HK1262659A1 - Anti cd25 fc gamma receptor bispecific antibodies for tumor specific cell depletion - Google Patents

Description

anti-CD 25 FC gamma receptor bispecific antibodies for tumor-specific cell depletion

Technical Field

The present invention is in the field of cancer immunotherapy and relates to a method of treating cancer, including a method of treating solid tumors, wherein said method involves the use of antibodies against CD 25.

Background

Cancer immunotherapy involves the use of the subject's own immune system to treat or prevent cancer. Immunotherapy makes use of the fact that: cancer cells typically have slightly different molecules on their surface that can be detected by the immune system. These molecules or cancer antigens are the most common proteins, but also include molecules such as carbohydrates. Immunotherapy therefore involves stimulating the immune system to attack tumor cells via these target antigens. However, malignant tumors (particularly solid tumors) can evade immune surveillance by a variety of mechanisms inherent to tumor cells and mediated by components of the tumor microenvironment. In the latter, regulatory T Cell (Treg Cell or Treg) tumor infiltration and more specifically, the adverse balance of effector T cells (Teff) and tregs (i.e., low ratio of Teff to Treg) has been proposed as a key factor (Smyth M et al, 2014, Immunol Cell biol.92, 473-4).

Since its discovery, tregs have been found to be critical in mediating immune homeostasis and promoting the establishment and maintenance of peripheral tolerance. However, in the context of cancer, their effects are more complex. Since cancer cells express both self and tumor-associated antigens, the presence of tregs seeking to suppress effector cell responses may lead to tumor progression. Infiltration of tregs in established tumors therefore represents one of the major obstacles to effective anti-tumor response and general cancer therapy. The mechanism of inhibition adopted by tregs is thought to significantly lead to the limitation or even failure of current therapies, in particular immunotherapy relying on the induction or enhancement of an anti-tumor response (Onishi H et al; 2012 anticancer. res.32, 997-1003).

Depletion of tregs as a therapeutic approach to the treatment of cancer is a method supported by studies showing the contribution of tregs to tumor determination and progression in murine models. Furthermore, tumor infiltration of tregs has also been correlated with a poor prognosis in several human cancers (Shang B et al, 2015, Sci rep.5: 15179). However, depletion of tregs in tumors is complex and the results of studies in this field differ. Accordingly, there is a need in the art for a method of treating cancer involving the depletion of tregs.

Among the potential molecular targets for achieving Treg depletion, the IL-2/CD25 interaction has been the subject of several studies in murine models, some of which involved the use of PC61 (a rat anti-mouse CD25 antibody) (Setiady Y et al, 2010.Eur J immunol.40: 780-6). The CD25 binding and functional activity of this antibody has been compared to the CD25 binding and functional activity of a panel of monoclonal antibodies produced by different authors (Lowentha1 J.W et al, 1985.J.Immunol., 135, 3988-3994; Moreau, J. -L et al, 1987.Eur. J.Immunol.17, 929-935; Volk HD et al, 1989Clin. exp. Immunol.76, 121-5; Dantal J et al, 1991, Transplantation 52: 110-5).

In this way, three epitopes within this target have been characterized that are different from or common to the mouse IL-2 binding site of anti-mouse CD 25. PC61 (having the mouse IgG1 isotype) blocks or inhibits the binding of IL-2 to CD25, as do many other hybridomas with anti-mouse CD25 antibodies (and most of those are disclosed as anti-human CD25 antibodies; see, e.g., WO2004/045512, WO2006/108670, WO1993/011238, and WO 1990/007861). Furthermore, the binding of PC61 to mouse CD25 was not affected by ADP-ribosylation of CD25 at the IL-2 binding site, as for other anti-mouse CD25 antibodies (e.g., 7D4) (Teege S et al, 2015, Sci Rep 5: 8959).

Some references mention the use of anti-CD 25, alone or in combination, in cancer or in conjunction with Treg depletion. (WO 2004/074437; WO 2006/108670; WO 2006/050172; WO 2011/077245; WO 2016/021720; WO 2004/045512; Grauer O et al, 2007int.J. cancer: 121: 95-105). However, when tested in a mouse cancer model, rat anti-mouse CD25PC61 failed to exhibit anti-tumor activity when delivered after tumor determination.

In the context of a murine autoimmune model, the anti-CD 25PC61 antibody was re-engineered to evaluate the effect of highly diverse Fc effector function within the anti-CD 25 antibody on IL-2 receptor blockade and peripheral Treg depletion (Huss D et al, 2016. immunol.148: 276-86). However, the ability of PC61, used alone or in combination with other antibodies or anti-cancer compounds, to deplete tregs in tumors or mediate anti-tumor therapeutic activity was never evaluated (thus, as an engineered antibody, or as an anti-human CD25 designed or characterized as having CD25 binding characteristics similar to those of PC61 to mouse CD 25).

Disclosure of Invention

The present invention provides novel anti-CD 25 antibodies and novel uses of anti-CD 25 antibodies, said anti-CD 25 antibodies being characterized by structural elements that allow efficient depletion of tregs, in particular within tumors. Within this scope, the structural and functional characteristics of rat IgG1 PC-61 (as described for mouse CD25) have been modified in order to provide antibodies that exhibit unexpectedly improved characteristics in terms of their efficacy as a depleting Treg and against tumors, either alone or in combination with other anti-cancer agents. These findings can be used to define and generate novel anti-human CD25 that provide comparable effects against tumors in human subjects.

Therefore, the key findings of the present inventors are the following unexpected findings: anti-mouse anti-CD 25PC61 was only able to deplete tregs in lymph nodes and circulation, but failed to do so within tumors. The lack of Treg depletion in tumors correlates with a lack of anti-tumor activity. This new and unexpected data prompted the present inventors to increase the subtractive activity of anti-mouse CD25 by Fc engineering, which resulted in efficient depletion of intratumoral tregs and anti-tumor activity.

In one broad aspect, the invention provides a method of treating a human subject having cancer, the method comprising the step of administering to the subject an anti-CD 25 antibody, wherein the subject has a tumor (preferably a solid tumor), wherein the anti-CD 25 antibody is an IgG1 antibody that binds with high affinity to at least one activated fcgamma receptor (preferably selected from the group consisting of fcyri, fcyriic, and fcyriiia) and depletes tumor-infiltrating regulatory T cells.

Such antibodies preferably have a dissociation constant (K) for CD25_d) Is less than 10^-8M, and/or a dissociation constant for at least one activated Fc gamma receptor that is smallAt about 10^-6And M. Most preferably, the anti-CD 25 antibody is characterized by other features associated with Fc γ receptors, in particular:

(a) binds to Fc γ receptors with an activation to inhibition ratio (a/I) higher than 1; and/or

(b) Binds at least one of Fc γ RI, Fc γ RIIc, and Fc γ RIIIa with a higher affinity than that of Fc γ RIIb.

It may provide further preferred features in view of the use of anti-CD 25 antibodies in the method of treatment. The anti-CD 25 antibody is preferably a monoclonal antibody, in particular a human or humanized antibody. Furthermore, the anti-CD 25 antibody may further elicit an enhanced CDC, ADCC and/or ADCP response, preferably an increased ADCC and/or ADCP response, more preferably an increased ADCC response, in view of its interaction with immune cells and/or other components of the immune system to exert its activity.

The anti-CD 25 antibodies of the invention (as defined in general and in further detail in the detailed description above) may be used in methods of treating a human subject, wherein the anti-CD 25 antibody is administered to a subject having a defined solid tumor (preferably for use in a method further comprising the step of identifying a subject having a solid tumor). Such methods may further comprise administering to the subject an immune checkpoint inhibitor, e.g., in the form of an antibody that binds to and inhibits an immune checkpoint protein. Preferred immune checkpoint inhibitors are PD-1 antagonists, which may be anti-PD-1 antibodies or anti-PD-L1 antibodies. More generally, an anti-CD 25 antibody can be used in a method of depleting regulatory T cells in a solid tumor in a subject, the method comprising the step of administering the anti-CD 25 antibody to the subject.

In another aspect, the anti-CD 25 antibodies of the invention can be used in the manufacture of a medicament for treating cancer in a human subject, wherein the subject has a solid tumor. Within this scope, the antibodies are for administration in combination with an immune checkpoint inhibitor, preferably a PD-1 antagonist.

