with, or substantially overlap with an epitope of the aforementioned anti-CD 73 antibody molecules.

In another aspect, disclosed herein are isolated nucleic acids (which encode any of the foregoing antibody molecules), vectors and host cells thereof. Nucleic acid molecules include, but are not limited to, RNA, genomic DNA, and cDNA.

In some embodiments, the isolated nucleic acid encodes an antibody heavy chain variable region, a light chain variable region, a heavy chain, and/or a light chain of any of the foregoing antibody molecules.

In some embodiments, an isolated nucleic acid encodes a heavy chain variable region, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO 45, 78, 85, 143, 152, 160, 67, 32, 11, or 169, or a nucleotide sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO 45, 78, 85, 143, 152, 160, 67, 32, 11, or 169.

In some embodiments, an isolated nucleic acid encodes a heavy chain, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO. 47, 80, 87, 69, 34, or 13, or a nucleotide sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO. 47, 80, 87, 69, 34, or 13.

In some embodiments, the isolated nucleic acid encodes a light chain variable region, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO. 56, 144, 22, or 170, or a nucleotide sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO. 56, 144, 22, or 170.

In some embodiments, the isolated nucleic acid encodes a light chain, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO. 58 or 24, or a nucleotide sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO. 58 or 24.

Exemplary antibody molecules that specifically bind human ENTPD2

In one embodiment of the dosage regimen of the invention, the anti-ENTPD 2 antibody is an anti-ENTPD 2 antibody molecule as described in WO 2019229658 (incorporated by reference in its entirety) published at 5, month 12 2019, entitled "ENTPD2 ANTIBODIES, COMBINATION THERAPIES, AND METHODS OF USING THE ANTIBODIES AND COMBINATION THERAPIES [ ENTPD2 antibody, combination therapy and methods of use of antibody and combination therapy ]".

In one aspect, provided herein are antibodies or antigen-binding fragments thereof, e.g., monoclonal antibodies or antigen-binding fragments thereof, that specifically bind to ENTPD2 protein ("ENTPD 2 antibodies or antigen-binding fragments" or "anti-ENTPD 2 antibodies or antigen-binding fragments"). In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to human ENTPD2 protein, e.g., monoclonal antibodies or antigen-binding fragments thereof ("human ENTPD2 antibodies or antigen-binding fragments" or "anti-human ENTPD2 antibodies or antigen-binding fragments"). In some embodiments, an anti-ENTPD 2 antibody or antigen-binding fragment thereof provided herein (e.g., an anti-human ENTPD2 antibody or antigen-binding fragment) comprises heavy chain CDR1 (HCDR 1), heavy chain CDR2 (HCDR 2), heavy chain CDR3 (HCDR 3), and light chain CDR1 (LCDR 1), light chain CDR2 (LCDR 2), and light chain CDR3 (LCDR 3). In some embodiments, an anti-ENTPD 2 antibody or antigen-binding fragment provided herein (e.g., an anti-human ENTPD2 antibody or antigen-binding fragment) comprises a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3, and a light chain variable region (VL) comprising CDR1, CDR2, and CDR 3. In some embodiments, an anti-ENTPD 2 antibody or antigen-binding fragment provided herein (e.g., an anti-human ENTPD2 antibody or antigen-binding fragment) comprises a full-length heavy chain sequence (HC) and a full-length light chain sequence (LC).

Table 9 lists the sequences of the ENTPD2 antibodies that specifically bind to the human ENTPD2 protein.

TABLE 9 sequences of exemplary anti-ENTPD 2 antibodies

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In some embodiments, an anti-human ENTPD2 antibody or antibody fragment (e.g., an antigen-binding fragment) comprises a VH domain having the amino acid sequence of any VH domain described in table 9. Other suitable anti-human ENTPD2 antibodies or antibody fragments (e.g., antigen-binding fragments) may include amino acids that have been mutated but have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity in the VH domain to the VH region depicted in the sequence depicted in table 9. In certain embodiments, the disclosure also provides antibodies or antibody fragments (e.g., antigen-binding fragments) that specifically bind to human ENTPD2, wherein the antibodies or antibody fragments (e.g., antigen-binding fragments) comprise VH CDRs having the amino acid sequences of any one of the VH CDRs listed in table 9. In particular embodiments, the invention provides antibodies or antibody fragments (e.g., antigen-binding fragments) that specifically bind to human ENTPD2, which antibodies or antibody fragments comprise (or alternatively consist of) one, two, three, four, five or more VH CDRs having the amino acid sequences of any one of the VH CDRs listed in table 9.

In some embodiments, an anti-human ENTPD2 antibody or antibody fragment (e.g., an antigen binding fragment) comprises a VL domain having the amino acid sequence of any of the VL domains described in table 9. Other suitable anti-human ENTPD2 antibodies or antibody fragments (e.g., antigen binding fragments) can include amino acids that have been mutated but have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity in the VL domain to the VL region depicted in the sequences described in table 9. The disclosure also provides antibodies or antibody fragments (e.g., antigen-binding fragments) that specifically bind to human ENTPD2, which antibodies or antibody fragments (e.g., antigen-binding fragments) comprise VL CDRs having the amino acid sequences of any one of the VL CDRs listed in table 9. In particular, the invention provides antibodies or antibody fragments (e.g., antigen-binding fragments) that specifically bind to human ENTPD2, which antibodies or antibody fragments comprise (or alternatively consist of) one, two, three or more VL CDRs having the amino acid sequences of any one of the VL CDRs listed in table 9.

Other anti-human ENTPD2 antibodies or antibody fragments (e.g., antigen binding fragments) disclosed herein include amino acids that have been mutated but have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity in CDR regions to CDR regions depicted in the sequences depicted in table 9. In some embodiments, it comprises a mutant amino acid sequence, wherein no more than 1, 2, 3, 4, or 5 amino acids in the CDR regions have been mutated when compared to the CDR regions depicted in the sequences described in table 9.

Also provided herein are nucleic acid sequences encoding VH, VL, full length heavy chain, and full length light chain of antibodies and antigen binding fragments thereof that specifically bind human ENTPD2, e.g., the nucleic acid sequences in table 9. Such nucleic acid sequences may be optimized for expression in mammalian cells.

Other anti-human ENTPD2 antibodies disclosed herein include those wherein the amino acids or nucleic acids encoding the amino acids have been mutated but have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequences described in table 9. In some embodiments, the antibody or antigen binding fragment thereof comprises a mutant amino acid sequence, wherein no more than 1, 2, 3, 4, or 5 amino acids have been mutated in the variable region when compared to the variable region depicted in the sequences described in table 9, but while retaining substantially the same therapeutic activity.

Because each provided antibody binds to human ENTPD2, VH, VL, full length light chain, and full length heavy chain sequences (amino acid sequences and nucleotide sequences encoding such amino acid sequences) can be "mixed and matched" to produce other ENTPD2 binding antibodies disclosed herein. Such "mixed and matched" ENTPD2 binding antibodies can be tested using binding assays known in the art (e.g., ELISA, assays described in the examples). When the chains are mixed and matched, the VH sequences from a particular VH/VL pairing should be replaced with structurally similar VH sequences. The full length heavy chain sequences from a particular full length heavy chain/full length light chain pairing should be replaced with structurally similar full length heavy chain sequences. The VL sequences from a particular VH/VL pairing should be replaced with structurally similar VL sequences. The full length light chain sequences from a particular full length heavy chain/full length light chain pairing should be replaced with structurally similar full length light chain sequences.

Thus, in one embodiment, the invention provides an isolated monoclonal antibody or antigen binding fragment thereof having: a heavy chain variable region (VH) comprising an amino acid sequence selected from any one of SEQ ID NOs 410, 425, 433, 446; and a light chain variable region (VL) comprising an amino acid sequence selected from any one of SEQ ID NOs 421, 429, 457, 464; wherein the antibody specifically binds to human ENTPD2.

In another embodiment, the invention provides (i) an isolated monoclonal antibody having: a full length Heavy Chain (HC) comprising an amino acid sequence selected from any one of SEQ ID NOs 412, 427, 435, 448; and a full length Light Chain (LC) comprising an amino acid sequence selected from any one of SEQ ID NOs 423, 431, 459, 466; or (ii) a functional protein comprising an antigen binding portion thereof.

In another embodiment, the present disclosure provides human ENTPD2 binding antibodies or antibody fragments thereof comprising heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3, or combinations thereof, as described in table 9. The amino acid sequences of VH CDR1 of these antibodies are shown in SEQ ID NOS 401, 404, 406, 407, 437, 440, 442, 443. The amino acid sequences of VH CDR2 of these antibodies are shown in SEQ ID NOS: 402, 405, 408, 438, 441, 444. The amino acid sequences of VH CDR3 of these antibodies are shown in SEQ ID NOS 403, 409, 439, 445. The amino acid sequences of VL CDR1 of these antibodies are shown in SEQ ID NOS 414, 417, 420, 450, 453, 456, 461, 462, 463. The amino acid sequences of VL CDR2 of these antibodies are shown in SEQ ID NOS 415, 418, 451, 454. The amino acid sequences of VL CDR3 of these antibodies are shown in SEQ ID NOS 416, 419, 452, 455.

Whereas each of these antibodies binds to ENTPD2 and antigen binding specificity is provided primarily by CDR1, CDR2, and CDR3 regions, VH CDR1, CDR2, and CDR3 sequences and VL CDR1, CDR2, and CDR3 sequences may be "mixed and matched" (i.e., CDRs from different antibodies may be mixed and matched), each antibody must contain VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3 to produce other human ENTPD2 binding molecules disclosed herein. Such "mixed and matched" ENTPD2 binding antibodies can be tested using binding assays known in the art and those described in the examples (e.g., ELISA). When VH CDR sequences are mixed and matched, CDR1, CDR2, and/or CDR3 sequences from a particular VH sequence should be replaced with one or more structurally similar CDR sequences. Likewise, when VL CDR sequences are mixed and matched, CDR1, CDR2, and/or CDR3 sequences from a particular VL sequence should be replaced with one or more structurally similar CDR sequences. It will be readily apparent to one of ordinary skill that new VH and VL sequences can be generated by substituting one or more VH and/or VL CDR region sequences having structurally similar sequences from the CDR sequences set forth herein for the monoclonal antibodies of the present disclosure.

Accordingly, the present disclosure provides an isolated monoclonal antibody, or antigen binding region thereof, comprising: a heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 401, 404, 406, 407, 437, 440, 442, 443; a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 402, 405, 408, 438, 441, 444; a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 403, 409, 439, 445; a light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 414, 417, 420, 450, 453, 456, 461, 462, 463; a light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 415, 418, 451, 454; and a light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 416, 419, 452, 455; wherein the antibody specifically binds to human ENTPD2.

In certain embodiments, the antibody that specifically binds human ENTPD2 is an antibody or antibody fragment (e.g., an antigen binding fragment) described in table 9.

In some embodiments, the antibody or antigen binding region thereof that specifically binds human ENTPD2 comprises: heavy chain complementarity determining region 1 (HCDR 1) comprising the amino acid sequence of SEQ ID NO:401, 404, 406 or 407; heavy chain complementarity determining region 2 (HCDR 2) comprising the amino acid sequence of SEQ ID NO:402, 405 or 408; heavy chain complementarity determining region 3 (HCDR 3) comprising the amino acid sequence of SEQ ID NO:403 or 409; light chain complementarity determining region 1 (LCDR 1) comprising the amino acid sequence of SEQ ID NO:414, 417 or 420; light chain complementarity determining region 2 (LCDR 2) comprising the amino acid sequence of SEQ ID NO:415 or 418; and light chain complementarity determining region 3 (LCDR 3) comprising the amino acid sequence of SEQ ID NO:416 or 419.

In some embodiments, the antibody or antigen binding region thereof that specifically binds human ENTPD2 comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO. 401; HCDR2 comprising the amino acid sequence of SEQ ID NO. 402; HCDR3 comprising the amino acid sequence of SEQ ID NO. 403; LCDR1 comprising the amino acid sequence of SEQ ID NO. 414; LCDR2 comprising the amino acid sequence of SEQ ID NO. 415; and LCDR3 comprising the amino acid sequence of SEQ ID NO. 416.

In some embodiments, the antibody or antigen binding region thereof that specifically binds human ENTPD2 comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO. 404; HCDR2 comprising the amino acid sequence of SEQ ID NO. 405; HCDR3 comprising the amino acid sequence of SEQ ID NO. 403; LCDR1 comprising the amino acid sequence of SEQ ID NO. 417; LCDR2 comprising the amino acid sequence of SEQ ID NO. 418; and LCDR3 comprising the amino acid sequence of SEQ ID NO: 419.

In some embodiments, the antibody or antigen binding region thereof that specifically binds human ENTPD2 comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO. 406; HCDR2 comprising the amino acid sequence of SEQ ID NO. 402; HCDR3 comprising the amino acid sequence of SEQ ID NO. 403; LCDR1 comprising the amino acid sequence of SEQ ID NO. 414; LCDR2 comprising the amino acid sequence of SEQ ID NO. 415; and LCDR3 comprising the amino acid sequence of SEQ ID NO. 416.

In some embodiments, the antibody or antigen binding region thereof that specifically binds human ENTPD2 comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 407; HCDR2 comprising the amino acid sequence of SEQ ID NO. 408; HCDR3 comprising the amino acid sequence of SEQ ID NO. 409; LCDR1 comprising the amino acid sequence of SEQ ID NO. 420; LCDR2 comprising the amino acid sequence of SEQ ID NO. 418; and LCDR3 comprising the amino acid sequence of SEQ ID NO. 416.

In some embodiments, the antibody or antigen binding region thereof that specifically binds human ENTPD2 comprises: HCDR1 comprising the amino acid sequence of SEQ ID No. 437, 440, 442 or 443; HCDR2 comprising the amino acid sequence of SEQ ID No. 438, 441 or 444; HCDR3 comprising the amino acid sequence of SEQ ID No. 439 or 445; LCDR1 comprising the amino acid sequence of SEQ ID NO. 450, 453 or 456; LCDR2 comprising the amino acid sequence of SEQ ID NO. 451 or 454; and LCDR3 comprising the amino acid sequence of SEQ ID NO 452 or 455.

In some embodiments, the antibody or antigen binding region thereof that specifically binds human ENTPD2 comprises: HCDR1 comprising the amino acid sequence of SEQ ID No. 437; HCDR2 comprising the amino acid sequence of SEQ ID NO. 438; HCDR3 comprising the amino acid sequence of SEQ ID No. 439; LCDR1 comprising the amino acid sequence of SEQ ID NO. 450; LCDR2 comprising the amino acid sequence of SEQ ID NO. 451; and LCDR3 comprising the amino acid sequence of SEQ ID NO. 452.

In some embodiments, the antibody or antigen binding region thereof that specifically binds human ENTPD2 comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO. 440; HCDR2 comprising the amino acid sequence of SEQ ID NO: 441; HCDR3 comprising the amino acid sequence of SEQ ID No. 439; LCDR1 comprising the amino acid sequence of SEQ ID NO. 453; LCDR2 comprising the amino acid sequence of SEQ ID NO. 454; and LCDR3 comprising the amino acid sequence of SEQ ID NO: 455.

In some embodiments, the antibody or antigen binding region thereof that specifically binds human ENTPD2 comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO. 442; HCDR2 comprising the amino acid sequence of SEQ ID NO. 438; HCDR3 comprising the amino acid sequence of SEQ ID No. 439; LCDR1 comprising the amino acid sequence of SEQ ID NO. 450; LCDR2 comprising the amino acid sequence of SEQ ID NO. 451; and LCDR3 comprising the amino acid sequence of SEQ ID NO. 452.

In some embodiments, the antibody or antigen binding region thereof that specifically binds human ENTPD2 comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO. 443; HCDR2 comprising the amino acid sequence of SEQ ID NO. 444; HCDR3 comprising the amino acid sequence of SEQ ID NO. 445; LCDR1 comprising the amino acid sequence of SEQ ID NO. 456; LCDR2 comprising the amino acid sequence of SEQ ID NO. 454; and LCDR3 comprising the amino acid sequence of SEQ ID NO. 452.

In some embodiments, the antibody or antigen binding region thereof that specifically binds human ENTPD2 comprises: HCDR1 comprising the amino acid sequence of SEQ ID No. 437, 440, 442 or 443; HCDR2 comprising the amino acid sequence of SEQ ID No. 438, 441 or 444; HCDR3 comprising the amino acid sequence of SEQ ID No. 439 or 445; LCDR1 comprising the amino acid sequence of SEQ ID NOS 461, 462 or 463; LCDR2 comprising the amino acid sequence of SEQ ID NO. 451 or 454; and LCDR3 comprising the amino acid sequence of SEQ ID NO 452 or 455.

In some embodiments, the antibody or antigen binding region thereof that specifically binds human ENTPD2 comprises: HCDR1 comprising the amino acid sequence of SEQ ID No. 437; HCDR2 comprising the amino acid sequence of SEQ ID NO. 438; HCDR3 comprising the amino acid sequence of SEQ ID No. 439; LCDR1 comprising the amino acid sequence of SEQ ID NO. 461; LCDR2 comprising the amino acid sequence of SEQ ID NO. 451; and LCDR3 comprising the amino acid sequence of SEQ ID NO. 452.

In some embodiments, the antibody or antigen binding region thereof that specifically binds human ENTPD2 comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO. 440; HCDR2 comprising the amino acid sequence of SEQ ID NO: 441; HCDR3 comprising the amino acid sequence of SEQ ID No. 439; LCDR1 comprising the amino acid sequence of SEQ ID NO. 462; LCDR2 comprising the amino acid sequence of SEQ ID NO. 454; and LCDR3 comprising the amino acid sequence of SEQ ID NO: 455.

In some embodiments, the antibody or antigen binding region thereof that specifically binds human ENTPD2 comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO. 442; HCDR2 comprising the amino acid sequence of SEQ ID NO. 438; HCDR3 comprising the amino acid sequence of SEQ ID No. 439; LCDR1 comprising the amino acid sequence of SEQ ID NO. 461; LCDR2 comprising the amino acid sequence of SEQ ID NO. 451; and LCDR3 comprising the amino acid sequence of SEQ ID NO. 452.

In some embodiments, the antibody or antigen binding region thereof that specifically binds human ENTPD2 comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO. 443; HCDR2 comprising the amino acid sequence of SEQ ID NO. 444; HCDR3 comprising the amino acid sequence of SEQ ID NO. 445; LCDR1 comprising the amino acid sequence of SEQ ID NO. 463; LCDR2 comprising the amino acid sequence of SEQ ID NO. 454; and LCDR3 comprising the amino acid sequence of SEQ ID NO. 452.

In some embodiments, the antibody or antigen binding region thereof that specifically binds human ENTPD2 comprises: a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:410 (or a sequence having at least about 90%, 95%, 99% or more identity thereto, and/or a sequence having one, two, three or more substitutions, insertions, deletions or modifications) and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:421 (or a sequence having at least about 90%, 95%, 99% or more identity thereto, and/or a sequence having one, two, three or more substitutions, insertions, deletions or modifications).

In some embodiments, the antibody or antigen binding region thereof that specifically binds human ENTPD2 comprises: a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:425 (or a sequence having at least about 90%, 95%, 99% or more identity thereto, and/or a sequence having one, two, three or more substitutions, insertions, deletions or modifications) and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:429 (or a sequence having at least about 90%, 95%, 99% or more identity thereto, and/or a sequence having one, two, three or more substitutions, insertions, deletions or modifications).

In some embodiments, the antibody or antigen binding region thereof that specifically binds human ENTPD2 comprises: a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID No. 433 (or a sequence having at least about 90%, 95%, 99% or more identity thereto, and/or a sequence having one, two, three or more substitutions, insertions, deletions or modifications) and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID No. 429 (or a sequence having at least about 90%, 95%, 99% or more identity thereto, and/or a sequence having one, two, three or more substitutions, insertions, deletions or modifications).

In some embodiments, the antibody or antigen binding region thereof that specifically binds human ENTPD2 comprises: a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID No. 446 (or a sequence having at least about 90%, 95%, 99% or more identity thereto, and/or a sequence having one, two, three or more substitutions, insertions, deletions or modifications) and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID No. 457 (or a sequence having at least about 90%, 95%, 99% or more identity thereto, and/or a sequence having one, two, three or more substitutions, insertions, deletions or modifications).

