EA043570B1 - ANTI-CD73 ANTIBODIES AND THEIR APPLICATIONS - Google Patents

Description

State of the art

CD73 (cluster of differentiation 73), also known as 5'-nucleotidase (5'-NT) or ecto-5'nucleotidase, is an enzyme that converts AMP to adenosine. CD73 catalyzes the formation of extracellular adenosine, which contributes to the formation of the immunosuppressive environment of the tumor. CD73 is overexpressed in stromal cells and tumor cells of various types, as well as in Treg cells, M2 Μφ and myeloid-derived suppressor cells (MDSC).

Preclinical data indicate that CD73 inhibition prevents adenosine-mediated lymphocyte suppression, increases CD8+ effector cell activity, and reduces both MDSC and Tregs activity. Several anti-CD73 antibodies are currently being developed as potential anticancer agents, but none have been approved for clinical use.

Short description

The present invention provides an anti-CD73 antibody having high binding affinity to human CD73 proteins and high activity to inhibit the enzymatic activity of CD73 on its own or on a cell. In addition, the binding of these antibodies may induce the internalization of CD73 by tumor cells, resulting in a further decrease in CD73 activity at the cell surface. Moreover, surprisingly, monovalent units, such as Fab fragments of these antibodies, have activity comparable to that of whole antibodies. At the same time, known antibodies against CD73, for example, MEDI-9447, developed by Medimmune, do not have such characteristics. Likewise, unlike MEDI-9447 and 11F11, which require a high density of cell surface expression of CD73 to exert their inhibitory effect, the antibodies of the present invention can achieve complete inhibition of CD73 at varying levels of cell surface expression. It is believed that these unexpected properties of the antibodies of the present invention are at least in part due to a different binding site for the CD73 protein. Unlike MEDI-9447 and 11F11, which bind the N-terminal domains of the CD73 protein, the antibodies of the present invention bind to the C-terminal domains and, more specifically, to amino acid residues Y345, D399, E400, R401 and R480. The properties of the antibodies of the present invention make them excellent candidates for therapeutic and diagnostic applications.

Thus, in accordance with one embodiment of the present invention there is provided an isolated antibody or antibody fragment having specificity for the human CD73 protein and binding to one or more amino acid residues selected in the C-terminal region of the human CD73 protein. The C-terminal portion of the human CD73 protein is known in the art to contain 238 amino acid residues, starting at residue 337, as shown in SEQ ID NO: 61.

In some embodiments, the antibody or fragment thereof binds to one or more of the C-terminal domains of the human CD73 protein. In some embodiments, the antibody or fragment thereof binds to one of the amino acid residues selected from the group consisting of or more of Y345, D399, E400, R401, and R480 of the human CD73 protein. In some embodiments, the antibody or fragment thereof binds to at least two of these amino acid residues.

In one embodiment, the present invention provides an anti-CD73 antibody or fragment thereof comprising a CDR1 VH of SEQ ID NO: 1, a CDR2 VH of SEQ ID NO: 2, a CDR3 VH of SEQ ID NO: 3, a CDR1 VL of SEQ ID NO: 4 , CDR2 VL according to SEQ ID NO: 5 and CDR3 VL according to SEQ ID NO: 6. In some embodiments, the antibody or fragment thereof may further comprise a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. In some embodiments, the light chain constant region may be a kappa or lambda chain constant region. In some embodiments, the antibody or fragment thereof is of the IgG, IgM, IgA, IgE, or IgD isotype, or more specifically, IgG1, IgG2, IgG3, or IgG4.

In some embodiments, the antibody or fragment thereof is a chimeric antibody, a humanized antibody, or a fully human antibody. In one embodiment, the antibody or fragment thereof is a humanized antibody.

In some embodiments, the humanized or human antibody or fragment thereof comprises a heavy chain variable region comprising one or more amino acid residues selected from the group consisting of: (a) Thr at position 30, (b) Lys at position 44, (c ) Met at position 48, (d) Ile at position 67 and (e) Arg at position 71 according to Kabat numbering and combinations thereof. In some embodiments, the humanized or human antibody or fragment thereof comprises a light chain variable region comprising one or more amino acid residues selected from the group consisting of: (a) Ser at position 5, (b) Pro at position 46, (c ) Tip at position 47, (d) Ser at position 70 and (f) Tyr at position 71 according to Kabat numbering and their combinations.

Examples of anti-CD73 antibodies or fragments thereof include antibodies or antibody fragments comprising a heavy chain variable region containing one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 7 and 9-13, or a peptide having at least 90 % identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and 9-13. In some embodiments, the heavy chain variable region

- 1 043570 contains the amino acid sequence of SEQ ID NO: 7 or 9. In some embodiments, the antibody or fragment thereof contains a light chain variable region containing one or more amino acid sequences selected from the group consisting of SEQ ID NO: 8, 15-20 and 22-24, or a peptide having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 15-20 and 22-24. In some embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8.

As shown in Experimental Example 7, the CDR regions may contain the addition, deletion or substitution of certain amino acids while maintaining or even improving the properties of the anti-CD73 antibody. Accordingly, some embodiments provide an isolated antibody or antibody fragment, wherein the antibody or antibody fragment has specificity for the human CD73 protein and contains: (a) a CDR1 VH (heavy chain variable region CDR1 region) of SEQ ID NO: 1 or a variant of SEQ ID NO: 1 containing a single substitution, deletion or insertion at position 1, 2 or 3 in SEQ ID NO: 1; (b) CDR2 VH according to SEQ ID NO: 2 or a variant of SEQ ID NO: 1 containing a single substitution, deletion or insertion at position 6, 7 or 8 in SEQ ID NO: 2; (c) CDR3 VH according to SEQ ID NO: 3 or a variant of SEQ ID NO: 3 containing a single substitution, deletion or insertion at position 7 or 8 in SEQ ID NO: 3; (d) CDR1 VL according to SEQ ID NO: 4 or a variant of SEQ ID NO: 4 containing a single substitution, deletion or insertion at position 3 or 4 in SEQ ID NO: 4; (f) CDR2 VL according to SEQ ID NO: 5 and (f) CDR3 VL according to SEQ ID NO: 6 or a variant of SEQ ID NO: 6 containing a single substitution, deletion or insertion at position 1, 2, 3 or 4 in SEQ ID NO: 6.

In some embodiments, an isolated antibody or fragment thereof is provided, wherein the antibody or antibody fragment is specific for the human CD73 protein and contains: (a) a CDR1 VH of SEQ ID NO: 1 or a variant of SEQ ID NO: 1 containing a single substitution at position 1, 2 or 3 in SEQ ID NO: 1; (b) CDR2 VH according to SEQ ID NO: 2 or a variant of SEQ ID NO: 1 containing a single substitution at position 6, 7 or 8 in SEQ ID NO: 2; (c) CDR3 VH according to SEQ ID NO: 3 or a variant of SEQ ID NO: 3 containing a single substitution at position 7 or 8 in SEQ ID NO: 3; (d) CDR1 VL according to SEQ ID NO: 4 or a variant of SEQ ID NO: 4 containing a single substitution at position 3 or 4 in SEQ ID NO: 4; (e) CDR2 VL as defined in SEQ ID NO: 5 and (f) CDR3 VL as defined in SEQ ID NO: 6 or a variant of SEQ ID NO: 6 containing a single substitution at position 1, 2, 3 or 4 in SEQ ID NO: 6.

In some embodiments, SEQ ID NO: 1 is selected from the group consisting of SEQ ID NO: 26-29. In some embodiments, SEQ ID NO: 2 is selected from the group consisting of SEQ ID NO: 30-36. In some embodiments, SEQ ID NO: 3 is selected from the group consisting of SEQ ID NO: 37-41. In some embodiments, SEQ ID NO: 4 is selected from the group consisting of SEQ ID NO: 42-45. In some embodiments, SEQ ID NO: 6 is selected from the group consisting of SEQ ID NO: 46-56.

Additionally, some embodiments provide a composition comprising an antibody or antibody fragment of the present invention and a pharmaceutically acceptable carrier. In addition, an isolated cell is provided containing one or more polynucleotides encoding an antibody or fragment thereof according to the present invention.

In yet another embodiment, the present invention provides a method of treating cancer in a patient in need thereof, comprising administering to the patient an antibody or fragment thereof of the present invention. In some embodiments, the cancer is selected from the group consisting of bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma , pancreatic cancer, prostate cancer and thyroid cancer. In some embodiments, the cancer is a solid tumor.

In some embodiments, the method further includes administering a second cancer treatment agent to the patient. In some embodiments, the second cancer treatment agent is an immune checkpoint inhibitor. In some embodiments, the inhibitor inhibits the expression or activity of programmed cell death protein-1 (PD-1), programmed cell death protein ligand-1 (PD-L1), cytotoxic T lymphocyte associated protein-4 (CTLA-4) , lymphocyte activation protein-3 (LAG-3), or combinations thereof. In some embodiments, the inhibitor is an anti-PD-1 or anti-PD-L1 antibody. In some embodiments, the inhibitor is selected from the group consisting of pembrolizumab, nivolumab, J43, RMP1-14, atezolizumab, ipilimumab, and combinations thereof.

In one embodiment, there is provided a method of treating cancer in a patient in need thereof, comprising: (a) treating a T cell in vitro with an antibody or fragment thereof according to the present invention; and (b) administering the treated T cell to the patient. In some embodiments, the method further includes isolating the T cell from the individual prior to step (a).

In some embodiments, the T cell is isolated from the patient. In some embodiments, the T cell is isolated from a non-patient donor individual. In some embodiments, the T cell is a tumor-infiltrating T lymphocyte, a CD4+ T cell, a CD8+ T cell, or a combination thereof.

In addition, in yet another embodiment, a method for detecting CD73 expression in a sample is provided, comprising contacting the sample with an antibody or fragment thereof of the present invention under conditions where the antibody or fragment thereof binds to CD73 and detecting the binding, which indicates expression of CD73 in the sample. Additionally, in one embodiment, a method is provided for identifying a patient with cancer suitable for treatment with an anti-CD73 antibody therapeutic agent, comprising isolating a cell from the patient with cancer and detecting the presence of CD73 protein using the antibody or fragment thereof of the present invention.

Brief description of the figures

In fig. 1 shows the binding of the 101-Mu antibody to recombinant human CD73 protein.

In fig. 2 shows the binding of antibody 101-Mu to recombinant human CD73 protein on the surface of a human ovarian cancer cell.

In fig. Figure 3 shows the kinetics of binding of the 101-Mu antibody to the recombinant CD73 protein.

In fig. 4 shows that antibody 101-Mu inhibits the enzymatic activity of CD73.

In fig. Figure 5 shows that the 101-Mu antibody inhibits the enzymatic activity of CD73 on the cell surface.

In fig. Figure 6 shows that 101-Mu reverses AMP-mediated suppression of CD4+ T cells, as indicated by IFN-γ production.

In fig. Figure 7 shows the binding kinetics of humanized antibodies.

In fig. 8 shows that humanized antibodies bind to cell surface proteins CD73.

In fig. 9 shows that humanized antibodies inhibited the enzymatic activity of CD73 proteins.

In fig. 10 shows that the humanized antibodies inhibited the enzymatic activity of cell surface proteins CD73.

In fig. 11 shows that humanized antibodies reverse AMP-mediated suppression of CD4+ T cells, as indicated by IFN-γ production.

In fig. 12 shows efficient binding of Hu101-28 to soluble CD73 and cell surface CD73.

In fig. 13 shows that binding of Hu101-28 to CD73 effectively blocks the enzymatic activity of CD73.

In fig. 14 shows that Hu101-28 non-competitively inhibits CD73 activity.

In fig. 15 shows that binding of Hu101-28 to CD73 induces CD73 internalization.

In fig. 16 shows that Hu101-28 reverses AMP-mediated suppression of the T cell response.

In fig. 17 shows that Hu101-28 reverses the suppression of T cell responses mediated by CD73+ tumor cells.

In fig. 18 shows staining images showing in vivo inhibition of CD73 enzyme by Hu101-28 in tumors in the A375 xenograft model.

In fig. 19 shows that Hu101-28 demonstrated efficacy as monotherapy in a tumor xenograft model.

In fig. 20 shows that Hu101-28 synergizes with anti-PD-L1 antibody in inhibiting tumor growth.

In fig. 21 shows binding and inhibition of cynomolgus CD73 activity by Hu101-28.

In fig. 22 shows that Hu101-28 does not compete with MEDI-9447 for binding to CD73.

In fig. Figure 23 shows a list of amino acid residues of CD73 that interact with Hu101-28.

In fig. 24 illustrates the epitopes of Hu101-28 and MEDI-9447.

In fig. 25 shows the higher activity of Hu101-28 compared to MEDI-9447.

In fig. 26 showed that Hu101-28 effectively inhibited CD73 on cells with different levels of CD73 expression.

Detailed description

Definitions

It should be noted that the singular forms referring to one or more objects (corresponding to the articles a and and in the English text), for example, antibody, represent one or more antibodies. In fact, the singular forms, as well as the terms one or more and at least one, can be used interchangeably herein.

As used herein, the term polypeptide is intended to include both the singular form polypeptide and the plural form polypeptides and refers to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term polypeptide refers to any chain or chains of two or more amino acids and does not refer to a product of a specific length. Thus, the definition of polypeptide includes peptides, dipeptides, oligopeptides, protein, chain of amino acids, or any other term used with respect to a chain or chains of two or more amino acids, and the term polypeptide may be used in place of any of these terms or in on equal footing with them. In addition, the term polypeptide is intended to refer to the products of post-expression modification of the polypeptide, including, without limitation, glycosylation, acetylation, phosphorylation, amidation, modification with known protecting/blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. The polypeptide may be isolated from a natural biological source or may be produced using recombinant technology, but it is not necessarily translated from a specialized nucleotide sequence. It can be obtained by any method, including through chemical synthesis.

As used herein, the term isolated is used to refer to cells, nucleic acids, for example, DNA or RNA, refers to molecules separated from other DNA or RNA, respectively, present in the natural source of the specified macromolecule. The term as used herein also refers to a nucleic acid or peptide substantially free of cellular material, viral material or culture medium when produced using recombinant DNA technology, or chemical precursors or other chemicals in chemical synthesis. In addition, the term isolated nucleic acid is intended to include fragments of nucleic acids that do not naturally occur as fragments and would not be present in their natural state. Additionally, the term isolated as used herein refers to cells or polypeptides isolated from other cellular proteins or tissues. Isolated polypeptides are intended to include both purified and recombinant polypeptides.

As used herein, the term recombinant per se refers to polypeptides or polynucleotides and refers to a form of polypeptide or polynucleotide not found in nature, a non-limiting example of which can be created by combining polynucleotides or polypeptides not normally found together.

The terms homology or identity or similarity refer to the sequence similarity of two peptides or two nucleic acid molecules. Homology can be determined by comparing positions within each sequence that can be aligned for comparison. If the position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at this position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An unrelated or non-homologous sequence has less than 40% identity, preferably less than 25% identity, to one of the sequences of the present invention.

A polynucleotide or region of a polynucleotide (or polypeptide or region of a polypeptide) characterized by some percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of identity sequence relative to another sequence means that in an alignment, the specified percentage of bases (or amino acids) is the same when comparing the two specified sequences. This alignment and percentage homology or sequence identity can be determined using software known in the art, for example, as described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. The default settings are preferably used for alignment. One program for alignment is BLAST using default parameters. In particular, the BLASTN and BLASTP programs using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides are polynucleotides having the above-mentioned specific percentage homology and encoding polypeptides having the same or similar biological activity.

The term equivalent nucleic acid or polynucleotide refers to a nucleic acid having a nucleotide sequence with a certain degree of homology or sequence identity to the nucleotide sequence of the specified nucleic acid or its complementary strand. A double-stranded nucleic acid homolog is intended to include nucleic acids having a nucleotide sequence exhibiting a certain degree of homology to each other or to complementary strands of each other. In one aspect, nucleic acid homologues are capable of hybridizing with said nucleic acid or a complementary strand thereof. Likewise, an equivalent polypeptide refers to a polypeptide characterized by a certain degree of homology or sequence identity with respect to the amino acid sequence of the reference polypeptide. In some aspects, the sequence identity is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some aspects, an equivalent polypeptide or polynucleotide

- 4 043570 contains one, two, three, four or five additions, deletions, substitutions and combinations thereof compared to the reference polypeptide or polynucleotide. In some aspects, the equivalent sequence retains the activity (eg, epitope binding) or structure (eg, ionic bonding) of the reference sequence.

Hybridization reactions can be performed under varying stringency conditions. In general, the low stringency hybridization reaction is performed at approximately 40° C. in approximately 10xSSC or equivalent ionic strength/temperature solution. Moderate stringency hybridizations are typically performed at about 50°C in about 6xSSC, and high stringency hybridization reactions are typically performed at 60°C in about 1xSSC. Hybridization reactions can also be performed under physiological conditions well known to one skilled in the art. A non-limiting example of physiological conditions is the temperature, ionic strength, pH and Mg ²⁺ concentration typically found in a cell.