In another aspect, the invention provides a combination of an anti-CD 25 antibody as defined above and another anti-cancer compound (preferably an immune checkpoint inhibitor or other compound as shown in the specific embodiments) for use in the treatment of cancer in a human subject, wherein the subject has a solid tumor and the anti-CD 25 antibody and the anti-cancer compound (e.g., an immune checkpoint inhibitor, such as a PD-1 antagonist) are administered simultaneously, separately or sequentially. Within this scope, the invention also provides a kit for treating cancer comprising an anti-CD 25 antibody as defined above and an anti-cancer compound (e.g., an immune checkpoint inhibitor, such as a PD-1 antagonist),

in another aspect, the present invention also provides a pharmaceutical composition comprising an anti-CD 25 antibody as defined above in a pharmaceutically acceptable medium. Such compositions may also comprise an anti-cancer compound (e.g., an immune checkpoint inhibitor, such as a PD-1 antagonist),

in yet another aspect, the invention also provides a bispecific antibody comprising:

(a) a first antigen binding portion that binds CD 25; and

(b) a second antigen-binding moiety that binds to another antigen;

wherein the bispecific antibody is an IgG1 antibody that binds with high affinity to at least one activated Fc gamma receptor and depletes tumor infiltrating regulatory T cells. Preferably, such second antigen-binding moiety binds to an antigen selected from an immune checkpoint protein, a tumor-associated antigen, or is (or is based on) an anti-human activated Fc receptor antibody (anti-Fc γ RI, anti-Fc γ RIIc or anti-Fc γ RIIIa antibody) or is (or is based on) an antagonistic anti-human Fc γ RIIb antibody.

Preferably, such a bispecific antibody comprises a second antigen-binding moiety that binds an immune checkpoint protein selected from the group consisting of: PD-1, CTLA-4, BTLA, KIR, LAG3, VISTA, TIGIT, TIM3, PD-L1, B7H3, B7H4, PD-L2, CD80, CD86, HVEM, LLT1, GAL9, GITR, OX40, CD137, and ICOS. Such immune checkpoint proteins are preferably expressed on tumor cells and are most preferably selected from the group consisting of PD-1, PD-L1 and CTLA-4. The second antigen-binding moiety that binds to an immune checkpoint protein may be comprised by or based on commercially available antibodies that act as immune checkpoint inhibitors, such as:

(a) in the case of PD-1, the anti-PD-1 antibody may be nivolumab or pembrolizumab.

(b) In the case of PD-L1, anti-PD-L1 is atlizumab;

(c) in the case of CTLA-4, the anti-CTLA-4 is ipilimumab.

Such bispecific antibodies can be provided in any commercially available form, including Duobody, BiTE DART, crossMab, knob-hole (Knobs-in-holes), Triomab, or other suitable molecular forms of bispecific antibodies and fragments thereof.

Alternatively, such bispecific antibodies comprise a second antigen-binding moiety that binds a tumor-associated antigen. In this alternative embodiment, such antigens and corresponding antibodies include, but are not limited to, CD22 (breninitumumab), CD20 (rituximab, tositumomab), CD56 (lovolumab), CD66e/CEA (labezumab), CD152/CTLA-4 (ipilimumab), CD221/IGF1R (MK-0646), CD326/Epcam (ibritumomab), CD340/HER2 (trastuzumab, pertuzumab), and EGFR (tuximab, panitumumab).

The combination of the anti-CD 25 antibody of the invention with another anti-cancer compound or the bispecific antibody as defined above can be used in a method of treating cancer, comprising the step of administering said combination or said bispecific antibody to a subject, in particular when said subject has a solid tumor and is used to treat cancer in a subject.

Other objects of the invention, including further definitions of the anti-human CD25 antibodies of the invention and their use in methods of treating cancer, in pharmaceutical compositions, in combination with other anti-cancer compounds, in bispecific antibodies are provided in the detailed embodiments and examples.

Detailed Description

The present invention provides a method of treating or preventing cancer (in particular solid tumors) in a subject, said method comprising the step of administering to said subject an antibody that binds CD25, wherein said anti-CD 25 antibody is characterized by structural elements that allow for efficient depletion of tregs, in particular within tumors. The invention also provides an antibody that binds CD25 as defined herein for use in the treatment or prevention of cancer, in particular solid tumors. Alternatively, the invention provides the use of an antibody that binds CD25 and allows efficient depletion of tregs for the manufacture of a medicament for the treatment or prevention of cancer, in particular solid tumors. The invention also provides the use of an antibody that binds CD25 and allows efficient depletion of tregs in the treatment or prevention of cancer, particularly solid tumors.

The present invention discloses a way to switch the isotype of anti-CD 25 antibody (exemplified by the rat anti-mouse CD25 antibody PC61) to a subtractive isotype (mouse IgG2 against PC61, but equivalent to IgG1 in humans) such that the depletion of regulatory T cells in the context of solid tumors is improved. Furthermore, the inventors have for the first time found that CD25 can be targeted to deplete regulatory T cells in a therapeutic context (e.g., in established solid tumors), and that CD25 is preferentially expressed in regulatory T cells. The present inventors have found that engineered anti-CD 25 antibodies with enhanced binding to activated Fc γ receptors produce efficient depletion of tumor-infiltrating regulatory T cells, which is a therapeutic approach that may be associated with other cancer targeting compounds such as those that target immune checkpoint proteins, tumor-associated antigens, or inhibitory Fc γ receptors (in combination with or within bispecific antibodies), for example.

The inventors have also for the first time found that inhibitory Fc γ receptor IIb is upregulated at the tumor site, thereby preventing efficient intratumoral regulatory T cell depletion by the original anti-mouse CD25 antibody PC 61. Accordingly, the present invention encompasses therapeutic applications involving a combination approach involving targeting CD25 and Fc γ receptor IIb.

CD25 is the α chain of the IL-2 receptor and is present on activated T cells, regulatory T cells, activated B cells, some thymocytes, bone marrow precursors and oligodendrocytes CD25 associates with CD122 and CD132 to form a heterotrimeric complex that acts as a high affinity receptor for IL-2 the consensus sequence for human CD25 is shown in SEQ ID NO: 1 below (Uniprot accession number P01589; the extracellular domain of mature human CD25 (corresponding to amino acids 22-240) is underlined and shown in SEQ ID NO: 2):

as used herein, "an antibody that binds to CD 25" refers to an antibody that is capable of binding to the CD25 subunit of the IL-2 receptor.

anti-CD 25 antibodies are antibodies capable of specifically binding to the CD25 subunit (antigen) of the IL-2 receptor. "specifically binds", "specifically binds" and "specifically binds" are understood to mean the dissociation constant (K) of an antibody for an antigen of interest_d) Is less than about 10^-6M、10^-7M、10^-8M、10^-9M、10^-10M、10^-11M or 10^-12And M. In preferred embodiments, the dissociation constant is less than 10^-8M, e.g. at 10^-9M、10^-10M、10^-11M or 10^-12M is in the range of.

As used herein, the term "antibody" refers to intact immunoglobulin molecules and fragments thereof that comprise an antigen binding site, and includes polyclonal, monoclonal, genetically engineered, and other modified forms of antibodies, including, but not limited to, chimeric, humanized, heteroconjugate, and/or multispecific antibodies (e.g., bispecific, diabodies, triabodies, and tetrabodies), and antigen-binding fragments of antibodiesSegments including, for example, Fab ', F (ab')₂Fab, Fv, rIgG, polypeptide-Fc fusion, single chain variants (scFv fragment, VHH, VH, VL, Fv, rIgG, Fc fusion, or a fusion of a single chain variant and a single chain variant,Shark single domain antibodies, single chain or tandem diabodiesVHH、A minibody,Bicyclic peptides and other alternative immunoglobulin scaffolds). In some embodiments, the antibody may lack the covalent modifications that it would have if it were naturally produced (e.g., attachment of glycans). In some embodiments, the antibody may contain covalent modifications (e.g., attachment of glycans, detectable moieties, therapeutic moieties, catalytic moieties, or other chemical groups such as polyethylene glycol that provide improved stability of the antibody or administration). "antibody" may also refer to camelid antibodies (heavy chain only antibodies) and antibody-like molecules such as antiporters (Skerra (2008) FEBS J275, 2677-83). In some embodiments, the antibodies are polyclonal or oligoclonal antibodies, i.e., produced as a panel of antibodies, each antibody being associated with a single antibody sequence and binding to a more or less different epitope within the antigen (e.g., a different epitope within the extracellular domain of human CD25 associated with a different reference anti-human CD25 antibody). Polyclonal or oligoclonal antibodies can be provided in a single formulation for medical use as described in the literature (Keams JD et al 2015.Mol Cancer ther. 14: 1625-36).

In one aspect of the invention, the antibody is a monoclonal antibody. The antibody may additionally or alternatively be a humanized or human antibody. In another aspect, the antibody is a human antibody, or in any case an antibody having a form and characteristics that allow its use and administration in a human subject.

Antibodies (abs) and immunoglobulins (igs) are glycoproteins with the same structural features. The immunoglobulin may be from any class, such as lgA, lgD, IgG, IgE, or IgM. The immunoglobulin may be of any subclass, such as IgG₁、lgG₂、lgG₃Or lgG₄. In a preferred aspect of the invention, the anti-CD 25 antibody is from the IgG class, preferably IgG₁Sub-classes. In one aspect, the anti-CD 25 antibody is from a human IgG₁Sub-classes.

The Fc region of IgG antibodies interacts with several cellular Fc γ receptors (Fc γ rs) to stimulate and regulate downstream effector mechanisms. There are five activating receptors, namely Fc γ RI (CD64), Fc γ RIIa (CD32a), Fc γ RIIc (CD32c), Fc γ RIIIa (CD16a) and Fc γ RIIIb (CD16 b), and an inhibitory receptor Fc γ RIIb (CD32 b). Communication of IgG antibodies to the immune system is controlled and mediated by Fc γ rs, which relay information sensed and collected by the antibodies to the immune system, thereby providing a link between the innate and adaptive immune systems, and particularly in the context of biological therapeutics (Hayes J et al, 2016.J inflam Res 9: 209-.