In some embodiments, the antibody or antigen binding region thereof that specifically binds human ENTPD2 comprises: a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID No. 446 (or a sequence having at least about 90%, 95%, 99% or more identity thereto, and/or a sequence having one, two, three or more substitutions, insertions, deletions or modifications) and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID No. 464 (or a sequence having at least about 90%, 95%, 99% or more identity thereto, and/or a sequence having one, two, three or more substitutions, insertions, deletions or modifications).

In some embodiments, an antibody that specifically binds human ENTPD2 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 412 (or a sequence having at least about 90%, 95%, 99% or more identity thereto, and/or a sequence having one, two, three or more substitutions, insertions, deletions or modifications) and a light chain comprising the amino acid sequence of SEQ ID NO. 423 (or a sequence having at least about 90%, 95%, 99% or more identity thereto, and/or a sequence having one, two, three or more substitutions, insertions, deletions or modifications).

In some embodiments, an antibody that specifically binds human ENTPD2 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 427 (or a sequence having at least about 90%, 95%, 99% or more identity thereto, and/or a sequence having one, two, three or more substitutions, insertions, deletions or modifications) and a light chain comprising the amino acid sequence of SEQ ID NO. 431 (or a sequence having at least about 90%, 95%, 99% or more identity thereto, and/or a sequence having one, two, three or more substitutions, insertions, deletions or modifications).

In some embodiments, an antibody that specifically binds human ENTPD2 comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 435 (or a sequence having at least about 90%, 95%, 99% or more identity thereto, and/or a sequence having one, two, three or more substitutions, insertions, deletions or modifications) and a light chain comprising the amino acid sequence of SEQ ID No. 431 (or a sequence having at least about 90%, 95%, 99% or more identity thereto, and/or a sequence having one, two, three or more substitutions, insertions, deletions or modifications).

In some embodiments, an antibody that specifically binds human ENTPD2 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO 448 (or a sequence having at least about 90%, 95%, 99% or more identity thereto, and/or a sequence having one, two, three or more substitutions, insertions, deletions or modifications) and a light chain comprising the amino acid sequence of SEQ ID NO 459 (or a sequence having at least about 90%, 95%, 99% or more identity thereto, and/or a sequence having one, two, three or more substitutions, insertions, deletions or modifications).

In some embodiments, an antibody that specifically binds human ENTPD2 comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 448 (or a sequence having at least about 90%, 95%, 99% or more identity thereto, and/or a sequence having one, two, three or more substitutions, insertions, deletions or modifications) and a light chain comprising the amino acid sequence of SEQ ID No. 466 (or a sequence having at least about 90%, 95%, 99% or more identity thereto, and/or a sequence having one, two, three or more substitutions, insertions, deletions or modifications).

In some embodiments, the invention provides antibodies or antigen-binding fragments thereof that bind human ENTPD2 protein, wherein the dissociation constant (KD) is less than 10nM, e.g., KD is less than 9nM, less than 8nM, less than 7nM, less than 6nM, less than 5nM, less than 4nM, less than 3nM, less than 2nM, less than 1nM, e.g., as measured by Biacore. In some embodiments, an antibody or antigen binding fragment provided herein binds to a human ENTPD2 protein, wherein the dissociation constant (KD) is less than 5nM, e.g., as measured by Biacore. In some embodiments, an antibody or antigen binding fragment provided herein binds to a human ENTPD2 protein, wherein the dissociation constant (KD) is less than 3nM, e.g., as measured by Biacore. In some embodiments, an antibody or antigen binding fragment provided herein binds to a human ENTPD2 protein, wherein the dissociation constant (KD) is less than 1nM, e.g., as measured by Biacore. In some embodiments, the dissociation constant of an antibody or antigen binding fragment thereof described herein for human ENTPD2 is measured by Biacore at 25 ℃.

Also provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an epitope in human ENTPD2, wherein the epitope comprises at least one of the following residues (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least twenty): his50, asp76, pro78, gly79, gly80, tyr85, asp87, asn88, gly91, gln94, ser95, gly98, glu101, gln102, gln105, asp106, arg245, thr272, gln273, leu275, asp278, arg298, ala347, ala350, thr351, arg392, ala393, arg394, or Tyr398. In some embodiments, such antibodies or antigen binding fragments include, but are not limited to, MAb1, MAb2, and MAb3 as disclosed in table 9.

Also provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an epitope in human ENTPD2, wherein the epitope comprises at least one of the following residues (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least twenty): gly79, gln250, leu253, trp266, arg268, gly269, phe270, ser271, thr272, gln273, val274, leu275, asp278, arg298, ser300, ser302, gly303, thr380, trp381, ala382, gly390, gln391, arg392, ala393, arg394, or Asp397. In some embodiments, such antibodies or antigen binding fragments include, but are not limited to, MAb4 and MAb5 as disclosed in table 9.

Once the desired epitope on the antigen is determined, it is possible to produce antibodies directed against the epitope, for example, using the techniques described in the present invention. Alternatively, during the discovery process, the production and characterization of antibodies can elucidate information about the desired epitope. Based on this information, antibodies can then be competitively screened to bind to the same epitope. The method of achieving this is to conduct cross-competition studies to find antibodies that competitively bind to each other, e.g., antibodies compete for binding to antigen. A high throughput method of "binning" these antibodies based on cross-competition of the antibodies is described in international patent application No. WO 2003/48731. As will be appreciated by those skilled in the art, virtually anything to which an antibody can specifically bind can be an epitope. Epitopes may comprise those residues to which antibodies bind.

In general, antibodies specific for a particular target antigen will preferentially recognize epitopes on the target antigen in a complex mixture of proteins and/or macromolecules.

Any number of epitope mapping techniques known in the art may be used to identify the region of a given polypeptide that contains an epitope. See, e.g., epitope Mapping Protocols in Methods in Molecular Biology [ epitope mapping protocol in molecular biology methods ], volume 66 (Glenn e.Morris, edit, 1996,Humana Press [ Hu Mana press ], totowa, N.J.). For example, a linear epitope may be determined by, for example, parallel synthesis of a large number of peptides on a solid support, which peptides correspond to portions of a protein molecule and which peptides react with antibodies while the peptides remain attached to the support. Such techniques are known in the art and are described, for example, in U.S. Pat. nos. 4,708,871; geysen et al, (1984) Proc.Natl.Acad.Sci.USA [ Proc. Natl.Acad.Sci.USA, U.S. national academy of sciences ]8:3998-4002; geysen et al, (1985) Proc.Natl.Acad.Sci.USA [ Proc. Natl.Acad.Sci.USA ]82:78-182; geysen et al, (1986) mol. Immunol [ molecular immunology ]23:709-715. Similarly, conformational epitopes can be readily identified by determining the spatial conformation of amino acids, such as by x-ray crystallography and two-dimensional nuclear magnetic resonance. See, e.g., epitope Mapping Protocols [ epitope mapping protocol ], supra. Standard antigenicity and hydrophilicity maps can also be used to identify antigenic regions of proteins, such as those calculated using, for example, the Omiga version 1.0 software program available from oxford molecular group (Oxford Molecular Group). The computer program was used to determine the antigenicity curve using the Hopp/Woods method (Hopp et al, (1981) Proc. Natl. Acad. Sci USA [ Proc. Natl. Acad. Sci. USA U.S. Sci. U.S. Sci. A. 78:3824-3828) and the hydrophilicity map using the Kyte-Doolittle technique (Kyte et al, (1982) J.mol. Biol. [ J. Mol. 157:105-132).

The antibody molecule may be a polyclonal antibody or a monoclonal antibody. Monoclonal antibodies can be prepared by hybridoma technology or methods that do not use hybridoma technology (e.g., recombinant methods). In some embodiments, antibodies may be produced recombinantly, e.g., by phage display or by combinatorial methods.

Phage display and combinatorial methods for producing antibodies are known in the art (as described, for example, in Ladner et al, U.S. Pat. No. 5,223,409; kang et al, international publication No. WO 92/18619; dower et al, international publication No. WO 91/17271; winter et al, international publication No. WO 92/15679; breitling et al, international publication No. WO 93/01188; mcCafferty et al, international publication No. WO 92/01047; gargarard et al, international publication No. WO 92/09809; ladner et al, international publication No. WO 90/02809; fuchs et al (1991) Bio/Technology [ biotechnology ]9:1370-1372; hay et al (1992) Hum Antibod Hybridomas [ human antibody hybridoma ]3:81-85; huklin Science [ Science ] 246:5-1281; grifs et al (1993) J [ J3:35:35:35:35 ] J-35:35, J-19:35:35, J-1991; bioJ-37, J-35:35, J-1991, J-35:35, et al, J-1991, J-Mol, J-35:35:37, J-Mol, J-1996:35, J-1991; and Barbas et al (1991) PNAS [ Proc. Natl. Acad. Sci. USA ] 88:7978-7982).

In one embodiment, the antibody is a fully human antibody (e.g., an antibody prepared in a mouse that has been genetically engineered to produce antibodies from human immunoglobulin sequences), or a non-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g., monkey), camel antibody.

The antibody may be an antibody that produces a variable region or a portion thereof (e.g., CDR) in a non-human organism (e.g., rat or mouse). Chimeric antibodies, CDR-grafted antibodies, and humanized antibodies are within the scope of the invention. Antibodies produced in a non-human organism (e.g., a rat or mouse) and then modified in, for example, a variable framework or constant region to reduce antigenicity in humans are within the present invention.

Chimeric and/or humanized antibodies may be engineered to minimize the immune response of a human patient to antibodies produced in a non-human subject or antibodies derived from the expression of non-human antibody genes. Chimeric antibodies comprise a non-human animal antibody variable region and a human antibody constant region. Such antibodies retain the epitope binding specificity of the original monoclonal antibody, but may be less immunogenic when administered to humans and therefore more likely to be tolerated by patients. For example, one or all (e.g., one, two, or three) of the variable regions of one or more light chains and/or one or all (e.g., one, two, or three) of the variable regions of one or more heavy chains of a mouse antibody (e.g., a mouse monoclonal antibody) may each be linked to a human constant region, such as, but not limited to, an IgG1 human constant region. Chimeric monoclonal antibodies can be produced by recombinant DNA techniques known in the art. For example, the gene encoding the constant region of a non-human antibody molecule may be replaced with a gene encoding a human constant region (see Robinson et al, PCT patent publication PCT/US86/02269; akira et al, european patent application 184,187; or Taniguchi, M., european patent application 171,496). In addition, other suitable techniques that may be used to generate chimeric antibodies are described, for example, in U.S. Pat. nos. 4,816,567;4,978,775;4,975,369; and 4,816,397.

Further "humanized" chimeric antibodies can be obtained by replacing portions of the variable region that are not involved in antigen binding with equivalent portions from human variable regions. Humanized antibodies comprise one or more human framework regions in the variable region and non-human (e.g., mouse, rat, or hamster) Complementarity Determining Regions (CDRs) of the heavy and/or light chain. In some embodiments, the humanized antibody comprises sequences that are fully human except for the CDR regions. Humanized antibodies are typically less immunogenic to humans than non-humanized antibodies and thus provide therapeutic benefits in some cases. Humanized ENTPD2 antibodies can be produced using methods known in the art. See, e.g., hwang et al, methods [ Methods ]36:35,2005; queen et al, proc.Natl.Acad.Sci.U.S.A. [ Proc.Natl.Acad.Sci.Sci.A. ]86:10029-10033,1989; jones et al Nature [ Nature ]321:522-25,1986; riechmann et al Nature [ Nature ]332:323-27,1988; verhoeyen et al Science [ Science ]239:1534-36,1988; orlandi et al, proc.Natl. Acad. Sci. U.S. A. [ Proc. Natl. Acad. Sci. USA ]86:3833-3837,1989; U.S. Pat. nos. 5,225,539;5,530,101;5,585,089;5,693,761;5,693,762; and 6,180,370; and WO 90/07861.

The human ENTPD2 antibodies can be produced using methods known in the art. For example, ergonomic techniques are used to convert non-human antibodies to engineered human antibodies. U.S. patent publication No. 20050008625 describes an in vivo method for replacing a non-human antibody variable region with a human variable region in an antibody while maintaining the same binding characteristics or providing better binding characteristics relative to a non-human antibody. The method relies on epitope-directed replacement of the variable region of a non-human reference antibody with a fully human antibody. The resulting human antibodies are generally not structurally related to the reference non-human antibodies, but bind to the same epitope on the same antigen as the reference antibody. Briefly, in the presence of a reporter system responsive to the binding of a test antibody to an antigen, a continuous epitope-directed complementation replacement method is achieved by establishing competition between libraries of multiple hybrids of a "competitor" and a reference antibody ("test antibody") in a cell for binding to a limited amount of antigen. The competitor may be a reference antibody or a derivative thereof, such as a single chain Fv fragment. The competitor may also be a natural or artificial ligand of the antigen that binds to the same epitope as the reference antibody. The only requirement for the competitor is that it bind to the same epitope as the reference antibody and that it competes for antigen binding with the reference antibody. The test antibodies have one common antigen-binding V region from a non-human antibody, and another V region randomly selected from a variety of sources (e.g., a library of human antibodies). The common V region from the reference antibody is used as a guide to localize the test antibody to the same epitope on the antigen and in the same orientation, biasing the selection towards the highest antigen binding fidelity to the reference antibody.

Many types of reporting systems can be used to detect the desired interaction between the test antibody and the antigen. For example, complementary reporter fragments may be linked to an antigen and a test antibody, respectively, such that activation of the reporter gene by fragment complementarity occurs only upon binding of the test antibody to the antigen. When the test antibody and antigen reporter fusion are co-expressed with a competitor, reporter activation becomes dependent on the ability of the test antibody to compete with the competitor, which is proportional to the affinity of the test antibody for the antigen. Other reporter systems that may be used include a self-inhibiting reporter reactivation system (RAIR) as disclosed in U.S. patent application Ser. No. 10/208,730 (publication No. 20030198971) or a reactivator of a competing activation system as disclosed in U.S. patent application Ser. No. 10/076,845 (publication No. 20030157579).

Selection was performed to identify cells expressing the individual test antibodies as well as competitors, antigens and reporter components using a sequential epitope-directed complementation substitution system. In these cells, each test antibody competes one-to-one with the competitor for binding to a limited amount of antigen. The activity of the reporter is proportional to the amount of antigen bound to the test antibody, while the amount of antigen bound to the test antibody is proportional to the affinity of the test antibody for the antigen and the stability of the test antibody. When expressed as a test antibody, the test antibody is initially selected based on its activity relative to a reference antibody. The result of the first round of selection is a set of "hybrid" antibodies, where each antibody consists of the same non-human V region from the reference antibody and human V region from the library, and each antibody binds the same epitope as the reference antibody. The one or more hybrid antibodies selected in the first round will have an affinity for the antigen that is comparable to or higher than the reference antibody.

In the second V-region replacement step, the human V-region selected in the first step is used as a guide for selecting the remaining non-human reference antibody V-regions for human replacement of the diverse library of homologous human V-regions. Hybrid antibodies selected in the first round may also be used as competitors for the second round of selection. The result of the second round of selection is a set of fully human antibodies that are structurally different from the reference antibodies, but which compete with the reference antibodies for binding to the same antigen. Some selected human antibodies bind to the same epitope on the same antigen of the reference antibody. In these selected human antibodies, one or more binds to the same epitope with an affinity comparable to or higher than that of the reference antibody.

Using mouse or chimeric ENTPD2 antibodies, human antibodies with the same binding specificity and the same or better binding affinity to human ENTPD2 can be produced. In addition, such human ENTPD2 antibodies are also commercially available from companies that commonly produce human antibodies, such as kalofos corporation (kalofos, inc.) (Mountain View, california (calif.)).

In some embodiments, the invention provides antibodies or antigen-binding fragments thereof that bind to human ENTPD2 protein and modulate one or more ENTPD2 activities/functions, e.g., inhibit, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the enzymatic activity of human ENTPD 2. In some embodiments, the enzymatic activity of human ENTPD2 is measured using an in vitro FRET assay that measures ATP to ADP hydrolysis by recombinant ENTPD2 or ENTPD2 expressed on the cell surface.

In some embodiments, an anti-human ENTPD2 antibody or antigen-binding fragment thereof described herein inhibits the ability of ENTPD2 to hydrolyze Adenosine Triphosphate (ATP). In some embodiments, the ability of ENTPD2 to hydrolyze ATP is measured using an in vitro FRET assay that measures ATP to ADP hydrolysis by recombinant ENTPD2 or ENTPD2 expressed on the cell surface.

In some embodiments, an anti-human ENTPD2 antibody or antigen-binding fragment thereof described herein interferes with the binding of ATP to ENTPD2 or captures ATP within the catalytic domain of ENTPD 2. In some embodiments, the interference of ATP binding to ENTPD2 or capture of ATP within the ENTPD2 catalytic domain is measured using an in vitro FRET assay that measures ATP to ADP hydrolysis by recombinant ENTPD2 or ENTPD2 expressed on the cell surface.

Pharmaceutical compositions, kits and administration

In an embodiment of the antibody used according to the invention or the method according to the invention, the antibody molecule that binds to human CD73 or human ENTPD2 is in the form of a pharmaceutical composition. A composition (e.g., a pharmaceutically acceptable composition) comprising an anti-CD 73 antibody molecule or an anti-ENTPD 2 antibody molecule described herein may be formulated with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion).

The compositions described herein may take a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, and suppositories. The appropriate form depends on the intended mode of administration and therapeutic application. Typical suitable compositions are in the form of injectable or infusible solutions. One suitable mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the antibody is administered by intravenous infusion or injection. In certain embodiments, the antibody is administered by intramuscular or subcutaneous injection.

As used herein, the phrases "parenteral administration" and "parenteral administration" mean modes of administration other than enteral and topical administration, typically by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

Therapeutic compositions should typically be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, dispersions, liposomes or other ordered structures suitable for high antibody concentrations. Sterile injectable solutions can be prepared by: the active compound (i.e., antibody or antibody portion) is incorporated in the desired amount, as desired, with one or a combination of the ingredients listed above, in a suitable solvent, and then filter sterilized. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. For example, proper fluidity of the solution may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. The absorption of the injectable composition may be prolonged by including agents (e.g., monostearates and gelatins) in the composition that delay absorption.

Antibody molecules can be administered by a variety of methods. Several are known in the art and for many therapeutic applications, a suitable route/mode of administration is intravenous injection or infusion. In embodiments, the antibody molecule may be administered by intravenous infusion at a rate of greater than 20mg/min (e.g., 20-40 mg/min). In embodiments, the antibody molecule may be administered by intravenous infusion at a rate of greater than or equal to 40mg/min to achieve a dose of about 35 to 440mg/m2, about 70 to 310mg/m2, or about 110 to 130mg/m 2. In embodiments, the antibody molecule may be administered by intravenous infusion at a rate of less than 10mg/min, e.g., less than or equal to 5mg/min, to achieve a dose of about 1 to 100mg/m2, about 5 to 50mg/m2, about 7 to 25mg/m2, or about 10mg/m 2. As will be appreciated by those skilled in the art, the route and/or manner of administration will vary depending on the desired result. In certain embodiments, the active compounds may be prepared with carriers that will protect the compounds from rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid may be used. Many methods for preparing such formulations are patented or generally known to those skilled in the art. See, e.g., sustained and Controlled Release Drug Delivery Systems [ sustained release drug delivery system ], J.R. Robinson, marcel Dekker, inc. [ Marselde Kerr, new York, 1978.

In one aspect of the invention there is provided an antibody molecule that binds human CD73 or human ENTPD2 for use in a method of treating cancer in a subject or in a method of treating cancer in a subject, wherein the antibody molecule is administered in a ascending dosing regimen such that in a primary phase the antibody molecule dose is administered in a primary dose according to a primary dosing period having a primary dosing period frequency, the primary phase being preceded by an initial phase in which the antibody molecule is administered at a dosing period frequency higher than the primary dosing period frequency and administered via a fractionated dose of the primary dose, wherein the sum of the fractionated doses should not exceed the amount of the primary phase dose in the initial phase over a period of time equal to the primary dosing period. The divided doses may be, for example, 1/6, 1/3, 1/2, 2/3 of the primary dose. In an embodiment of the antibody used according to the invention or of the method according to the invention, the sum of the divided doses added in an initial phase within a period of time equal to the main dosing period is equal to the main dose.