A polynucleotide consists of a specific sequence of four nucleic bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) instead of thymine if the polynucleotide is RNA. Thus, the term polynucleotide sequence is the literal representation of a polynucleotide molecule. This letter representation can be entered into databases on a computer containing a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. The term polymorphism refers to the simultaneous existence of more than one form of a gene or its fragment. A gene fragment for which there are at least two different forms, i.e. two different nucleotide sequences are called a polymorphic region of a gene. A polymorphic region may be a single nucleotide whose identity differs between alleles.

The terms polynucleotide and oligonucleotide are used interchangeably and refer to the polymeric form of nucleotides of any length, both deoxyribonucleotides and ribonucleotides or their analogues. Polynucleotides can have any three-dimensional structure and perform any known or unknown function. The following are non-limiting examples of polynucleotides: gene or gene fragment (e.g., probe, primer, EST or SAGE marker), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, microRNA , recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleotide probes and primers. The polynucleotide may contain modified nucleotides, such as methylated nucleotides, and nucleotide analogues. If present, modifications to the nucleotide structure can be made before or after assembly of the polynucleotide. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide may also be modified after polymerization, for example, by conjugation with a tag component. The term also refers to both double- and single-chain molecules. Unless otherwise specified or required, any embodiment of the present invention that is a polynucleotide includes both the double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.

The term encode, with respect to polynucleotides, refers to a polynucleotide that is said to encode a polypeptide if, in its native form or by manipulation using methods well known to those skilled in the art, it can be transcribed and/or translated to produce mRNA for polypeptide and/or fragment thereof. The antisense strand is the complementary strand of such a nucleic acid, and the coding sequence can be derived from it.

As used herein, the term antibody or antigen-binding polypeptide refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen binding fragment or single chain of an antibody. Thus, the term antibody includes any protein or peptide containing molecule containing at least a fragment of an immunoglobulin molecule having antigen binding biological activity. Examples of such fragments include a heavy or light chain complementarity determining region (CDR) or a ligand binding fragment thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework region (FR), or any fragment thereof, or at least one fragment of a binding protein, but is not limited to them.

The terms antibody fragment or antigen binding fragment as used herein are an antibody fragment, for example, F(ab') ₂ , F(ab) ₂ , Fab', Fab, Fv, scFv and the like. Regardless of the structure, the antibody fragment binds to the same antigen that the intact antibody recognizes. The term antibody fragment includes aptamers, spiegelmers and diabodies. The term antibody fragment also includes any synthetic or genetically engineered protein that acts similarly to an antibody by binding to a specific antigen to form a complex.

- 5 043570

The term single chain variable fragment or scFv refers to a fusion protein of the heavy (VH) and light chain ( _VL ) variable regions of immunoglobulins. In some aspects, these regions are connected by a short peptide linker containing from ten to about 25 amino acids. This linker is typically rich in glycine (for flexibility) and serine or threonine (for solubility), and can connect the N-terminus of VH to the C-terminus of _VL or vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of constant regions and the introduction of a linker. ScFv molecules are known in the art and are described, for example, in US Pat. No. 5,892,019.

The term antibody covers different classes of polypeptides, which may differ from a biochemical point of view. Those skilled in the art will understand that heavy chains are divided into gamma, mu, alpha, delta or epsilon chains (γ,μ,α,δ,ε), within which subclasses exist (e.g. γ1-γ4) . It is the nature of a given chain that determines the class of the antibody, eg IgG, IgM, IgA, IgG or IgE, respectively. Subclasses (isotypes) of immunoglobulins, for example, IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgG ₅ , etc. have been well studied and are known to be characterized by functional specialization. Modified versions of each of these classes and isotypes will readily be recognized by one skilled in the art in light of the present invention and are accordingly within the scope of the present invention. All classes of immunoglobulins are expressly included within the scope of the present invention, and the following discussion will generally focus on the IgG class of immunoglobulin molecules. With respect to IgG, a standard immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons, and two identical heavy chain polypeptides with a molecular weight of approximately 53,000-70,000. These four chains are typically linked by disulfide bonds in a Y'' configuration, where the light chains are grouped with the heavy chains, starting at the neck of the Y'' configuration and continuing throughout the variable region.

Antibodies, antigen binding polypeptides, variants or derivatives thereof according to the present invention include polyclonal, monoclonal, multispecific, humanized, primatized or chimeric antibodies or human antibodies, single chain antibodies, epitope binding fragments, for example, Fab, Fab' and F(ab') ₂ , Fd , Fv, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), VK or VH domain-containing fragments, Fab expression library-produced fragments, and anti-idiotypic (anti-Id) antibodies (including, e.g. anti-Id antibodies to, but not limited to, the LIGHT antibodies described herein. The immunoglobulins or antibody molecules of the present invention may be of any type (eg, IgG, IgE, IgM, IgD, IgA and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecules.

Light chains are classified as kappa or lambda (K, λ). The heavy chain of each class can bind to a kappa or lambda light chain. In general, the light and heavy chains are covalently linked to each other, and the tail portions of the two heavy chains are linked to each other through covalent disulfide bonds or non-covalent bonds if the immunoglobulins are produced using hybridomas, B cells or recombinant host cells. In the heavy chain, the amino acid sequence runs from the N-terminal region at the forked ends of the Y configuration to the C-terminal region at the bottom of each chain.

Both the light and heavy chains are divided into regions that share structural and functional homology. The terms constant and variable are used in a functional sense. In this regard, it should be taken into account that the variable domains of the fragments of both the light (VK) and heavy (VH) chains determine antigen recognition and specificity. Conversely, the light chain (CK) and heavy chain constant domains (CH1, CH2 or CH3) impart important biological properties to the antibody, such as secretion, ability to cross the placental barrier, Fc receptor binding, complement binding, etc. According to conventional numbering, the numbers of constant region domains increase as they become more distal from the antigen binding site or N-terminal region of the antibody. The N-terminal fragment is the variable region, and the C-terminal fragment is the constant region; The CH3 and CK domains actually constitute the C-terminus of the heavy and light chain, respectively.

As stated above, the variable region allows the antibody to specifically recognize and specifically bind epitopes of antigens. Those. The VK domain and VH domain or set of complementarity determining regions (CDRs) of an antibody together form a variable region that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms an antigen-binding site present at the end of each arm of the Y-shaped structure. More specifically, the antigen binding site is defined by three CDRs on each of the VH and VK chains (ie, CDR-H1, CDR-E2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3). In some cases, such as some immunoglobulin molecules derived from camelid species or engineered from camelid immunoglobulins, the complete immunoglobulin molecule may consist of only heavy chains and no light chains. See, for example, Hamers-Casterman et al, Nature 363:446-448 (1993).

Natural antibodies contain six complementarity determining regions, or CDRs.

- 6 043570 present in each antigen binding domain are short non-contiguous sequences of amino acids specifically arranged to form the antigen binding domain of the antibody, since the antibody is designed to form a three-dimensional configuration in an aqueous environment. The remaining amino acids in the antigen-binding domains, called framework regions, show less intermolecular variability. The framework regions mostly adopt a β-sheet conformation, and the CDRs form loops that connect to the P-sheet and in some cases form part of its structure. Thus, the framework regions act as a scaffold to position the CDRs in the desired orientation through non-covalent interactions between the chains. The antigen binding domain formed by the placed CDRs defines a surface complementary to the epitope of the immunologically reactive antigen. The complementary surface stimulates non-covalent binding of the antibody to its epitope. The amino acids constituting the CDRs and framework regions, respectively, can be readily identified by one skilled in the art for any given heavy or light chain variable region because they are precisely defined (see Sequences of Proteins of Immunological Interest, Kabat, E., et al. U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol, 196:901-917(1987)).

Where there are two definitions of a term used and/or accepted in the art, the definition of that term as used herein is intended to include all such meanings unless otherwise expressly stated. A specific example is the use of the term complementarity determining region (CDR) to describe non-contiguous antigen-binding sites within the variable region of heavy and light chain polypeptides. This specific region is described in Kabat et al, U.S. Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and Chothia et al., J. Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference in their entirety.The Kabat and Chothia CDR definitions include overlapping sets of amino acid residues when compared to each other However, it is intended that the application of any definition to the CDR of an antibody or variants thereof is within the scope of the term as defined and used herein.The corresponding amino acid residues constituting the CDR as defined in each of the above references are given below in the table for comparison.The exact residue numbers that make up a particular CDR vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues constitute a particular CDR given the amino acid sequence of the antibody variable region.

Kabat Chothia CDR-H1 31-35 26-32 CDR-H2 50-65 52-58 CDR-H3 95-102 95-102 CDR-L1 24-34 26-32 CDR-L2 50-56 50-52 CDR-L3 89-97 91-96

Kabat et al. also specified a numbering system for variable domain sequences applicable to any antibody. One skilled in the art can uniquely assign this Kabat numbering system to any variable domain sequence, without regard to experimental data other than the sequence itself. As used herein, Kabat numbering refers to the numbering system defined in Kabat et al., U.S. Dept. of Health and Human Services, Sequence of Proteins of Immunol ogical Interest (1983).

In addition to the above table, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins at approximately amino acid position 31 (i.e., approximately 9 residues after the first cysteine residue), contains approximately 5-7 amino acids, and ends at the next residue tryptophan. CDR-H2 begins at the fifteenth residue after the end of CDR-H1, contains approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins approximately at the thirty-third amino acid residue after the end of CDR-H2; contains 3-25 amino acids and ends with the sequence W-G-X-G, where X is any amino acid. CDR-L1 begins at approximately residue position 24 (ie, after the cysteine residue); contains approximately 10-17 amino acids and ends at the next tryptophan residue. CDR-L2 begins approximately sixteenth residue after the end of CDR-L1 and contains approximately 7 residues. CDR-L3 begins approximately thirty residues after the end of CDRL2 (ie, after the cysteine residue), contains approximately 7-11 residues, and ends with the sequence F or W-G-X-G, where X is any amino acid.

The antibodies described herein can be obtained from any animal, including birds and mammals. The antibodies are preferably human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken. In yet another embodiment, the variable region may be derived from cartilaginous fish (eg, sharks).

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As used herein, the term heavy chain constant region includes amino acid sequences derived from the immunoglobulin heavy chain. A heavy chain constant region-containing polypeptide contains at least one of a CH1 domain, a hinge (eg, upper, middle and/or lower hinge regions) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen binding polypeptide for use in the present invention may comprise a polypeptide chain comprising a CH1 domain; a polypeptide chain containing a CH1 domain, at least a fragment of a hinge domain, and a CH2 domain; a polypeptide chain containing a CH1 domain and a CH3 domain; a polypeptide chain containing a CH1 domain, at least a fragment of a hinge domain, and a CH3 domain, or a polypeptide chain containing a CH1 domain, at least a fragment of a hinge domain, a CH2 domain and a CH3 domain. In yet another embodiment, the polypeptide of the present invention comprises a polypeptide chain comprising a CH3 domain. In addition, an antibody for use in the present invention may lack at least a portion of the CH2 domain (eg, the CH2 domain may be completely or partially absent). As stated above, one skilled in the art will appreciate that the heavy chain constant region can be modified such that its amino acid sequence differs from the native immunoglobulin molecule.

The heavy chain constant region of the antibody described herein may be derived from various immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide may comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In yet another example, the heavy chain constant region of a polypeptide may comprise a hinge region derived in part from an IgG1 molecule and in part derived from an IgG3 molecule. In yet another example, the heavy chain fragment may comprise a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG4 molecule.

As used herein, the term light chain constant region includes amino acid sequences derived from the light chain of an antibody. The light chain constant region preferably contains at least one of a kappa constant domain or a lambda constant domain.

A light chain-heavy chain pair refers to a combination of a light chain and a heavy chain that can form a dimer through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

As stated previously, the subunit structure and three-dimensional configuration of the constant regions of the various classes of immunoglobulins are well known. As used herein, the term VH domain includes the N-terminal variable domain of an immunoglobulin heavy chain, and the term CH1 domain includes the first (proximal to the N-terminus) domain of the immunoglobulin heavy chain constant region. The CH1 domain is located adjacent to the VH domain and on the N-terminal side of the heavy chain hinge region of the immunoglobulin molecule.

As used herein, the term CH2 domain includes a fragment of a heavy chain molecule that extends, for example, from approximately residue 244 to residue 360 of an antibody according to conventional numbering schemes (residues 244-360, Kabat numbering system; and residues 231-340, EU numbering system; see Kabat et al, U.S. Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983).The CH2 domain is unique in that it is not directly coupled to another domain, but instead is between two CH2 domains of the intact native molecule IgG contains two N-linked branched carbohydrate chains.In addition, it is well documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues.

As used herein, the term hinge region includes the portion of the heavy chain molecule connecting the CH1 domain to the CH2 domain. This hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge regions can be divided into three distinct domains: the upper, middle and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).

As used herein, the term disulfide bond includes a covalent bond formed between two sulfur atoms. The amino acid cysteine contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In most natural IgG molecules, the CH1 and CK regions are connected by a disulfide bond, and the two heavy chains are connected by two disulfide bonds at positions corresponding to residues 239 and 242 according to the Kabat numbering system (positions 226 or 229 according to the EU numbering system).

As used herein, the term chimeric antibody means any antibody in which the immunoreactive region or site is derived from or originates from a first animal species and the constant region (which may be intact, partial, or modified in accordance with the present invention) is derived from an antibody from another animal species. In some embodiments, the target binding region or site is of non-human origin (eg, derived from a mouse or primate) and the constant region is derived from a human antibody.

Herein, the percentage of humanization is calculated by determining the number of amino acid differences in the framework region (ie, differences in non-CDR regions) between the humanized domain and the germline domain, subtracting this amount from the total number of amino acids, and then dividing it by the total number of amino acids and multiplying by 100.

The words specifically bind or have specificity to generally mean that the antibody binds to an epitope via its antigen binding domain and that said binding involves some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to specifically bind to an epitope if it binds to said epitope through its antigen binding domain more readily than if it binds to a random, nonspecific epitope. The term specificity is used herein to assess the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody A may be said to have higher specificity for a given epitope than antibody B, or antibody A may be said to bind to epitope C with higher specificity than to the related epitope D.

As used herein, the terms treat and treatment refer to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow (attenuate) an undesirable physiological change or disorder, such as the progression of cancer. Beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction in disease severity, stabilized (i.e., not worsening) disease state, delay or slowing of disease progression, alleviation or temporary relief of disease state, and remission (partial or complete), as detectable (detectable) and undetectable (undetectable). The term treatment can also mean an increase in life expectancy compared to life expectancy without treatment. Persons in need of treatment include persons already suffering from a condition or disorder, as well as persons prone to conditions or disorders, or persons in whom the condition or disorder should be prevented.

The term subject or individual or animal or patient or mammal means any subject, particularly a mammalian subject, for whom diagnosis, prognosis or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals and zoo animals, sporting animals or pets, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, etc. .d.

As used herein, phrases such as patient in need of treatment or subject in need of treatment include subjects, for example, mammalian subjects, for whom administration of an antibody or composition according to the present invention may benefit, for example, detection, diagnostic procedure and/or treatment.

Antibodies against CD73

The present invention provides anti-CD73 antibodies with high affinity and inhibitory activity against human CD73 protein. These antibodies can effectively bind to both free CD73 and CD73 on the surface of cells. Binding to the cell surface protein CD73 can cause internalization; this leads to decreased expression of CD73 protein on the cell surface, which reduces extracellular adenosine levels and weakens the immunosuppressive environment of the tumor. Additionally, because these antibodies do not compete with the AMP substrate for binding to the active site of CD73, but act allosterically or through other noncompetitive mechanisms, these antibodies do not interfere with the binding of CD73 to these endogenous AMP substrates, limiting the potential adverse effects of these antibodies.

In addition, as demonstrated in the experimental examples, these antibodies have some unique properties not observed in known anti-CD73 antibodies, such as MEDI-9447 from Medimmune. As shown in Example 12, although MEDI-9447 and 11F11 bind to the N-terminal domains of the CD73 protein, the target amino acids of the present antibodies (eg, Y345, D399, E400, R401 and R480) are located in the C-terminal domains.

The CD73 enzyme consists of a dimer of two identical 70 kDa subunits linked by a glycosylphosphatidylinositol linkage to the outer side of the plasma membrane. Crystal structures of dimeric human CD73 reveal an intense conformational transition between the open and closed forms of the enzyme, which is required for proper enzyme function. The contact surface during dimerization is formed by C-terminal domains. If the C-terminal domains are involved in other types of binding, it is speculated that dimerization and/or conformational transition may be blocked as a result of inhibition of CD73 activity. In contrast, binding to the antibody at the N-terminal domain may not lead to such effects.

Thus, it is expected that if the antibodies of the present invention bind the CD73 protein at its C-terminal domains, they can block dimerization of the protein and effectively inhibit its activity. Thus, the antibodies of the present invention are preferred over previously known anti-CD73 antibodies that bind to N-terminal domains.

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Thus, in accordance with one embodiment of the present invention there is provided an isolated antibody or antibody fragment having specificity for the human CD73 protein and binding to one or more amino acid residues selected in the C-terminal region of the human CD73 protein. The C-terminal portion of the human CD73 protein is known in the art to contain 238 amino acid residues, starting at residue 337, as shown in SEQ ID NO: 61 in the table below.