The IgG subclasses differ in their ability to bind Fc γ R, and this differential binding determines their ability to elicit a range of functional responses. For example, in humans, Fc γ RIIIa is the primary receptor involved in the activation of antibody-dependent cell-mediated cytotoxicity (ADCC), and IgG3, closely followed IgG1, exhibit the highest affinity for this receptor, reflecting their ability to efficiently induce ADCC.

In a preferred embodiment of the invention, the antibody binds Fc γ R with high affinity, preferably binds to an activating receptor with high affinity. Preferably, the antibody binds with high affinity to Fc γ RI and/or Fc γ RIIa and/or Fc γ RIIIa. In a particular embodiment, the antibody binds Fc γ R with a dissociation constant of less than about 10^-6M、10^-7M、10^{- 8}M、10^-9M or 10^-10M。

In one aspect, the antibody is an IgG₁Antibodies, preferably human IgG₁An antibody capable of binding to at least one Fc activating receptor. For example, the antibody can bind to one or more receptors selected from the group consisting of Fc γ RI, Fc γ RIIa, Fc γ RIIc, Fc γ RIIIa, and Fc γ RIIIb. In one aspect, the antibody is capable of binding Fc γ RIIIa. In one aspect, the antibody is capable of binding Fc γ RIIIa and Fc γ RIIa and optionally Fc γ RI. In one aspect, the antibodies are capable of binding these receptors with high affinity, e.g., a dissociation constant of less than about 10^-7M、10^-8M、10^-9M or 10^-10M，

In one aspect, the antibody binds the inhibitory receptor Fc γ RIIb with low affinity. In one aspect, the antibody binds Fc γ RIIb with a dissociation constant greater than 10^-7M, higher than 10^-6M is greater than 10^-5M。

In a preferred embodiment of the invention, the anti-human CD25 antibody is derived from human IgG₁A subset, and preferably has ADCC and/or ADCP activity, as discussed herein, particularly with respect to human-derived cells. Indeed, as previously described (Nimmerjahn F et al, 2005.Science, 310: 1510-2), the mIgG2a isotype (which corresponds to the human IgG1 isotype) binds all Fc γ R subtypes with a high activation to inhibition ratio (a/I) at least higher than 1. In contrast, other isotypes (such as the rgig 1 isotype) bind with similar affinity to only a single activating Fc γ R (Fc γ RIII) and inhibitory Fc γ RIIb, resulting in a low a/I ratio (< 1). As shown in the examples, this lower a/I ratio correlates with lower intratumoral Treg depletion and lower antitumor therapeutic activity of the isoforms.

In preferred embodiments, an anti-CD 25 antibody as described herein preferably binds human CD25 with high affinity. Still preferably, the anti-CD 25 antibody binds to the extracellular region of human CD25, as shown above. In one aspect, the invention provides an anti-CD 25 antibody as described herein. In particular, the examples provide experimental data generated with antibodies secreted by the PC-61.5.3 hybridoma and generally identified as PC61 or PC-61. Assays involving PC-61 and mouse CD25 in the literature (e.g., Setiady Y et al, 2010.eur.j. immunol.40: 780-6; McNeill a et al, 2007.Scand jimmunol.65: 63-9; Teege S et al, 2015, Sci Rep 5: 8959) and those disclosed in the examples (including recombinant antibodies comprising the CD25 binding domain of PC61) may be useful in characterizing, when appropriately isotype-related, those human antibodies that recognize human CD25 having the same functional characteristics of PC61 at the level of interaction with CD25 (in particular, by blocking IL-2 binding) and with fcgamma receptor (in particular, by preferentially binding to human activated fcgamma receptor and effectively depleting Treg), as described in the examples. Suitable methods will be known to those skilled in the art to achieve the desired functional characteristics of the antibodies as described herein.

In a preferred embodiment, the method of treating a human subject having cancer comprises the step of administering to the subject an anti-CD 25 antibody, wherein the subject preferably has a solid tumor, and wherein the anti-CD 25 antibody is preferably a human IgG1 antibody that binds with high affinity to at least one activated fcgamma receptor selected from the group consisting of fcyri (CD64), fcyriic (CD32c), and fcyriiia (CD16a) and depletes tumor-infiltrating regulatory T cells. Preferably, the dissociation constant (K) of the anti-CD 25 antibody to CD25_d) Is less than 10^-8And M. More preferably, the anti-CD 25 antibody binds to human CD25, thereby providing an effect on IL-2 binding and Treg depletion similar to that on mouse CD 25. In another embodiment, the anti-CD 25 antibody binds Fc γ RI (CD64), Fc γ RIIc (CD32c), Fc γ RIIIa (CD16a) with an activation to inhibition ratio (a/I) greater than 1, and/or with a higher affinity than that of binding Fc γ RIIb (CD32 b).

The CD25 binding domain of the PC-61 antibody has been cloned and expressed as a recombinant protein fused to an appropriate constant region. The sequence of the CD25 binding domain of the PC-61 antibody, as well as its specificity for different epitopes within the extracellular domain of CD25 and/or its other functional activity, can be used to compare candidate anti-CD 25 antibodies generated and screened by any suitable technique (e.g., by culturing groups of hybridomas or generating recombinant antibody libraries from CD 25-immunized rodents, and then screening these antibody lineages for functional characterization as described herein with CD25 fragments). The anti-CD 25 antibodies thus identified may also be produced as recombinant antibodies, particularly as intact antibodies or as fragments or variants as described herein.

Natural antibodies and immunoglobulins are typically heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has a variable domain at the amino terminus (V)_H) Followed by multiple constant domains. Each light chain has a variable domain at the amino terminus (V)_L) And a constant domain at the carboxy terminus.

The variable region is capable of interacting with structurally complementary antigen targets and is characterized by differences in amino acid sequence from antibodies with different antigen specificities. The variable region of the H chain or L chain contains an amino acid sequence capable of specifically binding to an antigen target. Within these sequences are smaller sequences called "hypervariable" because they have extreme variability between antibodies with different specificities. Such hypervariable regions are also referred to as "complementarity determining regions" or "CDR" regions.

These CDR regions explain the basic specificity of an antibody for a particular antigenic determinant structure. CDRs represent non-contiguous amino acid segments within the variable region, but regardless of the species, the positional positioning of these key amino acid sequences within the variable heavy and light chain regions has been found to have similar positioning in the amino acid sequences of the variable chains. The variable heavy and light chains of all antibodies each have 3 CDR regions, each of which is not contiguous with the other CDR regions of the respective light (L) and heavy (H) chains (designated L1, L2, L3, H1, H2, H3). The accepted CDR regions have been previously described (Kabat et al, 1977.J Biol Chem 252, 6609-.

The antibodies of the invention may act through Complement Dependent Cytotoxicity (CDC) and/or antibody dependent cell mediated cytotoxicity (ADCC) and/or antibody dependent cell mediated phagocytosis (ADCP) as well as any other mechanism that allows targeting, blocking proliferation and/or depleting Treg cells.

"Complement Dependent Cytotoxicity (CDC)" involves lysis of antigen-expressing cells by the antibody of the present invention in the presence of complement.

"antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (fcrs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on target cells and thereby cause lysis of the target cells.

"antibody-dependent cell-mediated phagocytosis" (ADCP) refers to a cell-mediated response in which Fc receptor (FcR) expressing phagocytic cells (e.g., macrophages) recognize bound antibody on target cells and thereby cause phagocytosis of the target cells.

CDC, ADCC and ADCP can be measured using assays known and available in the art (Clynes et al (1998) Proc Natl Acad Sci USA 95, 652-6). The constant regions of antibodies are important in the ability of the antibody to fix complement and mediate cell-dependent cellular cytotoxicity and phagocytosis. Thus, as discussed herein, the isotype of an antibody can be selected based on whether it is desired that the antibody mediate cytotoxicity/phagocytosis.

As discussed herein, in one embodiment of the invention, an anti-CD 25 antibody that causes Treg cell depletion is used. For example, anti-CD 25 antibodies that elicit a strong CDC response and/or strong ADCC and/or strong ADCP response may be used. Methods for increasing CDC, ADCC and/or ADCP are known in the art. For example, the CDC response may be increased with mutations in the antibody that increase the affinity of C1q binding (Idusogie et al (2001) J lmmunol 166, 2571-5).

ADCC can be increased by eliminating fucose moieties from the antibody glycans, e.g., by producing antibodies in the YB2/0 cell line, or by producing antibodies in human IgG₁By introducing specific mutations (e.g., S298A/E333A/K334A, S239D/I332E/A330L, G236A/S239D/A330L/I332E) on the Fc portion of (e.g., S298A/E333A/K334) in the Fc portion of (e.g., Lazar et al (2006) Proc Natl Acad Sci USA 103, 2005-2010; Smith et al (2012) Proc Natl Acad Sci USA 109, 6181-6). ADCP may also be increased by introducing specific mutations in the Fc portion of human IgG1 (Richards et al (2008) Mol Cancer Ther7, 2517-27).