In some embodiments, an anti-CD 73 antibody molecule or an anti-ENTPD 2 antibody molecule disclosed herein is administered in the main phase by injection (e.g., subcutaneously or intravenously) at a dose (e.g., a plateau dose) of about 60mg to 2400mg, e.g., about 100mg to 2400mg, about 100mg to 2200mg, about 100mg to 2000mg, about 100mg to 1800mg, about 100mg to 1600mg, about 100mg to 1400mg, about 100mg to 1200mg, about 100mg to 1000mg, about 100mg to 800mg, about 100mg to 600mg, about 100mg to 400mg, about 100mg to 200mg. In some embodiments, an anti-CD 73 antibody molecule or an anti-ENTPD 2 antibody molecule disclosed herein is administered in the main phase by (e.g., subcutaneous or intravenous) injection at a dose (e.g., a plateau dose) of about 100mg, about 150mg, 200mg, about 250mg, about 300mg, about 350mg, about 400mg, about 450mg, about 500mg, about 550mg, 600mg, about 650mg, about 700mg, about 750mg, about 800mg, about 850mg, about 900mg, about 950mg, about 1000mg, about 1050mg, about 1100mg, about 1150mg, about 1200mg, and about 2400mg. The dosing schedule in the main phase (e.g., a plateau dosing schedule) may vary from, for example, once a week to once every 2, 3, or 4 weeks. In one embodiment, the anti-CD 73 antibody molecule or anti-ENTPD 2 antibody molecule disclosed herein is administered in a dose of about 100mg to 1200mg once every two weeks during the main phase. In one embodiment, the anti-CD 73 antibody molecules disclosed herein are administered in a dose of at least about 600mg once every two weeks during the primary phase. In one embodiment, the anti-ENTPD 2 antibody molecules disclosed herein are administered in a dose of at least about 300mg once every two weeks during the primary phase.

In certain embodiments, in the main phase, the antibody molecule is administered at a dose, e.g., QW, Q2W, or Q4W, (e.g., intravenously): about 200mg, about 400mg, about 600mg, about 800mg, about 1000mg, about 1200mg, about 2400mg, about 3000mg, or about 3600mg. In certain embodiments, the antibody molecule is administered at a dose Q2W of about 200mg (e.g., intravenously). In certain embodiments, the antibody molecule is administered at a dose Q2W of about 300mg (e.g., intravenously). In certain embodiments, the antibody molecule is administered at a dose Q2W of about 400mg (e.g., intravenously). In certain embodiments, the antibody molecule is administered at a dose Q2W of about 600mg (e.g., intravenously). In certain embodiments, the antibody molecule is administered at a dose Q2W of about 800mg (e.g., intravenously). In certain embodiments, the antibody molecule is administered at a dose Q2W of about 1000mg (e.g., intravenously). In certain embodiments, the antibody molecule is administered at a dose Q2W of about 1200mg (e.g., intravenously). In certain embodiments, the antibody molecule is administered at a dose of about 2400mg Q2W (e.g., intravenously). In certain embodiments, the antibody molecule is administered at a dose Q2W of about 3000mg (e.g., intravenously). In certain embodiments, the antibody molecule is administered at a dose Q2W of about 3600mg (e.g., intravenously).

In an embodiment of the antibody used according to the invention or the method according to the invention, the main dose of antibody molecules binding to human ENTPD2 is 300mg. In an embodiment, the primary dose frequency is Q2W.

In an embodiment of the antibody used according to the invention or the method according to the invention, in the initial phase, the antibody molecule binding to human ENTPD2 is administered at the frequency of QW or Q2W.

In embodiments of the antibodies used according to the invention or the methods according to the invention, the initial phase of administration of the anti-CD 73 antibody or the anti-ENTPD 2 antibody (or both) is two weeks.

In an embodiment of the antibody used according to the invention or the method according to the invention, in this initial phase the sum of the divided doses administered over a period equal to the main dosing period is equal to the main dose. In an embodiment, these initial stage doses are administered first in a lower amount than the main stage dose, and then in an intermediate amount that alters the main stage dose. The lower amount is lower than the main stage dose and the intermediate dose is a value between these two amounts. In an embodiment, in the initial phase, the divided doses are about 200mg and about 400mg, and the divided doses are administered within two weeks. In another embodiment, in the initial phase, the divided doses are about 100mg and about 500mg, and the divided doses are administered within two weeks. In an alternative embodiment, in the initial phase, the sum of the divided doses administered over a period equal to the main dosing period is equal to the main dose, and wherein the divided doses are equal amounts, e.g. about 300mg and about 300mg. In these embodiments, these amounts refer to the amount of anti-CD 73 antibody.

For example, an antibody molecule that binds human CD73 may be administered as follows: in the initial phase, about 200mg was administered on day 1, about 400mg was administered on day 8, then about 600mg was administered at the beginning of the main phase on day 15, after which about 600mg was administered with Q2W continued.

In some embodiments, an antibody molecule that binds human ENTPD2 may be administered as follows: in the initial phase, about 100mg was administered on day 1, then about 300mg was administered at the beginning of the main phase on day 15, after which Q2W administration was continued for about 300mg.

In some embodiments, an antibody molecule that binds human ENTPD2 may be administered as follows: in the initial phase, about 100mg was administered on day 1, about 100mg was administered on day 8, then about 300mg was administered at the beginning of the main phase on day 15, after which about 300mg was administered with Q2W continued.

In one embodiment, an anti-CD 73 antibody molecule or an anti-ENTPD 2 antibody molecule disclosed herein is administered, for example, by infusion over a period of 30 minutes, a period of 1 hour, or a period of up to 2 hours. In one embodiment, an anti-CD 73 antibody molecule or an anti-ENTPD 2 antibody molecule disclosed herein is administered, for example, by infusion over a period of 1 to 2 hours.

Pharmaceutical compositions useful in the dosage regimen of the invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody molecule useful in the dosage regimen of the invention. "therapeutically effective amount" means an amount effective to achieve the desired therapeutic result at the necessary dosage and for the necessary period of time. The therapeutically effective amount of the modified antibody or antibody fragment may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also an amount of an antibody molecule that has a therapeutic benefit that exceeds any toxic or detrimental effect. The "therapeutically effective dose" preferably inhibits a measurable parameter, such as inhibiting by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to an untreated subject. The measurable parameter may be, for example, tumor growth rate or pathogen growth rate. The ability of antibody molecules to inhibit a measurable parameter can be assessed in an animal model system that predicts efficacy in a corresponding human disease. Alternatively, such properties of the composition may be assessed by examining the ability of the compound to inhibit, such inhibition being performed in vitro by assays known to those of skill in the art.

"prophylactically effective amount" refers to an amount effective in dosimetry and for the period of time required to achieve the desired prophylactic result. Typically, such prophylactically effective amounts will be less than therapeutically effective amounts because the prophylactic dose is administered in the subject prior to or early in the disease.

Also described herein are kits comprising the antibody molecules described herein. The kit may include one or more other elements, including: instructions for use; other agents, such as labels, therapeutic agents, or agents for chelating or otherwise coupling antibodies to labels or therapeutic agents or radioprotective compositions; a device or other material for preparing antibody molecules for administration; a pharmaceutically acceptable carrier; and devices or other materials for administration to a subject.

Therapeutic use

As used herein, the term "subject" is intended to include both human and non-human animals. In one embodiment, the subject is a human subject, e.g., a human patient suffering from a disease or disorder characterized by CD73 or ENTPD2 dysfunction. The term "non-human animal" includes mammals and non-mammals, such as non-human primates. In one embodiment, the subject is a human. In one embodiment, the subject is a human patient in need of an enhanced immune response. In one embodiment, the subject is immunocompromised, e.g., the subject is undergoing or has undergone chemotherapy or radiation therapy. Alternatively or in combination, the subject is immunocompromised or at risk of being immunocompromised due to infection. The methods and compositions described herein are useful for treating human patients suffering from disorders treatable by enhancing T cell mediated immune responses. For example, the methods and compositions described herein can enhance a variety of immune activities. In one embodiment, the number or activity of tumor-infiltrating T lymphocytes (TILs) of the subject is increased.

Cancer of the human body

In one embodiment, the dosage regimen according to the invention involves treating a subject in vivo with an anti-CD 73 antibody molecule or an anti-ENTPD 2 antibody molecule such that the growth of a cancerous tumor is inhibited or reduced. anti-CD 73 antibodies alone may be used to inhibit the growth of cancerous tumors. Alternatively, an anti-CD 73 antibody molecule or an anti-ENTPD 2 antibody may be used in combination with one or more of the following: standard of care treatment (e.g., against cancer), another antibody molecule, an immunomodulatory agent (e.g., an activator of a co-stimulatory molecule or an inhibitor of a co-inhibitory molecule); vaccines, such as therapeutic cancer vaccines; or other forms of cell therapy as described below.

Thus, in one embodiment, a dosage regimen according to the invention provides a method of inhibiting tumor cell growth in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-CD 73 antibody molecule or an anti-ENTPD 2 antibody molecule described herein according to a dosage regimen as described herein.

In one embodiment, these methods are applicable to in vivo cancer treatment. When an anti-CD 73 antibody molecule or an anti-ENTPD 2 antibody is administered in combination with one or more agents, the combination may be administered in any order or simultaneously according to a dosage regimen as described herein.

Type of cancer

In another aspect of the dosage regimen according to the invention, methods of treating a subject, e.g., reducing or ameliorating a hyperproliferative condition or disorder (e.g., cancer), e.g., a solid tumor, hematological cancer, soft tissue tumor, or metastatic lesion, in a subject are provided. The method comprises administering to the subject one or more of the anti-CD 73 antibody molecules or anti-ENTPD 2 antibody molecules described herein, alone or in combination with other agents or therapeutic modalities.

As used herein, the term "cancer" is meant to include all types of cancerous growth or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of the histopathological type or stage of invasion. Examples of cancerous disorders include, but are not limited to, solid tumors, hematological cancers, soft tissue tumors, and metastatic lesions. Examples of solid tumors include malignant tumors of various organ systems, such as those affecting the liver, lung, breast, lymph, gastrointestinal (e.g., colon), genitourinary tract (e.g., renal cells, urothelial cells), prostate and pharynx, for example sarcomas and carcinomas (including adenocarcinomas and squamous cell carcinomas). Adenocarcinomas include malignant tumors such as most colon, rectal, renal cell, liver, non-small cell lung, small intestine and esophagus cancers. Squamous cell carcinoma includes, for example, malignant tumors in the lung, esophagus, skin, head and neck area, mouth, anus, and cervix. In one embodiment, the cancer is melanoma, such as advanced melanoma. The methods and compositions of the dosage regimen of the invention can also be used to treat or prevent metastatic lesions of the aforementioned cancers.

Exemplary cancers for which growth may be inhibited using a dosage regimen according to the invention include cancers that typically respond to immunotherapy. Non-limiting examples of preferred cancers to be treated include melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate cancer), breast cancer, colon cancer, and lung cancer (e.g., non-small cell lung cancer). In an embodiment of the dosage regimen of the invention, the cancer to be treated is non-small cell lung cancer, pancreatic ductal adenocarcinoma, triple negative breast cancer, microsatellite stabilized (MSS) colorectal cancer, metastatic castration-resistant prostate cancer, ovarian cancer or renal cell carcinoma. In addition, refractory or recurrent malignant tumors can be treated using the antibody molecules described herein. Metastatic castration-resistant prostate cancer is also known as hormone refractory prostate cancer or non-androgen dependent prostate cancer.

Examples of other cancers that may be treated include bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastroesophageal cancer, gastric cancer (stomach cancer), testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, merck (Merkel) cell cancer, hodgkin's lymphoma, non-hodgkin's lymphoma, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urinary tract cancer, penile cancer, chronic or acute leukemia (including acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), childhood solid tumors, lymphocytic lymphomas, bladder cancer, multiple myeloma, myelodysplastic syndrome, renal or ureteral cancer, renal pelvis cancer, central nervous system tumors (CNS), primary CNS lymphomas, tumor angiogenesis, spinal cord axis tumors, stem gliomas, pituitary adenomas, karst, epidermoid sarcoma, sarcomas, cancer, T-cell-induced cancers (including squamous cell carcinoma, cancer such as those of the environment), and combinations thereof.

In some embodiments, the therapies herein can be used to treat patients suffering from (or identified as suffering from) cancer associated with an infection (e.g., a viral or bacterial infection). Exemplary cancers include cervical cancer, anal cancer, HPV-associated head and neck squamous cell carcinoma, HPV-associated esophageal papilloma, HHV 6-associated lymphoma, EBV-associated lymphoma (including burkitt's lymphoma), gastric MALT lymphoma, other infection-associated MALT lymphoma, HCC, and kaposi's sarcoma.

In other embodiments, the cancer is a hematological malignancy or cancer, including but not limited to leukemia or lymphoma. For example, anti-CD 73 antibody molecules or anti-ENTPD 2 antibody molecules may be used to treat cancers and malignancies, including but not limited to, e.g., acute leukemia, including but not limited to, e.g., B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("tal"), acute Lymphoblastic Leukemia (ALL); one or more chronic leukemias including, but not limited to, e.g., chronic Myelogenous Leukemia (CML), chronic Lymphocytic Leukemia (CLL); additional hematological cancers or hematological disorders include, but are not limited to, for example, B cell prolymphocytic leukemia, a blast plasmacytoid dendritic cell tumor, burkitt lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic and myelodysplastic syndrome, non-hodgkin's lymphoma, plasmablastoid lymphoma, plasmacytoid dendritic cell tumor, waldenstrom's macroglobulinemia, and "pre-leukemia" which is a collection of various hematological disorders that are linked together by the ineffective production (or dysplasia) of myeloblasts, and the like.

In one embodiment, the cancer is selected from lung cancer (e.g., non-small cell lung cancer (NSCLC) (e.g., NSCLC with squamous and/or non-squamous histology, or NSCLC adenocarcinoma)), melanoma (e.g., advanced melanoma), renal cancer (e.g., renal cell carcinoma such as clear cell renal cell carcinoma), liver cancer, myeloma (e.g., multiple myeloma), prostate cancer, breast cancer (e.g., breast cancer that does not express one, two, or all of estrogen receptor, progesterone receptor, or Her 2/neu), such as triple negative breast cancer), colorectal cancer (e.g., microsatellite stabilized (MSS) colorectal cancer), ovarian cancer), cholangiocarcinoma (intrahepatic or extrahepatic), pancreatic cancer, head and neck cancer (e.g., head and Neck Squamous Cell Carcinoma (HNSCC)), anal cancer, gastroesophageal cancer, thyroid cancer, cervical cancer, lymphoproliferative disease (e.g., post-transplant lymphoproliferative disease) or blood cancer, T-cell lymphoma, non-hodgkin lymphoma, or leukemia (e.g., myelogenous leukemia).

In one embodiment, the cancer is selected from lung cancer (e.g., non-small cell lung cancer), pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), breast cancer (e.g., triple negative breast cancer), melanoma, head and neck cancer (e.g., squamous head and neck cancer), colorectal cancer (e.g., microsatellite stabilized (MSS) colorectal cancer), ovarian cancer, metastatic castration-resistant prostate cancer, or renal cancer (e.g., renal cell carcinoma).

In one embodiment, the cancer is selected from bladder cancer, leukemia, lymphoma, glioma, glioblastoma, ovarian cancer, thyroid cancer, esophageal cancer, prostate cancer, uterine/cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, colon cancer, renal cancer, gastric cancer, germ cell cancer, bone cancer, liver cancer, skin cancer, central nervous system tumor, myeloma, sarcoma, and virus-associated cancer.

In one embodiment, the cancer is selected from colon cancer (e.g., colorectal cancer (CRC) or colorectal adenocarcinoma), gastric cancer (e.g., gastric adenocarcinoma, gastric cancer (gastric carcinoma)), esophageal cancer (e.g., esophageal Squamous Cell Carcinoma (ESCC), esophageal Gastric Junction (EGJ) cancer), lung cancer (e.g., small cell lung cancer), breast cancer (e.g., breast adenocarcinoma), or ovarian cancer.

In one embodiment, the cancer is colorectal cancer (CRC) or colorectal adenocarcinoma. In another embodiment, the cancer is MSS colorectal cancer (CRC).

In another embodiment, the cancer is esophageal cancer.

In further embodiments, the cancer is gastric cancer.

In yet another embodiment, the cancer is Esophageal Gastric Junction (EGJ) cancer.

In yet another embodiment, the cancer is Esophageal Squamous Cell Carcinoma (ESCC).

In further embodiments, the cancer is cholangiocarcinoma. Bile duct cancer may be intrahepatic or extrahepatic.

In another embodiment, the cancer is pancreatic cancer.

Combinations of anti-CD 73 antibody molecules and/or anti-ENTPD 2 antibody molecules

In a dosage regimen according to the invention, an anti-CD 73 antibody molecule or an anti-ENTPD 2 antibody molecule may be used in combination with other therapies and/or with each other. For example, combination therapies may include a composition comprising an anti-CD 73 antibody molecule or an anti-ENTPD 2 antibody as described herein co-formulated and/or co-administered with one or more additional therapeutic agents (e.g., one or more anticancer agents, cytotoxic or cytostatic agents, hormonal therapy, vaccines and/or other immunotherapies). In some embodiments, an anti-CD 73 antibody molecule or an anti-ENTPD 2 antibody molecule as described herein is co-formulated and/or co-administered with each other. In other embodiments, the anti-CD 73 antibody molecule or the anti-ENTPD 2 antibody molecule is administered in combination with other therapeutic treatment modalities (including surgery, radiation, cryosurgery, and/or hyperthermia). Such combination therapies may advantageously use lower doses of the administered therapeutic agent, thereby avoiding the possible toxicity or complications associated with various monotherapy.

"in combination with … …" is not intended to imply that the therapy or therapeutic agent must be administered simultaneously and/or formulated for delivery together, although such delivery methods are also within the scope described herein. The anti-CD 73 antibody molecule or anti-ENTPD 2 antibody molecule may be administered concurrently with, or before or after, one or more other additional therapies or therapeutic agents. The anti-CD 73 antibody molecule or anti-ENTPD 2 antibody molecule and other agents or treatment regimens may be administered in any order. Typically, each agent will be administered at a dosage and/or schedule determined for that agent. It will be further understood that the additional therapeutic agents used in the combination may be administered together in a single composition or separately in different compositions. In general, it is contemplated that the additional therapeutic agents used in the combination are used at levels not exceeding those used alone. In some embodiments, the level used in the combination will be lower than the level used alone.

Exemplary adenosine A2A receptor antagonists

In certain embodiments of dosage regimens according to the invention, an anti-CD 73 antibody molecule and/or an anti-ENTPD 2 antibody molecule described herein is administered in combination with an adenosine A2A receptor (A2 AR) antagonist. Exemplary A2AR antagonists include, for example, PBF509 (Palobioframa/Nohua Co., ltd. (also referred to as NIR 178), CPI444/V81444 (Kawok/Genencom (Corvus/Genntech)), AZD4635/HTL-1071 (Aspirinotecan/Haplotai Co., astrazeneca/Heptares)), vepatadian (Redox/Juno Co.), GBV-2034 (Globavir Co.), AB928 (Axwell biosciences (Arcus Biosciences)), theophylline, itracine (Kyowa Co., ltd. (Kyowa Hakko Kogyo)), tozadan/SYN-115 (Acorda Co., ltd.), KW-6356 (KW-6356), mardan-6 (Padan/Schradem (Sch37 Xie Lin/SCH), and Primant (Scheark/42023).