Table A

CD73 sequence

Name Sequence (SEQ ID NO: 61; C-terminal fragment in bold underline) Human CD73 protein MCPRAARAPATLLLALGAVLWPAAGAWELTILHTNDVHSRLEQTSEDSSKCVNASRCMGGVARLFTKVQQ IRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNALRYDAMALGNHEFDNGVEGLIEPLLKEAKFPIL SANIKAKGPLASQISGLYLPYKVLPVGDEWGIVGYTSKETPFLSNPGTNLVFEDEIT ALQPEVDKLKTL NVNKIIALGHSGFEMDKLIAQKVRGVDVWGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPWQA YAFGKYLGYLKIEFDERGNVIS SHGNPILLNS SIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSS QSCRFRECNMGNLICDAMINNNLRHTDEMFWNHVSMCILNGGGIR SPIDERNNGTITWENLAAVLPFGGT FDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHWYDLSRKPGDRWKLDVLCTKCRVPSYDPLKMD EVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINWSTYISKMKVIYPAVEGRIKFSTGSHCHGSFSL IFLS LWAVIFVLYQ

In some embodiments, the antibody or fragment thereof binds to one or more of the C-terminal domains of the human CD73 protein. In some embodiments, the antibody or fragment thereof binds to one of the amino acid residues selected from the group consisting of or more of Y345, D399, E400, R401, and R480 of the human CD73 protein. In some embodiments, the antibody or fragment thereof binds to at least two of these amino acid residues.

In some embodiments, the antibody or fragment thereof binds to at least Y345 and D399. In some embodiments, the antibody or fragment thereof binds to at least Y345 and E400. In some embodiments, the antibody or fragment thereof binds to at least Y345 and R401. In some embodiments, the antibody or fragment thereof binds to at least Y345 and R480. In some embodiments, the antibody or fragment thereof binds to at least D399 and E400. In some embodiments, the antibody or fragment thereof binds to at least D399 and R401. In some embodiments, the antibody or fragment thereof binds to at least D399 and R480. In some embodiments, the antibody or fragment thereof binds to at least E400 and R401. In some embodiments, the antibody or fragment thereof binds to at least E400 and R480. In some embodiments, the antibody or fragment thereof binds to at least R401 and R480.

In some embodiments, the antibody or fragment thereof binds to at least Y345, D399, and E400. In some embodiments, the antibody or fragment thereof binds to at least Y345, D399, and R401. In some embodiments, the antibody or fragment thereof binds to at least Y345, D399, and R480. In some embodiments, the antibody or fragment thereof binds to at least Y345, E400, and R401. In some embodiments, the antibody or fragment thereof binds to at least Y345, E400, and R480. In some embodiments, the antibody or fragment thereof binds to at least Y345, R401, and R480. In some embodiments, the antibody or fragment thereof binds to at least D399, E400, and R401. In some embodiments, the antibody or fragment thereof binds to at least D399, E400, and R480. In some embodiments, the antibody or fragment thereof binds to at least E400, R401, and R480.

In some embodiments, the antibody or fragment thereof binds to at least Y345, D399, E400, and R401. In some embodiments, the antibody or fragment thereof binds to at least Y345, D399, E400, and R480. In some embodiments, the antibody or fragment thereof binds to at least Y345, D399, R401, and R480. In some embodiments, the antibody or fragment thereof binds to at least Y345, E400, R401, and R480. In some embodiments, the antibody or fragment thereof binds to at least D399, E400, R401, and R480. In some embodiments, the antibody or fragment thereof binds to each of residues Y345, D399, E400, R401, and R480.

In accordance with one embodiment of the present invention, there is provided an antibody comprising heavy chain and light chain variable domains with the CDR regions set forth in SEQ ID NOs: 1-6.

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Table 1

CDR region sequences

Name Sequences SEQ ID NO: VH CDR1 SGYYWN 1 VH CDR2 YINYGGSNGYNPSLKS 2 VH CDR3 DYDAYYEALDD 3 VL CDR1 RASSRVNYMH 4 VL CDR2 ATSNLAS 5 VL CDR3 QQWSSNPPT 6

As demonstrated in the experimental examples, murine, human, or humanized antibodies containing these CDR regions have potent CD73 binding and inhibitory activity. Further computer modeling showed that certain residues within the CDR could be modified to maintain or improve the properties of the antibodies. Such residues are called hot spots; they are highlighted in Table 1. In some embodiments, the anti-CD73 antibody of the present invention comprises the VH and VL CDRs listed in Table 1. 1, with one, two or three additional modifications. Such modifications may be additions, deletions or substitutions of amino acids.

In some embodiments, the modification is a replacement at at most one hot spot position in each of the CDRs. In some embodiments, the modification is a replacement at one, two, or three such hot spot positions. In one embodiment, the modification is a replacement at one of the hot spot positions. In some embodiments, such substitutions are conservative substitutions.

A conservative amino acid substitution is a substitution in which an amino acid residue is replaced with an amino acid residue containing a similar side chain. The art has identified families of amino acid residues containing similar side chains, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar 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 (eg, tyrosine, phenylalanine, tryptophan, histidine). Thus, an amino acid residue that is not an essential amino acid in an immunoglobulin polypeptide is preferably replaced by another amino acid residue from the same side chain family. In yet another embodiment, the amino acid chain may be replaced by a structurally similar chain that differs in the order and/or composition of the side chain family members.

Non-limiting examples of conservative amino acid substitutions are shown in the table below, where a similarity score of 0 or higher indicates a conservative substitution between two amino acids.

table 2

Amino acid similarity matrix

C G P s A T D E N Q H TO R V M I L F Y w W -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17 Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10 F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 0 1 2 9 L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6 I -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5 M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6 V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4 R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6 TO -5 -2 -1 0 -1 0 0 0 1 1 0 5 H -3 -2 0 -1 -1 -1 1 1 2 3 6 Q -5 -1 0 -1 0 -1 2 2 1 4 N -4 0 -1 1 0 0 2 1 2 E -5 0 -1 0 0 0 3 4 D -5 1 -1 0 0 0 4 T -2 0 0 1 1 3 A -2 1 1 1 2 S 0 1 1 1 P -3 -1 6 G -3 5 C 12

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Table 3

Conservative amino acid substitutions

For amino acid Replacement Alanin D-Ala, Gly, Aib, β-Ala, L-Cys, D-Cys Arginine D-Arg, Lys, D-Lys, Om D-Om Asparagine D-Asn, Asp, D-Asp, Glu, D-Glu Gin, D-Gln Aspartic acid D-Asp, D-Asn, Asn, Glu, D-Glu, Gin, D-Gln Cysteine D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr, L-Ser, D-Ser Glutamine D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic acid D-Glu, D-Asp, Asp, Asn, D-Asn, Gin, D-Gln Glycine Ala, D-Ala, Pro, D-Pro, Aib, β-Ala Isoleucine D-Ile, Vai, D-Val, Leu, D-Leu, Met, D-Met Leucine Vai, D-Val, Met, D-Met, D-Ile, D-Leu, He Lysine D-Lys, Arg, D-Arg, Om, D-Om Methionine D-Met, S-Me-Cys, He, D-Ile, Leu, D-Leu, Vai, D-Val Phenylalanine D-Phe, Tyr, D-Tyr, His, D-His, Trp, D-Trp Proline D-Pro Serin D-Ser, Thr, D-Thr, allo-Thr, L-Cys, D-Cys Threonine D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Vai, D-Val Tyrosine D-Tyr, Phe, D-Phe, His, D-His, Trp, D-Trp Valin D-Val, Leu, D-Leu, He, D-Ile, Met, D-Met

Specific examples of CDRs with suitable substitutions are provided in SEQ ID NO: 26-56 of Example 7. Thus, in some embodiments, an antibody of the present invention comprises a VH CDR1 of SEQ ID NO: 1 or any of 26-29. In some embodiments, an antibody of the present invention comprises a CDR2 VH of SEQ ID NO: 2 or any of 30-36. In some embodiments, an antibody of the present invention comprises a CDR3 VH of SEQ ID NO: 1 or any of 37-41. In some embodiments, an antibody of the present invention comprises CDR1 VL of SEQ ID NO: 4 or any of 42-45. In some embodiments, an antibody of the present invention comprises a CDR2 VL of SEQ ID NO: 5. In some embodiments, an antibody of the present invention comprises a CDR3 VL of SEQ ID NO: 6 or any of 46-56.

In some embodiments, the antibody or antibody fragment contains no more than one, no more than two, or no more than three of the above substitutions. In some embodiments, the antibody or antibody fragment comprises a CDR1 VH of SEQ ID NO: 1 or any of SEQ ID NOs: 26-29, a CDR2 VH of SEQ ID NO: 2, a CDR3 VH of SEQ ID NO: 3, a CDR1 VL of SEQ ID NO: 4, CDR2 VL according to SEQ ID NO: 5 and CDR3 VL according to SEQ ID NO: 6.

In some embodiments, the antibody or antibody fragment comprises a CDR1 VH as per SEQ ID NO: 1, a CDR2 VH as per SEQ ID NO: 2, or any of SEQ ID NOs: 30-36, a CDR3 VH as per SEQ ID NO: 3, a CDR1 VL as per SEQ ID NO: 3 ID NO: 4, CDR2 VL according to SEQ ID NO: 5 and CDR3 VL according to SEQ ID NO: 6.

In some embodiments, the antibody or antibody fragment comprises a CDR1 VH as per SEQ ID NO: 1, a CDR2 VH as per SEQ ID NO: 2, a CDR3 VH as per SEQ ID NO: 3, or any of SEQ ID NOs: 37-41, a CDR1 VL as per SEQ ID NO: 4, CDR2 VL according to SEQ ID NO: 5 and CDR3 VL according to SEQ ID NO: 6.

In some embodiments, the antibody or antibody fragment comprises a CDR1 VH of SEQ ID NO: 1, a CDR2 VH of SEQ ID NO: 2, a CDR3 VH of SEQ ID NO: 3, a CDR1 VL of SEQ ID NO: 4, or any of the SEQ ID NOs : 42-45, CDR2 VL according to SEQ ID NO: 5 and CDR3 VL according to SEQ ID NO: 6.

In some embodiments, the antibody or antibody fragment comprises a CDR1 VH according to

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SEQ ID NO: 1, CDR2 VH according to SEQ ID NO: 2, CDR3 VH according to SEQ ID NO: 3, CDR1 VL according to

SEQ ID NO: 4, CDR2 VL as per SEQ ID NO: 5 and CDR3 VL as per SEQ ID NO: 6 or any of SEQ

ID NO: 46-56.

Non-limiting examples of VH are provided in SEQ ID NO: 7 and 9-13, of which SEQ ID NO: 10 is mouse VH and SEQ ID NO: 7, 9 and 11-13 are humanized sequences. Additionally, among the humanized VHs, SEQ ID NOs: 7, 9 and 12-13 contain one or more back mutations to the mouse version. Likewise, non-limiting examples of VL (VK) are provided in SEQ ID NOs: 8, 15-20 and 22-24. SEQ ID NO: 15 is the mouse sequence, SEQ ID NO: 16 and 22 are the original modified humanized sequences shown in the examples. SEQ ID NOs: 8, 17-20 and 22-24 are humanized VLs with back mutations.

Back mutations have been shown to be useful in maintaining some of the characteristics of anti-CD73 antibodies. Accordingly, in some embodiments, the anti-CD73 antibodies of the present invention, particularly human or humanized antibodies, contain one or more back mutations. In some embodiments, the back mutation in VH (i.e., an amino acid inserted at a specified position) is one or more mutations selected from (a) Thr at position 30, (b) Lys at position 44, (c) Met at position 48, (d) Ile at position 67 and (e) Arg at position 71 according to Kabat numbering and combinations thereof. In some embodiments, the back mutation in VL is one or more mutations selected from (a) Ser at position 5, (b) Pro at position 46, (c) Trp at position 47, (d) Ser at position 70, and ( f) Tyr in position 71 according to Kabat numbering and combination thereof.

In some embodiments, the anti-CD73 antibody of the present invention comprises VH of SEQ ID NO: 7 or any of 9-13, VL of SEQ ID NO: 8 or any of 15-20 and 22-24, or their respective biological equivalents. A biological equivalent of VH or VL is a sequence containing the specified amino acids and having 80%, 85%, 90%, 95%, 98%, or 99% overall sequence identity. The biological equivalent of SEQ ID NO: 7 may thus be a VH having 80%, 85%, 90%, 95%, 98%, or 99% overall sequence identity to SEQ ID NO: 7 but retaining the CDR ( SEQ ID NO: 1-6 or variants thereof), and optionally retaining one or more or all of the back mutations.

In one embodiment, VH contains the amino acid sequence of SEQ ID NO: 7 and VL contains the amino acid sequence of SEQ ID NO: 8. In one embodiment, VH contains the amino acid sequence of SEQ ID NO: 9 and VL contains the amino acid sequence of SEQ ID NO: 8.

In addition, one skilled in the art will appreciate that the antibodies described herein can be modified such that their amino acid sequence differs from the natural binding polypeptide from which they are derived. For example, the polypeptide or amino acid sequence derived from said protein may be similar, e.g. may have a certain percentage identity to the original sequence, e.g. may be 60%, 70%, 75%, 80%, 85%, 90 %, 95%, 98% or 99% identical to the original sequence.

In some embodiments, the antibody contains an amino acid sequence or one or more groups not normally associated with the antibody. Typical modifications are detailed below. For example, an antibody of the present invention may contain a flexible linker sequence or may be modified by the addition of a functional group (eg, PEG, drug, toxin, or tag).

Antibodies, variants or derivatives thereof of the present invention include derivatives modified, for example, by covalently attaching any type of molecule to the antibody such that the covalent attachment does not prevent the antibody from binding to the epitope. By way of example, and not by way of limitation, antibodies can be modified, for example, by glycosylation, acetylation, PEGylation, phosphorylation, amidation, modification with known protecting/blocking groups, proteolytic cleavage, attachment to a cellular ligand or other protein, etc. Any of numerous chemical modifications can be accomplished using known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, antibodies may contain one or more non-classical amino acids.

In some embodiments, antibodies may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEGs.

The antibodies may be conjugated or combined with a therapeutic agent, which may include a detectable label, for example, a radiolabel, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent, which may be a drug or a toxin, a contrast agent for Ultrasound, a non-radioactive tracer, a combination thereof, and other similar agents known in the art.

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Antibodies can be labeled with a detectable label by attaching them to a chemiluminescent compound. The presence of a chemiluminescent-labeled antigen-binding polypeptide is then determined by detecting the presence of luminescence produced by the chemical reaction. Examples of particularly useful chemiluminescent tracer compounds are luminol, isoluminol, acridinium ester, imidazole, acridinium salt and oxalate ester.

In addition, antibodies can be labeled with metal atoms capable of fluorescence, such as ¹⁵² Eu, or other metals of the lanthanide family. These metals can be attached to the antibody using metal chelate groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating various groups to an antibody are well known, see, for example, Arnon et al., Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., Antibodies For Drug Delivery, in Controlled Drug Delivery (2nd Ed.), Robinson et al., (eds.), Marcel Dekker, Inc. , pp. 623-53 (1987); Thorpe, Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 ( 1985); Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibodies In Cancer Therapy, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates, Immunol. Rev. (52:119-58 (1982)).

Antibody-encoding polynucleotides and methods for producing antibodies

The present invention also provides isolated polynucleotides or nucleic acid molecules encoding antibodies, variants and derivatives thereof according to the present invention. Examples of polynucleotides include SEQ ID NO: 57-60. The polynucleotides of the present invention may encode entire heavy and light chain variable regions of antigen binding polypeptides, variants and derivatives thereof, on the same polynucleotide molecule or on separate polynucleotide molecules. In addition, the polynucleotides of the present invention may encode fragments of the heavy and light chain variable regions of antigen binding polypeptides, variants and derivatives thereof, on the same polynucleotide molecule or on separate polynucleotide molecules.

Methods for producing antibodies are well known in the art and are described herein. In some embodiments, both the variable and constant regions of the antigen binding polypeptides of the present invention are entirely human in origin. Fully human antibodies can be obtained using techniques described in the art and herein. For example, fully human antibodies against a specific antigen can be obtained by introducing the antigen into a transgenic animal modified to produce such antibodies in response to antigenic stimulation, wherein the endogenous loci of the animal are disabled. Typical techniques that can be used to obtain such antibodies are described in US patents 6150584; 6458592; 6420140, incorporated herein by reference in its entirety.