In a preferred embodiment of the invention, the antibody is optimized to elicit an ADCC response, i.e. the ADCC response is enhanced, increased or improved relative to other anti-CD 25 antibodies or the exemplary unmodified anti-CD 25 monoclonal antibody.

As used herein, "chimeric antibody" may refer to an antibody having variable sequences derived from an immunoglobulin from one species (e.g., a rat or mouse antibody) and immunoglobulin constant regions from another species (e.g., from a human antibody). In some embodiments, the chimeric antibody may have a constant region that is enhanced to induce ADCC.

The antibodies according to the invention may also be partially or wholly synthetic, wherein at least a portion of the polypeptide chains of the antibody are synthetic and possibly optimized for binding to their cognate antigen. Such antibodies may be chimeric or humanized antibodies, and may be fully tetrameric in structure, or may be dimeric and comprise only a single heavy chain and a single light chain.

The antibody of the present invention may also be a monoclonal antibody. As used herein, "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and does not refer to the method by which the antibody is produced.

The antibody of the present invention may also be a human antibody. As used herein, "human antibody" refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

anti-CD 25 antibodies exhibiting the characteristics as described herein represent another object of the invention. In another embodiment, the invention provides a nucleic acid molecule encoding an anti-CD 25 antibody as defined herein. In some embodiments, such provided nucleic acid molecules may contain codon-optimized nucleic acid sequences, and/or may be comprised in an expression cassette within an appropriate nucleic acid vector for expression in a host cell, such as, for example, a bacteria, yeast, insect, fish, mouse, simian, or human cell. In some embodiments, the invention provides a host cell comprising a heterologous nucleic acid molecule (e.g., a DNA vector) that expresses a desired antibody.

In some embodiments, the present invention provides methods of making an isolated anti-CD 25 antibody as defined above. In some embodiments, such methods can include culturing a host cell comprising a nucleic acid (e.g., a heterologous nucleic acid that can be contained by a vector and/or delivered to the host cell). Preferably, the host cell (and/or heterologous nucleic acid sequence) is arranged and constructed such that the antibody or antigen-binding fragment thereof is secreted from the host cell and isolated from the cell culture supernatant.

The antibodies of the invention may be monospecific, bispecific or multispecific. A "multispecific antibody" may have specificity for different epitopes of one target antigen or polypeptide, or may contain antigen-binding domains specific for more than one target antigen or polypeptide (Kufer et al (2004) Trends Biotechnol 22, 238-44).

In one aspect of the invention, the antibody is a monospecific antibody. As discussed further below, in an alternative aspect, the antibody is a bispecific antibody.

As used herein, "bispecific antibody" refers to an antibody that has the ability to bind to two different epitopes on a single antigen or polypeptide or on two different antigens or polypeptides.

The bispecific antibodies of the invention as discussed herein can be produced by the following method: biological methods, such as somatic cell hybridization; or genetic methods such as expression of non-native DNA sequences encoding the desired antibody structure in a cell line or organism; chemical means (e.g., by chemical coupling, genetic fusion, non-covalent association, or other means to one or more molecular entities, such as another antibody or antibody fragment); or a combination thereof.

Techniques and products allowing the generation of monospecificity or bispecific are known in the art, as in the literature, and are also extensively reviewed with respect to alternatives, antibody-drug conjugates, antibody design methods, in vitro screening methods, constant regions, post-translational and chemical modifications, improved features for triggering Cancer cell death, such as Fc engineering (Tiller K and tesser P, 2015Annu Rev Biomed eng.17: 191-216; Speiss C et al 2015.Molecular Immunology 67: 95-106; Weiner G2015. Nat Rev Cancer, 15: 361-370; Fan G et al 2015.J hematocol 8: 130).

As used herein, "epitope" or "antigenic determinant" refers to a site on an antigen to which an antibody binds. As is well known in the art, epitopes may be formed from contiguous amino acids (linear epitopes) or from noncontiguous amino acids juxtaposed by tertiary folding of a protein (conformational epitopes). Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically comprise at least 3, and more typically at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining the spatial conformation of an epitope are well known in the art and include, for example, x-ray crystallography and 2-D nuclear magnetic resonance. See, e.g., epipope Mapping Protocols in methods in Molecular Biology, vol 66, Glenn E.Morris, eds (1996).

In some embodiments, the anti-CD 25 antibody can be included in a medicament further comprising a conjugated payload, such as a therapeutic or diagnostic agent, particularly for cancer therapy or diagnosis. anti-CD 25 antibody conjugates with radionuclides or toxins may be used. Of radionuclides in generalExamples are, for example,⁹⁰Y、¹³¹i and⁶⁷cu, etc., and examples of commonly used toxins are doxorubicin and calicheamicin. In another embodiment, the anti-CD 25 antibody may be modified to have an altered half-life. Methods for achieving altered half-lives are known in the art.

In one embodiment, the antibody preferably can block the function of human CD25 in addition to promoting depletion (via ADCC, ADCP and/or CDC) of cells expressing CD 25. Preferably, it also blocks the binding of human IL-2 to human CD25, and most preferably blocks human IL-2 signaling in CD25 expressing cells.

In a preferred embodiment of the invention, the subject of any aspect of the invention as described herein is a mammal, preferably a cat, dog, horse, donkey, sheep, pig, goat, cow, hamster, mouse, rat, rabbit or guinea pig, but most preferably the subject is a human. Thus, in all aspects of the invention as described herein, the subject is preferably a human.

As used herein, the terms "cancer," "cancerous," and "malignant" refer to or describe the physiological condition in mammals that is generally characterized by unregulated cell growth.

Examples of cancer include, but are not limited to, carcinoma, lymphoma, leukemia, blastoma, and sarcoma. More specific examples of such cancers include squamous cell cancer, myeloma, small-cell lung cancer, non-small cell lung cancer, glioma, hepatocellular carcinoma (HCC), hodgkin's lymphoma, non-hodgkin's lymphoma, Acute Myelogenous Leukemia (AML), multiple myeloma, gastrointestinal (tract) cancer, kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, gastric cancer, bladder cancer, liver cancer, breast cancer, colon cancer, and head and neck cancer.

In one aspect, the cancer involves a solid tumor. Examples of solid tumors are sarcomas (including cancers caused by transformed cells of mesenchymal origin in tissues such as cancellous bone, cartilage, fat, muscle, blood vessels, hematopoietic cells or fibrous connective tissue), carcinomas (including tumors caused by epithelial cells), mesotheliomas, neuroblastomas, retinoblastomas, and the like. Cancers involving solid tumors include, but are not limited to, brain cancer, lung cancer, stomach cancer, duodenal cancer, esophageal cancer, breast cancer, colon and rectal cancer, kidney cancer, bladder cancer, kidney cancer, pancreatic cancer, prostate cancer, ovarian cancer, melanoma, oral cancer, sarcoma, eye cancer, thyroid cancer, urinary tract cancer, vaginal cancer, neck cancer, lymphoma, and the like.

In one aspect of the invention, the cancer is selected from melanoma, non-small cell lung cancer, renal cancer, ovarian cancer, bladder cancer, sarcoma, and colon cancer. In a preferred aspect of the invention, the cancer is selected from melanoma, ovarian cancer, non-small cell lung cancer and renal cancer. In one embodiment, the cancer is not melanoma, ovarian cancer, or breast cancer. In a preferred aspect, the cancer is sarcoma, colon cancer, melanoma or colorectal cancer, or more generally any human cancer for which the MCA205, CT26, B16 or MC38 cell lines (as identified in the examples) may represent a preclinical model useful for validating a compound as being a useful for its therapeutic management.

As used herein, the term "tumor" as it applies to a subject diagnosed with or suspected of having cancer refers to a malignant or potentially malignant neoplasm or tissue mass of any size, and includes primary tumors and secondary neoplasms. The terms "cancer," "malignant tumor," "neoplasm," "tumor," and "carcinoma" are also used interchangeably herein to refer to tumors and tumor cells that exhibit relatively abnormal, uncontrolled, and/or autonomous growth such that they exhibit an abnormal growth phenotype characterized by a significant loss of control over cell proliferation. Generally, target cells for detection or treatment include precancerous (e.g., benign), malignant, pre-metastatic, and non-metastatic cells. The teachings of the present disclosure may be associated with any and all cancers.

As used herein, a "solid tumor" is an abnormal growth or mass of tissue that generally does not contain cysts or fluid regions, particularly tumors and/or metastases (wherever located) other than leukemia or non-solid lymphoma. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them and/or the tissue or organ in which they reside. Examples of solid tumors are sarcomas (including cancers caused by transformed cells of mesenchymal origin in tissues such as cancellous bone, cartilage, fat, muscle, blood vessels, hematopoietic cells or fibrous connective tissue), carcinomas (including tumors caused by epithelial cells), melanomas, lymphomas, mesotheliomas, neuroblastomas and retinoblastomas.