In certain embodiments, the A2AR antagonist is PBF509. The PBF509 is also referred to as NIR178.PBF509 and other A2AR antagonists are disclosed in US 8,796,284 and WO 2017/025918, which applications are incorporated herein by reference in their entirety. PBF509 refers to 5-bromo-2, 6-di- (1H-pyrazol-1-yl) pyrimidin-4-amine having the structure:

in certain embodiments, the A2AR antagonist is CPI444/V81444.CPI-444 and other A2AR antagonists are disclosed in WO 2009/156737, which is incorporated herein by reference in its entirety. In certain embodiments, the A2AR antagonist is (S) -7- (5-methylfuran-2-yl) -3- ((6- (((tetrahydrofuran-3-yl) oxy) methyl) pyridin-2-yl) methyl) -3H- [1,2,3] triazolo [4,5-d ] pyrimidin-5-amine. In certain embodiments, the A2AR antagonist is (R) -7- (5-methylfuran-2-yl) -3- ((6- (((tetrahydrofuran-3-yl) oxy) methyl) pyridin-2-yl) methyl) -3H- [1,2,3] triazolo [4,5-d ] pyrimidin-5-amine, or a racemate thereof. In certain embodiments, the A2AR antagonist is 7- (5-methylfuran-2-yl) -3- ((6- (((tetrahydrofuran-3-yl) oxy) methyl) pyridin-2-yl) methyl) -3H- [1,2,3] triazolo [4,5-d ] pyrimidin-5-amine. In certain embodiments, the A2AR antagonist has the following structure:

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In certain embodiments, the A2AR antagonist is AZD 4635/HTL-1071. A2AR antagonists are disclosed in WO 2011/095625, which is incorporated herein by reference in its entirety. In certain embodiments, the A2AR antagonist is 6- (2-chloro-6-methylpyridin-4-yl) -5- (4-fluorophenyl) -1,2, 4-triazin-3-amine. In certain embodiments, the A2AR antagonist has the following structure:

in certain embodiments, the A2AR antagonist is ST-4206 (Lidean biosciences). In certain embodiments, the A2AR antagonist is an A2AR antagonist described in US 9,133,197, which is incorporated herein by reference in its entirety. In certain embodiments, the A2AR antagonist has the following structure:

in certain embodiments, the A2AR antagonist is an A2AR antagonist described in US 8114845, US 9029393, US20170015758, or US20160129108, which are incorporated herein by reference in their entirety.

In certain embodiments, the A2AR antagonist is itrafylline (CAS registry number 155270-99-8). Itratheophylline is also known as KW-6002 or 8- [ (E) -2- (3, 4-dimethoxyphenyl) vinyl ] -1, 3-diethyl-7-methyl-3, 7-dihydro-1H-purine-2, 6-dione. For example, itratheophylline is disclosed in LeWitt et al, (2008) Annals of Neurology [ neurological yearbook ]63 (3): 295-302.

In certain embodiments, the A2aR antagonist is tolzadi nan (Biotie). Tolzadir is also known as SYN115 or 4-hydroxy-N- (4-methoxy-7-morpholin-4-yl-1, 3-benzothiazol-2-yl) -4-methylpiperidine-1-carboxamide. Tolzadi's blockade of endogenous adenosine at the A2a receptor results in enhanced dopamine effect at the D2 receptor and inhibition of glutamate at the mGluR5 receptor. In some embodiments, the A2aR antagonist is Pryidi NAN (CAS registry number 377727-87-2). Pralidoxime is also known as SCH 420814 or 2- (2-furyl) -7- [2- [4- [4- (2-methoxyethoxy) phenyl ] -1-piperazinyl ] ethyl ] 7H-pyrazolo [4,3-e ] [1,2,4] triazolo [1,5-c ] pyrimidin-5-amine. Pryidi's south was developed as a drug that acts as a potent and selective antagonist of the adenosine A2A receptor.

In certain embodiments, the A2aR antagonist is verapamil. Wipatadine is also known as BIIB014, V2006, or 3- [ (4-amino-3-methylphenyl) methyl ] -7- (furan-2-yl) triazolo [4,5-d ] pyrimidin-5-amine.

Other exemplary A2aR antagonists include, for example, ATL-444, MSX-3, SCH-58261, SCH-412,348, SCH-442,416, VER-6623, VER-6947, VER-7835, CGS-15943, or ZM-241,385.

Exemplary PD-1 inhibitors

In certain embodiments of the dosage regimen of the invention, the anti-CD 73 antibody molecules and/or anti-ENTPD 2 antibody molecules described herein are administered in combination with a PD-1 inhibitor. The PD-1 inhibitor may be an antibody, antigen-binding fragment thereof, immunoadhesin, fusion protein, or oligopeptide. In some embodiments, the PD-1 inhibitor is selected from Stadalimumab (PDR 001) (Norhua Co., ltd.), nawuzumab (Bristol-Myers Squibb), pembrolizumab (merck), pilimumab (Curetech), MEDI0680 (England Mei Dimiao Si (Medimmune)), REGN2810 (regenerator), TSR-042 (Tasal Luo Gongsi (Tesaro)), PF-06801591 (Pfizer), tiarelizumab (B-A317, beigene), BGB-108 (Beigene), INCSHR1210 (Saint Co (Incyte)), or AMP-224 (An Puli).

Exemplary anti-PD-1 antibody molecules

In one embodiment of the dosage regimen of the invention, the PD-1 inhibitor is an anti-PD-1 antibody molecule. In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody molecule, as described in US2015/0210769 (incorporated by reference in its entirety) published at 30, 7, 2015, titled "antibody molecule of Antibody Molecules to PD-1and Uses Thereof[PD-1 and uses thereof".

In one embodiment, the anti-PD-1 antibody molecule comprises at least one, two, three, four, five, or six Complementarity Determining Regions (CDRs) (or generally all CDRs) from a heavy and light chain variable region comprising an amino acid sequence set forth in table 5 (e.g., a heavy and light chain variable region sequence from BAP 049-clone-E or BAP 049-clone-B disclosed in table 5), or encoded by a nucleotide sequence set forth in table 5. In some embodiments, the CDRs are according to the cabazite definition (e.g., as set forth in table 5). In some embodiments, CDRs are defined according to Qiao Xiya (e.g., as listed in table 5). In some embodiments, the CDRs are defined according to the combined CDRs of both cabazite and Qiao Xiya (e.g., as listed in table 5). In one embodiment, the combination of the carboplatin and Qiao Xiya CDRs of VH CDR1 comprises the amino acid sequence GYTFTTYWMH (SEQ ID NO: 541). In one embodiment, one or more of the CDRs (or generally all CDRs) have one, two, three, four, five, six or more changes, such as amino acid substitutions (e.g., conservative amino acid substitutions) or deletions, relative to the amino acid sequences shown in table 5, or the amino acid sequences encoded by the nucleotide sequences shown in table 5.

In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain variable region (VH) comprising the VH CDR1 amino acid sequence of SEQ ID NO. 501, the VH CDR2 amino acid sequence of SEQ ID NO. 502 and the VH CDR3 amino acid sequence of SEQ ID NO. 503; and a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO:510, the VLCDR2 amino acid sequence of SEQ ID NO:511 and the VLCDR3 amino acid sequence of SEQ ID NO:512, each as disclosed in Table 5.

In one embodiment, the antibody molecule comprises a VH comprising a VHCDR1 encoded by the nucleotide sequence of SEQ ID No. 524, a VHCDR2 encoded by the nucleotide sequence of SEQ ID No. 525, and a VHCDR3 encoded by the nucleotide sequence of SEQ ID No. 526; and a VL comprising a VLCDR1 encoded by the nucleotide sequence of SEQ ID NO. 529, a VLCDR2 encoded by the nucleotide sequence of SEQ ID NO. 530, and a VLCDR3 encoded by the nucleotide sequence of SEQ ID NO. 531, each as disclosed in Table 5.

In one embodiment, the anti-PD-1 antibody molecule comprises: a VH comprising the amino acid sequence of SEQ ID No. 506, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% or more sequence identity to SEQ ID No. 506. In one embodiment, the anti-PD-1 antibody molecule comprises: VL comprising the amino acid sequence of SEQ ID No. 520, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% or more sequence identity to SEQ ID No. 520. In one embodiment, the anti-PD-1 antibody molecule comprises: a VL comprising the amino acid sequence of SEQ ID No. 516, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% or more sequence identity to SEQ ID No. 516. In one embodiment, the anti-PD-1 antibody molecule comprises: VH comprising the amino acid sequence of SEQ ID No. 506 and VL comprising the amino acid sequence of SEQ ID No. 520. In one embodiment, the anti-PD-1 antibody molecule comprises: VH comprising the amino acid sequence of SEQ ID No. 506 and VL comprising the amino acid sequence of SEQ ID No. 516.

In one embodiment, the antibody molecule comprises: a VH encoded by the nucleotide sequence of SEQ ID No. 507, or a nucleotide sequence having at least about 85%, 90%, 95%, or 99%, or more, sequence identity to SEQ ID No. 507. In one embodiment, the antibody molecule comprises: VL encoded by the nucleotide sequence of SEQ ID No. 521 or 517, or a nucleotide sequence having at least about 85%, 90%, 95%, or 99% or more sequence identity to SEQ ID No. 521 or 517. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO:507 and a VL encoded by the nucleotide sequence of SEQ ID NO:521 or 517.

In one embodiment, the anti-PD-1 antibody molecule comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO. 508, or an amino acid sequence having at least about 85%, 90%, 95%, or 99%, or more, sequence identity to SEQ ID NO. 508. In one embodiment, the anti-PD-1 antibody molecule comprises: a light chain comprising the amino acid sequence of SEQ ID NO. 522, or an amino acid sequence having at least about 85%, 90%, 95%, or 99%, or more, sequence identity to SEQ ID NO. 522. In one embodiment, the anti-PD-1 antibody molecule comprises: a light chain comprising the amino acid sequence of SEQ ID NO. 518, or an amino acid sequence having at least about 85%, 90%, 95%, or 99%, or more, sequence identity to SEQ ID NO. 518. In one embodiment, the anti-PD-1 antibody molecule comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO. 508 and a light chain comprising the amino acid sequence of SEQ ID NO. 522. In one embodiment, the anti-PD-1 antibody molecule comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO. 508 and a light chain comprising the amino acid sequence of SEQ ID NO. 518.

In one embodiment, the antibody molecule comprises: heavy chains encoded by the nucleotide sequence of SEQ ID NO. 509, or a nucleotide sequence having at least about 85%, 90%, 95%, or 99%, or more, sequence identity to SEQ ID NO. 509. In one embodiment, the antibody molecule comprises: a light chain encoded by the nucleotide sequence of SEQ ID NO. 523 or 519, or a nucleotide sequence having at least about 85%, 90%, 95%, or 99% or more sequence identity to SEQ ID NO. 523 or 519. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO. 509 and a light chain encoded by the nucleotide sequence of SEQ ID NO. 523 or 519.

The antibody molecules described herein may be made by vectors, host cells, and methods described in US 2015/0210769 (which is incorporated by reference in its entirety).

TABLE 5 amino acid and nucleotide sequences of exemplary anti-PD-1 antibody molecules

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Other exemplary PD-1 inhibitors

In one embodiment, the anti-PD-1 antibody molecule is Nawuzumab (BASEMERIUM Guibao), also known as MDX-1106, MDX-1106-04, ONO-4538, BMS-936558, orNivolumab (clone 5C 4) and other anti-PD-1 antibodies are disclosed in US 8,008,449 and WO 2006/121168, which are incorporated by reference in their entirety. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the following: the CDR sequences (or generally all CDR sequences) of nivolumab, the heavy or light chain variable region sequences, or the heavy or light chain sequences, e.g., as disclosed in table 6.

In one embodiment, the anti-PD-1 antibody molecule is pembrolizumab (Merck, inc&Co)), also known as Lanrolizumab (Lambrolizumab), MK-3475, MK03475, SCH-900475, orPembrolizumab and other anti-PD-1 antibodies are disclosed in Hamid, o.et al (2013) New England Journal of Medicine [ journal of new england medicine ]]369 (2) 134-44, US 8,354,509 and WO 2009/114335, which are incorporated by reference in their entirety. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the following: the CDR sequences (or generally all CDR sequences) of pembrolizumab, the heavy or light chain variable region sequences, or the heavy or light chain sequences, e.g., as disclosed in table 6.

In one embodiment, the anti-PD-1 antibody molecule is Pidazumab (therapeutic technology Co.), also known as CT-011. Pittuzumab and other anti-PD-1 antibodies are disclosed in Rosenblatt, J.et al, (2011) J Immunotherapy [ J.Immunotherapy ]34 (5): 409-18; US 7,695,715; US 7,332,582; and US 8,686,119, which are incorporated by reference in their entirety. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the following: the CDR sequences (or generally all CDR sequences) of pilzumab, the heavy or light chain variable region sequences, or the heavy or light chain sequences, e.g., as disclosed in table 6.

In one embodiment, the anti-PD-1 antibody molecule is MEDI0680 (England Mei Dimiao, inc.), also known as AMP-514.MEDI0680 and other anti-PD-1 antibodies are disclosed in US 9,205,148 and WO 2012/145493 (which are incorporated by reference in their entirety). In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the following: the CDR sequence (or overall all CDR sequences) of MEDI0680, the heavy or light chain variable region sequence, or the heavy or light chain sequence.

In one embodiment, the anti-PD-1 antibody molecule is REGN2810 (regenerator corporation). In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the following: the CDR sequence (or overall all CDR sequences) of REGN2810, the heavy or light chain variable region sequence, or the heavy or light chain sequence.

In one embodiment, the anti-PD-1 antibody molecule is PF-06801591 (pyro). In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the following: the CDR sequence (or generally all CDR sequences) of PF-06801591, the heavy or light chain variable region sequence, or the heavy or light chain sequence.

In one embodiment, the anti-PD-1 antibody molecule is tirelimumab (BGB-A317) or BGB-108 (BAIJISHENZHI). In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the following: the CDR sequences (or generally all CDR sequences) of tirelimumab (BGB-a 317) or BGB-108, the heavy or light chain variable region sequences, or the heavy or light chain sequences.

In one embodiment, the anti-PD-1 antibody molecule is INCSHR1210 (Saint Co.), also known as INCSHR01210 or SHR-1210. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the following: the CDR sequence (or generally all CDR sequences) of incsshr 1210, the heavy or light chain variable region sequence, or the heavy or light chain sequence.

In one embodiment, the anti-PD-1 antibody molecule is TSR-042 (Tassa Luo Gongsi), also known as ANB011. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the following: the CDR sequence (or generally all CDR sequences) of TSR-042, the heavy or light chain variable region sequence, or the heavy or light chain sequence.

Other known anti-PD-1 antibodies include those described, for example, in the following: WO 2015/112800, WO 2016/092419, WO 2015/085847, WO 2014/179664, WO 2014/194302, WO 2014/209404, WO 2015/200119, US 8,735,553, US 7,488,802, US 8,927,697, US 8,993,731, and US 9,102,727, which are incorporated by reference in their entirety.

In one embodiment, the anti-PD-1 antibody is an antibody that competes with one of the anti-PD-1 antibodies described herein for binding to the same epitope on PD-1 and/or binds to the same epitope on PD-1.

In one embodiment, the PD-1 inhibitor is a peptide that inhibits the PD-1 signaling pathway, e.g., as described in US 8,907,053, which is incorporated by reference in its entirety. In one embodiment, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence), hi one embodiment, the PD-1 inhibitor is AMP-224 (B7-DCIg (An Puli, inc.), e.g., as disclosed in WO 2010/027827 and WO 2011/066342, which are incorporated by reference in their entirety).

TABLE 6 amino acid sequences of other exemplary anti-PD-1 antibody molecules

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Exemplary PD-L1 inhibitors

In certain embodiments of the dosage regimen of the invention, the anti-CD 73 antibody molecules and/or anti-ENTPD 2 antibody molecules described herein are administered in combination with a PD-L1 inhibitor. The PD-L1 inhibitor may be an antibody, antigen-binding fragment thereof, immunoadhesin, fusion protein, or oligopeptide. In some embodiments, the PD-L1 inhibitor is selected from FAZ053 (nohua corporation), alemtuzumab (genetec/Roche), avistuzumab (Merck Serono and pyroxene), divaruzumab (imperforate Mei Dimiao/aslican), or BMS-936559 (bai meishi precious).

Exemplary anti-PD-L1 antibody molecules

In one embodiment of the dosage regimen of the invention, the PD-L1 inhibitor is an anti-PD-L1 antibody molecule. In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody molecule, as disclosed in U.S. Pat. No. 2016/0108123 (incorporated by reference in its entirety) published at 21/2016 entitled "Antibody Molecules to PD-L1 and Users Theeoof [ antibody molecule to PD-L1 and Uses Thereof ]".

In one embodiment, the anti-PD-L1 antibody molecule comprises at least one, two, three, four, five, or six Complementarity Determining Regions (CDRs) (or generally all CDRs) from a heavy and light chain variable region comprising an amino acid sequence set forth in table 7 (e.g., heavy and light chain variable region sequences from BAP 058-clone O or BAP 058-clone N disclosed in table 7), or encoded by a nucleotide sequence set forth in table 7. In some embodiments, the CDRs are according to the cabazite definition (e.g., as set forth in table 7). In some embodiments, CDRs are defined according to Qiao Xiya (e.g., as listed in table 7). In some embodiments, the CDRs are defined according to the combined CDRs of both cabazite and Qiao Xiya (e.g., as listed in table 7). In one embodiment, the combination of the carboplatin and Qiao Xiya CDRs of VH CDR1 comprises amino acid sequence GYTFTSYWMY (SEQ ID NO: 647). In one embodiment, one or more of the CDRs (or generally all CDRs) have one, two, three, four, five, six or more changes, such as amino acid substitutions (e.g., conservative amino acid substitutions) or deletions, relative to the amino acid sequences shown in table 7, or the amino acid sequences encoded by the nucleotide sequences shown in table 7.

In one embodiment, the anti-PD-L1 antibody molecule comprises a heavy chain variable region (VH) comprising the VH CDR1 amino acid sequence of SEQ ID NO. 601, the VH CDR2 amino acid sequence of SEQ ID NO. 602, and the VH CDR3 amino acid sequence of SEQ ID NO. 603; and a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO. 609, the VLCDR2 amino acid sequence of SEQ ID NO. 610, and the VLCDR3 amino acid sequence of SEQ ID NO. 611, each as disclosed in Table 7.

In one embodiment, the anti-PD-L1 antibody molecule comprises a VH comprising a VHCDR1 encoded by the nucleotide sequence of SEQ ID NO. 628, a VHCDR2 encoded by the nucleotide sequence of SEQ ID NO. 629, and a VHCDR3 encoded by the nucleotide sequence of SEQ ID NO. 630; and a VL comprising a VLCDR1 encoded by the nucleotide sequence of SEQ ID NO. 633, a VLCDR2 encoded by the nucleotide sequence of SEQ ID NO. 634, and a VLCDR3 encoded by the nucleotide sequence of SEQ ID NO. 635, each as disclosed in Table 7.

In one embodiment, the anti-PD-L1 antibody molecule comprises: a VH comprising the amino acid sequence of SEQ ID No. 606, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID No. 606. In one embodiment, the anti-PD-L1 antibody molecule comprises: VL comprising the amino acid sequence of SEQ ID NO. 616 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% or more sequence identity to SEQ ID NO. 616. In one embodiment, the anti-PD-L1 antibody molecule comprises: a VH comprising the amino acid sequence of SEQ ID No. 620, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID No. 620. In one embodiment, the anti-PD-L1 antibody molecule comprises: VL comprising the amino acid sequence of SEQ ID No. 624, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID No. 624. In one embodiment, the anti-PD-L1 antibody molecule comprises: VH comprising the amino acid sequence of SEQ ID No. 606 and VL comprising the amino acid sequence of SEQ ID No. 616. In one embodiment, the anti-PD-L1 antibody molecule comprises: VH comprising the amino acid sequence of SEQ ID No. 620 and VL comprising the amino acid sequence of SEQ ID No. 624.