In some embodiments, the resulting antibodies do not induce a harmful immune response in the animal being treated, such as a human. In one embodiment, the antigen-binding polypeptides, variants or derivatives thereof of the present invention are modified to reduce their immunogenicity using techniques recognized in the art. For example, antibodies can be humanized, primatized, deimmunized, or produced as chimeric antibodies. These types of antibodies are derived from a non-human animal antibody, usually a mouse or primate antibody, and retain the antigen-binding properties of the original antibody but are less immunogenic in humans. This can be achieved in a variety of ways, including (a) complete transplantation of non-human variable domains onto human antibody constant regions to produce chimeric antibodies; (b) transplanting at least a fragment of one or more complementarity determining regions (CDRs) of non-human origin onto the framework and constant regions of a human antibody, with or without retention of critical framework residues; or (c) complete transplantation of variable domains of non-human origin, but masking them by a section similar to the human antibody sequence by replacing surface residues. Such methods are described in Morrison et al., Proc. Natl. Acad. Sci. USA 57:6851-6855 (1984); Morrison et al., Adv. Immunol. 44:65-92 (1988); Verhoeyen et al, Science 239:1534-1536 (1988); Padlan, Molec. Immun. 25:489-498 (1991); Padlan, Molec. Immun. 31:169-217 (1994), and US Patent Nos. 5,585,089, 5,693,761, 5,693,762, and 6,190,370, all of which are incorporated herein by reference in their entirety.

Deimmunization can also be used to reduce the immunogenicity of the antibody. As used herein, the term deimmunization includes modification of an antibody by modification of T cell epitopes (see, for example, International Patent Application Publications Nos. WO/9852976 A1 and WO/0034317 A2). For example, the sequences of the variable regions of the heavy chain and light chain of the parent antibody are analyzed and a map of human T-cell epitopes of each V region is generated

- 14 043570 indicating the location of epitopes in relation to complementarity determining regions (CDRs) and other key residues within the sequence. Individual T cell epitopes from the T cell epitope map are analyzed to identify alternative low-risk amino acid substitutions in terms of altering the activity of the final antibody. A range of alternative heavy chain and light chain variable region sequences containing combinations of amino acid substitutions are constructed and these sequences are then inserted into a series of binding polypeptides. Typically, 12 to 24 variant antibodies are prepared and tested for binding and/or function. The complete heavy and light chain genes containing the modified human variable regions and constant regions are then cloned into expression vectors, and the resulting plasmids are introduced into cell lines to produce the complete antibody. The antibodies are then compared using appropriate biochemical and biological assays to determine which option is best.

The binding specificity of the antigen-binding polypeptides of the present invention can be determined by in vitro assays, for example, immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

Alternatively, the procedures described for the production of single chain units (US Patent No. 4694778; Bird, Science 242:423-442 (1988); Huston et al, Proc. Natl. Acad. Sci. USA 55:5879-5883 (1988) ; and Ward et al, Nature 334:544-554 (1989)) can be adapted to produce single chain units according to the present invention. Single-chain units are formed by joining heavy and light chain fragments of the Fv region through an amino acid bridge, resulting in a single-chain hybrid peptide. In addition, techniques for assembling functional Fv fragments in E. coli can be used (Skerra et al., Science 242: 1038-1041 (1988)).

Examples of techniques that can be used to produce single chain Fv (scFv) and antibodies include those described in US Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., Proc. Natl. Sci. USA 90:1995-1999 (1993); and Skerra et al, Science 240:1038-1040 (1988). For some applications, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric or humanized antibodies or human antibodies. A chimeric antibody is a molecule in which different antibody fragments are derived from different animal species, for example, antibodies containing a variable region derived from a mouse monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, Science 229:1202 (1985); Oi et al, BioTechniques 4:214 (1986); Gillies et al, J. Immunol. Methods 125:191-202 (1989); U.S. Patent Nos. 5,807,715, 4,816,567, and 4,816,397, which are incorporated herein by reference in their entirety.

Humanized antibodies are antibody molecules derived from antibodies from a non-human animal species that bind a desired antigen and contain one or more complementarity determining regions (CDRs) of a non-human animal antibody and framework regions from a human immunoglobulin molecule. Often, framework residues in human framework regions can be replaced with a corresponding residue from the donor antibody CDR to modify, preferably improve, binding to the antigen. These framework substitutions are identified by methods well known in the art, for example, by modeling the interactions of CDRs and framework residues to identify framework residues important for antigen binding, and by comparing sequences to identify unusual framework residues at certain positions. (See, for example, Queen et al, US Pat. No. 5,585,089; Riechmann et al, Nature 332:323 (1988), incorporated herein by reference in their entirety.) Antibodies can be humanized using various techniques known in the art, including, for example, CDR grafting (EP 239400; PCT publication WO 91/09967; US patent No. 5225539, 5530101 and 5585089), lining or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., Proc. Natl. Sci. USA 91:969-973 (1994)), and circuit recombination (US Pat. No. 5,565,332, incorporated herein by reference in its entirety).

Fully human antibodies are particularly desirable for the therapeutic treatment of human patients. Human antibodies can be produced using a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See also US Pat. Nos. 4,444,887 and 4,716,111; and PCT Publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.

Human antibodies can also be produced using transgenic mice that are unable to express functional endogenous immunoglobulins but are able to express human immunoglobulin genes. For example, human immunoglobulin heavy or light chain gene complexes can be introduced into mouse embryonic stem cells randomly or through homologous recombination. Alternatively, the human variable region, constant region and D region can be introduced into mouse embryonic stem cells in addition to the genes

- 15 043570 human heavy and light chain. Mouse immunoglobulin heavy or light chain genes can be rendered nonfunctional individually or simultaneously by introducing human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. Modified embryonic stem cells are expanded and microinjected into blastocysts to create chimeric mice. The chimeric mice are then crossed to produce homozygous offspring that express human antibodies. Transgenic mice are immunized with the selected antigen in a conventional manner, for example, using the full-length desired target polypeptide or a fragment thereof. Monoclonal antibodies against an antigen can be obtained from immunized transgenic mice using conventional hybridoma technology. Human immunoglobulin transgenes carried by transgenic mice are rearranged during B cell differentiation and subsequently undergo class switching and somatic mutations. Thus, the use of this technique makes it possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For a review of this technology for human antibody production, see Lonberg and Huszar Int. Rev. Immunol. 73:65-93 (1995). For a detailed discussion of this technology for the production of human antibodies and human monoclonal antibodies and protocols for the production of such antibodies, see, for example, PCTWO 98/24893; WO 96/34096; WO 96/33735; US Pat. Nos. 5,413,923, 5625,126, 5633425, 5569825, 5661016, 5545806, 5814318, and 5939598, all of which are incorporated herein by reference in their entirety. Additionally, companies such as Abgenix, Inc. can be used to produce human antibodies against a selected antigen using technology similar to that described above. (Fremont, California, USA) and GenPharm (San Jose, California, USA).

Fully human antibodies that recognize a selected epitope can also be produced using a technique called directed selection. In this approach, a selected non-human animal monoclonal antibody, such as a mouse antibody, is used to guide the selection of a fully human antibody recognizing the same epitope. (Jespers et al., Bio/Technology 72:899-903 (1988). See also US Pat. No. 5,565,332, incorporated herein by reference in its entirety.)

In yet another embodiment, DNA encoding the desired monoclonal antibodies can be easily isolated and sequenced using standard procedures (eg, using oligonucleotide probes capable of specifically binding to genes encoding mouse antibody heavy and light chains). The preferred source of such DNA is isolated and subcloned hybridoma cells. Once isolated, the DNA can be placed into expression vectors, which are then transfected into prokaryotic or eukaryotic host cells, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulins. More specifically, isolated DNA (which may be synthetic, as described herein) can be used to clone constant and variable region sequences for the production of antibodies, as described in Newman et al., US patent No. 5658570, filed January 25, 1995, included incorporated into this document by reference. Essentially, this involves isolating RNA from selected cells, converting it to cDNA, and amplifying it through PCR using Ig-specific primers. Suitable primers for this purpose are also described in US Pat. No. 5,658,570. In the detailed discussion below, transformed cells expressing the desired antibody can be grown in relatively large quantities to provide clinical and commercial supplies of immunoglobulins.

In addition, using conventional recombinant DNA technology, one or more CDRs of the antigen binding polypeptides of the present invention can be inserted into framework regions, for example, into human framework regions to humanize a non-human animal antibody. The framework regions can be natural or consensus framework regions and, preferably, human framework regions (for a list of human framework regions, see, for example, Chothia et al, J. Mol. Biol. 278:457-479 (1998)). The polynucleotide obtained by combining framework regions and CDRs preferably encodes an antibody that specifically binds to at least one epitope of the desired polypeptide, for example, LIGHT. Preferably, one or more amino acid substitutions can be made in the framework regions, and these amino acid substitutions preferably improve the binding of the antibody to its antigen. In addition, such methods can be used to produce amino acid substitutions or deletions of one or more cysteine residues in the variable region involved in intrachain disulfide bond formation to produce antibody molecules without one or more intrachain disulfide bonds. Other polynucleotide modifications are included in the present invention and are known to those skilled in the art.

In addition, techniques developed for the production of chimeric antibodies can be used (Morrison et al, Proc. Natl. Acad. Sci. USA:851-855 (1984); Neuberger et al, Nature 372:604-608 (1984); Takeda et al. al, Nature 314:452-454 (1985)) by splicing the genes of a mouse antibody molecule having the corresponding antigen specificity together with the genes of a human antibody molecule having the corresponding biological activity. As used herein, a chimeric antibody is a molecule in which various fragments are derived from antibodies of different animal species, for example, a molecule comprising a variable region derived from a mouse monoclonal antibody and a human immunoglobulin constant region.

Another highly effective means of producing recombinant antibodies is described in Newman, Biotechnology 10: 1455-1460 (1992). Specifically, this technique allows the production of primatized antibodies containing monkey variable domains and human constant sequences. This source is incorporated herein by reference in its entirety. In addition, this technique is also described in commonly cited US Patent Nos. 5,658,570, 5,693,780, and 5,756,096, each of which is incorporated herein by reference.

Alternatively, antibody-producing cell lines can be selected and cultured using techniques well known in the art. Such techniques are described in various laboratory manuals and primary publications. Accordingly, techniques suitable for use in the present invention are described in Current Protocols in Immunology, Coligan et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991), incorporated in its entirety incorporated herein by reference together with the supplementary materials.

In addition, those skilled in the art are aware of standard techniques that can be used to introduce mutations into the nucleotide sequence encoding an antibody of the present invention, including, but not limited to, site-directed mutagenesis and PCR-mediated mutagenesis that result in amino acid substitutions. . The variants (including derivatives) preferably encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions compared to the reference heavy chain variable region, CDR-H1, CDR-H2, CDR-H3, light chain variable region, CDR-L1, CDR-L2, or CDR-L3. Alternatively, one can introduce random mutations throughout all or part of the coding sequence, for example by saturation mutagenesis, and screen the resulting mutants for biological activity to identify mutants that retain activity.

Treatment methods

As described herein, the antibodies, variants or derivatives of the present invention can be used in various methods of treatment and diagnosis.

The present invention further relates to antibody therapeutic methods comprising administering antibodies of the present invention to a patient, eg, an animal, mammal and human, for the treatment of one or more of the disorders or conditions described herein. Therapeutic compounds of the present invention include, but are not limited to, antibodies of the present invention (including variants and derivatives thereof described herein) and nucleic acids or polynucleotides encoding antibodies of the present invention (including variants and derivatives thereof described herein). .

In addition, the antibodies of the present invention can be used to treat or suppress cancer. As presented above, CD73 can be overexpressed in tumor cells. Tumor-derived CD73 may function as an extracellular adenosine-producing ectoenzyme, which stimulates tumor growth by limiting antitumor T-cell immunity through the adenosine receptor signaling pathway. Results obtained using small molecule inhibitors or monoclonal antibodies targeting CD73 in mouse tumor models indicate that CD73-targeting therapeutics represent an important alternative and realistic approach to effectively control tumor growth. In particular, it promotes the action of T cell-based therapeutics by enhancing adaptive immune response mechanisms, which may enhance the effects of tumor-infiltrating T lymphocytes and lead to improved survival in cancer patients.

Accordingly, some embodiments provide methods for treating cancer in a patient in need thereof. In one embodiment, the method comprises administering to a patient an effective amount of an antibody of the present invention. In some embodiments, at least one of the cancer cells (eg, stromal cells) in the patient overexpresses CD73.

Non-limiting examples of cancer include bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer glands and thyroid cancer.

Cellular therapeutics, and more specifically chimeric antigen receptor (CAR) T cell therapeutics, are also provided by the present invention. A suitable T cell that contacts the anti-CD73 antibody of the present invention (or, alternatively, engineered to express an anti-CD73 antibody) may be used.

- 17 043570

CD73 according to the present invention). Once contacted or engineered, the T cell can be administered to a cancer patient in need of treatment. A patient with cancer may have any type of cancer described herein. The T cell may be, for example, a tumor infiltrating T lymphocyte, a CD4+ T cell, a CD8+ T cell, or a combination thereof, without limitation.

In some embodiments, a patient with cancer independently releases a T cell from his or her body. In some embodiments, the T cell is provided by a donor or cell bank. When isolating T cells from a patient with cancer, it is possible to minimize unwanted immune reactions.

Additional diseases or conditions associated with increased cell survival that may be treated, prevented, diagnosed and/or predicted using the antibodies or variants and derivatives thereof of the present invention include progression and/or metastasis of malignancies and related disorders, such as leukemia (including acute leukemias (eg, acute lymphoblastic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic leukemia and erythroleukemia) and chronic leukemias (eg, chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia leukemia)), polycythemia vera , lymphomas (e.g., Hodgkin's disease and non-Hodgkin's lymphomas), multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease and solid tumors, including, but not limited to, sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, dimfangioendothelial sarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat jelly carcinoma h, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma , bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma.

The specific dosage and treatment regimen for any given patient depends on various factors, including the specific antibodies used, their variants and derivatives, the patient's age, body weight, general health, gender, and nutritional status, as well as the timing of administration, rate of elimination, and drug combinations. and the severity of the specific disease being treated. Expert assessment of such factors by medical professionals is within the normal skill of one skilled in the art. The amount also depends on the individual patient being treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease and the desired effect. The amount used can be determined using pharmacological and pharmacokinetic principles well known in the art.

Methods of administering antibodies and variations thereof include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral administration. Antigen-binding polypeptides or compositions thereof can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous membranes (for example, oral mucosa, anal or intestinal mucosa, etc.) and can be administered together with other biologically active agents. Thus, pharmaceutical compositions containing the antigen-binding polypeptides of the present invention can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (in the form of powders, ointments, drops or transdermal patch), buccally, or as an oral or nasal aerosol.

The term parenteral as used herein refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrathoracic, subcutaneous and intra-articular injection and infusion.

Administration may be systemic or local. In addition, it may be desirable to administer the antibodies of the present invention to the central nervous system by any suitable route, including intraventricular or intrathecal injection; intraventricular injection can be facilitated by using an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. In addition, pulmonary administration can be used, for example, using an inhaler or nebulizer and a formulation with an aerosolizing agent.

It may be desirable to locally administer the antigen binding polypeptides or compositions of the present invention to the area in need of treatment; this can be achieved, for example (but not by way of limitation), by local infusion during surgery, external application, for example in combination with a wound dressing after surgery, by injection,

- 18 043570 by means of a catheter, suppository or implant, wherein said implant is a porous, non-porous or jelly-like material, including membranes, for example, sialastic membranes or fibers. When administering a protein, including an antibody of the present invention, care should be taken to use materials that do not absorb the protein.

In yet another embodiment, the antigen binding polypeptide or composition can be delivered in vesicles, particularly liposomes (see Langer, 1990, Science 249:1527-1533; Treat et al, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez- Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see mainly ibid.).

In yet another embodiment, the antigen binding polypeptide or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al, 1980, Surgery 88:507; Saudek et al, 1989, N. Engl. J. Med. 321:574). In yet another embodiment, polymeric materials may be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al, 1985, Science 228: 190; During et al, 1989, Ann Neurol 25:351; Howard et al, 1989, J Neurosurg 71:105). In yet another embodiment, a controlled release system can be placed near the target of a therapeutic agent, such as the brain, such that only a portion of the systemic dose needs to be administered (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2 , pp. 115-138 (1984)). Other controlled release systems are discussed in the review by Langer (1990, Science 249:1527-1533).

In a particular embodiment, where the composition of the present invention contains a nucleic acid or polynucleotide encoding a protein, the nucleic acid can be administered in vivo to stimulate the expression of the encoded protein by constructing it in an appropriate nucleic acid expression vector and introducing it so that it becomes intracellular, for example, using a retroviral vector (see US Pat. No. 4,980,286) or by direct injection or using microparticle bombardment (eg, through a gene gun; Biolistic, Dupont), or coating with lipids or cell surface receptors or transfection agents, or by introducing it in connection with a homeobox-like peptide, which obviously penetrates the nucleus (see, for example, Joliot et al, 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), etc. Alternatively, the nucleic acid can be introduced into the cell and incorporated into the DNA of the host cell for expression by homologous recombination.

The amount of antibodies of the present invention that may be effective in the treatment, suppression and prevention of an inflammatory, immune or malignant disease, disorder or condition can be determined using standard clinical techniques. In addition, in vitro assays may optionally be used to facilitate determination of the optimal dose range. The exact dosage used in the formulation also depends on the route of administration and the severity of the disease, disorder or condition, and should be selected according to the judgment of the practitioner and taking into account the circumstances of each patient. Effective doses can be extrapolated from dose response curves obtained in vitro or in animal model test systems.