Particularly preferred cancers according to the present invention include cancers characterized by the presence of a solid tumor, i.e. the subject does not have a non-solid tumor. In all aspects of the invention as discussed herein, preferably the cancer is a solid tumor, i.e. the subject has a solid tumor (and does not have a non-solid tumor).

Reference to "treating" or "treating" cancer as used herein defines the achievement of at least one positive therapeutic effect, such as, for example, a reduction in the number of cancer cells, a reduction in the size of a tumor, a reduction in the rate of cancer cell infiltration into peripheral organs, or a reduction in the rate of tumor metastasis or tumor growth.

Positive therapeutic effects in cancer can be measured in a number of ways (e.g., Weber (2009) J nuclear Med 50, 1S-10S). By way of example, with respect to tumor growth inhibition, T/C ≦ 42% is the lowest level of anti-tumor activity according to the National Cancer Institute (NCI) standard. T/C < 10% is considered a high level of anti-tumor activity, where T/C (%) ═ median tumor volume of treatment/median tumor volume of control x 100. In some embodiments, the treatment achieved by the therapeutically effective amount is any of Progression Free Survival (PFS), Disease Free Survival (DFS), or Overall Survival (OS). PFS (also referred to as "tumor progression time") refers to the length of time during and after treatment that cancer does not grow, and includes the amount of time a patient experiences a complete response or a partial response, as well as the amount of time a patient experiences stable disease. DFS refers to the length of time a patient remains disease-free during or after treatment. OS refers to an increase in life expectancy compared to the original or untreated individual or patient.

As used herein, reference to "preventing" (or preventing) refers to delaying or preventing the onset of cancer symptoms. Prevention may be absolute (so that no disease occurs) or may be effective in only certain individuals or for a limited amount of time.

In a preferred aspect of the invention, the subject has a defined tumor, i.e. a subject already having a tumor, e.g. is classified as a solid tumor. Thus, the invention as described herein can be used when the subject already has a tumor (e.g., a solid tumor). Thus, the present invention provides a treatment option that can be used to treat existing tumors. In one aspect of the invention, the subject has an existing solid tumor. The invention may be used as prophylaxis, or preferably as treatment, of a subject already suffering from a solid tumor. In one aspect, the invention is not used as a prophylactic or preventative.

In one aspect, using the invention as described herein, tumor regression can be enhanced, tumor growth can be impaired or reduced, and/or survival time can be enhanced, e.g., as compared to other cancer treatments (e.g., standard of care treatments for a given cancer).

In one aspect of the invention, a method of treating or preventing cancer as described herein further comprises the step of identifying a subject having cancer, in particular identifying a subject having a tumor, such as a solid tumor.

The dosage regimen of a therapy described herein effective to treat a cancer patient can vary depending on factors such as the disease state, age, and weight of the patient, and the ability of the therapy to elicit an anti-cancer response in the subject. The selection of an appropriate dosage will be within the ability of those skilled in the art. For example, 0.01, 0.1, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or 50 mg/kg. In some embodiments, such an amount is an amount of a unit dose (or an entire portion thereof) suitable for administration according to a dosing regimen that has been determined to correlate with a desired or beneficial result (i.e., with a therapeutic dosing regimen) when administered to a relevant population.

An antibody according to any aspect of the invention as described herein may be in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, diluent or excipient. These compositions include, for example, liquid, semi-solid, and solid dosage formulations, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, or liposomes. In some embodiments, the preferred form may depend on the intended mode of administration and/or therapeutic application. The antibody-containing pharmaceutical composition can be administered by any suitable method known in the art, including, but not limited to, oral, transmucosal, by inhalation, topical, buccal, nasal, rectal, or parenteral (e.g., intravenous, infusion, intratumoral, intranodal, subcutaneous, intraperitoneal, intramuscular, intradermal, transdermal, or other types of administration, involving physical disruption of the subject's tissue and administration of the pharmaceutical composition through a gap in the tissue). Such formulations may, for example, be in the form of injectable or infusible solutions suitable for intradermal, intratumoral or subcutaneous administration or for intravenous infusion. Administration may involve intermittent dosing. Alternatively, administration can involve continuous administration (e.g., perfusion) for at least a selected period of time, either simultaneously with or in between administration of the other compound.

In some embodiments, the antibody can be prepared with carriers that protect it from rapid release and/or degradation, such as controlled release formulations, such as implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used.

One skilled in the art will appreciate that, for example, the route of delivery (e.g., oral versus intravenous versus subcutaneous versus intratumoral, etc.) can affect the amount of the dose and/or the amount of the dose required can affect the route of delivery. For example, when a particular high concentration of an agent is of interest at a particular site or location (e.g., within a tumor), focused delivery (e.g., in this example, intra-tumor delivery) may be desirable and/or useful. Other factors to be considered in optimizing the route and/or dosing regimen of a given treatment regimen may include, for example, the particular cancer being treated (e.g., type, stage, location, etc.), the clinical condition of the subject (e.g., age, general health, etc.), the presence or absence of combination therapy, and other factors known to the practitioner.

The pharmaceutical compositions should generally be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, dispersions, liposomes or other ordered structures suitable for higher drug concentrations. Sterile injectable solutions can be prepared by incorporating the antibody in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions, pastes, and implantable sustained release or biodegradable formulations in oily or aqueous vehicles as discussed herein. Sterile injectable preparations may be prepared using non-toxic parenterally acceptable diluents or solvents. Each pharmaceutical composition used according to the invention may include pharmaceutically acceptable dispersing, wetting, suspending, isotonicity, coating, antibacterial and antifungal agents, carriers, excipients, salts or stabilizers that are non-toxic to the subject at the dosages and concentrations used. Preferably, such compositions may further comprise a pharmaceutically acceptable carrier or excipient for use in the treatment of cancer, which is compatible with a given method and/or site of administration, e.g. for parenteral (e.g. subcutaneous, intradermal or intravenous injection), intratumoral or peritumoral administration.

While one embodiment of a therapeutic method or composition used in accordance with the present invention may not be effective in achieving a positive therapeutic effect in every subject, it should do so in using a pharmaceutical composition and dosing regimen that is compatible with good medical practice and as tested by any statistical test known in the art (e.g., student's t-test, X-test)²Test, according to Mann andwhimey's U test, Kruskal-Wallis test (H test), Jonckheere-Terpsra test, and Wilcoxon test) consistent with a statistically significant number of subjects.

In the above and following, reference to a tumor, a tumor disease, a cancerous tumor, or a cancer may alternatively or additionally imply metastasis in the original organ or tissue and/or in any other location, regardless of the location of the tumor and/or metastasis.

As discussed herein, the present invention relates to depleting regulatory T cells (tregs). Thus, in one aspect of the invention, the anti-CD 25 antibody depletes or reduces tumor infiltrating regulatory T cells. In one aspect, the depleting is by ADCC. In another aspect, the subtraction is by ADCP. anti-CD 25 antibodies may also deplete or reduce circulating regulatory T cells. In one aspect, the depleting is by ADCC. In another aspect, the subtraction is by ADCP.

Accordingly, the present invention provides a method for depleting regulatory T cells in a tumor in a subject, the method comprising administering to the subject an anti-CD 25 antibody. In preferred embodiments, the tregs are depleted in solid tumors. By "depleting" is meant that the number, rate, or percentage of tregs is reduced relative to when the anti-CD 25 antibody is not administered. In particular embodiments of the invention as described herein, more than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 99% of the tumor-infiltrating regulatory T cells are depleted.

As used herein, "regulatory T cells" ("Tregs", "Treg cells" or "Tregs") refer to the CD4+ T lymphocyte lineage that specifically controls autoimmunity, allergic reactions, and infection. Typically, they modulate the activity of T cell populations, but they may also affect certain innate immune system cell types. Tregs are typically identified by the expression of the biomarkers CD4, CD25 and Foxp 3. Naturally occurring Treg cells typically account for about 5% -10% of peripheral CD4+ T lymphocytes. However, within the tumor microenvironment (i.e. tumor-infiltrating Treg cells), they may constitute up to 20% -30% of the total CD4+ T lymphocyte population.

Activated human Treg cells can directly kill target cells such as effector T cells and APCs through perforin or granzyme B-dependent pathways, cytotoxic T lymphocyte-associated antigen 4(CTLA4+) Treg cells induce indoleamine 2, 3-dioxygenase (IDO) expression by APCs and these in turn inhibit T cell activation by reducing tryptophan, Treg cells can release interleukin-10 (IL-10) and transforming growth factor (TGF β) in vivo and thus directly inhibit T cell activation and inhibit APC function by inhibiting expression of MHC molecules, CD80, CD86 and IL-12 Treg cells can also inhibit immunity by expressing high levels of CTLA4, CTLA4 can bind to CD80 and CD86 on antigen presenting cells and prevent proper activation of effector T cells.

In a preferred embodiment of the invention, the ratio of effector T cells to regulatory T cells in a solid tumor is increased. In some embodiments, the ratio of effector T cells to regulatory T cells in the solid tumor is increased to greater than 5, 10, 15, 20, 40, or 80.

Immune effector cells refer to immune cells involved in the effector phase of an immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T Cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils.