In one embodiment, the antibody molecule comprises: VH encoded by the nucleotide sequence of SEQ ID No. 607, or a nucleotide sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID No. 607. In one embodiment, the antibody molecule comprises: VL encoded by the nucleotide sequence of SEQ ID No. 617, or a nucleotide sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID No. 617. In one embodiment, the antibody molecule comprises: a VH encoded by the nucleotide sequence of SEQ ID No. 621, or a nucleotide sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID No. 621. In one embodiment, the antibody molecule comprises: VL encoded by the nucleotide sequence of SEQ ID No. 625, or a nucleotide sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID No. 625. In one embodiment, the antibody molecule comprises: a VH encoded by the nucleotide sequence of SEQ ID NO:607 and a VL encoded by the nucleotide sequence of SEQ ID NO: 617. In one embodiment, the antibody molecule comprises: a VH encoded by the nucleotide sequence of SEQ ID NO:621 and a VL encoded by the nucleotide sequence of SEQ ID NO: 625.

In one embodiment, the anti-PD-L1 antibody molecule comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO. 608, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO. 608. In one embodiment, the anti-PD-L1 antibody molecule comprises: a light chain comprising the amino acid sequence of SEQ ID NO. 618, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO. 618. In one embodiment, the anti-PD-L1 antibody molecule comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO. 622, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO. 622. In one embodiment, the anti-PD-L1 antibody molecule comprises: a light chain comprising the amino acid sequence of SEQ ID NO. 626, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO. 626. In one embodiment, the anti-PD-L1 antibody molecule comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO. 608 and a light chain comprising the amino acid sequence of SEQ ID NO. 618. In one embodiment, the anti-PD-L1 antibody molecule comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO. 622 and a light chain comprising the amino acid sequence of SEQ ID NO. 626.

In one embodiment, the antibody molecule comprises: a heavy chain encoded by the nucleotide sequence of SEQ ID NO. 615, or a nucleotide sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO. 615. In one embodiment, the antibody molecule comprises: a light chain encoded by the nucleotide sequence of SEQ ID NO. 619, or a nucleotide sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO. 619. In one embodiment, the antibody molecule comprises: heavy chain encoded by the nucleotide sequence of SEQ ID NO:623, or a nucleotide sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 623. In one embodiment, the antibody molecule comprises: a light chain encoded by the nucleotide sequence of SEQ ID NO. 627, or a nucleotide sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO. 627. In one embodiment, the antibody molecule comprises: a heavy chain encoded by the nucleotide sequence of SEQ ID NO. 615 and a light chain encoded by the nucleotide sequence of SEQ ID NO. 619. In one embodiment, the antibody molecule comprises: a heavy chain encoded by the nucleotide sequence of SEQ ID NO. 623 and a light chain encoded by the nucleotide sequence of SEQ ID NO. 627.

The antibody molecules described herein may be made by vectors, host cells, and methods described in US 2016/0108123 (which is incorporated by reference in its entirety).

TABLE 7 amino acid and nucleotide sequences of exemplary anti-PD-L1 antibody molecules

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Other exemplary PD-L1 inhibitors

In one embodiment, the anti-PD-L1 antibody molecule is alemtuzumab (GeneTek/Roche), also known as MPDL3280A, RG7446, RO5541267, YW243.55.S70, or TECENTRIQ ^TM . Alet Li Zhushan anti-and other anti-PD-L1 antibodies are disclosed in US 8,217,149, which antibodies are incorporated by reference in their entirety. In one embodiment, the anti-PD-L1 antibody molecule comprises one or more of the following: the CDR sequences (or generally all CDR sequences) of alemtuzumab, the heavy or light chain variable region sequences, or the heavy or light chain sequences, e.g., as disclosed in table 8.

In one embodiment, the anti-PD-L1 antibody molecule is avermectin (mercaptitude and febrile), also known as MSB0010718C. Avstuzumab and other anti-PD-L1 antibodies are disclosed in WO 2013/079174 (which is incorporated by reference in its entirety). In one embodiment, the anti-PD-L1 antibody molecule comprises one or more of the following: the CDR sequences (or generally all CDR sequences) of avermectin, the heavy or light chain variable region sequences, or the heavy or light chain sequences, e.g., as disclosed in table 8.

In one embodiment, the anti-PD-L1 antibody molecule is dimaruzumab (imperial Mei Dimiao s/aslicon inc), also known as MEDI4736. Devaluzumab and other anti-PD-L1 antibodies are disclosed in US 8,779,108 (which is incorporated by reference in its entirety). In one embodiment, the anti-PD-L1 antibody molecule comprises one or more of the following: the CDR sequences (or generally all CDR sequences) of cerluzumab, the heavy or light chain variable region sequences, or the heavy or light chain sequences, e.g., as disclosed in table 8.

In one embodiment, the anti-PD-L1 antibody molecule is BMS-936559 (BASEMERIUM Guibao), also known as MDX-1105 or 12A4.BMS-936559 and other anti-PD-L1 antibodies are disclosed in US 7,943,743 and WO 2015/081158 (which are incorporated by reference in their entirety). In one embodiment, the anti-PD-L1 antibody molecule comprises one or more of the following: the CDR sequences (or generally all CDR sequences) of BMS-936559, the heavy or light chain variable region sequences, or the heavy or light chain sequences, e.g., as disclosed in table 8.

Other known anti-PD-L1 antibodies include those described, for example, in the following: WO2015/181342, WO 2014/100079, WO 2016/000619, WO 2014/022758, WO 2014/055897, WO 2015/061668, WO 2013/079174, WO 2012/145493, WO 2015/112805, WO 2015/109124, WO 2015/195163, US 8,168,179, US 8,552,154, US 8,460,927, and US 9,175,082, which are incorporated by reference in their entirety.

In one embodiment, the anti-PD-L1 antibody is an antibody that competes with one of the anti-PD-L1 antibodies described herein for binding to the same epitope on PD-L1 and/or binding to the same epitope on PD-L1.

TABLE 8 amino acid sequences of other exemplary anti-PD-L1 antibody molecules

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Exemplary ENTPD2 inhibitors

In one embodiment of the dosage regimen according to the invention, the additional therapeutic agent is an ENTPD2 agent. In embodiments, the agent of ENTPD2 is an ENTPD2 inhibitor.

The ENTPD2 inhibitor may be an antibody, immunoadhesin, fusion protein, or oligopeptide. In one embodiment, the agent of ENTPD2 or the agent of ENTPD2 inhibitor is an ENTPD2 antibody molecule. In one embodiment of the dosage regimen of the invention, the agent of ENTPD2 is an ENTPD2 antibody molecule as described in WO 2019229658 (incorporated by reference in its entirety) published at 5/12/2019 entitled "ENTPD2 Antibodies, combination Therapies, and Methods of Using the Antibodies and Combination Therapies [ ENTPD2 Antibodies, combination therapies, methods of use of Antibodies and combination therapies ]".

In one embodiment of a dosage regimen according to the invention, the additional therapeutic agent is an anti-ENTPD 2 antibody molecule comprising at least one, two, three, four, five, or six Complementarity Determining Regions (CDRs) (or generally all CDRs) from a heavy chain and a light chain variable region comprising an amino acid sequence set forth in Table 9 (e.g., from the heavy chain and light chain variable region sequences of anti-human ENTPD2 MAB1, anti-human ENTPD2 MAB2, anti-human ENTPD2 MAB3, anti-human ENTPD2 MAB4, or anti-human ENTPD2 MAB5 disclosed in Table 9), or encoded by a nucleotide sequence set forth in Table 9. In some embodiments, the CDRs are according to the cabazite definition (e.g., as set forth in table 9). In some embodiments, CDRs are defined according to Qiao Xiya (e.g., as listed in table 9). In some embodiments, the CDRs are defined according to the combined CDRs of both cabazite and Qiao Xiya (e.g., as listed in table 9). In one embodiment, one or more of the CDRs (or all of the CDRs in general) have one, two, three, four, five, six or more changes, such as amino acid substitutions (e.g., conservative amino acid substitutions) or deletions, relative to the amino acid sequences shown in table 9, or the amino acid sequences encoded by the nucleotide sequences shown in table 9.

In one embodiment, the anti-ENTPD 2 antibody molecule comprises a heavy chain variable region (VH) comprising the VH CDR1 amino acid sequence of SEQ ID NO. 401, the VH CDR2 amino acid sequence of SEQ ID NO. 402 and the VH CDR3 amino acid sequence of SEQ ID NO. 403; and a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO:414, the VLCDR2 amino acid sequence of SEQ ID NO:415 and the VLCDR3 amino acid sequence of SEQ ID NO:416, each as disclosed in Table 9.

In one embodiment, the anti-ENTPD 2 antibody molecule comprises: a VH comprising the amino acid sequence of SEQ ID No. 410, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% or more sequence identity to SEQ ID No. 410. In one embodiment, the anti-ENTPD 2 antibody molecule comprises: VL comprising the amino acid sequence of SEQ ID No. 421, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% or more sequence identity to SEQ ID No. 421. In one embodiment, the anti-ENTPD 2 antibody molecule comprises: VH comprising the amino acid sequence of SEQ ID No. 410 and VL comprising the amino acid sequence of SEQ ID No. 421.

In one embodiment, the anti-ENTPD 2 antibody molecule comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO. 412, or an amino acid sequence having at least about 85%, 90%, 95%, or 99%, or more, sequence identity to SEQ ID NO. 412. In one embodiment, the anti-ENTPD 2 antibody molecule comprises: a light chain comprising the amino acid sequence of SEQ ID NO:423, or an amino acid sequence having at least about 85%, 90%, 95%, or 99%, or more, sequence identity to SEQ ID NO: 423. In one embodiment, the anti-ENTPD 2 antibody molecule comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO. 412 and a light chain comprising the amino acid sequence of SEQ ID NO. 423.

The antibody molecules described herein may be made by vectors, host cells, and methods described in WO 2019229658 (which is incorporated by reference in its entirety).

Exemplary TGF-beta inhibitors

In certain embodiments of dosage regimens according to the invention, the additional therapeutic agent is a transforming growth factor beta (TGF-beta) inhibitor. In some embodiments, the combination is used to treat cancer, e.g., a cancer as described herein.

TGF-beta belongs to a large family of structurally related cytokines including, for example, bone Morphogenic Proteins (BMPs), growth and differentiation factors, activin, and inhibin. In some embodiments, a TGF-beta inhibitor described herein may bind to and/or inhibit one or more isoforms of TGF-beta (e.g., one, two, or all of TGF-beta 1, TGF-beta 2, or TGF-beta 3).

In some embodiments, the TGF-beta inhibitor is non-sappan monoclonal antibody (fresolimumab) (CAS registry number: 948564-73-6). Non-sappan mab is also known as GC1008. Non-hematoxylin monoclonal antibodies are human monoclonal antibodies that bind and inhibit TGF- β isoforms 1, 2 and 3.

The heavy chain of non-sappan mab has the following amino acid sequence:

the light chain of non-sappan mab has the following amino acid sequence:

Non-sappan monoclonal antibodies are disclosed, for example, in WO 2006/086469, US 8,383,780, and US 8,591,901.

In some embodiments, the TGF- β inhibitor is XOMA 089.XOMA 089 is also known as xpa.42.089.XOMA 089 is a fully human monoclonal antibody that binds to and neutralizes TGF- β1 and 2 ligands.

The heavy chain variable region of XOMA 089 has the following amino acid sequence:

(SEQ ID NO: 6) is disclosed in WO 2012/167143.

The light chain variable region of XOMA 089 has the following amino acid sequence:

(SEQ ID NO: 8) is disclosed in WO 2012/167143.

In certain embodiments, the dosage regimen comprises an inhibitor of ENTPD2 (e.g., an anti-ENTPD 2 antibody molecule described herein) and an inhibitor of CD73 (e.g., an anti-CD 73 antibody molecule described herein) and a TGF- β inhibitor (e.g., a TGF- β inhibitor described herein).

In one embodiment, the dosage regimen includes a compound disclosed in the TGF- β inhibitor XOMA 089 or PCT publication No. WO 2012/167143, and an inhibitor of ENTPD2 (e.g., an anti-ENTPD 2 antibody described herein) and an inhibitor of CD73 (e.g., an anti-CD 73 antibody described herein).

In one embodiment, a TGF- β inhibitor XOMA 089 or a compound disclosed in PCT publication No. WO 2012/167143 is administered with an inhibitor of ENTPD2 (e.g., an anti-ENTPD 2 antibody described herein) and an inhibitor of CD73 (e.g., an anti-CD 73 antibody molecule) in a dosage regimen to treat cancer, wherein the cancer is MSS colorectal cancer (CRC), cholangiocarcinoma (intrahepatic or extrahepatic), pancreatic cancer, esophageal-gastric junction (EGJ) cancer, or gastric cancer.

Triptans

In an embodiment of the antibody for use according to the invention or the method according to the invention, the triptan is administered together with one or more doses of an antibody molecule that binds to human CD73 or human ENTPD 2. The triptan may be administered, for example, prior to or concurrently with the anti-CD 73 antibody or the anti-ENTPD 2 antibody. Triptans are a family of tryptamine-based drugs that act as 5-hydroxytryptamine 5-HT at cerebral blood vessels and nerve endings _IB And 5-HT _ID Agonists of the receptor. In an embodiment, the triptan is selected from almotriptan (axit), eletriptan (Relpax), frovatriptan (Frova), naratriptan (Amerge), rizatriptanPravastatin (Maxalt), sumatriptan (Imitrex), zolmitriptan (zemig), lamiditan (Reyvow), which may be combined with additional agents, such as sumatriptan with sodium naproxen (Treximet). In a preferred embodiment, the triptan is sumatriptan or zolmitriptan. Triptan may be administered, for example, on day 1, prior to infusion of anti-CD 73 antibody. Triptan may be used according to standard dosages for a particular triptan (e.g. 25mg, 50mg, or 100mg sumatriptan tablet).

Examples

The following examples are provided to aid in the understanding of the present invention, but are not intended to, and should not be construed to, limit its scope in any way.

Example 1: generation and characterization of anti-CD 73 antibodies

Selection and optimization of anti-CD 73 antibodies from synthetic yeast antibody libraries

anti-CD 73 monoclonal antibodies representing five different epitope bins (epi bins) were selected from eight initial human synthetic yeast libraries using the following method.

Materials and methods

The antigen was biotinylated using the EZ-Link sulfo-NHS-biotinylated kit from Pierce. Goat F (ab') ₂ Anti-human kappa-FITC (LC-FITC), extrAvidin-PE (EA-PE) and streptavidin-AF 633 (SA-633) were obtained from Southern Biotech, sigma and Molecular Probes, respectively. Streptavidin microbeads (MicroBead) and MACS LC separation columns were purchased from the meitian and gentle biotechnology company (Miltenyi Biotec). Goat anti-human IgG-PE (human-PE) was obtained from south biotechnology company.

Preliminary findings

8 initial human synthetic yeast libraries were propagated as described previously, each library being about 10 ⁹ Diversity of (see, e.g., Y. Xu et al Addressing polyspecificity of antibodies selected from an in vitro yeast presentation system: a FACS-based, high-throughput selection and analytical tool. [ solving the problem of multi-specificity of antibodies selected from in vitro yeast presentation systems: FACS-based, high throughput screening and analysis tools ] ]PEDS26.10,663-70 (2013); WO 2009036379; WO 2010105256; and WO 2012009568, incorporated herein by reference in its entirety). For the first two rounds of selection, a magnetic bead sorting technique using the MACS system of meitian-whirl company was performed as described previously (see, e.g., siegel et al High efficiency recovery and epitope-specific sorting of an scFv yeast display library [ efficient recovery of scFv yeast display library and epitope-specific sorting]J Immunol Methods journal of immunological methods]286 (1-2), 141-153 (2004), which is incorporated herein by reference in its entirety). Briefly, yeast cells (about 10 ¹⁰ Individual cells/library) were incubated with 3ml of 100nM biotinylated antigen in wash buffer (phosphate buffered saline (PBS)/0.1% Bovine Serum Albumin (BSA)) at 30℃for 30min. After washing once with 40mL ice-cold wash buffer, the cell pellet was resuspended in 20mL wash buffer and streptavidin microbeads (500 μl) were added to the yeast and incubated for 15min at 4 ℃. Next, yeast cells were pelleted, resuspended in 20mL wash buffer, and loaded onto a meitian gentle company LS column. After 20mL loading, the column was washed 3 times with 3mL of wash buffer. The column was then removed from the magnetic field and the yeast cells were eluted with 5mL of growth medium and then grown overnight. The following rounds of selection were performed using flow cytometry. Precipitation of about 2X 10 ⁷ The individual yeast cells are washed three times with wash buffer and incubated with biotinylated antigen (100 to 1 nM) at reduced concentration under equilibrium conditions, 30nM of the different species at 30℃to obtain species cross-reactivity, or non-specific antibodies are removed from the selection with a multi-specific depletion reagent (PSR). For PSR depletion, the library is incubated with a 1:10 dilution of biotinylated PSR reagent as described previously (see, e.g., Y. Xu et al Addressing polyspecificity of antibodies selected from an in vitro yeast presentation system: a FACS-based, high-throughput selection and analytical tool. [ solving the problem of multi-specificity of antibodies selected from in vitro yeast presentation systems: FACS-based, high throughput screening and analytical tools ]]PEDS 26.10,663-70 (2013), which is incorporated herein by reference in its entirety). The yeast cells were then washed twice with wash buffer and at 4℃with LCFITC (1:100 dilution) and SA-633 (1:500 dilution) or EAPE (1:50 dilution) secondary reagent staining for 15min. After washing twice with wash buffer, the cell pellet was resuspended in 0.3mL wash buffer and transferred to a sorting tube capped with a filter (cartridge). Sorting was performed using a FACS ARIA sorter (BD Biosciences) and sorting gates were determined to select antibodies with the desired characteristics. The selection round is repeated until a population with all the desired characteristics is obtained. After the last round of sorting, yeast cells were plated and individual colonies were selected for characterization.

Light chain diversification protocols were used during the preliminary discovery phase to further discover and improve antibodies.

Light chain lot diversification scheme: heavy chain plasmids from the initial selection output were extracted from yeast by crushing and grabbing (grab), propagated in E.coli and subsequently purified, and then transformed to have a size of 5X 10 ⁶ A diverse library of light chains. The same conditions as the initial findings were used for selection by one round MACS and four rounds FACS.

Antibody optimisation

Optimization of antibodies was performed by introducing diversity into the heavy and light chain variable regions, as described below.

CDRH1 and CDRH2 selection: recombination of CDRH3 of Single antibody to have 1X 10 ⁸ Diverse pre-made libraries of CDRH1 and CDRH2 variants were selected using one round MACS and four rounds FACS as described in the initial findings. In different FACS runs, the library was examined for PSR binding, species cross-reactivity, and affinity pressure by titration or parental Fab pre-complexing, and sorted to obtain populations with the desired characteristics.

Antibody production and purification

Yeast clones were grown to saturation and then induced by shaking at 30℃for 48h. After induction, the yeast cells were precipitated and the supernatant was collected for purification. IgG was purified using a Protein a (Protein a) column and eluted with acetic acid at pH 2.0. Fab fragments were produced by papain digestion and purified by kappa select (GE medical life sciences group (GE Healthcare LifeSciences)).

ForteBio K _D Measurement of

ForteBio affinity measurements (see, e.g., estep et al, high throughput solution-based measurement of antibody-antigen affinity and epitope binning, [ solution-based high throughput measurements of antibody-antigen affinity and epitope boxing ] Mabs [ monoclonal antibody ]5 (2), 270-278 (2013), which are incorporated herein by reference in their entirety) were performed on Octet RED384 substantially as described previously. Briefly, forteBio affinity measurements were performed by on-line loading of IgG onto the AHQ sensor. The sensor was equilibrated offline in assay buffer for 30min, then monitored online for 60 seconds to establish a baseline. The sensor with loaded IgG was exposed to 100nM antigen for 3 minutes and then transferred to assay buffer for 3 minutes for off-rate (off-rate) measurement. All kinetics were analyzed using a 1:1 binding model. The antigens used were:

human CD73-His: recombinant human 5' -nucleotidase/CD 73 protein, CF from R & D Systems catalog: 5795-EN

Mouse CD73-His: recombinant mouse 5' -nucleotidase/CD 73 protein, CF from R & D systems company catalogue: 4488-EN

Cynomolgus monkey CD73-His: cynomolgus monkey CD73/NT5E protein (His tag), from the catalogue of the company san francisco (Sino Biological): 90192-C08H-50

ForteBio epitope boxing (epitope binding)/ligand blocking

Epitope boxing/ligand blocking was performed using a standard sandwich format cross-blocking assay. Control anti-target IgG was loaded onto the AHQ sensor and the unoccupied Fc binding sites on the sensor were blocked with an unrelated human IgG1 antibody. The sensor is then exposed to 100nM target antigen, then to a second anti-target antibody or ligand. Additional binding of the secondary antibody or ligand after antigen association indicates an unoccupied epitope (non-competitor), while no binding indicates that the epitope is blocked (competitor or ligand blocking).