As a general suggestion, the dosage of the antigen binding polypeptides of the present invention administered to a patient is typically from 0.1 mg/kg to 100 mg/kg of the patient's body weight, from 0.1 mg/kg to 20 mg/kg of the patient's body weight, or 1 mg/kg to 10 mg/kg patient body weight. Generally, human antibodies have a longer half-life in the human body compared to antibodies of other species due to the immune response to foreign polypeptides. Thus, lower dosages and less frequent administration can often be used. In addition, the dosage and frequency of administration of the antibodies of the present invention can be reduced by enhancing the uptake and penetration of the antibodies into tissues (eg, the brain) through modifications such as lipidation.

Methods of treating an infectious or malignant disease, condition or disorder involving administration of an antibody, variant or derivative thereof of the present invention are typically tested in vitro and then in vivo in an appropriate animal model for desired therapeutic or prophylactic activity prior to use in humans. Suitable animal models, including transgenic animals, are well known to those skilled in the art. For example, in vitro assays to demonstrate the therapeutic effect of an antigen binding polypeptide described herein involve the effect of the antigen binding polypeptide on a cell line or tissue sample from a patient. The effect of an antigen binding polypeptide on a cell line and/or tissue sample can be determined using techniques known to those skilled in the art, for example, the assays described elsewhere herein. In accordance with the present invention, in vitro assays that can be used to determine whether administration of a specific antigen binding polypeptide is indicated include in vitro cell culture assays in which a sample of patient tissue is grown in culture, exposed to, or otherwise treated compound and observe the effect of such a compound on a tissue sample.

Various delivery systems are known that can be used to administer an antibody of the present invention or a polynucleotide encoding an antibody of the present invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, for example, Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part of a retroviral or other vector, etc.

In a further embodiment, the composition of the present invention is administered in combination with an antitumor agent, an antiviral agent, an antibacterial agent, or an antibiotic or antifungal agents. Any of these agents known in the art can be administered in the compositions of the present invention.

In yet another embodiment, the compositions of the present invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that can be administered with the compositions of the present invention include antibiotic derivatives (eg, doxorubicin, bleomycin, daunorubicin and dactinomycin); antiestrogens (for example, tamoxifen); antimetabolites (eg, fluorouracil, 5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (eg, carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cisplatin, and vincristine sulfate); hormones (eg, medroxyprogesterone, estramustine sodium phosphate, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene and testolactone); nitrogen mustard derivatives (eg, mephalen, chlorambucil, mechlorethamine (nitrogen mustard) and thiotepa); steroids and combinations (eg, betamethasone sodium phosphate); and other agents (eg, but not limited to, dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

In a further embodiment, the compositions of the present invention are administered in combination with cytokines. Cytokines that can be administered with the compositions of the present invention include IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL- 15, antibody against, but not limited to, CD40, CD40L and TNF-α.

In additional embodiments, the compositions of the present invention are administered in combination with other therapeutic or prophylactic regimens, such as radiation therapy.

Combination Therapeutics

In addition, the present invention provides combination therapeutic agents comprising the use of one or more anti-CD73 antibodies of the present invention together with a second anti-cancer (chemotherapeutic) agent. Chemotherapeutic agents can be classified according to their mechanism of action, for example into the following groups:

antimetabolites/anticancer agents such as the pyrimidine analogues floxuridine, capecitabine and cytarabine;

purine analogues, folate antagonists and related inhibitors;

antiproliferative/antimitotic agents, including natural products such as vinca alkaloids (vinblastine, vincristine), and microtubule disrupting agents such as taxane (paclitaxel, docetaxel), vinblastine, nocodazole, epothilones, vinorelbine (NAVELBINE®) and epipodophyllotoxins (etoposide, teniposide);

DNA damaging agents, eg actinomycin, amsacrine, busulfan, carboplatin, chlorambucil, cisplatin, cyclophosphamide, (CYTOXAN®), dactinomycin, daunorubicin, doxorubicin, epirubicin, ifosfamide, melphalan, merchlorethamine, mitomycin, mitoxantrone, nitrosourea, procarbazine , taxol, taxotere, teniposide, etoposide and triethylene thiophosphoramide;

antibiotics, for example, dactinomycin, daunorubicin, doxorubicin, idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin;

enzymes, for example, L-asparaginase, which systemically metabolizes L-asparagine and deprives cells of it that are unable to synthesize their own asparagine;

antiplatelet agents;

antiproliferative/antimitotic alkylating agents, for example, nitrogen mustard derivatives, cyclophosphamide and analogs (melphalan, chlorambucil, hexamethylmelamine and thiotepa), alkyl nitrosourea derivatives (carmustine) and analogs, streptozocin and triazenes (dacarbazine);

antiproliferative/antimitotic antimetabolites, such as folic acid analogues (methotrexate);

platinum coordination complexes (cisplatin, oxaliplatin and carboplatin), procarbazine, hydroxyurea, mitotane and aminoglutethimide;

hormones, hormone analogues (estrogen, tamoxifen, goserelin, bicalutamide and nilutamide) and aromatase inhibitors (letrozole and anastrozole);

- 20 043570 anticoagulants, for example heparin, synthetic heparin salts and other thrombin inhibitors;

fibrinolytics, for example tissue-type plasminogen activator, streptokinase, urokinase, aspirin, dipyridamole, ticlopidine and clopidogrel;

anti-migration agents;

antisecretory agents (brefeldin);

immunosuppressants - tacrolimus, sirolimus, azathioprine and mycophenolate;

compounds (TNP-470, genistein) and growth factor inhibitors (vascular endothelial growth factor inhibitors and fibroblast growth factor inhibitors);

angiotensin receptor blockers, nitric oxide donors;

antisense oligonucleotides;

antibodies, such as trastuzumab and rituximab;

cell cycle inhibitors and differentiation inducers, such as tretinoin;

inhibitors, topoisomerase inhibitors (doxorubicin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan, mitoxantrone, topotecan and irinotecan) and corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone and prednisolone);

inhibitors of growth factor signaling pathway kinases;

inducers of dysfunction;

toxins such as cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin, diphtheria toxin and caspase activators;

and chromatin.

Additional examples of chemotherapeutic agents include:

alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®);

alkylsulfonates, for example busulfan, improsulfan and piposulfan;

aziridines, for example benzodopa, carboquone, meturedopa and uredopa;

ethylenimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylmelamine;

acetogenins, especially bullatacin and bullatacinone;

camptothecin, including the synthetic analogue of topotecan;

bryostatin;

kallistatin;

CC-1065, including its synthetic analogues adozelesin, carzelesin and bizelesin;

cryptophycins, in particular cryptophycin 1 and cryptophycin 8;

dolastatin;

duocarmycin, including synthetic analogues KW-2189 and CBI-TMI;

eleutherobin;

pancratistatin;

sarcodictyin;

spongistatin;

nitrogen mustard derivatives, for example, chlorambucil, chlornaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiquin, phenesterine, prednimustine, trofosfamide and uracil mustard;

nitrosourea derivatives, for example, carmustine, chlorosotocin, fotemustine, lomustine, nimustine and ranimustine;

antibiotics, e.g. enediyne antibiotics (e.g. calichemycin, especially calichemycin gamma-II and calichemycin phi-P), dinemycin, including dinemycin A, bisphosphonates, e.g. clodronate, esperamycin, neocarcinostatin chromophore and related chromoprotein enediyne chromophore antibiotics, aclacinomysins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carcinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxoA-norleucine, doxorubicin (including morpholindoxorubicin, cyanmorpholindoxorubicin, 2- pyrrolindoxorubicin and deoxydoxorubicin), epirubicin, ezorubicin, idarubicin, marcellomycin, mitomycins, for example, mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porphyromycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;

antimetabolites such as methotrexate and 5-fluorouracil (5-FU);

folic acid analogues, such as demopterin, methotrexate, pteropterin and trimetrexate;

purine analogues, for example, fludarabine, 6-mercaptopurine, thiamniprin, thioguanine;

pyrimidine analogues, for example, ancytabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocytabine and floxuridine;

androgens, such as calusterone, dromostanolone propionate, epithiostanol, mepithiostane and testolactone;

anti-adrenal agents such as aminoglutethimide, mitotane and trilostane;

folic acid compensators, for example, folinic acid;

- 21 043570 trichothecenes, especially T-2 toxin, verracurin A, roridin A and anguidin;

taxoids, such as paclitaxel (TAXOL®) and docetaxel (TAXOTERE®);

platinum analogues, such as cisplatin and carboplatin;

aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatrexate; defofamine; demecolcine; diaziquon; elformitine; elliptinium acetate; epothilone; ethoglucide; gallium nitrate; hydroxyurea; lentinan; leucovorin; lonidamine; maytansinoids, eg maytansine, and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitracrine; pentostatin; phenomet; pirarubicin; losoxantrone; fluoropyrimidine; folinic acid; podophyllic acid; 2-ethylhydrazide; procarbazine; polysaccharide-K (PSK); razoxane; rhizoxin; sisofuran; spirogermanium; tenuazonic acid; triaziquon; 2,2',2-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustin; mitobronitol; mitolactol; pipobromance; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepu; chlorambucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (NAVELBINE®); Novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; SRT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DFMO); retinoids, such as retinoic acid; capecitabine; FOLFIRI (fluorouracil, leucovorin and irinotecan);

and pharmaceutically acceptable salts, acids or derivatives of any of the above compounds.

Additionally, the definition of a chemotherapeutic agent includes hormone antagonists, such as antiestrogens and selective estrogen receptor modulators (SERMs), aromatase inhibitors, antiandrogens, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing that regulate or inhibit the effects of hormones on tumors.

Examples of antiestrogens and SERMs include, for example, tamoxifen (including NOLVADEX™), raloxifene, droloxifene, 4-hydroxytamoxifene, trioxifene, keoxifene, LY117018, onapristone and toremifene (FARESTON®).

Aromatase inhibitors regulate estrogen production in the adrenal glands. Examples include 4(5)-imidazoles, aminoglutethimide, megestrol acetate (MEGACE®), exemestane, formestane, fadrozole, vorozole (RIVISOR®), letrozole (FEMARA®), and anastrozole (ARIMIDEX®).

Examples of antiandrogens include flutamide, nilutamide, bicalutamide, leuprolide and goserelin.

Examples of chemotherapeutic agents also include antiangiogenic agents, including retinoic acid and its derivatives, 2-methoxyestradiol, ANGIOSTATIN®, ENDOSTATIN®, suramin, squalamine, tissue metalloproteinase inhibitor-1, tissue metalloproteinase inhibitor-2, plasminogen activator inhibitor-1, inhibitor -2 plasminogen activator, cartilage inhibitor, paclitaxel (nab-paclitaxel), platelet factor 4, protamine sulfate (clupein), sulfated chitin derivatives (derived from snow crab shells), sulfated polysaccharide peptidoglycan complex (sp-pg), staurosporine, metabolic modulators matrix, including proline analogs ((1-azetidine-2-carboxylic acid (LACA)), cis-hydroxyproline, d,I-3,4dehydroproline, thiaproline, α,α'-dipyridyl, beta-aminopropionitrile fumarate, 4- propyl-5-(4pyridinyl)-2(3h)-oxazolone, methotrexate, mitoxantrone, heparin, interferons, serum macroglobulin 2, chicken metalloproteinase-3 (ChLMP-3) inhibitor, chymostatin, betacyclodextrin tetradecasulfate, eponemycin, fumagillin, gold-thiomalate sodium, d-penicillamine, serum beta-1 anticollagenase, alpha-2 antiplasmin, bisantrene, disodium lobenzarite, n2-carboxyphenyl-4-chloroanthronilic acid disodium salt or CCA, thalidomide, angiostatic steroid, carboxyaminoimidazole and metalloproteinase inhibitors, e.g. BB-94 , but are not limited to them. Other anti-angiogenic agents include antibodies, preferably monoclonal antibodies, against the angiogenic growth factors: beta-FGF, alpha-FGF, FGF-5, VEGF isoforms, VEGF-S, HGF/GF and Ang1/Ang-2.

Examples of chemotherapeutic agents also include antifibrotic agents, including compounds such as beta-aminopropionitrile (BAPN), as well as compounds described in US Pat. No. 4,965,288 (Palfreyman, et al), which relates to lysyl oxidase inhibitors and their use in the treatment of diseases and conditions associated with pathological collagen accumulation, and in US patent No. 4997854 (Kagan et al), which relates to LOX inhibitory compounds for the treatment of various pathological fibrotic conditions, incorporated herein by reference, but not limited to. Other exemplary inhibitors are described in US Pat. No. 4,943,593 (Palfreyman et al), which covers compounds such as 2-isobutyl-3-fluoro-, chloro-, or bromoallylamine, US Pat. No. 5,021,456 (Palfreyman et al), 5,059,714 (Palfreyman et al). al), 5120764 (McCarthy et al), 5182297 (Palfreyman et al), 5252608 (Palfreyman et al), which relate to 2-(1-naphthyloxymethyl)-3fluoroallylamine, and US Patent Publication No. 2004/0248871 (Farjanel et al) , which are incorporated herein by reference.

Typical antifibrotic agents also include primary amines that react with the carbonyl group of the active site of lysyl oxidases, and more specifically, amines that, upon binding with the carbonyl group, form a resonance stabilized product, for example the following primary amines: ethyleneamine, hydrazine, phenylhydrazine and their derivatives; semicarbazide and urea derivatives;

aminonitriles, for example BAPN or 2-nitroethylamine; unsaturated or saturated halogenated amines, for example, 2-bromoethylamine, 2-chloroethylamine, 2-trifluoroethylamine, 3-bromopropylamine and rhalobenzylamines; and selenhomocysteine lactone.

Other antifibrotic agents are copper chelates with or without cell permeability. Typical compounds include indirect inhibitors that block aldehyde derivatives formed during the oxidative deamination of lysyl and hydroxyl residues by lysyl oxidases. Examples include thiolamines, in particular D-penicillamine and its analogs, for example, 2-amino-5-mercapto-5-methylhexanoic acid, D-2-amino-3methyl-3-((2-acetamideethyl)dithio)butanoic acid, p- 2-amino-3-methyl-3-((2-aminoethyl)dithio)butanoic acid, sodium 4-((p-1-dimethyl-2-amino-2-carboxyethyl)dithio)butanesulfurate, 2-acetamide ethyl-2acetamide ethanethiol sulfonate and Sodium 4-mercaptobutanesulfinate trihydrate.

Examples of chemotherapeutic agents also include immunotherapeutic agents, including, but not limited to, therapeutic antibodies suitable for treating patients. Some examples of therapeutic antibodies include simtuzumab, abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab, bavituximab, bestumomab, bevacizumab, bivatuzumab, blinatumomab, brentuximab, santuzumab, catumaxomab, cetuximab, cituzumab, cixutumumab, clivatuzumab, conatumumab, daratumumab , drozitumab, duligotumab, dusigitumab, detumomab, dacetuzumab, dalotuzumab, ecromeximab, elotuzumab, ensituximab, ertumaxomab, etaracizumab, farletuzumab, ficlatuzumab, figitumumab, flanvotumab, futuximab, ganitumab, gemtuzumab, gi rentuximab, glembatumumab, ibritumomab, igovomab, imgatuzumab, indatuximab, inotuzumab , intetumumab, ipilimumab, iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, minretumomab, mitumomab, moxetumomab, narnatumab, naptumomab, necitumumab, nimotuzumab, nofetumomab, oka ratuzumab, ofatumumab, olaratumab, onartuzumab, oportuzumab, oregovomab , panitumumab, parsatuzumab, patritumab, pemtumomab, pertuzumab, pintumomab, pritumumab, racotumomab, radretumab, rilotumumab, rituximab, robatumumab, satumomab, sibrotuzumab, siltuximab, solitomab, takatuzumab, taplitumomab, tenatumomab, those protumumab, tigatuzumab, tositumomab, trastuzumab, tucotuzumab, ublituximab , veltuzumab, vorsetuzumab, votumumab, zalutumumab, CC49 and 3F8. Rituximab can be used to treat refractory B-cell malignancies, including border zone lymphoma, WM, CLL, and small lymphocytic lymphoma. The combination of rituximab and chemotherapy agents is particularly effective.

Typical therapeutic antibodies can be further labeled or combined with radioisotope species, such as indium-111, yttrium-90 or iodine-131.

In one embodiment, the additional therapeutic agent is an alkylating agent derived from nitrogen mustard. Non-limiting examples of nitrogen mustard derivative alkylating agents include chlorambucil.