For example, monocytes, macrophages, neutrophils, eosinophils, and lymphocytes that express Fc α R are involved in the specific killing of target cells and present antigens to other components of the immune system, or bind antigen-presenting cells.

In some embodiments, different agents directed to cancer may be administered by the same or different delivery routes and/or according to different regimens in combination with the antibody. Alternatively or additionally, in some embodiments, one or more doses of the first active agent are administered substantially simultaneously with one or more other active agents, and in some embodiments by a common route and/or as part of a single composition. One skilled in the art will further appreciate that synergy is achieved according to some embodiments of the combination therapies provided herein; in some such embodiments, the dosage of one or more agents used in the combination may be substantially different (e.g., lower) and/or may be delivered by an alternative route than is standard, preferred, or necessary when the agents are used in a different treatment regimen (e.g., as a monotherapy and/or as part of a different combination therapy).

In some embodiments, when two or more active agents are used according to the present invention, such agents may be administered simultaneously or sequentially. In some embodiments, administration of one agent is specifically timed relative to administration of another agent. For example, in some embodiments, a first agent is administered such that a particular effect is observed (or expected to be observed, e.g., based on a population study showing a correlation between a given dosing regimen and a target particular effect). In some embodiments, the desired relative dosing regimen for the agents administered in the combination can be empirically assessed or determined, e.g., using ex vivo, in vivo, and/or in vitro models; in some embodiments, such assessment or empirical determination is made in vivo, in a patient population (e.g., such that correlations are established), or alternatively in a particular target patient.

In another aspect of the invention, the inventors have shown that anti-CD 25 antibodies show improved therapeutic effect when combined with immune checkpoint inhibitors. As shown in this example, combination therapy with an anti-CD 25 antibody and an immune checkpoint inhibitor may have a synergistic effect in the treatment of established tumors. The data in this example for PD-1/PD-L1 relate to interfering with the PD-1/PD-L1 interaction. Thus, the interaction between the PD-1 receptor and the PD-L1 ligand can be blocked, resulting in "PD-1 blockade". In one aspect, the combination may result in enhanced tumor regression, enhanced impaired or reduced tumor growth and/or enhanced survival time using the invention as described herein, e.g., as compared to the anti-CD 25 antibody alone or the PD-1/PD-L1 blocking (either directly using the anti-PD 1 antibody, or indirectly using the anti-PD-L1 antibody).

As used herein, an "immune checkpoint" or "immune checkpoint protein" refers to a protein that belongs to an inhibitory pathway in the immune system, in particular for modulating T cell responses. Under normal physiological conditions, immune checkpoints are critical to prevent autoimmunity, particularly during responses to pathogens. Cancer cells are able to alter the regulation of immune checkpoint protein expression in order to avoid immune surveillance.

Examples of immune checkpoint proteins include, but are not limited to, PD-1, CTLA-4, BTLA, KIR, LAG3, TIGIT, CD155, B7H3, B7H4, VISTA, and TIM3, and also OX40, GITR, ICOS, 4-1BB, and HVEM. An immune checkpoint protein may also refer to a protein that binds to other immune checkpoint proteins that modulate an immune response in an inhibitory manner. Such proteins include PD-L1, PD-L2, CD80, CD86, HVEM, LLT1 and GAL 9.

By "immune checkpoint protein inhibitor" is meant any protein that can interfere with signal transduction and/or protein-protein interactions mediated by an immune checkpoint protein. In one aspect of the invention, the immune checkpoint protein is PD-1 or PD-L1. In a preferred aspect of the invention as described herein, the immune checkpoint inhibitor interferes with the PD-1/PD-L1 interaction by an anti-PD-1 or anti-PD-L1 antibody.

Accordingly, the present invention also provides a method of treating cancer comprising administering to a subject an anti-CD 25 antibody and a checkpoint inhibitor. The invention also provides an anti-CD 25 antibody and an immune checkpoint inhibitor for use in the treatment of cancer.

The invention further provides the use of an anti-CD 25 antibody and an immune checkpoint inhibitor for the manufacture of a medicament for the treatment of cancer. Administration of the anti-CD 25 antibody and the immune checkpoint inhibitor may be simultaneous, separate or sequential.

The present invention provides a combination of an anti-CD 25 antibody and an immune checkpoint inhibitor for use in the treatment of cancer in a subject, wherein the anti-CD 25 antibody and the immune checkpoint inhibitor are administered simultaneously, separately or sequentially. Such anti-human CD25 antibodies are preferably human IgG1, and may be used in particular in combination with antibodies targeting immune checkpoints, either in the presence or absence of sequences that allow ADCC, ADCP and/or CDC.

In an alternative aspect, the invention provides an anti-CD 25 antibody for use in the treatment of cancer, wherein the antibody is for administration in combination with an immune checkpoint inhibitor. The invention also provides the use of an anti-CD 25 antibody in the manufacture of a medicament for the treatment of cancer, wherein the medicament is for administration in combination with an immune checkpoint inhibitor.

The present invention provides a pharmaceutical composition comprising an anti-CD 25 antibody and an immune checkpoint inhibitor in a pharmaceutically acceptable medium. As discussed above, the immune checkpoint inhibitor may be an inhibitor of PD-1, i.e., a PD-1 antagonist.

PD-1 (programmed cell death protein 1) (also known as CD279) is a cell surface receptor expressed on activated T cells and B cells. Interactions with its ligands have been shown to attenuate T cell responses in vitro and in vivo. PD-1 binds two ligands, PD-L1 and PD-L2. PD-1 belongs to the immunoglobulin superfamily. PD-1 signaling requires PD-1 ligand binding in close proximity to peptide antigens presented by the Major Histocompatibility Complex (MHC) (Freeman (2008) Proc Natl Acad Sci USA105, 10275-6). Thus, proteins, antibodies or small molecules that prevent co-ligation of PD-1 and TCR on T cell membranes are useful PD-1 antagonists.

In one embodiment, the PD-1 receptor antagonist is an anti-PD-1 antibody, or antigen-binding fragment thereof, that specifically binds to PD-1 and blocks the binding of PD-L1 to PD-1. The anti-PD-1 antibody may be a monoclonal antibody. The anti-PD-1 antibody may be a human antibody or a humanized antibody. An anti-PD-1 antibody is an antibody that is capable of specifically binding to the PD-1 receptor. anti-PD-1 antibodies known in the art include nivolumab and pembrolizumab.

The PD-1 antagonists of the present invention also include compounds or agents that bind and/or block the PD-1 ligand to interfere with or inhibit the binding of the ligand to the PD-1 receptor, or that directly bind and block the PD-1 receptor without inducing inhibitory signal transduction through the PD-1 receptor. Alternatively, the PD-1 receptor antagonist can directly bind to the PD-1 receptor without triggering inhibitory signal transduction, and also bind to a ligand of the PD-1 receptor to reduce or inhibit the ligand from triggering signal transduction through the PD-1 receptor. By reducing the number and/or amount of ligands that bind to PD-1 receptors and trigger transduction of inhibitory signals, fewer cells are attenuated by the negative signal delivered by PD-1 signaling, and a more robust immune response can be achieved.

In one embodiment, the PD-1 receptor antagonist is an anti-PD-L1 antibody, or antigen-binding fragment thereof, that specifically binds to PD-L1 and blocks the binding of PD-L1 to PD-1. The anti-PD-L1 antibody may be a monoclonal antibody. The anti-PD-L1 antibody can be a human antibody or a humanized antibody, such as atuzumab (MPDL 3280A).

The invention also provides a method of treating cancer comprising administering to a subject an anti-CD 25 antibody and an antibody that is an agonist of a T cell activation co-stimulatory pathway. Antibody agonists for the T cell activation co-stimulatory pathway include, but are not limited to, agonist antibodies to ICOS, GITR, OX40, CD40, LIGHT, and 4-1 BB.

Surprisingly, the inventors have identified that the level of the inhibitory Fc receptor Fc γ RIIb (CD32b) can be increased in solid tumors. Thus, another method of treating cancer comprises administering an anti-CD 25 antibody and a compound that reduces, blocks, inhibits and/or antagonizes Fc γ RIIb (CD32 b). Such Fc γ RIIb antagonists may be small molecules that interfere with Fc γ RIIb-induced intracellular signaling, modified antibodies that do not engage inhibitory Fc γ RIIb receptors, or anti-human Fc γ RIIb (anti-CD 32b antibodies, for example, antagonistic anti-human Fc γ RIIb antibodies have also been characterized for their anti-tumor properties (Roghanian a et al, 2015, Cancer cell.27, 473-488; Rozan C et al, 2013, Mol Cancer ther.12: 1481-91; WO 2015173384; WO 0220080933).

In another aspect, the invention provides a bispecific antibody comprising:

(a) a first antigen binding portion that binds CD 25; and

(b) a second antigen-binding portion that binds an immune checkpoint protein, a tumor-associated antigen, is (or is based on) an anti-human activated Fc receptor antibody (e.g., anti-FcgRI, anti-fcgriiia, anti-fcgriiii), or is (or is based on) an antagonistic anti-human fcyriib antibody;

wherein said bispecific antibody is preferably an IgG1 antibody that binds with high affinity to at least one activated fey receptor and depletes tumor infiltrating regulatory T cells.