MSD-SET kinetic assay

Equilibrium affinity measurements were performed as described previously (Estep et al, 2013). Solution Equilibrium Titration (SET) was performed in PBS+0.1% IgG free BSA (PBSF), the antigen was kept constant at 10-100pM, and incubated with 3-to 5-fold serial dilutions of the antibody starting from 5-100nM (experimental conditions depend on the sample). Antibodies (20 nM in PBS) were coated onto standard binding MSD-ECL plates overnight at 4℃or 30min at room temperature. Plates were then blocked for 30min with shaking at 700rpm, and then washed 3 times with wash buffer (pbsf+0.05% tween 20). SET samples were applied and incubated on the plate with shaking at 700rpm for 150s, followed by one wash. The antigen captured on the plate was detected by incubation on the plate for 3min with 250ng/mL sulphur-tagged streptavidin in PBSF. Plates were washed 3 times with wash buffer and then read on a MSD Sector Imager 2400 instrument using 1x read buffer with surfactant. The percentage of free antigen was plotted as a function of titrated antibody in Prism and fitted to a quadratic equation to extract KD. To increase throughput, a liquid handling robot was used throughout the MSD-SET experiment (including SET sample preparation).

Cell binding assays

Approximately 100,000 antigen-overexpressing cells were washed with wash buffer and incubated with 100 μl 100nM IgG for 5 min at room temperature. The cells were then washed twice with wash buffer and incubated with 100. Mu.l of 1:100 human-PE for 15 minutes on ice. The cells were then washed twice with wash buffer and analyzed on a FACS Canto II analyzer (BD biosciences).

Results

Yeast cells were screened for libraries expressing human antibodies on their surface for binding to human CD73. The two antibodies from epitope boxes 4, 918 and 930 bound well to CD73 and inhibited the enzymatic activity of CD73 (data not shown). These two antibodies were affinity matured to yield two related antibody lineages, termed lineages 1 and 3, respectively (table 10). These anti-CD 73 antibodies are expressed in three different forms: igG1 antibodies (referred to as.c constructs, e.g., 350. C), igG4 antibodies comprising an S228P mutation in the Fc region (IgG 4S228P, referred to as.a constructs, e.g., 350. A), or IgG4 antibodies comprising S228P and L235E mutations in the Fc region (IgG 4S228P/L235E, referred to as.b constructs, e.g., 350. B), are numbered according to Eu numbering. The sequences of these antibodies are disclosed in table 1. For antibody 350.A, two batches of antibodies, hereinafter 350.A1 and 350.A2, were generated.

TABLE 10 two lineages of anti-CD 73 antibodies

Pedigree 1	Antibodies to	IgG1 forms	IgG 4S 228P form	IgG 4S 228P/L235E forms
					Parent strain	918	918.C	918.A	918.B
Children' s	350	350.C	350.A1、350.A2	350.B
					Children' s	356	356.C	356.A	356.B
Children' s	358	358.C	358.A	358.B
					Pedigree 3	Antibodies to	IgG1 forms	IgG 4S 228P form	IgG 4S 228P/L235E forms
Parent strain	930	930.C	930.A	930.B
					Children' s	373	373.C	373.A	373.B
Children' s	374	374.C	374.A	374.B
					Children' s	376	376.C	376.A	376.B
Children' s	377	377.C	377.A	377.B
					Children' s	379	379.C	379.A	379.B

All anti-CD 73 antibodies tested bound to human and cynomolgus CD73. Lineage 1 antibodies also bind murine CD73. The Kd values of these antibodies measured using Octet as described above are provided in table 11.

TABLE 11 affinity of anti-CD 73 antibodies

* Kd measurement approaches the upper limit

Next, using epitope boxing/ligand blocking studies, it was shown that parent antibody 918 competes with progeny antibodies 350, 356 and 358 for binding to CD73. Similarly, parent antibody 930 competes with progeny antibodies 373, 374, 376, 377, and 379 for binding to CD73. 918 and 930 compete with the internal reference anti-CD 73 antibody, indicating that these antibodies share the same epitope box.

Fab and antibody affinity measurements using surface plasmon resonance

Fab for mabs 350 and 373 were generated by engineering a terminator (stop) between the two proline residues above the core hinge region of the heavy chains of 350 and 373. Both were expressed in Expi293F (ThermoFisher) cells and purified using CaptureSelect IgG CH affinity resin (samfeier).

Biacore was used to measure cross-species affinity with Fab material of mabs 350 and 373. The proteins used were as follows: recombinant human CD73 (R & D systems 5795-EN); recombinant cynomolgus monkey CD73 (yi qiao shenzhou company 90912-C08H); recombinant mouse CD73 (R & D systems Co 4488-EN); recombinant rat CD73 (Yiqiaoshenzhou 80375-R08H). Anti-human Fab (GE healthcare life sciences) was immobilized on all 4 flow cells (Fc) of a CM5 chip (GE). Fab 350 and 373 were captured at about 20RU on Fc2 and Fc 4. 0.01nM to 90nM CD73 (3-fold dilution series) was flowed over all 4 Fc. All samples were diluted in running buffer HBS-EP+ (pH 7.4,0.01M HEPES,150mM NaCl,3mM EDTA and 0.05% (v/v) P20).

The results of Kd (M) affinity for cross-species binding for 350 and 373Fab are shown in table 12.

TABLE 12 affinity for anti-CD 73Fab

Fab	Antigens	K _d (M)
			Fab 350	hCD73	≤1E-10
Fab 350	cCD73	≤1E-10
			Fab 350	mCD73	1.728E-8
Fab 350	rCD73	2.829E-8
			Fab 373	hCD73	1.304E-8
Fab 373	cCD73	9.465E-9
			Fab 373	mCD73	Unbound material
Fab 373	rCD73	Unbound material

In another study, the affinity of full length antibody 373.A or 373.A Fab fragments for human, cynomolgus monkey, mouse and rat CD73 was determined by using the Surface Plasmon Resonance (SPR) method using anti-histidine (His) antibody capture Biacore. anti-His abs were immobilized directly on CM5 chip surface by amine coupling. His-tagged human CD73/His, cynomolgus CD73/His, mouse CD73/His or rat CD73/His were flowed through and captured in the desired Resonance Unit (RU) (Rmax 20). Serial dilutions of IgG or Fab antibody analyte concentrate were flowed through at 60 μl/min. The sensor map was analyzed for a 1:1 binding model using the manufacturer's software. The 373.A and 373.A Fab failed to detect binding to the mouse CD73/His protein and the rat CD73/His protein, indicating that 373.A did not cross-react with rodents.

The affinities of human and cynomolgus CD73 to both 373.A and 373.A Fab were established. Hydrogen-deuterium mass spectrometry and size exclusion chromatography studies support a model of locking the CD73 dimer conformation to an open-open (inactive-inactive) conformation by 373.A, supporting 1:1 bidentate binding of 1ab:1CD73 dimer. Thus, the entire Ab affinity was used instead of Fab measurement, considering that 1:1 bidentate binding would be beneficial for avidity. Full length antibody 373.A bound recombinant human CD73 with a Kd of 0.991+ -0.267 nM and cross-reacted with recombinant cynomolgus monkey CD73 with a Kd of 0.068+ -0.009 nM as determined by Biacore kinetic binding studies.

anti-CD 73 antibody binding to whole blood target

Whole blood target engagement was assessed by flow cytometry using whole blood from healthy human donors. Briefly, biotinylated antibodies were incubated with whole blood for 30 minutes prior to erythrocyte lysis and fixation. Fixed cells were stained for CD3 and CD8 to identify cd8+ T cells, and streptavidin-APC was stained to detect biotin. After staining, the cells were washed and analyzed by flow cytometry.

For the anti-CD 73 antibodies tested, a dose-dependent binding was observed as measured by Median Fluorescence Intensity (MFI) of APC signals (data not shown). Biotinylated isotype control antibodies did not show binding to cd8+ T cells.

CD73 target occupancy on human whole blood samples

The conceptual demonstration of Target Occupancy (TO) of CD73 on human whole blood samples was performed by ex vivo treatment of donor blood with unlabeled 373. A. Titration of 373.A or DNP-IgG4sm isotype control was performed from 10. Mu.g/mL to 0.17 ng/mL. Samples from both donors were treated with higher doses (10. Mu.g/mL to about 0.1. Mu.g/mL) of unlabeled 373.A, preventing biotinylated 373.A from binding to cells, reducing the geometric mean fluorescence intensity (gMFI) value to a stable value at fluorescent background levels (about 550gMFI for both donors). This indicates total CD73 target occupancy. In contrast, the gMFI values of cells pretreated with a smaller amount of unlabeled 373.A (0.17 ng/mL and 0.51 ng/mL) were similar to those of samples pretreated with DNP-IgG4sm isotype control (about 1600gMFI for donor 1 and about 2200gMFI for donor 2). Isotype control treated samples mimic zero target occupancy blood.

Inhibition of enzymatic Activity of soluble recombinant CD73

The 5' extracellular nucleotidase CD73 is the rate limiting step in the conversion of AMP to adenosine. The ability of anti-CD 73 antibodies to inhibit the enzymatic activity of CD73 was measured using a malachite green phosphate assay. Briefly, 25ng/ml recombinant human CD73 was incubated with a dose-titrated (0-500. Mu.M) substrate Adenosine Monophosphate (AMP) in the presence of buffer alone or in the presence of 1. Mu.g/ml isotype control antibody or anti-CD 73 antibody 350.C at a concentration of 1, 0.3 or 0.1. Mu.g/ml. The release of inorganic phosphate (Pi) was measured using a malachite green phosphate assay kit (enzolife sciences (Enzo Life Sciences), catalog number BML-AK 111).

The control antibody at the test concentration had no effect on the Miq constant (Km) of recombinant human CD 73. In contrast, anti-CD 73 antibody 350.C caused a dose-dependent decrease in Vmax on the Km curve (data not shown), indicating that antibody 350.C is a non-competitive inhibitor of human CD 73.

Next, the ability of anti-CD 73 antibodies 350, 356, 373 and 374 expressed in the form a or B to inhibit the enzymatic activity of recombinant human and cynomolgus monkey CD73 was tested using malachite green phosphate assay similar to that described above. Briefly, anti-CD 73 antibodies were incubated with 25ng/ml recombinant human or cynomolgus monkey CD73 in the presence of 25. Mu.M AMP for 10 minutes. The release of inorganic phosphate (Pi) was measured using a malachite green phosphate assay kit (en zodiac life sciences, catalog No. BML-AK 111). Normalized percent inhibition (% INH) was determined using zero-time control as 100% INH and no-antibody control as 0% INH.

All tested anti-CD 73 antibodies inhibited the enzymatic activity of soluble recombinant human and cynomolgus monkey CD73 (data not shown).

Inhibition of enzymatic activity of soluble endogenous CD73

In addition, anti-CD 73 antibodies were tested for their enzyme inhibitory activity on soluble endogenous CD73 (e.g., CD73 shed from the cell surface).

In the first study, anti-CD 73 antibodies 350 and 373, expressed in the form of. A or. B, or isotype control antibodies, were incubated with MDA-MB-231 (human breast cancer cell line) in serum-free conditioned medium (conditioned serum free media) for 240 minutes in the presence of 100. Mu.M AMP. The disappearance of AMP was measured by a modified Cell Titer Glo (CTG) assay (Promega, catalog number G9242/3). AMP inhibits luciferase signal in CTG kits. As the added AMP is enzymatically consumed by CD73, the luciferase signal increases. Normalized percent inhibition (% INH) was determined using zero-time control as 100% INH and no-antibody control as 0% INH.

The anti-CD 73 antibody dose-dependently inhibited the enzymatic activity of CD73 shed from the breast cancer cell line MDA-MB-231 (data not shown).

In a second study, anti-CD 73 antibodies 350, 356, 358, 373, 374, 377 and 379, all expressed in the form of. B, were incubated with diluted (12.5% v: v in PBS) serum from pancreatic cancer patients for 60 minutes in the presence of 100. Mu.M AMP. Similar to the first study, AMP disappearance was measured by a modified Cell Titer Glo (CTG) assay and normalized percent inhibition (%inh) was determined using zero-time control as 100% INH and no-antibody control as 0% INH.

anti-CD 73 antibodies also inhibited CD73 enzymatic activity in serum from pancreatic cancer patients in a dose-dependent manner (data not shown).

Inhibition of cell surface expressed CD73 enzymatic activity

First, the ability of anti-CD 73 antibodies 350, 356, 358, 373, 374, 377 and 379 (all in the.b form) to inhibit CD73 expressed on breast cancer cell line MDA-MB-231 was examined using a malachite green phosphate assay. Briefly, antibodies were incubated with cells for 180 minutes in the presence of 100. Mu.M AMP. The release of inorganic phosphate from AMP was measured using malachite green phosphate assay kit (enzocine biosciences, catalog number BML-AK 111). Normalized percent inhibition (% INH) was determined using zero-time control as 100% INH and no-antibody control as 0% INH.

All tested anti-CD 73 antibodies inhibited the surface-expressed CD73 enzyme activity of the breast cancer cell line MDA-MB-231 (data not shown).

Next, since the lineage 1 antibodies cross-reacted with mouse CD73, whereas the lineage 3 antibodies did not cross-react, antibodies from both lineages were tested against CD73 expressed on the surface of human or murine breast cancer cell lines. The anti-CD 73 antibody was incubated with the human breast cancer cell line MDA-MB-231 or the murine breast cancer cell line 4T1 in the presence of 100. Mu.M AMP for 240 minutes. The disappearance of AMP was measured by the modified Cell Titer Glo (CTG) assay described above, and normalized percent inhibition (% INH) was determined using zero-time control as 100% INH and no-antibody control as 0% INH.

Consistent with their binding profile, lineage 1 antibodies 918, 350, 356 and 358 inhibited human and murine CD73, while lineage 3 antibodies 930, 373, 374, 376, 377 and 379 inhibited human CD73 but not murine CD73 (data not shown).

In addition, two modified Cell Titer Glo (CTG) assays were performed to test the enzyme inhibitory activity of anti-CD 73 antibodies against CD73 expressed on human breast cancer Cell line MDA-MB-231 or human ovarian cancer Cell line SKOV 3. In both studies, 1000ng/ml of anti-CD 73 antibody was incubated with 20,000 cells/ml cells for 240 minutes at 37℃in the presence of 100. Mu.M AMP. The disappearance of AMP was measured by the modified Cell Titer Glo (CTG) assay described above, and normalized percent inhibition (% INH) was determined using zero-time control as 100% INH and no-antibody control as 0% INH.

In both studies, all tested anti-CD 73 antibodies were able to inhibit surface CD73 expressed on the human breast cancer cell line MDA-MB-231 or the human ovarian cancer cell line SKOV3 (data not shown).

Next, a similar Cell Titer Glo (CTG) assay was performed to examine the ability of anti-CD 73 antibodies to inhibit human CD73 expressed on HEK 293 Cell lines. Briefly, HEK 293 cell lines were engineered to stably overexpress human CD73 and incubated with anti-CD 73 antibodies in the presence of 100 μm AMP for 150 minutes. The disappearance of AMP was measured by the modified Cell Titer Glo (CTG) assay described above, and normalized percent inhibition (% INH) was determined using zero-time control as 100% INH and no-antibody control as 0% INH.

anti-CD 73 antibodies 350, 356, 373, and 374 in either the a or B forms inhibited membrane-bound human CD73 in a dose-dependent manner (data not shown).

In addition, the enzyme inhibition activity of anti-CD 73 antibodies was also examined using human PBMC. Briefly, primary human PBMCs were isolated from two separate donors and incubated with anti-CD 73 antibodies for 480 minutes in the presence of 25 μm AMP. The disappearance of AMP was measured by the modified Cell Titer Glo (CTG) assay described above, and normalized percent inhibition (% INH) was determined using zero-time control as 100% INH and no-antibody control as 0% INH.

The anti-CD 73 antibodies tested inhibited the enzymatic activity of CD73 expressed on primary human PBMCs from two donors (data not shown).

Restoration of CD4+ and CD8+ T cell proliferation in the presence of AMP

Next, anti-CD 73 antibodies were tested for their ability to alleviate AMP-mediated inhibition of CD4+ T cells. Briefly, cd4+ T cells were isolated from Peripheral Blood Mononuclear Cells (PBMCs) pooled from healthy human donors. CD4+ T cells were stained with CellTrace Violet (CTV) (Semerfeichi technologies (Thermo Fisher Scientific), catalog number C34557) to follow cell division prior to stimulation with anti-CD 3/28 beads in the presence of 800. Mu.M AMP. Proliferation was determined by CTV dilution using flow cytometry on day 4. CTV stained cells lost approximately half of the fluorescent signal per division, as measured by flow cytometry. Proliferation index was calculated as a measure of the level of T cell division under each condition, where 100 represents maximum proliferation and 0 represents no proliferation.

The anti-CD 73 antibodies tested were all able to restore cd4+ T cell proliferation in the presence of AMP (data not shown).

Inhibition of CD73 enzymatic Activity in vivo

In addition, the anti-CD 73 antibody was examined for its enzyme inhibitory activity in vivo. The CD73 highly expressed MDA-MB231 breast cancer cell line (ATCC HTB-26) was implanted into athymic nude mice female mice (6-8 weeks old) at 10X106 cells/mouse/200. Mu.l. When the tumors were 200mm3, five mice per group were randomly allocated and were treated intraperitoneally with 20 or 200 micrograms/mouse of control polyclonal human IgG or a group of anti-CD 73 mabs. The antibodies tested were anti-CD 73 antibodies 350, 356, 373 and 374, expressed in the form a or B.

Three days after administration, plasma was collected at a rate of one plasma to five methanol by volume. The methanol quenched samples were stored at-80 ℃ prior to use and centrifuged after use. The pellet was discarded and the supernatant transferred to a new Ai Bende tube. Stock solutions of internal standards (IS, C-13 labeled adenosine and N-15 labeled inosine, cambridge isotope laboratories (Cambridge Isotope Laboratories, MA)) were added to a final concentration of 50nM. The prepared samples were then analyzed using an LC/MS system (coupled with Shimadzu LC pump (LC-20 AD) and CTC autosampler with DLW wash) of API-6500QTrap (AB Sciex Co., USA). For each sample, 5. Mu.L was injected and separated using a SeQuant ZIC-pHILIC column (5 μm,150x 2.1mm, milibore, mass.) maintained at 40 ℃. Elution was performed using a binary gradient, where mobile phase B was 100% acetonitrile (no additive) and mobile phase a was 12mM ammonium formate and 12mM formic acid in a 1:1 (v/v) mixture of water and acetonitrile. The elution procedure was set to (0, 85,0.6), (0.5, 85,0.4), (2, 10, 0.4), (4.5, 10, 0.4), (5, 85,0.4), (5.5, 85,0.6), where the values in brackets are time (in minutes), percentage of mobile phase B and flow rate (in mL/min). In ESI positive mode, adenosine and C13-adenosine were monitored from 0.5 to 4.5 minutes with mass transitions of 268- >136 and 273- >136, respectively. In ESI negative mode, inosine and N15-inosine were monitored from 0.5 to 4.5 minutes with mass transitions 267- >135 and 271- >139, respectively. The results are reported as nM adenosine or inosine.

All tested anti-CD 73 antibodies effectively reduced the accumulation of adenosine and inosine in serum of immunocompromised mice implanted with CD73 highly expressed MDA-MB231 breast cancer cell lines (data not shown).