In one embodiment, the compounds and compositions described herein can be used or combined with one or more additional therapeutic agents. Said one or more therapeutic agents include Ab1 inhibitor, activated CDC kinase (ACK), adenosine receptor A2B (A2B), apoptosis signaling kinase (ASK), Aurora kinase, Bruton's tyrosine kinase (BTK), BET bromodomain (BRD) ), for example, BRD4, c-Kit, c-Met,

CDK-activating kinase (CAK), calmodulin-dependent protein kinase (CaMK), cyclin-dependent kinase (CDK), casein kinase (CK), discoidin domain receptor (DDR), epidermal growth factor receptors (EGFR), focal adhesion kinase (FAK), Flt-3, FYN, glycogen synthase kinase (GSK), HCK, histone deacetylase (HDAC), IKK, e.g. IKKβε, isocitrate dehydrogenase (IDH), e.g. IDH1, Janus kinase (JAK), KDR, lymphocyte-specific protein tyrosine kinase (LCK), protein lysyl oxidases, lysyl oxidase-like protein (LOXL), LYN, matrix metalloprotease (MMP), MEK, mitogen-activated protein kinase (MAPK), NEK9, NPM-ALK, p38 kinase, platelet-derived growth factor (TGF), phosphorylase kinase (PK), polo-like kinase (PLK), phosphatidylinositol 3-kinase (PI3K), protein kinase (PK), e.g. protein kinase A, B and/or C, PYK, spleen tyrosine kinase (SYK), serine/threonine kinase TPL2, serine/threonine kinase STK, protein signal transduction and activator of transcription (STAT), SRC, serine/threonine protein kinase (TBK), such as, but not limited to, TBK1, TIE, tyrosine kinase (TK), vascular endothelial growth factor receptor (VEGFR), YES, or any combination thereof.

ASK inhibitors include ASK1 inhibitors. Examples of ASK1 inhibitors include, but are not limited to, those described in WO 2011/008709 (Gilead Sciences) and WO 2013/112741 (Gilead Sciences).

Examples of BTK inhibitors include, but are not limited to, ibrutinib, HM71224, ONO-4059, and CC-292.

DDR inhibitors include DDR1 and/or DDR2 inhibitors. Examples of DDR inhibitors include those described in WO 2014/047624 (Gilead Sciences), US 2009/0142345 (Takeda

- 23 043570

Pharmaceutical), US 2011/0287011 (Oncomed Pharmaceuticals), WO 2013/027802 (Chugai Pharmaceutical) and

WO 2013/034933 (Imperial Innovations), but not limited to them.

Examples of HDAC inhibitors include, but are not limited to, pracinostat and panobinostat.

JAK inhibitors include JAK1, JAK2 and/or JAK3. Examples of JAK inhibitors include, but are not limited to, filgotinib, ruxolitinib, fedratinib, tofacitinib, baricitinib, lestavrtinib, pacritinib, XL019, AZD1480, INCB039110, LY2784544, BMS911543 and NS018.

LOXL inhibitors include inhibitors of LOXL1, LOXL2, LOXL3, LOXL4 and/or LOXL5. Examples of LOXL inhibitors include, but are not limited to, the antibodies described in WO 2009/017833 (Arresto Biosciences).

Examples of LOXL2 inhibitors include, but are not limited to, those described in WO 2009/017833 (Arresto Biosciences), WO 2009/035791 (Arresto Biosciences) and WO 2011/097513 (Gilead Biologies).

MMP inhibitors include MMP inhibitors 1 to 10. Examples of MMP9 inhibitors include, but are not limited to, marimastat (BB-2516), cipemastat (Ro 32-3555), and the inhibitors described in WO 2012/027721 (Gilead Sciences).

PI3K inhibitors include PI3Ky, PI3Kδ, PI3Ke, PI3Ka and/or a general PI3K inhibitor. Examples of PI3K inhibitors include, but are not limited to, wortmannin, BKM120, CH5132799, XL756, and GDC-0980.

Examples of PI3Ky inhibitors include, but are not limited to, ZSTK474, AS252424, LY294002, and TG100115.

Examples of PI3Kδ inhibitors include PI3KII, TGR-1202, AMG-319, GSK2269557, X-339, X-414, RP5090, KAR4141, XL499, OXY111A, IPI-145, IPI-443 and compounds described in WO 2005/113556 (ICOS ), WO 2013/052699 (Gilead Calistoga), WO 2013/116562 (Gilead Calistoga), WO 2014/100765 (Gilead Calistoga), WO 2014/100767 (Gilead Calistoga) and WO 2014/201409 (Gilead Sciences), but not limited to them.

Examples of PI3Ke inhibitors include, but are not limited to, GSK2636771, BAY 10824391, and TGX221.

Examples of PI3Ka inhibitors include, but are not limited to, buparlisib, BAY 80-6946, BYL719, PX-866, RG7604, MLN1117, WX-037, AEZA-129 and PA799.

Examples of common PI3K inhibitors include, but are not limited to, LY294002, BEZ235, XL147 (SAR245408) and GDC0941.

Examples of SYK inhibitors include, but are not limited to, tamatinib (R406), fostamatinib (R788), PRT062607, BAY61-3606, NVP-QAB 205 AA, R112, R343, and the compounds described in US Pat. No. 8,450,321 (Gilead Connecticut).

TKI targets may include epidermal growth factor receptors (EGFR) and fibroblast growth factor receptors (FGF), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF). Examples of TKIs that target EGFR include, but are not limited to, gefitinib and erlotinib. Sunitinib is a non-limiting example of a TKI that targets the FGF, TGF and VEGF receptors.

Combinations with an immune checkpoint inhibitor

In some embodiments, the anti-CD73 antibodies of the present invention may be used in conjunction with an immune checkpoint inhibitor. Immune checkpoints are immune system molecules that turn a signal on (co-stimulatory molecules) or off. Many cancers defend themselves against the immune system by inhibiting T cell signaling. An immune checkpoint inhibitor can help stop this cell defense mechanism. The target of an immune checkpoint inhibitor can be any or any of the following checkpoint molecules - PD-1, PD-L1, CTLA-4, LAG-3 (also known as CD223), CD28, CD122, 4-1BB (also known as CD137) or BTLA (also known as CD272).

Programmed T cell death protein 1 (PD-1) is a transmembrane protein found on the surface of T cells that, when bound to programmed T cell death protein ligand 1 (PD-L1) on tumor cells, results in suppressive activity T cells and reduction of T cell-mediated cytotoxicity. Thus, PD-1 and PD-L1 are inhibitors of the immune system or immune checkpoint switches. Representative PD-1 inhibitors include, but are not limited to, nivolumab (Opdivo) (BMS-936558), pembrolizumab (Keytruda), pidilizumab, AMP-224, MEDI0680 (AMP-514), PDR001, MPDL3280A, MEDI4736, BMS-936559, and MSB0010718C.

Programmed cell death protein ligand-1 (PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homolog-1 (B7-H1), is a protein encoded by the CD274 gene in humans. Non-limiting examples of PD-L1 inhibitors include atezolizumab (Tecentriq), durvalumab (MEDI4736), avelumab (MSB0010718C), MPDL3280A, BMS935559 (MDX-1105) and AMP-224.

CTLA-4 is a protein receptor that suppresses the immune system. Non-limiting examples of CTLA-4 inhibitors include ipilimumab (Yervoy) (also known as BMS734016, MDX-010, MDX-101) and tremelimumab (formerly known as ticilimumab, CP-675206).

Lymphocyte activation gene 3 (LAG-3) is an immune checkpoint receptor on the cell surface that suppresses the immune response through its effects on Tregs as well as a direct effect on CD8+ T cells. LAG-3 inhibitors include, but are not limited to, LAG525 and/or BMS-986016.

CD28 is constitutively expressed on almost all CD4+ T cells and approximately half of all CD8 T cells and stimulates T cell expansion. Non-limiting examples of CD28 inhibitors include TGN1412.

CD122 enhances the proliferation of effector CD8+ T cells. Non-limiting examples include NKTR-214.

4-1BB (also known as CD137) is involved in T cell proliferation. The CD37-mediated signaling pathway is also known to protect T cells and, in particular, CD8+ T cells from activation-induced cell death. PF-05082566, urelumab (BMS-663513) and lipocalin are examples of CD137 inhibitors.

When using any of the above therapeutic agents, the anti-CD73 antibody can be administered simultaneously or separately from another anti-cancer agent. When administered separately, the anti-CD73 antibody may be administered before or after the other anticancer agent.

Diagnostic methods

Overexpression of CD73 is observed in some tumor samples, and patients with cells overexpressing CD73 are likely to respond to anti-CD73 antibody therapeutics of the present invention. Accordingly, the antibodies of the present invention can also be used for diagnostic or prognostic purposes.

The sample, which preferably contains the cell, can be obtained from a patient, which may be a cancer patient or a patient for whom it is desired to perform a diagnosis. The cell may be a cell from tumor tissue or a tumor block, a blood sample, a urine sample, or any sample from the patient's body. With optional sample pre-preparation, the sample can be incubated with an antibody of the present invention under conditions that allow the antibody to react with CD73 protein potentially present in the sample. Methods such as ELISA can be used to detect the presence of CD73 protein in a sample, taking full advantage of the anti-CD73 antibody.

The presence of CD73 protein in a sample (not necessarily in a specific amount or concentration) can be used to diagnose cancer, as an indication that a patient is suitable for treatment with an antibody, or as an indication that a patient has responded (or not responded) to cancer treatment. For a prognostic method, detection is performed one, two or more times at certain stages after initiation of cancer treatment to indicate the progress of treatment.

Compositions

The present invention also provides pharmaceutical compositions. Such a composition contains an effective amount of an antibody and a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises a second anticancer agent (eg, an immune checkpoint inhibitor).

In a specific embodiment, the term pharmaceutically acceptable means approved by a regulatory agency of the federal or state government or listed in the United States Pharmacopoeia or other generally accepted pharmacopoeia for use in animals and, more specifically, in humans. In addition, a pharmaceutically acceptable carrier is generally a non-toxic solid, semi-solid or liquid excipient, diluent, encapsulation material or excipient of any type.

The term carrier refers to the diluent, adjuvant, excipient or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, for example, water and oils, including petroleum, vegetable, animal or synthetic oils, for example, peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is the preferred carrier for intravenous administration of a pharmaceutical composition. Saline solutions and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, particularly for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene glycol, water, ethanol, etc. P. If desired, the composition may also contain small amounts of humectants or emulsifiers or pH buffering agents, for example acetates, citrates or phosphates. In addition, the use of antibacterial agents is envisaged, for example, benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, for example, ethylenediaminetetraacetic acid; and agents to adjust tonicity, such as sodium chloride or dextrose. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The composition can be formulated as a suppository using conventional vehicles and carriers, for example triglycerides. Formulations for oral administration may contain standard carriers, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. pharmaceutical grade. Examples of suitable pharmaceutical carriers are described by E. W. Martin in Remington's Pharmaceutical Sciences, incorporated herein by reference. Such a composition may contain a therapeutically effective amount of the antigen binding polypeptide, preferably in purified form, together with a suitable amount of carrier to formulate for proper administration to a patient. The composition must correspond to the method of administration. The parent drug can be placed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

In one embodiment, the composition is formulated according to conventional procedures into a pharmaceutical composition suitable for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also contain a solubilizing agent and a local anesthetic, for example, lignocaine, to relieve pain at the injection site. In general, the ingredients are administered separately or in mixture with each other in a dosage form, for example, as a dry lyophilized powder or an anhydrous concentrate in a sealed container, such as an ampoule or sachet, indicating the amount of active agent. If the composition is to be administered by infusion, it may be dispensed into an infusion bottle containing sterile water or pharmaceutical grade saline. If the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided to mix the ingredients before administration.

The compounds of the present invention can be formulated in neutral or salt form. Pharmaceutically acceptable salts include salts formed by anions, for example, hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and salts formed by cations, for example, hydroxides of sodium, potassium, ammonium, calcium, iron (III), isopropylamine , triethylamine, 2ethylamine ethanol, histidine, procaine, etc.

Examples

Example 1: Cloning of mouse antibody 101-Mu.

This example describes the process of producing a mouse monoclonal antibody against human CD73 using hybridoma technology. Human CD73 protein was produced using a recombinant CD73-expressing CHOK1 cell line (CD73-CHOK1). To generate mouse monoclonal antibodies against human CD73, 6-8 week old female BALB/c mice were first immunized with 1.5 x 10 ⁷ CD73-CHOK1 cells. On days 14 and 33 after the first immunization, immunized mice were boosted with 1.5 x 10 ⁷ CD73-CHOK1 cells. To select mice that produce antibodies that bind to the CD73 protein, sera from immunized mice were analyzed by ELISA. Briefly, microtiter plates were coated with 1 μg/ml human CD73 protein in PBS in a volume of 100 μl/well at room temperature (RT) overnight and then blocked with 100 μl/well 5% BSA. Dilutions of plasma from immunized mice were added to each well and incubated for 1-2 hours at RT. The plates were washed with PBS/Tween and then incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody for 1 h at RT. After washing, the plates were developed with ABTS substrate and analyzed on a spectrophotometer at an OD of 405 nm. Mice with sufficient anti-CD73 IgG titers were boosted with 50 μg of human CD73-Fc protein on day 54 postimmunization. The resulting mice were used for cell fusion. Hybridoma supernatants were tested for the presence of anti-CD73 IgG by ELISA. Eight different hybridoma clones were identified, among which 101-Mu was selected for further analysis. The VH sequence of 101-MU is shown in Table 6 as SEQ ID NO: 10, and the VL sequence is shown in Table 7 as SEQ ID NO: 15. The corresponding DNA sequences are shown in Table 1. 10 as SEQ ID NO: 57 and 58.

Example 2: Binding of 101-Mu to CD73.

In this example, the binding of the mouse anti-human CD73 mAb 101-Mu to recombinant human CD73 protein (1 μg/ml in 100 μl) was tested by itself or on the cell surface in a dose-dependent manner in an ELISA.

Microtiter plates were coated with recombinant human CD73 protein (Novoprotein) at a concentration of 1 μg/ml in PBS for 2 h at room temperature (RT). After antigen coating, wells were blocked with PBS/0.05% Tween (PBST) with 1% BSA for 1 h at RT. After washing the wells with PBST, various concentrations of anti-CD73 antibodies were added to the wells and incubated for 1 h at RT. To detect antibody binding, anti-mouse Fc secondary antibodies conjugated to HRP (Jackson Immuno Research) were added, followed by the addition of fluorogenic substrates (Roche). Between all incubation steps, the plate wells were washed three times with PBST. Fluorescence was measured using a TECAN Spectrafluor plate reader.

- 26 043570 human anti-CD73 mAb1 was used as a positive control. mAb1 was prepared according to the sequence described in US2016/0194407. The results for comparison are presented in Fig. 1, which shows that the EC50 for 101-Mu is 0.08 nM and the EC50 for mAb1 is 0.03 nM.

In fig. 2 shows the results of a binding assay using a human ovarian cancer cell line (SK-OV-3 cells) endogenously expressing human CD73 on the surface. After incubation with the indicated antibodies, cells were stained with various concentrations of control IgG, mouse anti-CD73 antibody (101-Mu), and reference antibody (mAb1) at 4°C for 30 min. Cells were then washed three times with PBS followed by incubation with APC-labeled anti-mouse Fc specific antibody (Invitrogen) at 4°C for 30 min. Binding was measured using FACSCanto (Becton-Dickinson). As in FIG. 1, these figures show that 101-Mu had equivalent binding affinity to CD73 compared to mAb1 (EC50 for 101-Mu was 0.54 nM; EC50 for mAb1 was 0.30 nM).

In fig. Figure 3 shows graphs of the binding kinetics of 101-Mu and mAb1 to recombinant human CD73 (recombinant human CD73 was used as the analyte at successive concentrations (100, 50, 25, 12.5, 6.25, 3.125 nM)). Antibody-antigen binding kinetics analysis was performed using the Biacore T200 system via a human antibody capture approach. Anti-mouse Fc IgG was immobilized on a CM5 sensor chip according to the manufacturer's instructions. The test antibody was administered and captured by immobilized anti-human Fc IgG. Successive concentrations of antigen were then administered individually and the binding profile was recorded for each concentration of antigen tested, respectively. The assay system was regenerated by injecting 10 mM glycine-HCl, pH 1.5 for 30 seconds. The running buffer was HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% P20). The assay temperature was 25°C, and the association and dissociation times were 180 and 600 seconds, respectively. Biacore data were fitted using Biacore T200 evaluation 1.0 software according to a 1:1 binding model to calculate association (ka) and dissociation (kd) rate constants as well as equilibrium constant (KD). In addition to FIG. 3 some summary data is presented in the table below.

Sample ka (1/Ms) kd (1/s) KD (M) 101-Mi 8DZE+04 3.42E-05 4.21E-10 mat! 2.08E+05 2.73E-05 1.31E-10

Example 3. Inhibition of CD73 enzymatic activity by 101-Mu.

In this example, the ability of the 101-Mu antibody to inhibit the enzymatic activity of CD73 was tested.

Recombinant human CD73 protein (0.3 μg/ml) was incubated in 96-well flat-bottom microplates in the presence of varying doses of anti-CD73 antibody or isotype control Ab. The plates were incubated for 15 min at 37°C. ATP (100 μM) and AMP (100 μM) were then added to each well and incubated for another 30 min at 37°C. CellTiterGlo containing luciferase (Promega) was added to the wells, and light emission inhibition was measured using a luminometer (Molecular Device).

Excess AMP is known to block ATP-dependent luciferase activity. Addition of CD73, which catalyzes the production of adenosine and inorganic phosphate from AMP, restored luciferase activity and light emission. Thus, an antibody that blocks CD73 enzymatic activity may reduce light emission. Percentage enzymatic activity was assessed as described below: Residual CD73 activity was calculated as follows: (CD73+At+ATP+AMP)-(ATP+AMP)/(CD73+ATP+AMP)-(ATP+AMP)*100. A graph of the results is shown in Fig. 4, which shows that, in contrast to the negative control IgG, 101 Mu inhibited CD73 enzymatic activity in a dose-dependent manner (IC50=3.89 nM).