As used herein, "tumor-associated antigen" refers to an antigen that is expressed on tumor cells, thereby distinguishing them from non-cancer cells adjacent to them, and includes, but is not limited to, CD20, CD38, PD-L1, EGFR, EGFRV3, CEA, TYRP1, and HER 2. Various review articles have been disclosed describing relevant tumor-associated antigens and corresponding therapeutically useful anti-tumor antibodies (see, e.g., Sliwkowski & Mellman (2013) Science 341, 192-8). Such antigens and corresponding antibodies include, but are not limited to, CD22 (brimonizumab), CD20 (rituximab, tositumomab), CD56 (lovozumab), CD66e/CEA (rabeprizumab), CD152/CTLA-4 (ipilimumab), CD221/IGF1R (MK-0646), CD326/Epcam (ibritumomab), CD340/HER2 (trastuzumab, pertuzumab), and EGFR (cetuximab, panitumumab).

In one aspect, a bispecific antibody according to the invention as described herein produces ADCC, or in one aspect, enhanced ADCC.

The bispecific antibody can bind to a specific epitope on CD25, as well as to a specific epitope on an immune checkpoint protein or tumor associated antigen as defined herein. In a preferred embodiment, the second antigen-binding moiety binds to PD-L1. In a preferred aspect, the present invention provides a bispecific antibody comprising:

(a) a first antigen binding portion that binds CD 25; and

(b) a second antigen-binding moiety that binds to an immune checkpoint protein expressed on a tumor cell.

In a particular embodiment, the immune checkpoint protein expressed on the tumor cell is PD-L1, VISTA, GAL9, B7H3 or B7H 4. Still preferably, the anti-CD 25 antibody is an IgG1 antibody that binds with high affinity to Fc γ receptors and depletes tumor infiltrating regulatory T cells.

One skilled in the art will be able to generate bispecific antibodies using known methods. Bispecific antibodies according to the invention may be used in any aspect of the invention as described herein. Preferably, the second antigen-binding moiety within the bispecific antibody according to the invention binds to human PD-1, human PD-L1 or human CTLA-4.

In one aspect, the bispecific antibody can bind CD25 and an immunomodulatory receptor expressed at high levels on tumor-infiltrating tregs, such as CTLA4, ICOS, GITR, 4-1BB, or OX 40.

The invention also provides a kit comprising an anti-CD 25 antibody as described herein, and an immune checkpoint inhibitor, preferably a PD-1 antagonist (directly using an anti-PD 1 antibody, or indirectly using an anti-PD-L1 antibody), as discussed herein. In one aspect, the immune checkpoint inhibitor is anti-PD-L1. In an alternative embodiment, the kit comprises an anti-CD 25 antibody as described herein and an antibody that is an agonist of a T cell activation co-stimulatory pathway. The kit may include instructions for use.

In another aspect, the kit may comprise an anti-CD 25 antibody and a compound that reduces, blocks, inhibits and/or antagonizes fcyriib (CD32b) as described herein, or alternatively an anti-CD 25 antibody and an anti-human activated Fc receptor antibody (anti-fcyri, anti-fcyriic, or anti-fcyriiia) as described herein.

Any aspect of the invention as described herein may be performed in combination with an additional cancer therapy. In particular, the anti-CD 25 antibody and optionally an immune checkpoint inhibitor (or any other combination therapy) according to the invention may be administered in combination with a co-stimulatory antibody, chemotherapy and/or radiotherapy, targeted therapy or monoclonal antibody therapy.

Contemplated chemotherapeutic agents include, but are not limited to, alkylating agents, anthracyclines, epothilones, nitrosoureas, ethylenimine/methylmelamines, alkyl sulfonates, alkylating agents, antimetabolites, pyrimidine analogs, epipodophyllotoxins, enzymes such as L-asparaginase, biological translation regulators such as IFN α, IFN- γ, IL-2, IL-12, G-CSF, and GM-CSF, platinum coordination complexes (such as cisplatin, oxaliplatin, and carboplatin), anthracenedione, substituted ureas such as hydroxyurea, methylhydrazine derivatives (including N-Methylhydrazine (MIH) and procarbazine), adrenocortical suppressants such as mitotane (o, p' -DDD) and aminoglutethimide, hormones and antagonists including adrenocortical antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide, progestins such as hydroxyprogesterone caproic acid, medroxyprogesterone acetate and megestrol acetate, testosterone and antiandrogen equivalents such as estradiol and antiandrogen, testosterone equivalents, and testosterone equivalents such as norgestagens and testosterone equivalents, and antiandrogen equivalents, and testosterone equivalents such as leuprolide, and testosterone equivalents.

Additional cancer therapies may also include administration of cancer vaccines. As used herein, "cancer vaccine" refers to a therapeutic cancer vaccine that is administered to a cancer patient and is intended to eradicate cancer cells by boosting the patient's own immune response. Cancer vaccines include tumor cell vaccines (autologous and allogeneic), dendritic cell vaccines (ex vivo generated and peptide activated), protein/peptide based cancer vaccines and genetic vaccines (DNA, RNA and virus based vaccines). Thus, in principle, therapeutic cancer vaccines can be used to inhibit further growth of advanced cancers and/or recurrent tumors that are refractory to conventional therapies (e.g., surgery, radiation therapy, and chemotherapy). Tumor cell-based vaccines (autologous and allogeneic) include those genetically modified to secrete soluble immune stimulators such as cytokines (IL2, IFN-g, IL12, GMCSF, FLT3L), single chain Fv antibodies against immunoregulatory receptors (PD-1, CTLA-4, GITR, ICOS, OX40, 4-1BB), and/or ligands expressing immune stimulatory receptors on their membranes such as ICOS ligands, 4-1BB ligands, GITR ligands, and/or OX40 ligands, and the like.

Additional cancer therapies may be other antibodies or small molecule agents that reduce immune regulation within the peripheral and tumor microenvironment, such as molecules targeting the TGFb pathway, IDO (indoleamine deoxyase), arginase, and/or CSF 1R.

'combination' may refer to the administration of an additional therapy prior to, concurrently with, or subsequent to the administration of any aspect according to the invention.

The invention will now be further described by the following examples, which are intended to assist those of ordinary skill in the art in carrying out the invention and are not intended to limit the scope of the invention in any way, with reference to the accompanying drawings, in which:

FIG. 1-shows that CD25pos of blood and lymph nodes regulates expression of T cells (A) CD25 (detection antibody clone 7D 4; anti-mouse CD25, IgM isotype) on the surface of T cell subsets present in Lymph Nodes (LN) and Tumor Infiltrating Lymphocytes (TIL) of different tumor models. The histogram represents one mouse per tumor model. (B) Percentage of CD25 positive cells and MFI of CD25 in PBMC and T cell subpopulations from pooled data (n ═ 10) from each experiment using MCA205 tumor model. The same evaluation (restricted to T cell subpopulations) has been performed in MC38, B16 and CT26 tumor models in (C) and (D). Error bars represent Standard Error (SE) of the mean. Statistical correlations between CD 4-positive cells, Foxp 3-positive cells, and CD 8-positive or CD 4-positive/Foxp 3-negative cells were indicated.

FIG. 2-shows anti-CD 25(α CD25) -mediated limitation of CD25 positivity in blood and lymph nodes, regulatory T cell depletion in MCA205 tumor model (A) expression of CD25 (detection antibody clone 7D 4; anti-mouse CD25, IgM isotype) and FoxP3 in CD4 positive T cells (B) mean fluorescence intensity of CD25 on Tregs (gated on CD4 positive, FoxP3 positive T cells). sub-cutaneous inoculation with 5x10⁵Tumor-bearing mice were injected with 200 μ g of anti-CD 25-r1(α CD25-r 1; anti-CD 25 rat IgG1), anti-CD 25-m2a (α CD25-m2 a; anti-CD 25 murine IgG2a), anti-CTLA-4 (α CTLA-4; anti-CTLA 4 clone B56), or untreated (no tx.) 5 days and 7 days after MCA205 cells, Peripheral Blood Mononuclear Cells (PBMC), Lymph Nodes (LN), and tumor-infiltrating lymphocytes (TIL) were harvested on day 9, processed and stained for flow cytometry analysis.

Figure 3-shows anti-CD 25(α CD25) mediated effects on T cell sub-populations in the MCA205 tumor model of figure 2: (a) percentage of FoxP3 positive cells from total CD4 positive T cells and (B) absolute number of FoxP3 positive, FoxP3 negative T cells in parallel with CD4 positive, FoxP3 negative T cells showing PBMC (cell number/mL), LN (total number of cells in three draining lymph nodes) and TIL (cell number/g tumor) CD4 positive, FoxP3 positive T cells (C) effect CD4 positive, ratio of FoxP3 positive T cells (Treg cells) and (D) ratio of effect CD8 positive T cells/Treg cells, gated on CD4 positive FoxP3 negative and CD8 positive T cells.