Example 2: anti-CD 73 antibody 373.A as single agent in combination with BAP 049-clone-E and/or PBF509 for phase I/Ib studies in patients with advanced malignancies

Immunotherapy (e.g., PD-1, PD-L1, and CTLA-4) against different immune checkpoints has shown efficacy in a variety of cancer indications over the past decade. However, while some patients achieved an objective and durable response to checkpoint blockade, most patients showed only modest or no clinical benefit, suggesting that tumors used alternative immunosuppressive mechanisms to achieve immune escape (Allar et al, clin Cancer Res. [ clinical Cancer research ]2013;19 (20): 5626-35; vesely et al, annu Rev Immunol [ immunological annual review ]2011; 29:235-271). Thus, it may be desirable to block multiple immunosuppressive pathways simultaneously to elicit clinically meaningful responses in a large number of patients.

Adenosine production and signaling have been potential therapeutic targets in cancer treatment for the past few years. Adenosine creates an immunosuppressive tumor microenvironment by reducing cytotoxic anti-tumor immune responses, enhancing proliferation and polarization of immunosuppressive cells, and increasing neovascularization (Young et al, cancer Discovery 2014;4 (8): 879-88). Preclinical data indicate that CD73 blockade significantly delays primary tumor growth and inhibits lung metastasis in immune competent isogenic mouse models (Stagg et al, 2010). Similar results were observed in one study, in which deletion of the A2aR gene in the host resulted in rejection of established immunogenic tumors in A2 aR-deficient mice, whereas no rejection was seen in control wild-type mice (Ohta et al, PNAS [ Proc. Natl. Acad. Sci. USA 2006;103 (35): 13132-37).

Phase I/Ib open-label multicenter studies aimed at assessing safety, tolerability, primary antitumor activity, pharmacokinetics (PK) and Pharmacodynamics (PD) of anti-CD 73 antibody 373.A as a single agent and in combination with the A2aR antagonist PBF509 (NIR 178) and/or anti-PD-1 antibody BAP 049-clone-E (swadazumab) for patients with advanced malignancy.

The sequence of anti-CD 73 antibody 373.A is given in table 1-see antibodies designated 373. It has an IgG4 form, which IgG4 form has the S228P mutation. The heavy chain of anti-CD 73 antibody 373.A is 373.A (SEQ ID NO:46, wherein X is K), and this anti-CD 73 antibody having this heavy chain sequence will be referred to as anti-CD 73 antibody 373.A for convenience.

The main objective was to characterize safety and tolerability and determine the Recommended Dose (RD) of anti-CD 73 antibody 373.A as single agent and in combination with PBF509 and/or BAP 049-clone-E. A secondary objective was to evaluate anti-CD 73 antibody 373.A as a single drug and primary anti-tumor activity and PK when combined with PBF509 and/or BAP 049-clone-E, to evaluate immunogenicity of anti-CD 73 antibody 373.A and BAP 049-clone-E and to characterize changes in immune infiltration in tumors after treatment, such as changes in Tumor Infiltrating Lymphocytes (TIL), tumor-associated macrophages (TAM), cd8+ T cells and PDL-1 expression from baseline.

BAP 049-clone-E (Stbadizumab) is a high affinity ligand blocking humanized anti-programmed death 1 (PD-1) IgG4 antibody that blocks PD-L1 and PD-L2 binding to PD-1. BAP 049-clone-E is being tested in advanced malignancy in phase I/II studies. The sequences of BAP 049-clone-E are disclosed in Table 5.

PBF509 (NIR 178) is a novel non-xanthine based compound and is a potent oral adenosine A2aR antagonist.

Two phase I/Ib and phase II studies are underway to evaluate PBF509 as a single agent and/or in combination with BAP 049-clone-E, respectively, for patients with advanced non-small cell lung cancer (NSCLC) and solid tumors, as well as non-hodgkin's lymphoma.

This I/Ib study will first recruit adult patients who have progressed to advanced malignancies that have progressed or for which the indication is intolerant to standard therapies, where moderate to high levels of CD73 expression are associated with poor prognosis, suggesting an adenosine-mediated immune escape (Wu et al, journal of Surgical Oncology J.Oncology 2012,106 (2): 130-137; gaudeau et al, oncoimmunology [ tumor immunology ];2016,5 (5): e1127496; inoue et al, oncotarget [ tumor target ];2017,8 (5): 8738-8751). These indications include non-small cell lung cancer (NSCLC), triple Negative Breast Cancer (TNBC), pancreatic cancer (PDAC), renal Cell Carcinoma (RCC), ovarian cancer, microsatellite stabilized (MSS) colorectal cancer and metastatic castration-resistant prostate cancer (mCRPC), although additional indications may be registered in addition to the emerging clinical data (e.g. efficacy data or confirmed pathway activation).

The study consisted of two parts: (1) The single agent anti-CD 73 antibody 373.A, the dual combination anti-CD 73 antibody 373.A/PBF509 and the anti-CD 73 antibody 373.A/BAP 049-clone-E or the triple combination anti-CD 73 antibody 373.A/PBF509/BAP 049-clone-E dose escalation portion, which results in the Recommended Dose (RD) statement for each treatment, and (2) the dose escalation portion, wherein the patient will receive the treatment at the single agent, dual combination and triple combination of RD. The incremental portion will recruit patients with advanced NSCLC, TNBC, PDAC, RCC, ovarian and colorectal cancers (MSS); and there is no limit to the number of previous treatments. The enlarged portion will recruit up to 3 lines of previously treated advanced malignant patients.

In the expanded section, patients for each indication were equally randomized to the combination treatment group. Random assignment will be made according to the indication and further stratification within certain indications by prior PD-1/PD-L1 treatment (initial or drug resistance).

Dose and regimen selection

anti-CD 73 antibody 373.A monotherapy

The initial dose of the 60mg flat dose anti-CD 73 antibody 373.A administered intravenously every 2 weeks (Q2W) was selected based on the observed preclinical safety, tolerability and PK data in cynomolgus monkeys and the published medical history of CD73 deficient patients.

The 60mg dose is considered the lowest pharmacologically active dose (mPAD) because it is expected to provide (1) about 20 hours >90% cd8+t cell CD73 occupancy, (2) about 22 hours >90% adenosine inhibition, and (3) about 17 hours estimated >90% overall CD73 occupancy.

From modeling of TK data from cynomolgus toxicology studies, ex vivo CD8+ T cell CD73 occupancy data, and in vitro data inhibiting adenosine formation, it is expected that a dose of > 1200mg Q2W will achieve >90% target occupancy on CD8+ T cells and a dose of > 600mg Q2W will achieve >90% inhibition of adenosine production throughout the dosing interval.

The dose of anti-CD 73 antibody 373.A will be incremented in sequential groups under the guidance of Bayesian Logistic Regression Model (BLRM) in combination with overdose control (EWOC) criteria until the Maximum Tolerated Dose (MTD) or the expanded Recommended Dose (RD) is identified. Preclinical data and models indicate that high antigen deposition may exist and that high doses (e.g., > 1200mg q2 w) may be required to achieve continuous target occupancy throughout the dose interval. Dose escalation will be performed primarily by the Q2W regimen. However, if this regimen showed rapid anti-CD 73 antibody 373.A elimination and no target saturation within the dosing interval, a more frequent QW regimen could be tested. On the other hand, if the Q4W regimen is predicted not to be rapidly eliminated within the dosing interval, the Q4W regimen may be studied instead.

For ascending dosing schedules, anti-CD 73 antibody 373.A will initially be administered on a weekly schedule (QW) at C1D1 and C1D8, then starting at C1D15, anti-CD 73 antibody 373.A will be administered on a bi-weekly schedule (Q2W).

anti-CD 73 antibody 373.A/PBF509 combination

The maximum initial dose of the anti-CD 73 antibody 373.A/PBF509 double combination was 200mg q2w anti-CD 73 antibody 373.A and 80mg BID PBF509.

200mg of Q2W anti-CD 73 antibody 373.A was a low dose of anti-CD 73 antibody 373.A, and it was expected that >90% target occupancy on CD8+ T cells would be achieved for about 2.3 days. The 373.A dose of 200mg of Q2W anti-CD 73 antibody was 16% of the 1200mg of Q2W dose, and it was expected that >90% CD8+ T cell target occupancy would be achieved throughout the dosing interval.

To determine the appropriate dose of each drug in the combination, the dose escalation method of anti-CD 73 antibody 373.A and PBF509 will be employed under the guidance of Bayesian Logistic Regression Model (BLRM) in combination with the dose overdose control (EWOC) principle standard.

anti-CD 73 antibody 373.A/BAP 049-clone-E combinations

The maximum initial doses for the dual A/BAP 049-clone-E combination were 200mg of Q2W anti-CD 73 antibody 373.A and 400mg of Q4W BAP 049-clone-E.

The basic principle of 200mg of the q2w anti-CD 73 antibody 373.A has been described above. 200mg of Q2W anti-CD 73 antibody 373.A will be used in combination with the RD of BAP 049-clone-E (i.e., 400mg of Q4W, which has proven to be safe and effective).

The anti-CD 73 antibody 373.A dose level will be escalated under the guidance of Bayesian Logistic Regression Model (BLRM) combined with overdose control (EWOC) principle criteria, wherein BAP 049-clone-E was used at a fixed dose.

A/BAP 049-clone-E/PBF 509 combinations

The maximum starting dose for the anti-CD 73 antibody 373.A/BAP 049-clone-E/PBF 509 triple combination would be 200mg of the Q2W anti-CD 73 antibody 373.A, 400mg of the Q4W BAP 049-clone-E and 80mg of BID PBF509.

The dose escalation method of fixed dose BAP 049-clone-E anti-CD 73 antibody 373.A/BAP 049-clone-E/PBF 509 will be employed under the guidance of the Bayesian Logistic Regression Model (BLRM) in combination with the overdose control (EWOC) standard to determine the appropriate dose of anti-CD 73 antibody 373.A and PBF509 in the triple combination.

anti-CD 73 antibody 373.A (100 mg powder for infusion solution) will be administered intravenously as a 1 hour (up to 2 hours if indicated clinically) infusion. BAP 049-clone-E (100 mg powder for infusion solution) will be administered intravenously as a 30 minute (up to 2 hours if indicated clinically) infusion. When administered in combination, the anti-CD 73 antibody 373.A and BAP 049-clone-E may be administered on the same day for each infusion using separate infusion materials (bag, line, filter). The same access site may be used for both infusions. anti-CD 73 antibody 373.A can be infused first, followed by a rest for 30 minutes, followed by BAP 049-clone-E infusion. PBF509 (40 mg and/or 80mg and/or 160mg capsules for oral administration) was orally administered twice daily (BID) continuously. In the visit where anti-CD 73 antibody 373.A and/or BAP 049-clone-E was to be administered, administration of PBF509 was first performed, followed by infusion of anti-CD 73 antibody 373. A. No interruption was required between PBF509 administration and anti-CD 73 antibody 373.a infusion.

Tables 13-16 describe the initial doses and dose levels that can be assessed during this trial. Patients treated with anti-CD 73 antibody 373.A single agent or anti-CD 73 antibody 373.A in combination with BAP 049-clone-E and/or PBF509 will begin study treatment on cycle 1 day 1. Each cycle consisted of 28 days. BAP 049-clone-E Q W will be administered on day 1 of the cycle. PBF509BID will be administered on each day of the cycle.

In the ascending dosing schedule of anti-CD 73 antibody 373.A, the initial dose of anti-CD 73 antibody 373.A, cycle 1, day 1, will be followed by the dose of anti-CD 73 antibody 373.A, cycle 1, day 8, and then Q2W was administered anti-CD 73 antibody 373.A starting on day 15. The sum of the first two doses of anti-CD 73 antibody 373.A (day 1 and day 8) should not exceed the dose level of anti-CD 73 antibody 373.A administered as a single agent or in combination with BAP 049-clone-E and/or PBF509, Q2W. The group with anti-CD 73 antibody 373.A ascending dosing will be studied at the dose level previously tested with anti-CD 73 antibody 373.A continuous dosing.

TABLE 13 temporary dosage level of anti-CD 73 antibody 373.A

TABLE 14 temporary dose level of anti-CD 73 antibody 373.A in combination with PBF509

TABLE 15 temporary dose level of anti-CD 73 antibody 373.A in combination with BAP 049-clone-E

TABLE 16 temporary dose level of PBF509 in combination with anti-CD 73 antibody 373.A and BAP 049-clone-E

Example 3: ascending dosing regimen

It was observed that the majority of patients experiencing any level of headache (> 90%) had this event occurred after the first dose of anti-CD 73 antibody 373. A. The maximum concentration (Cmax) of anti-CD 73 antibody 373.A after the first dose was studied by using logistic regression analysis to determine the relationship to headache probability. It was found that there was a statistically significant relationship between the 373.A Cmax of anti-CD 73 antibody after the first dose and the probability of experiencing any level of headache event. When Cmax of anti-CD 73 antibody 373.A was below 100 μg/ml, the probability of experiencing headache events on grade 2 or more was estimated to be below 25% and the probability of experiencing headache events on grade 3 or more was estimated to be below 10%.

A population PK model was developed to mimic the Cmax range following administration of different anti-CD 73 antibody 373.a dose levels. The results indicate that the Cmax of most patients at a dose of 373.A of 200mg anti-CD 73 antibody in cycle 1 was less than 100 μg/ml, indicating that the use of 200mg would significantly reduce the incidence of headache. Since headache is the first dose effect and regresses within 48 hours, an additional 400mg dose at the second week (cycle 1, day 8) is recommended to maintain a dose strength comparable to 600mg q2w regimen for two weeks. Population PK simulations show that dividing a dose of 600mg of anti-CD 73 antibody 373.a into 200mg (week 1) +400mg (week 2) will have a similar exposure (i.e. AUC) to 600mg q2w, with Cmax about 3 times lower at week 1.

Example 4 anti-ENTPD 2 mAb1 as a single agent and in combination with Studies' Beadizumab, anti-CD 73Ab and NIR178 in stage I/Ib, open label, multicenter study in patients with advanced solid tumors

Study design

This study is a FIH, open label, stage I/Ib, multicenter study consisting of an up-dosing portion of anti-ENTPD 2 mAb1 as a single agent combined with Studies' Beadizumab, anti-CD 73Ab or NIR178, followed by an extension. Furthermore, after evaluating all security and validity data and determining the MTD/RD of the double combination, the use of an optional triple combination may be considered. Recruitment will be limited to subjects with MSS CRC, cholangiocarcinoma, pancreatic cancer, esophageal cancer, EGJ, or gastric cancer.

During the ascending portion, the first dose of the first two subjects treated with untested anti-ENTPD 2 mAb1 (either as a single agent, or in combination with the swabber or NIR178 or anti-CD 73 Ab) dose levels will be staggered by 48 hours. After determining that the 3 dose levels of anti-ent pd2 mAb1 single agent are safe and tolerogenic, a combined dose escalation may be initiated. Once the RD or MTD of anti-ENTPD 2 mAb1 and anti-ENTPD 2 mAb1 in combination with the scada bead mAb, anti-CD 73Ab or NIR178 as a single agent are determined, the corresponding one or more expansion portions may be initiated.

"primary dose" is designated as C1D1 only (S2) or C1D1 and C1D8 doses (S3). In both cases, one or more of the primary doses will be lower than the experimental dose. The "experimental dose" is specified as the dose administered at C1D15 and later on the Q2W schedule. In S3, the initial C1D8 primary dose will be equal to the C1D1 primary dose, and may be further increased according to tolerability. The primary dose escalation protocol will focus on studying experimental doses using BLRM in combination with EWOC standards. However, if the initial dose of C1D1 and/or C1D8 needs to be modified, a separate BLRM will be used.

Study treatment

For the purposes of this study, the term "study drug" refers to anti-ENTPD 2 mAb1, anti-CD 73 Ab, studizumab (PDR 001) and NIR178. Study treatment was defined as anti-ENTPD 2 mAb1 alone or in combination with swabber mAb (PDR 001), anti-CD 73 Ab or NIR178.

All doses and all dose changes prescribed and assigned to subjects during the study must be recorded on the appropriate dose administration record eCRF.

Study and control drugs

TABLE 18 study drug

Initial dose

A lower starting dose may be recommended after all additional relevant data is included in the model and all available data is clinically reviewed prior to treatment of the first subject. The starting dose will meet EWOC standards.

anti-ENTPD 2 mAb1 single agent treatment:

in this FIH study, the initial dose of anti-ENTPD 2 mAb1 would be 100mg Q2W, and based on pre-clinical data of anti-ENTPD 2 mAb 1.

Combination therapy:

combination of anti-ENTPD 2 mAb1 with swabber: studizumab of RP2D (400 mg Q4W)

Combination of anti-ENTPD 2 mAb1 with NIR 178: NIR178 160mg BID (establishing RP2D in combination with Studizumab and establishing NIR178 MTD at 480mg BID)

Combination of anti-ENTPD 2 mAb1 with anti-CD 73 Ab: the temporary starting dose for anti-CD 73 Ab would be 100mg q2w.

The temporary initial experimental dose of anti-ENTPD 2 mAb1 was chosen as 300mg Q2W for all combined dose increments, irrespective of the dosing schedule. In practice, at the beginning of the respective combined dose escalation, a lower starting dose may be selected after reviewing all available safety data from the respective single agent to ensure that the combined dose based on the available DLT data for the respective single agent meets EWOC criteria.

It should be noted that the initial experimental dose of anti-ENTPD 2 mAb1 in combination therapy will not exceed the highest tolerated dose during single agent dose escalation meeting the EWOC criterion according to BHLRM/BLRM.

The initial primary dose in combination therapy will be selected based on the initial dose of the anti-ENTPD 2 mAb1 single agent. If one or more primary doses need to be incremented during the combined dose escalation, the following conditions must be met:

cumulative exposure for selection of one or more primary doses over the first 14 days must meet EWOC criteria according to BLRM for guiding primary dose selection;

the experimental dose must meet the EWOC standard according to BLRM for guiding experimental dose selection of single agent against ENTPD2 mAb 1.

Temporary dose level

The anti-ENTPD 2 mAb1 alone and in combination will use temporary dose levels shown in the table below. All information of the previous dose will be evaluated and a decision will be made based on an analysis of all relevant data available from all dose levels before any dose is incremented. The recommended dose of subjects in the next cohort will be guided by BLRM/BHLRM in combination with EWOC guidelines.

Table 19 describes the initial and temporary dose levels of anti-ent pd2 mAb1 monotherapy that can be evaluated during this trial. In schedule 1, anti-ent pd2 mAb1 will be administered once every two weeks (Q2W) or every 4 weeks (Q4W), without a primary dose. If the data emerging from the study supports a less frequent dosing regimen, the Q4W dosing regimen can be tested without initial dose. Two additional dosing schedules outlined in schedule 2 and schedule 3 contained one or more primary doses below the experimental dose administered at C1D 15.

Schedule 2 (S2) contains a single initial dose at C1D1 and an experimental dose starting at C1D15, and continuing the dose in the Q2W regimen. The initial primary dose will be fixed at 100mg of anti-ENTPD 2 mAb 1. Subsequently, adjustments may be made based on the newly emerging security/tolerability, PK and/or PD data. The C1D1 dose will not exceed a dose level that meets the EWOC guidelines for BLRM defined for primary dose modifications. The temporary initial experimental dose was set to 300mg.

Schedule 3 (S3) contains two initial doses (first at C1D1 and second at C1D 8) and experimental doses starting at C1D15, and continuing the dose in the Q2W regimen. The initial primary dose of C1D8 will be the same as the primary dose of C1D 1. Adjustments may then be made based on the emerging safety/tolerability, PK and/or PD data, and whether further immune tolerance is deemed required prior to administration of the first experimental dose. The initial experimental dose of schedule 3 will not be higher than the highest experimental dose in schedule 2 that meets the EWOC standard according to BLRM. The cumulative dose administered during the first 14 days of cycle 1 will not exceed a dose that meets the EWOC guidelines for BLRM defined for primary dose modification. In addition, the C1D8 dose will be selected such that the cumulative dose C1D1 plus C1D8 will not be increased by more than 100% of the previously tested cumulative dose considered well tolerated. The same principle applies to any higher C1D8 dose.

Table 19, table 20, table 21, table 22 and table 23 outline the proposed temporary experimental dose levels for anti-ent pd2mAb1 on day 15 of cycle 1.