CD73-expressing SK-OV-3 cells were seeded in a 96-well plate at 1 x 10 ⁵ cells per well in the presence of various doses of anti-CD73 Ab or isotype control Ab. The plates were incubated for 15 min at 37°C. AMP (100 μM) was added to each well and incubated for 1 h at 37°C. The supernatants were collected into a new 96-well plate and ATP was added to a final concentration of 100 μM. CellTiter-Glo reagent (Promega) was added at a 1:1 ratio and the enzymatic activity of cellular CD73 was determined by measuring light emission on a luminometer (Molecular Device). The percentage enzymatic activity was estimated as described below: Residual CD73 activity was calculated as follows: (SK-OV3 cells+At+ATP+AMP)-(ATP+AMP)/(SK-OV-3 cells+ATP+AMP)-( ATP+AMP)*100. A graph of the results is shown in Fig. 5, which shows that, in contrast to the negative control IgG, 101-Mu inhibited CD73 enzymatic activity in a dose-dependent manner (IC50=4.62 nM).

Example 4: Reversal of AMP-Mediated CD4+ T Cell Suppression by 101-Mu.

In this example, the ability of the antibody to reverse AMP-mediated suppression of CD4+ T cells was tested.

- 27 043570

Human CD4+ T cells were purified from PBMCs by positive selection using human CD4 microbeads (Miltenyi Biotech). Isolated CD4+ T cells were stimulated using preimmobilized anti-CD3 antibody (2 μg/ml) and soluble anti-CD28 antibody (1 μg/ml) in the presence or absence of AMP (500 μM). Serial dilutions of anti-CD73 and control IgG antibodies were added to each well, cultured for 72 h, and the supernatant was analyzed for the presence of IFN-γ by ELISA. As shown in FIG. 6, 101-Mu increased IFN-γ production by CD4+ cells in a dose-dependent manner, while control had no significant effect.

Example 5. Humanization of 101-Mu.

The above examples demonstrate that the 101-Mu antibody is a potent inhibitor of CD73 enzymatic activity and can completely reverse the immunosuppressive effect of adenosine on T cell activation. Accordingly, this antibody was chosen as the basis for humanization.

The 101-Mu variable region genes were used to create a humanized MAb. VH and VK 101-MU were compared with an available database of human Ig gene sequences to identify the highest matching human embryonic Ig gene sequences. The closest matches among human sequences for the light chain were the A10/Jk4 (construct 1) and L6/Jk4 (construct 2) genes, and for the heavy chain, the VH4-B/JH6 gene. Humanized variable domain sequences were then constructed, where CDR1 (SEQ ID NO.4), 2 (SEQ ID NO.5) and 3 (SEQ ID NO.6) of the 101-MU light chain were grafted onto the framework sequences of the corresponding light chain genes, and the sequences CDR1 (SEQ ID NO.1), 2 (SEQ ID NO.2) and 3 (SEQ ID NO.3) of VH 101-MU were grafted onto the framework sequences of the corresponding VH gene.

A 3D model was then generated to determine the presence of positions in the framework regions where substitution of a mouse amino acid for a human amino acid could affect CDR binding and/or conformation. The identified beneficial reverse mutations, as well as the peptide sequences containing such mutations, are shown in the tables below (highlighted).

Table 3

VH backmutations and VH sequences with backmutations

VH design: VH4-b/JH6 Construct Mutation VH 101-Mi Chimera VH.1 101-Mi Transplanted CDRs VH.la 101-Mi V71R VH.lb 101-Mi V71R, V67I VH.lc 101-Mi V71R, V67I, I48M VH.ld 101-Mi V71R, V67I, I48M, S30T, G44K

Table 4

VK backmutations and VK sequences with backmutations (construct 1)

Design 1 VK: A10/Jk4 Construct Mutation Vk 101-Mu Chimera Vk.l 101-Mu Transplanted CDRs Vk.la 101-Mu F71Y, D70S Vk.lb 101-Mu F71Y, D70S, K49S Vk.lc 101-Mu F71Y, D70S, K49S, L46P, L47W Vk.ld 101-Mu F71Y, D70S, K49S, L46P, L47W, T5S

Table 5

VK backmutations and VK sequences with backmutations (construct 1)

Design 2 VK: L6/Jk4 Construct Mutation VK.2 101-Mu Transplanted CDRs VK.2a 101-Mu F71Y, D70S VK.2b 101-Mu F71Y, D70S, K49S, I58V VK.2c 101-Mu F71Y, D70S, K49S, L46P, L47W, T5S, A43S, I58V

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Table 6

Humanized VH and humanized VH with back mutations

Name (SEQ ID NO:) Sequences Kabat no. VH 101-Mu (10) VH.l 101-Mu (11) VH.IA 101-Mu (9) VH.1B 101-Mu (12) VH.1C 101-Mu (13) VH.1D 101-Mu (7) VH4-B/JH6 (14) 12 3 4 1234567890123456789012345678901A234567890123456789 DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMG EVQLQESGPGLVKPSETLSLTCAVSGYSISSGYYWNWIRQPPGKGLEWIG EVQLQES GPGLVKPSETLSLTCAVSGYSISSGYYWNWIRQPPGKGLEWIG EVQLQESGPGLVKPSETLSLTCAVSGYSISSGYYWNWIRQPPGKGLEWIG EVQLQESGPGLVKPSETLSLTCAVSGYSISSGYYWNWIRQPPGKGLEWMG EVQLQESGPGLVKPSETLSLTCAVSGYSITSGYYWNWIRQPPGKKLE WMG QVQLQESGPGLVKPSETLSLTCAVSGYSISSGYYWGWIRQPPGKGLEWIG No. no Kabat VH 101-Mu 5 6 7 8 9 012345678901234567890123456789012ABC34567890123456 YINYGGSNGYNPSLKSRISITRDTSKNQFFLKLNSVTTEDTATYYCARDY VH.l 101-Mu VH.IA 101-Mu VH.1B 101-Mu VH.1C 101-Mu VH.1D 101-Mu VH4-B/JH6 YINYGGSNGYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDY YINYGGSNGYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARDY YINYGGSNGYNPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARDY YINYGGSNGYNPSLKSRITIISRDTSKNQFSLKLSSVTAAD TAVYYCARDY YINYGGSNGYNPSLKSRITIISRDTSKNQFSLKLSSVTAADTAVYYCARDY SIYHSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR— No. no Kabat VH 101-Mu VH.l 101-Mu VH.IA 101-Mu VH.1B 101-Mu VH.1C 101-Mu VH.1D 101-Mu VH4-B/JH6 0 1 7890ABC1234567890123 DAYYEALDDWGQGT SVTVS S DAYYEALDDWGQGTTVTVSS DAYYEALDDWGQGTTVTVSS DAYYEALDDWGQGTTVTVS S DAYYEALDDWGQGTTVTVS S DAYYEALDDWGQGTTVTVS S --------- _{WGQGTTVTV SS}

Bold: CDR sections; underlined font: back mutations

Table 7

Humanized VK and humanized VK with back mutations (design 1)

Name (SEQ ID NO: ) Sequences No. no Kabat 1 2 3 4 5 123456789012345678901234567890123456789012345678901 VK 101-Mu (15) QIVLSQSPAILSASPGEKVTMTCRASSRVN-YMHWYQQKPGSSPKPWISAT VK.l 101-Mu (16) EIVLTQSPDFQSVTPKEKVTITCRASSRVN-YMHWYQQKPDQSPKLLIKAT VK.IA 101-Mu (17) EIVLTQSPDFQSVTPKEKVTITCRASSRVN-YMHWYQQKPDQSPKLLIKAT VK.1B 101-Mu (18) EIVLTQSPDFQSVTPKEKVTITCRASSRVN-YMHWYQQKPDQSPKLLISAT VK.1C 101-Mu (19) EIVLTQSPDFQSVTPKEKVTITCRASSRVN-YMHWYQQKPDQSPKPWISAT VK.1D 101-Mu (20) EIVLSQSPDFQSVTPKEKVTITCRASSRVN-YMHWYQQKPDQSPKPWISAT A10/JK4 (21) EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSLHWYQQKPDQSPKLLIKYA No. no Kabat 6 7 8 9 0 234567890123456789012345678901234567890123456789012 Vk 101-Mu SNLASGVPARFSGSGSGTSYSLTISRVETEDAATYYCQQWSSNPPTFGGGT VK.l 101-Mu SNLASGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQWSSNPPTFGGGT VK.IA 101-Mu SNLASGVPSRFSGSGSGTSYTLTINSLEAEDAATYYCQQWSSNPPTFGGGT VK.1B 101-Mu SNLASGVPSRFSGSGSGTSYTLTINSLEAEDAATYYCQQWSSNPPTFGGGT VK.1C 101-Mu SNLASGVPSRFSGSGSGTSYTLTINSLEAEDAATYYCQQWSSNPPTFGGGT VK.1D 101-Mu SNLASGVPSRFSGSGSGTSYTLTINSLEAEDAATYYCQQWSSNPPTFGGGT A10/JK4 SQSFSGVPSRFSGSGSGTDFTLTINSLEAEDAATYYC---------FGGGT No. no Kabat 34567 Vk 101-Mu KLEIK VK.l 101-Mu KVEIK VK.IA 101-Mu KVEIK VK.1B 101-Mu KVEIK VK.1C 101-Mu KVEIK VK.1D 101-Mu KVEIK A10/JK4 KVEIK

Bold: CDR sections; underlined font: back mutations

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Humanized VK and humanized VK with back mutations (design 2)

Table 8

Name (SEQ ID NO: ) Sequences Kabat no. 1 2 3 4 5 123456789012345678901234567890123456789012345678901 VK 101-Mu (15) QIVLSQSPAILSASPGEKVTMTCRASSRVN-YMHWYQQKPGSSPKPWISAT VK.2 101-Mu (22) EIVLTQSPATLSLSPGERATLSGRASSRVN-YMHWYQQKPGQAPRLLIYAT VK.2A 101-Mu (23) EIVLTQSPATLSLSPGERATLSCRASSRVN-YMHWYQQKPGQAPRLLIYAT VK.2B 101-Mu (24) EIVLTQSPATLSLSPGERATLSCRASSRVN-YMHWYQQKPGQAPRLLISAT VK.2C 101-Mu (8) EIVLSQSPATLSLSPGERATLSCRASSRVN-YMHWYQQKPGQSPRPWISAT L6/JK4 (25) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDA No. no Kabat 6 7 8 9 0 234567890123456789012345678901234567890123456789012 Vk 101-Mu SNLASGVPARFSGSGSGTSYSLTISRVETEDAATYYCQQWSSNPPTFGGGT VK.2 101-Mu SNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSSNPPTFGGGT VK.2A 101-Mu SNLASGIPARFSGSGSGTSYTLTISSLEPEDFAVYYCQQWSSNPPTFGGGT VK.2B 101-Mu SNLASGVPARFSGSGSGTSYTLTISSLEPEDFAVYYCQQWSSNPPTFGGGT VK.2C 101-Mu SNLASGVPARFSGSGSGTSYTLTISSLEPEDFAVYYCQQWSSNPPTFGGGT L6/JK4 SNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC---------FGGGT No. no Kabat 34567 Vk 101-Mu KLEIK VK.2 101-Mu KVEIK VK.2A 101-Mu KVEIK VK.2B 101-Mu KVEIK VK.2C 101-Mu KVEIK L6/JK4 KVEIK

Bold: CDR sections; underlined font: back mutations

Humanized VH and VK genes were produced synthetically and respectively cloned into vectors containing human gamma 1 kappa constant domains. The conjugation of human VH and VK allowed the creation of a number of humanized antibodies, listed in the table below.

Table 9

Humanized antibodies

VK.la 101- Mu VK.lb 101Mu VK.lc 101- Mu VK.ld 101Mu VK.2a 101Mu VK.2b 101Mu VK.2c 101Mu VK 101-Mu VH.la 101-Mu HulOl-l Hui 01-2 Hui 01-3 Hui 01-4 HulOl-17 Hul01-21 Hul01-25 VH.lb 101-Mu HulOl-5 Hui 01-6 Hui 01-7 Hui 01-8 Hul01-18 Hul01-22 Hui 01-26 VH.lc 101-Mu HulOl-9 HulOl-10 HulOl-ll Hul01-12 Hul01-19 Hul01-23 Hui 01-27 VH.ld 101-Mu Hul01-13 HulOl-14 Hul01-15 Hul01-16 Hul01-20 Hul01-24 Hul01-28 VH 101Mu 101-Mu chimera

In table 10 shows several examples of nucleotide sequences encoding the VH and VL regions of antibodies 101-MU and Hu101-28.

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Table 10

Nucleotide sequences

Name SEQ ID NO: Subsequence VH 101-Mi 57 A GTTTTTCCTGAAGCTGA ATTCTGTGACTACTGAGGACACAGCTACATATTACTGTGCAAGAGACTAT GATGGTTACTACGAAGCTCTGGACGACTGGGGTCAAGGAACCTCAGTCAC CGTCTCCTCA VL 101-Mi 58 CAAATTGTTCTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGA GAAGGTCACAATGACTTGCAGGGCCAGCTCACGTGTAAATTACATGCACT GGTACCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTCTGCCACA TCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGG GACCTCTTACTCTCTCACAATTAGCAGAGTAGAGACT GAAGATGCTGCCA CTTATTACTGCCAGCAGTGGAGTAGTAACCCACCCACGTTCGGAGGGGGG AC CAAG CT G GAAATAAAA VHHul01-28 59 GAGGTACAGCTTCAGGAGTCAGGACCTGGCCTCGTGAAACCTTCTGAGAC TCTGTCTCTCACCTGCGCTGTCTCTGGCTACTCCATCACCAGTGGTTATT ACTGGAACTGGATCCGGCAGCCTCCAGGAAAGAAGCTGGAATGGATGGGC TACATCAACTACGGCGGTAGCAATGG CTACAACCCAT CT CTCAAAAGTCG GATCACCATCTCTAGGGACACATCTAAGAAC CAGTTTTCCCTGAAGCTGA GTTCTGTGACTGCTGCCGACACAGCTGTGTATTACTGTGCAAGAGACTAT GATGCTTACTACGAAGCTCTGGACGACTGGGGTCAAGGAACCACAGTCAC CGTCTCCTCA VLHulOl-28 60 GAAATTGTTCTCTCCCAGTCTCCAGCAACCCTGTCTCTGTCTCCAGGGGA GAGGGCCACACTGTCTTGCAGGGCCAGCTCACGTGTAAATTACATGCACT GGTACCAGCAGAAGCCAGGACAGTCCCCCCAGACCCTGGATTTCTGCCACA TCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGG GACCTCTTACACTCTCACAATTAGCAGCCTGGA GCCAGAAGATTTCGCCG TGTATTACTGCCAGCAGTGGAGTAGTAACCCACCCACGTTCGGAGGGGGG AC CAAG GT G GAAAT CAAA

Example 6: Testing of Humanized Antibodies.

In this example, humanized antibodies were tested for their binding affinity and inhibitory activity. The testing procedures are similar to those described in examples 1-4, the results are presented in Fig. 7-11.

The Biacore T200 system was used for binding analysis using a human antibody capture approach. Anti-human Fc IgG was immobilized on the CM5 sensor chip according to the manufacturer's instructions. The test antibody was administered and captured by immobilized anti-human Fc IgG. Successive concentrations of antigen were then administered individually and the binding profile was recorded for each concentration of antigen tested, respectively. The assay system was regenerated by injecting 10 mM glycine-HCl, pH 1.5 for 30 seconds. The running buffer was HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% P20). The assay temperature was 25°C, and the association and dissociation times were 180 and 600 seconds, respectively. Biacore data were fitted using Biacore T200 evaluation 1.0 software according to a 1:1 binding model to calculate association (ka) and dissociation (kd) rate constants as well as equilibrium constant (KD).

Among all antibodies tested, Hu101-25 and Hu101-28 showed the highest binding affinity (see Fig. 7 and table below); these were used for further analysis.

Sample ka (1/Mc) kd(1/c) KD(M) HulOl-25 7.37E+04 5.51E-05 7.47E-10 Hul01-28 5.89E+05 3.47E-05 5.90E-10

A human ovarian cancer cell line (SK-OV-3 cells) endogenously expressing human CD73 on the surface was stained with various concentrations of control IgG, a chimeric anti-CD73 antibody (101-Mu-chimeric, or 101-Chi), and a humanized anti-CD73 antibody (Hu101 -25 and Hu101-28) at °C for 30 minutes. Cells were then washed three times with PBS followed by incubation with APC-labeled anti-human Fc specific antibody (Invitrogen) at 4°C for 30 min. Binding was measured using FACSCanto (Becton-Dickinson) and the results are shown in FIG. 8 (see also table below).