The anti-ENTPD 2mAb1 dosing strategy described above can be evaluated against the single agents anti-ENTPD 2mAb1, a combination of anti-ENTPD 2mAb1 with Stadalimumab (see Table 21), a combination of anti-ENTPD 2mAb1 with NIR178 (see Table 22) and a combination of anti-ENTPD 2mAb1 with anti-CD 73 Ab (see Table 23).

TABLE 19 temporary dose level timetable 1 for anti-ENTPD 2mAb1 (S1)

* Additional and/or intermediate/lower (dose levels-1, -2) dose levels may be added during the study. Enriched groups at any dose level below the MTD can be added to better understand safety, PK or PD.

TABLE 20 temporary dose level for anti-ENTPD 2mAb1 schedules 2 and 3 (S2 and S3)

* For S2, a primary dose of 100mg of anti-ENTPD 2mAb1 will be administered only on cycle 1 day 1. The primary dose may be modified if deemed necessary and based on safety and tolerability; for S3, an additional primary dose will be administered on cycle 1 day 8 and will initially be the same as cycle 1 day 1 dose. Subsequently, the cumulative dose on cycle 1, day 1 plus day 8 of cycle 1 may be incremented such that it is not >100% of the cumulative dose previously assessed as tolerable.

* As shown in this table 20, the initial experimental dose of anti-ENTPD 2mAb1 for the Q2W regimen was temporary and will begin on cycle 1, day 15. It does not increase by 100% over the previously tested and proven safe experimental dose. Table 21 depicts temporary dose levels of anti-ENTPD 2mAb1 in combination with swabber that can be assessed during this trial. There was no dose reduction of the swabber.

Table 21 temporary dose levels of anti-ENTPD 2mAb1 in combination with Stbada zumab

* Additional and/or intermediate/lower (dose level-1) dose levels/schedules may be added during the study. Enriched groups at any dose level below the MTD can be added to better understand safety, PK or PD.

* The initial dose of the combined anti-ENTPD 2mAb1 shown in this table was temporary. After reviewing all available safety data from both single agents and ensuring that the combined dose based on the available DLT data for both single agents meets EWOC criteria, a lower starting dose may be selected.

Table 22 describes temporary dose levels of anti-ent pd2mAb1 in combination with NIR178 that can be evaluated during this trial.

Table 22 temporary dose levels of anti-ent pd2mAb1 combined with NIR178

* The initial dose of the combined anti-ENTPD 2 mAb1 shown in this table was temporary. After reviewing all available safety data from both single agents and ensuring that the combined dose based on the available DLT data for both single agents meets EWOC criteria, a lower starting dose may be selected.

* Based on PK/PD and safety data, incremental doses of NIR178 (up to 480mg BID) can be considered.

Table 23 describes temporary dose levels of anti-ENTPD 2 mAb1 in combination with anti-CD 73 Ab that can be assessed during this trial.

TABLE 23 temporary dose level of combination of anti-CD 73 Ab and anti-ENTPD 2 mAb1

* Additional and/or intermediate/lower (-1 dose level) dose levels/schedules may be added during the study. Enriched groups at any dose level below the MTD can be added to better understand safety, PK or PD.

Incorporated by reference

All publications, patents, and accession numbers mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

Equivalents (Eq.)

While specific embodiments of the invention have been discussed, the above description is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the following claims. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification.

Claims

1. An antibody molecule that binds human CD73 for use in treating cancer in a subject, wherein the antibody molecule is administered in a ascending dosing regimen such that in a main phase an antibody molecule dose is administered in a main dose according to a main dosing period having a main dosing period frequency, the main phase being preceded by an initial phase in which the antibody molecule is administered at a dosing period frequency higher than the main dosing period frequency and via a split dose of the main dose, wherein in the initial phase within a period equal to the main dosing period the sum of the split doses should not exceed the amount of the main phase dose, wherein the antibody molecule comprises (i) a heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (VHCDR 1) amino acid sequence of SEQ ID NO:38, the VHCDR2 amino acid sequence of SEQ ID NO:36 and the VHCDR3 amino acid sequence of SEQ ID NO: 37; and (ii) a light chain variable region (VL) comprising the light chain complementarity determining region 1 (VLCDR 1) amino acid sequence of SEQ ID NO. 48, the VLCDR2 amino acid sequence of SEQ ID NO. 49 and the VLCDR3 amino acid sequence of SEQ ID NO. 50.

2. A method of treating cancer in a subject, the method comprising administering to the subject an antibody molecule that binds human CD73 in an amount effective to treat the cancer, wherein the antibody molecule is administered in a ascending dosing regimen such that in a main phase an antibody molecule dose is administered in a main dose according to a main dosing period having a main dosing period frequency, the main phase being preceded by an initial phase in which the antibody molecule is administered at a dosing period frequency that is higher than the main dosing period frequency and via a fractionated dose of the main dose, wherein the sum of the fractionated doses should not exceed the amount of the main phase dose in the initial phase over a period of time equal to the main dosing period, wherein the antibody molecule comprises (i) a heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (VHCDR 1) amino acid sequence of SEQ ID NO:38, the cdr2 amino acid sequence of SEQ ID NO:36, and the vh3 amino acid sequence of SEQ ID NO: 37; and (ii) a light chain variable region (VL) comprising the light chain complementarity determining region 1 (VLCDR 1) amino acid sequence of SEQ ID NO. 48, the VLCDR2 amino acid sequence of SEQ ID NO. 49 and the VLCDR3 amino acid sequence of SEQ ID NO. 50.

3. The antibody for use according to claim 1 or the method according to claim 2, wherein the antibody molecule that binds human CD73 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 44 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID No. 44 and/or a light chain variable region comprising the amino acid sequence of SEQ ID No. 55 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID No. 55.

4. The antibody for use of any one of claims 1 or 3 or the method of any one of claims 2 or 3, wherein the antibody molecule that binds human CD73 comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 46 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID No. 46, wherein X in SEQ ID No. 46 is K and the light chain comprises the amino acid sequence of SEQ ID No. 57 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID No. 57.

5. The antibody for use of any one of claims 1 or 3 to 4 or the method of any one of claims 2 to 4, wherein the antibody molecule that binds human CD73 comprises a heavy chain constant region of IgG4 and a light chain constant region of κ.

6. The antibody for use of any one of claims 1 or 3 to 5 or the method of any one of claims 2 to 5, wherein the antibody molecule that binds human CD73 comprises

7. The antibody for use of any one of claims 1 or 3 to 6 or the method of any one of claims 2 to 6, wherein in the initial phase within a period of time equal to the primary dosing period, the sum of the divided doses added equals the primary dose.

8. The antibody for use according to any one of claims 1 or 3 to 7 or the method according to any one of claims 2 to 7, such that the initial stage is used to prevent or reduce the incidence or severity of headache and/or migraine.

9. The antibody for use of any one of claims 1 or 3 to 8 or the method of any one of claims 2 to 8, wherein the primary dose is 600mg and/or the primary dose frequency is Q2W.

10. The antibody for use of any one of claims 1 or 3 to 9 or the method of any one of claims 2 to 9, wherein in the initial phase the antibody molecule is administered at the frequency of QW.

11. The antibody for use of any one of claims 1 or 3 to 10 or the method of any one of claims 2 to 10, wherein in the initial phase the sum of the divided doses administered over a period of time equal to the primary dosing period is equal to the primary dose, and the divided doses are administered first in a lower amount of the primary phase dose and then in an intermediate amount of the primary phase dose.

12. The antibody for use of any one of claims 1 or 3 to 11 or the method of any one of claims 2 to 11, wherein in the initial phase the divided dose is about 200mg and about 400mg and the divided dose is administered within two weeks.

13. The antibody for use according to any one of claims 1 or 3 to 12 or the method according to any one of claims 2 to 12, wherein the antibody molecule that binds human CD73 is administered as follows: in the initial phase, about 200mg was administered on day 1, about 400mg was administered on day 8, then the main phase was started on day 15, about 600mg was administered, after which Q2W administration was continued for about 600mg.

14. The antibody for use of any one of claims 1 or 3 to 13 or the method of any one of claims 2 to 13, wherein the antibody molecule is administered by IV infusion within 1 to 2 hours, the frequency of administration being dependent on the frequency of dosing periods for each stage.

15. The antibody for use of any one of claims 1 or 3 to 14 or the method of any one of claims 2 to 14, wherein the antibody molecule that binds human CD73 is administered in combination with one or more therapeutic agents or procedures.

16. The antibody for use of any one of claims 1 or 3 to 15 or the method of any one of claims 2 to 15, wherein the antibody molecule that binds human CD73 is administered in combination with triptan.

17. The antibody for use according to claim 16 or the method according to claim 16, wherein the triptan is selected from almotriptan, eletriptan, frotriptan, naratriptan, rizatriptan, sumatriptan, zolmitriptan, lamiditan, optionally in combination with another agent, such as sumatriptan in combination with naproxen sodium.

18. The antibody for use of any one of claims 15 to 17 or the method of any one of claims 15 to 17, wherein the antibody molecule that binds human CD73 is administered in combination with a PD-1 inhibitor.

19. The antibody for use of claim 18 or the method of claim 18, wherein the PD-1 inhibitor is selected from the group consisting of: stadalimumab, nivolumab, pembrolizumab, pidazumab, MEDI0680, REGN2810, TSR-042, PF-06801591 and AMP-224, preferably Stadalimumab.

20. The antibody for use of any one of claims 18 to 19 or the method of any one of claims 18 to 19, wherein the PD-1 inhibitor is an anti-PD-1 antibody molecule, wherein the anti-PD-1 antibody molecule is administered at a dose of about 400mg Q4W.

21. The antibody for use of any one of claims 15 to 20 or the method of any one of claims 15 to 20, wherein the antibody molecule that binds human CD73 is administered in combination with an adenosine A2AR antagonist.

22. The antibody for use of claim 21 or the method of claim 21, wherein

(ii) The adenosine A2AR antagonist is selected from the group consisting of: 5-bromo-2, 6-di- (1H-pyrazol-1-yl) pyrimidin-4-amine; (S) -7- (5-methylfuran-2-yl) -3- ((6- (((tetrahydrofuran-3-yl) oxy) methyl) pyridin-2-yl) methyl) -3H- [1,2,3] triazolo [4,5-d ] pyrimidin-5-amine; (R) -7- (5-methylfuran-2-yl) -3- ((6- (((tetrahydrofuran-3-yl) oxy) methyl) pyridin-2-yl) methyl) -3H- [1,2,3] triazolo [4,5-d ] pyrimidin-5-amine, or a racemate thereof; 7- (5-methylfuran-2-yl) -3- ((6- (((tetrahydrofuran-3-yl) oxy) methyl) pyridin-2-yl) methyl) -3H- [1,2,3] triazolo [4,5-d ] pyrimidin-5-amine; 6- (2-chloro-6-methylpyridin-4-yl) -5- (4-fluorophenyl) -1,2, 4-triazin-3-amine;

Preferably, wherein the adenosine A2AR antagonist is PBF509.

23. The antibody for use of any one of claims 21 to 22 or the method of any one of claims 21 to 22, wherein the adenosine A2AR antagonist is administered at a dose of about 80mg, about 160mg, 240mg or about 320mg, preferably wherein the adenosine A2AR antagonist is administered at a dose of about 240mg twice daily (BID).

24. The antibody for use of any one of claims 15 to 23 or the method of any one of claims 15 to 23, wherein the antibody molecule that binds human CD73 is administered in combination with an anti-human ENTPD2 antibody.

25. The antibody for use of claim 24 or the method of claim 24, wherein the antibody molecule that binds human CD73 is administered in combination with an anti-human ENTPD2 antibody comprising a VH comprising the VHCDR1 amino acid sequence of SEQ ID NO:401, the VHCDR2 amino acid sequence of SEQ ID NO:402, and the VHCDR3 amino acid sequence of SEQ ID NO: 403; and VL comprising the VLCDR1 amino acid sequence of SEQ ID NO. 414, the VLCDR2 amino acid sequence of SEQ ID NO. 415 and the VLCDR3 amino acid sequence of SEQ ID NO. 416.

26. The antibody for use of claim 24 or 25 or the method of claim 24 or 25, wherein the antibody molecule that binds human CD73 is administered in combination with an anti-human ENTPD2 antibody comprising a VH having the sequence of SEQ ID No. 410 and a VL having the sequence of SEQ ID No. 421.

27. The antibody for use of any one of claims 24 to 26 or the method of any one of claims 24 to 26, wherein the antibody molecule that binds human CD73 is administered in combination with an anti-human ENTPD2 antibody comprising a heavy chain having the sequence of SEQ ID No. 412 and a light chain having the sequence of SEQ ID No. 423.

28. The antibody for use of any one of claims 1 or 3 to 27 or the method of any one of claims 2 to 27, wherein the antibody molecule that binds human CD73 is administered in combination with swabber administered at about 400mg q4w and PBF509 administered at about 240mg BID as follows: in the initial phase about 200mg QW was administered, then about 400mg QW was administered, followed by about 600mg Q2W in the main phase.

29. The antibody for use of any one of claims 1 or 3 to 28 or the method of any one of claims 2 to 28, wherein the cancer is selected from non-small cell lung cancer, pancreatic ductal adenocarcinoma, triple negative breast cancer, microsatellite stabilized (MSS) colorectal cancer, metastatic castration-resistant prostate cancer, ovarian cancer or renal cell carcinoma.

30. The antibody for use of any one of claims 1 or 3 to 29 or the method of any one of claims 2 to 29, wherein the antibody molecule that binds human CD73 is in the form of a pharmaceutical composition comprising the antibody molecule of any one of claims 1 to 6 and a pharmaceutically acceptable carrier, excipient or stabilizer.

31. An antibody molecule that binds human ENTPD2 for use in treating cancer in a subject,

wherein the antibody molecule is administered on a ascending dosing regimen such that in a main phase the antibody molecule dose is administered in a main dose according to a main dosing period having a main dosing period frequency, the main phase being preceded by an initial phase in which the antibody molecule is administered at a dosing period frequency equal to or higher than the main dosing period frequency and via divided doses of the main dose,

wherein the sum of the divided doses added should not exceed the amount of the primary phase dose in the initial phase over a period of time equal to the primary dosing period.

32. A method of treating cancer in a subject, the method comprising administering to the subject an antibody molecule that binds human ENTPD2 in an amount effective to treat the cancer,

33. The antibody for use of claim 31 or the method of claim 32, wherein the antibody molecule comprises:

(i) A heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (VHCDR 1) amino acid sequence of SEQ ID No. 401, the VHCDR2 amino acid sequence of SEQ ID No. 402, and the VHCDR3 amino acid sequence of SEQ ID No. 403; and

(ii) A light chain variable region (VL) comprising the light chain complementarity determining region 1 (VLCDR 1) amino acid sequence of SEQ ID No. 414, the VLCDR2 amino acid sequence of SEQ ID No. 415, and the VLCDR3 amino acid sequence of SEQ ID No. 416.

34. The antibody for use of claim 31 or 33 or the method of claim 32 or 33, wherein the initial stage is for preventing or reducing the incidence or severity of Cytokine Release Syndrome (CRS).

35. The antibody for use of any one of claims 31 or 33-34 or the method of any one of claims 32-34, wherein the primary dose is 300mg, 600mg, 1200mg, or 2400mg and/or the primary dose frequency is Q2W.

36. The antibody for use of any one of claims 31 or 33-35 or the method of any one of claims 32-35, wherein in the initial phase the antibody molecule is administered at a frequency of QW or Q2W.

37. The antibody for use of any one of claims 31 or 33-36 or the method of any one of claims 32-36, wherein in the initial phase the fractionated dose is 100mg.

38. The antibody for use of any one of claims 31 or 33-37 or the method of any one of claims 32-37, wherein in the initial phase the antibody molecule is administered once or twice within two weeks.

39. The antibody for use of any one of claims 31 or 33-38 or the method of any one of claims 32-38, wherein the antibody molecule is administered as follows: in the initial phase, about 100mg is administered on day 1, then about 300mg is administered at day 15 starting the main phase, after which Q2W administration is continued for about 300mg.

40. The antibody for use of any one of claims 31 or 33-38 or the method of any one of claims 32-38, wherein the antibody molecule is administered as follows: in the initial phase, about 100mg was administered on day 1, about 100mg was administered on day 8, then the main phase was started on day 15, about 300mg was administered, after which Q2W administration was continued about 300mg.

41. The antibody for use of any one of claims 31 or 33-40 or the method of any one of claims 32-40, wherein the antibody molecule is administered intravenously to the subject as an infusion over 1 hour (up to 2 hours if clinically indicated).

42. The antibody for use of any one of claims 31 or 33-41 or the method of any one of claims 32-41, wherein the antibody molecule is administered in combination with one or more therapeutic agents or procedures.

43. The antibody or method for use of claim 42, wherein the antibody molecule is administered in combination with a PD-1 inhibitor.

44. The antibody or method for use of claim 43, wherein the PD-1 inhibitor is selected from the group consisting of: tirilizumab, stbadizumab, nivolumab, pembrolizumab, pierizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224.

45. The antibody or method for use of claim 43 or 44, wherein the PD-1 inhibitor is an anti-PD-1 antibody molecule, wherein the anti-PD-1 antibody molecule is administered at a dose of about 400mg Q4W.

46. The antibody or method for use of claim 42, wherein the antibody molecule is administered in combination with an adenosine A2AR antagonist.

47. The antibody or method of claim 46, wherein

preferably, wherein the adenosine A2AR antagonist is PBF509.

48. The antibody or method for use of claim 46 or 47, wherein the adenosine A2AR antagonist is administered at a dose of about 80mg, about 160mg, 240mg, or about 320mg, preferably wherein the adenosine A2AR antagonist is administered at a dose of about 160mg twice daily (BID).

49. The antibody or method for use of claim 42, wherein the antibody molecule is administered in combination with an anti-human CD73 antibody.

50. The antibody or method for use of claim 49, wherein the anti-human CD73 antibody comprises: (i) A heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (VHCDR 1) amino acid sequence of SEQ ID No. 38, the VHCDR2 amino acid sequence of SEQ ID No. 36, and the VHCDR3 amino acid sequence of SEQ ID No. 37; and (ii) a light chain variable region (VL) comprising the light chain complementarity determining region 1 (VLCDR 1) amino acid sequence of SEQ ID NO. 48, the VLCDR2 amino acid sequence of SEQ ID NO. 49 and the VLCDR3 amino acid sequence of SEQ ID NO. 50.

51. The antibody or method for use of claim 49, wherein the anti-human CD73 antibody comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 44 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO. 44 and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO. 55 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO. 55.

52. The antibody or method for use of claim 49, wherein the anti-human CD73 antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO. 46 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO. 46, wherein X in SEQ ID NO. 46 is K, and/or a light chain comprising the amino acid sequence of SEQ ID NO. 57 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO. 57.

53. The antibody for use of any one of claims 31 or 33-52 or the method of any one of claims 32-52, wherein the cancer is MSS colorectal cancer (CRC), cholangiocarcinoma (intrahepatic or extrahepatic), pancreatic cancer, esophageal-gastric junction (EGJ) cancer, or gastric cancer.

54. The antibody for use of any one of claims 31 or 33-53 or the method of any one of claims 32-53, wherein the antibody molecule that binds human ENTPD2 is in the form of a pharmaceutical composition comprising the antibody molecule of claim 33 and a pharmaceutically acceptable carrier, excipient or stabilizer.

55. The antibody for use of any one of claims 31 or 33-54 or the method of any one of claims 32-54, wherein the antibody molecule comprises:

A heavy chain variable region (VH) comprising SEQ ID No. 410 or a sequence having at least about 95% or more identity thereto and a light chain variable region (VL) comprising SEQ ID No. 421 or a sequence having at least about 95% or more identity thereto.

56. The antibody for use of any one of claims 31 or 33-54 or the method of any one of claims 32-54, wherein the antibody molecule comprises:

a heavy chain comprising SEQ ID No. 412 or a sequence having at least about 95% or more identity thereto and a light chain comprising SEQ ID No. 423 or a sequence having at least about 95% or more identity thereto.