101-chimeric EC50 = 1.35 hM HulOl-25 EC50 = 2.38 hM Hul01-28 EC50 = 1.35 hM

Recombinant human CD73 protein (0.3 μg/ml) was incubated in 96-well flat-bottom microplates in the presence of varying doses of anti-CD73 antibody or isotype control Ab. The plates were incubated for 15 min at 37°C. ATP (100 μM) and AMP (100 μM) were then added to each well and incubated for another 30 min at 37°C. CellTiter-Glo containing luciferase (Promega) was added to the wells and light emission inhibition was measured using a luminometer

- 31 043570 (Molecular Device). Excess AMP is known to block ATP-dependent luciferase activity.

Addition of CD73, which catalyzes the production of adenosine and inorganic phosphate from AMP, restored luciferase activity and light emission. Thus, an antibody that blocks CD73 enzymatic activity may reduce light emission. The percentage enzymatic activity was assessed as described below: Residual CD73 activity: (CD73+At+ATP+AMP)-(ATP+AMP)/(CD73+ATP+AMP)-(ATP+AMP)*100.

In fig. 9 shows that all three antibodies tested showed strong inhibitory activity (see also table below).

IC50 (nM) Max inhibition (%) 101-chimeric 4.25 98% Ni101-25 4.72 99% Hul01-28 9.35 98%

In fig. 10 shows the results of testing the inhibition of CD73 enzymatic activity by humanized antibodies. CD73-expressing SK-OV-3 cells were seeded in a 96-well plate at 1 x 10 ^L 5 cells per well in the presence of various doses of anti-CD73 Ab or isotype control Ab. The plates were incubated for 15 min at 37°C. AMP (100 μM) was added to each well and incubated for 1 hour at 37°C. Supernatants were collected in a new 96-well plate and ATP was added to a final concentration of 100 μM. CellTiterGlo reagent (Promega) was added at a 1:1 ratio and the enzymatic activity of cellular CD73 was determined by measuring light emission on a luminometer (Molecular Device). Percentage enzymatic activity was assessed as described below: Residual CD73 activity: (SK-OV3 cells+At+ATP+AMP)-(ATP+AMP)/(SK-OV-3 cells+ATP+AMP)-(ATP+AMP )*100. The results are shown in Fig. 10 and below in the table and demonstrate the powerful inhibitory effect of these antibodies.

IC50 (nM) Max inhibition (%) 101-chimeric 1.82 100% Ni101-25 2.98 100% Hul01-28 2.52 100%

To test the ability of antibodies to reverse AMP-mediated CD4+ T cell suppression, human CD4+ T cells were purified from PBMCs by positive selection using human CD4 microbeads (Miltenyi Biotech). Isolated CD4+ T cells were stimulated using preimmobilized anti-CD3 antibody (2 μg/ml) and soluble anti-CD28 antibody (1 μg/ml) in the presence or absence of AMP (500 μM). Serial dilutions of anti-CD73 and control IgG antibodies were added to each well, cultured for 72 h, and the supernatant was analyzed for the presence of IFN-γ by ELISA. As shown in FIG. 11, all three antibodies tested reversed AMP-mediated CD4+ T cell suppression in a dose-dependent manner.

Example 7: Computer simulation of further changes and optimization of humanized antibodies.

It was believed that certain amino acid residues in the CDR regions or framework regions could be changed to further improve or maintain the activity and/or stability of the antibodies. Variants were tested using a computer tool (VectorNTI, available at www.ebi.ac.uk/tools/msa/clustalo/) against their structural, conformational and functional properties, and promising sequences (within CDR regions) are listed below in the tables.

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Table 11

CDRs VH and VL and variants thereof suitable for inclusion in humanized antibodies

Name Subsequence SEQ ID NO: VH CDR1 SGYYWN 1 Option TGYYWN 26 Option CGYYWN 27 Option SAYYWN 28 Option SPYYWN 29

Name Subsequence SEQ ID NO: VH CDR2 YINYGGSNGY NPSLKS 2 Option YINYGASNGY NPSLKS thirty Option YINYGPSNGY NPSLKS 31 Option YINYGGTNGY NPSLKS 32 Option YINYGGCNGY NPSLKS 33 Option YINYGGSDGYNPSLKS 34 Option YINYGGSEGY NPSLKS 35 Option YINYGGSQGY NPSLKS 36

Name Subsequence SEQ ID NO: VH CDR3 DYDAYYEALD 3 Option DYDAYYDALD 37 Option DYDAYYQALD 38 Option DYDAYYNALD 39 Option DYDAYYEGLD D 40 Option DYDAYYECLD D 41

Name Subsequence SEQ ID NO: VL CDR1 RASSRVNYM N 4 Option RATSRVNYM N 42 Option RACSRVNYM N 43 Option RASTRVNYM N 44 Option RASCRVNYM N 45

Name Subsequence SEQ ID NO: VL CDR3 QQWSSNPPT 6 Option NQWSSNPPT 46 Option DQWSSNPPT 47 Option EQWSSNPPT 48 Option QNWSSNPPT 49 Option QDWSSNPPT 50 Option QEWSSNPPT 51 Option QQYSSNPPT 52 Option QQWTSNPPT 53 Option QQWCSNPPT 54 Option QQWSTNPPT 55 Option QQWSCNPPT 56

Underlined (font): hot spot mutation residues and their substitutions Example 8: Hu101-28 is an anti-CD73 antibody with a dual mechanism of action.

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This example shows that Hu101-28 has a dual mechanism of action. It not only blocks the enzymatic activity of CD73 in a non-competitive manner, but also induces CD73 internalization to the cell surface.

CD73 protein binding was tested using soluble CD73 and cell surface CD73, and the results are shown in FIG. 12. In the left panel, 100 μl of a solution containing recombinant human CD73 (2 μg/ml) was used in an ELISA binding assay, and the graph shows the binding of Hu101-28. In the right panel, human ovarian carcinoma cell line (SK-OV-3) was stained with various concentrations of Hu101-28 and surface binding analyzed by flow cytometry. As shown, the EC ₅₀ in the tests was 88.7 pM and 0.67 nM, respectively, highlighting the high affinity of the antibody.

The ability of Hu101-28 to block CD73 activity was then tested. Recombinant human CD73 (0.3 μg/ml) was incubated with Hu101-28 for 15 min. ATP (100 μM) and AMP (100 μM) were then added for another 30 min. CellTiter-Glo (Promega) was added and light emission inhibition was measured using a luminometer. As shown in FIG. 13, left panel, the IC ₅₀ in solution was 2.84 nM. With respect to CD73 present on the cells, SK-OV-3 or MDA-MB-231 cells (1χ10 ^Λ 5 cells) were incubated with Hu101-28 for 15 min, and then AMP (100 μM) and ATP (100 μM) followed by incubation for 1 hour. CellTiter-Glo (Promega) was added and light emission inhibition was measured using a luminometer. As shown in FIG. 13, right panel, the IC ₅₀ was 2.52 nM and 8.21 nM for SK-OV-3 and MDA-MB-231, respectively.

Further experiments showed that Hu101-28 did not compete with the AMP substrate for binding to the active site, but acted allosterically or through other non-competitive mechanisms, which is preferable from the point of view of avoiding the need to compete with multiple endogenous AMP substrates. As shown in FIG. 14, increasing the concentration of the AMP substrate did not prevent hydrolysis by Hu101-28, indicating that Hu101-28 acts as a non-competitive inhibitor. In contrast, the inhibitory activity of ADP can be overcome by increasing the substrate concentration to eliminate the binding of ADP to the active site.

Interesting and unexpected data are shown in FIG. 15 Internalization of CD73 following binding to Hu101-28 was demonstrated by measuring the cell surface level of CD73 by flow cytometry or the viability of MDA-MB-231 cells following binding of Hu309 in the presence of a toxin-conjugated secondary antibody. Compared to IgG, which did not change cell surface CD73 levels, Hu101-28 resulted in more than half the reduction in cell surface CD73 levels within 6 hours (left panel), and even greater cell killing (right panel).

Thus, the above experiments demonstrated that Hu101-28 has a dual mechanism of action (MoA): it noncompetitively blocks the enzymatic activity of CD73 and induces CD73 cell surface internalization.

Example 9 Hu101-28 completely reverses the suppression of CD4+ and CD8+ T-cell responses mediated by AMP or tumor cells.

This example demonstrates that Hu101-28 completely reverses the suppression of CD4+ and CD8+ T cell responses mediated by AMP or tumor cells.

Human CD4+ or CD8+ T cells were labeled with CFSE and stimulated with anti-CD3/CD28 antibodies in the presence or absence of AMP. Hu101-28 was added to the culture for 72 hours. The culture supernatant was analyzed for IFN-g production using solid-phase ELISA. In fig. 16, left panel, Hu101-28 increased IFN-γ production in CD4+ cells in a dose-dependent manner. Similarly, as shown in the right panel, Hu101-28 increased IFN-γ production in CD8+ cells in a dose-dependent manner.

To test the effect of Hu101-28 on tumor cell-mediated suppression of T cell responses, CD73-expressing SK-OV-3 cells were treated with mitomycin C and cocultured with purified CD4+ or CD8+ T cells in the presence of anti-CD3/CD28 antibodies. Hu101-28 was added to the culture for 72 h, and the culture supernatant was analyzed for IFN-γ production using ELISA. As shown in FIG. 17, left panel, Hu101-28 increased IFN-γ production in CD4+ cells in a dose-dependent manner. Similarly, as shown in the right panel, Hu101-28 increased IFN-γ production in CD8+ cells in a dose-dependent manner.

Example 10 Hu101-28 suppresses tumor growth.

This example demonstrated that Hu101-28 effectively suppressed tumor CD73 activity, resulting in tumor growth inhibition in monotherapy or combination therapy.

In vivo CD73 enzymatic activity was measured by enzymatic histochemistry in A375 xenograft model tumors, and the staining results are shown in FIG. 18. Brown staining indicates the presence of active CD73, while the absence of brown staining indicated inhibition of CD73 enzymatic activity by the antibody. A375 xenograft model tumors treated with Hu101-28 or control IgG were collected and the results showed that CD73 activity in tumor sections was significantly reduced by Hu101-28 treatment compared to the control IgG group.

The efficacy of Hu101-28 as monotherapy was also evaluated in a tumor xenograft model. A375 melanoma cells and human PBMCs were transplanted into NSG mice with immunodeficiency. On the day of transplantation, tumor-bearing mice were given different doses of Hu101-28 or control IgG by intraperitoneal injection every other day. As shown in FIG. 19, Hu101-28 consistently suppressed tumor growth in a dose-dependent manner.

The combinatorial effect of Hu101-28 and anti-PD-L1 antibodies was assessed. HCC827 cells were transplanted into NSG mice. When tumor volume reached 100-150 mm ³ , tumor-bearing mice were injected with freshly isolated human PBMC via tail vein injection and then treated with control IgG, anti-PD-L1 antibody alone, Hu101-28 alone, and anti-PD-L1 antibody in combination with Hu101-28 at the indicated doses every two days, starting from the day of PBMC transplantation. In fig. 20 shows the synergistic effect of Hu101-28 and anti-PD-L1 antibody.

Example 11 Hu101-28 cross-reacts with cynomolgus CD73.

This example demonstrates that the humanized antibody Hu101-28 cross-reacted with cynomolgus CD73 and had an acceptable PK and safety profile in an exploratory clinical study.

In fig. 21 shows binding and inhibition of cynomolgus CD73 activity by Hu101-28. Hu101-28 had comparable efficacy in binding and inhibiting the activity of cynomolgus CD73 compared to human CD73 in an in vitro biochemical assay, with no binding of Hu101-28 to rodent CD73. These results support the use of the cynomolgus macaque as a non-clinical study model to evaluate the toxicity, PK and TK of Hu101-28.

Example 12: Hu101-28 binds to the C-terminal domains of CD73.

This example shows that one of the humanized antibodies, Hu101-28, bound to the C-terminal domains of the CD73 protein, in contrast to known anti-CD-73 antibodies that bind to the N-terminal domains.

Evaluation of epitopes of anti-CD73 antibodies was performed using competitive ELISA. CD73 ECD protein and Hu101-28 were preincubated and then added with biotinylated MEDI9447 (Medimmune), a streptavidin-HRP detection antibody. The results showed that the addition of biotinylated R9447 did not compete with Hu101-28 binding, indicating that both antibodies were located at non-overlapping epitopes (Figure 22). As a control, addition of biotinylated MEDI-9447 could result in binding competition with itself.

To identify the Hu101-28 epitope, a library of clones of CD73 variants with single amino acid mutations was obtained. The binding of Fab Hu101-28 to each variant in the library was determined in duplicate using high-throughput flow cytometry. For each CD73 variant, the average binding value was plotted against CD73 expression (represented by the reactivity of the control) and critical residues were identified on the plot. Critical residues for CD73 - Fab binding (in red) were identified as residues whose mutations were negative to the Fab binding assay but positive to the control mAbs. Average binding reactivity values (and ranges) are listed for the critical residues indicated in the screens and shown in FIG. 23. Hu309 bound to the C-terminal region of CD73 at epitope Y345, D399, E400, R401 and R480 (visualized in FIG. 24, top left panel), while MEDI-9447 bound to the N-terminal region (see comparison in Fig. 24).

The unique binding properties of Hu101-28 were thought to be responsible for its superior CD73 inhibition profile compared to known antibodies. This is demonstrated in FIG. 25. The top comparison shows that Hu101-28 showed complete inhibition of CD73 activity without a high-dose effect, while MEDI-9447 at high doses showed a significant high-dose effect. In the lower comparison panel, Fab Hu101-28 demonstrated CD73 activity inhibition potency comparable to whole IgG, while Fab MEDI9447 did not inhibit soluble CD73.

This indicates that CD73 inhibition requires binding at both binding sites on the full MEDI-9447 antibody, whereas monovalent binding is sufficient for Hu101-28. It was hypothesized that MEDI-9447 bound to two different CD73 dimers to exert inhibitory activity, whereas Hu101-28 does not have such a requirement. To test this hypothesis, the inhibitory effect of Hu101-28 was tested using cells with different levels of CD73 expression. As shown in FIG. 26, Hu101-28 could achieve complete inhibition of CD73 activity on cells expressing varying levels of CD73 on the surface, including cells with low expression levels where the distance between CD73 molecules is likely to be significant.

The scope of the present invention is not limited to the specific embodiments described, which should be considered as a single illustration of individual aspects of the present invention, and any functionally equivalent compositions and methods are included within the scope of the present invention. It will be apparent to one skilled in the art that the methods and compositions of the present invention

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Claims (15)

Various modifications and changes can be made without departing from the spirit of the invention. Thus, it is intended that the present invention include modifications and variations of the present invention provided they fall within the spirit of the appended claims and their equivalents. All publications and patent applications referenced herein are incorporated herein by reference in their entirety to the same extent as if each individual publication or patent application had been specifically stated to be incorporated herein by reference. CLAIM 1. An isolated antibody or antigen-binding fragment thereof, wherein said antibody or fragment thereof has specificity for the human CD73 protein and contains a CDR1 VH containing the amino acid sequence of SEQ ID NO: 1, a CDR2 VH containing the amino acid sequence of SEQ ID NO: 2, CDR3 VH containing the amino acid sequence SEQ ID NO: 3; CDR1 VL containing the amino acid sequence SEQ ID NO: 4; CDR2 VL containing the amino acid sequence of SEQ ID NO: 5, and CDR3 VL containing the amino acid sequence of SEQ ID NO: 6. 2. The antibody or antigen-binding fragment thereof according to claim 1, further comprising a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. 3. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that the light chain constant region is a kappa or lambda chain constant region. 4. An antibody or an antigen-binding fragment thereof according to claim 1, characterized in that the antibody or fragment thereof belongs to the IgG, IgM, IgA, IgE or IgD isotype. 5. The antibody or antigen-binding fragment thereof according to claim 4, characterized in that the isotype is IgG1, IgG2, IgG3 or IgG4. 6. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5, characterized in that the antibody or fragment thereof is a chimeric antibody, a humanized antibody or a fully human antibody. 7. The antibody or antigen-binding fragment thereof according to claim 6, characterized in that the antibody or fragment thereof is a humanized antibody. 8. The antibody or antigen-binding fragment thereof according to claim 6, comprising a heavy chain variable region containing one or more amino acid residues selected from the group consisting of: (a) Thr at position 30, (b) Lys at position 44, (c) Met at position 48, (d) Ile at position 67, and (e) Arg at position 71, according to Kabat numbering, and combinations thereof. 9. The antibody or antigen-binding fragment thereof according to claim 6, comprising a light chain variable region containing one or more amino acid residues selected from the group consisting of: (a) Ser at position 5, (b) Pro at position 46, (c) Trp at position 47, (d) Ser at position 49, (e) Ser at position 70, (f) Tyr at position 71, according with Kabat numbering, and their combinations. 10. An antibody or antigen binding fragment thereof according to any one of claims 1 to 9, comprising a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 9 to 13, or a peptide having at least 90 % identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and 9-13. 11. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 7 or 9. 12. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, comprising a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 15-20 and 22-24, or a peptide having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 15-20 and 22-24. 13. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that the light chain variable region contains the amino acid sequence of SEQ ID NO: 8. 14. A bispecific antibody comprising an antigen binding fragment according to any one of claims 1 to 13 and a second antibody or antigen binding fragment. 15. A pharmaceutical composition containing an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier. -