FIG. 4-shows representative histograms of expression of Fc γ R on B cells (CD19 positive), T cells (CD3 positive, CD5 positive), NK cells (NK1.1 positive), granulocytes (CD11B + Ly6G +), conventional dendritic cells (cDC; CD11 c-high MHCII positive) and monocytes/macrophages (Mono/M φ; CD11B positive, Ly6G negative, NK1.1 negative, CD11 c-low/negative), as assessed by flow cytometry 10 days after tumor challenge in the untreated MCA205 tumor model (see FIG. 2). Error bar denotes SEM (n ═ 3); the data corresponded to one of three independent experiments, which were found to be consistent across the three independent experiments.

Figure 5-shows how Treg depletion depends on the expression of activated Fc-gamma receptors. C57BL/6 wild-type mice (wt) and Fcer1 g-/-mice were injected subcutaneously with 5x10 on day 0⁵MCA205 cells, and then injection of 200 μ g of anti-cd25 on day 5 and day 7 tumor, draining lymph nodes and blood were harvested on day 9, processed and stained for flow cytometry analysis modulated T cells were identified by CD4 and FoxP3 expression in PBMC, LN and til.showing the percentage of FoxP3+ from total CD4+ cells (a). applying the same method in wild type (wt), Fcgr3-/-, Fcgr 4-/-or Fcgr 2B-/-demonstrating inhibition of α CD25-r1 mediated Treg in tumors Fc γ RIIb.

Figure 6-shows that the synergistic effect of the combination of anti-CD 25-m2a and anti-PD-1 results in eradication of established tumors. Growth curves (a) of individual mice and mean MCA205 tumor volume over time (B) for each treatment group are shown. The number of tumor-free survivors or statistical significance after 100 days is indicated in each graph. Error bars represent SE of the mean. Kaplan-meier survival curves with cumulative data from two independent experiments are also shown. Survival curves for mice (each condition n-10) injected with MC38(C) or CT26(D) tumor cells and treated as described in the MCA205 model are also shown. Mice were injected subcutaneously with 5x10⁵MCA205, MC38 or CT26 cells and then treated with indicated anti-CD 25(200 μ g intraperitoneally) on day 5, followed (or not) by administration of anti-PD-1 (α PD-1, anti-PD 1, clone RMP 1-14; 100 μ g intraperitoneally) on days 6, 9 and 12.

Figure 7-shows functional analysis of MCA205 tumor model determined as described in figure 6 using immune cells harvested at day 14. The ratio (%) of tumor-infiltrating CD4+ Foxp3-T cells in MCA205 tumor to Ki67+ cells in CD8+ T cells. (A) And CD4 positive, FoxP3 negative Teff/Treg ratio and CD8 positive/Treg ratio (B) in tumors are shown for each treatment group. For the same treatment groups in CD 4-positive and CD 8-positive cells, the frequency (D) of tumor infiltrating lymphocytes against intracellular staining for IFNg expression (C) and interferon gamma producing effector T cells (IFN γ) after ex vivo re-stimulation with PMA and ionomycin is also shown. (B) The histograms in (a) correspond to representative mice for each treatment group. Representative plots and statistical significance from two independent experiments (n ═ 10) are provided in (a), (B), and (D).

Figure 8-shows that tumor ablation of anti-CD 25-m2 a/anti-PD 1 is CD8+ T cell dependent MCA205 tumor growth curve (no tx, a) in untreated individual mice, treatment with a combination of anti-CD 25-m2a and anti-PD-1 (α PD-1+ α CD25-m2 a; B), or also including the same combination of anti-CD 8(α PD-1+ α CD25-m2a + α CD 8; C)⁵MCA205 cells and treated with 200 μ g of anti-CD 25-m2a (α CD25-m2a, clone PC61, mouse IgG2a isotype) on day 5, followed by 100 μ g of anti-PD-1 (α PD-1, clone RMP1-14) intraperitoneally on days 6, 9 and 12 in mice of the indicated group, CD8 positive cells were depleted by intraperitoneal injection of 200 μ g of anti-CD 8(α CD8, clone 2.43) on days 4, 9, 12 and 17.

Figure 9-shows that anti-CD 25-m2 a/anti-PD-1 therapy induces at least partial tumor control against B16 melanoma tumors. As defined in fig. 6(a), B16 tumor growth curves of individual mice treated with Gvax alone or in combination with the indicated antibodies. The corresponding kaplan-meier survival curves (B) were also generated. Intradermal (i.d.) injection of 5X10 into mice⁴B16 melanoma cells, and then treated with 200 μ g of anti-CD 25(α CD25-r1, clone PC61 rat IgG1 isotype or α CD25-m2a, clone PC61 mouse IgG2a isotype) on day 5, followed by intraperitoneal injections on days 6, 9 and 12Injection of 200 μ g of anti-PD-1 (α PD-1, clone RMP1-14) and intradermal injection of 1X 10⁶Tumor growth was followed for each irradiated (150Gy) B16-Gvax, and mice were euthanized when any orthogonal diameter reached 150mm or on day 80 of the study (whichever was reached first.) the median survival days for the different groups (n, number of mice) were for Gvax21 days only (n-14), for Gvax + α PD-127 days (n-15), for Gvax + α CD25-r 121 days (n-7), for Gvax + α CD25-m2a 33 days (n-8), for Gvax + α PD-1+ α CD25-r 129 days (n-13), and for Gvax + α PD-1+ α CD25-m2a 39 days (n-12).

FIG. 10-CT 26 tumor growth curves of individual mice untreated (PBS, vehicle only), treated with either IgG1(PC61m 1; mouse IgG1 isotype, thus having low Fc receptor-mediated killing activity, low ADCC and CDC activity) or IgG2a (PC61m 2; mouse IgG2a, thus having high Fc receptor-mediated activity, high ADCC and CDC activity) and further with or without anti-mouse PD1(α PD1RMP 1-14.) anti-mouse CD 25. CT26 cells for implantation were harvested during log phase growth and resuspended in cold PBS. on day 1 of the study, each mouse was injected subcutaneously in the right flank in 3x 10 in 0.1mL cell suspension⁵And (4) cells. Anti-mouse CD25(10mg/kg) was injected intraperitoneally on day 6 (when palpable tumors were detected). Anti-mouse PD1(100 μ g/injection) was injected intraperitoneally on days 7, 10, 14, and 17. Tumors were measured two-dimensionally twice weekly to monitor growth. Tumor size (mm)³) The calculation is as follows: tumor volume (w2 xl)/2, where w is the width of the tumor and 1 is the length of the tumor (mm). The end point of the study was 2000mm³The tumor volume of (a) or (b) for 60 days, whichever comes first.

Figure 11-shows CT26 tumor growth curves of individual mice that were not treated (PBS, vehicle only), treated with anti-mouse CD25 with IgG1 or IgG2a (PC61m1 and PC61m2, respectively), and further with or without combination with anti-mouse PD-L1 clone 10f.9g2 (adpdl110f.9g2) the model, protocol and analysis were performed as for the α PD 1-based combination experiment of figure 10.

FIG. 12-shows untreated (PB)S, vehicle only), MC38 tumor growth curves of individual mice treated with anti-mouse CD25 with IgG1 or IgG2a (PC61m1 and PC61m2, respectively) and further with or without anti-mouse PD1 clone RMP1-14(aPD 1RMP1-14), as described for the CT26 tumor model in fig. 10. MC38 colon cancer cells for implantation were harvested during log phase growth and resuspended in cold PBS. Each mouse was injected subcutaneously in the right flank with 5x10 in 0.1mL cell suspension⁵And (4) tumor cells. The volume of the tumor is approximately 100 to 150mm³The tumor is monitored at the target range. 22 days after tumor implantation, on study day 1, will have a size of 63-196mm³Animals with individual tumor volumes within the range were divided into 9 groups (n-10), with the group mean tumor volume at 104-108mm³Within the range of (1). In mice carrying established MC38 tumors, treatment was started at D1. The effect of each treatment was compared to vehicle-treated control groups receiving intraperitoneal (i.p.) PBS on day 1, day 2, day 5, day 9, and day 12. anti-PD 1 was administered intraperitoneally at 100 μ g/animal starting on day 2, twice weekly for two weeks. PC61-m1 and PC61-m2a were administered intraperitoneally at 200. mu.g/animal once on day 1. Tumor measurements were taken twice weekly until day 45, where individual animals reached 1000mm³The study was withdrawn at the end of tumor volume.

Figure 13-shows MC38 tumor growth curves of individual mice that were not treated (PBS, vehicle only), treated with anti-mouse CD25 with IgG1 or IgG2a (PC61m1 and PC61m2, respectively), and further with or without combination with anti-mouse PD-L1 clone 10f.9g2 (adpdl110f.9g2.) the models, protocols, and analyses were performed as for the α PD 1-based combination experiments of figure 12.

Figure 14-shows CD25 expression in the periphery of tumor-localized immune cells in samples from different types of human cancers. Representative histograms show CD25 expression in TIL subpopulations from stage IV human ovarian cancer (peritoneal metastasis; A) and human bladder cancer (B). Representative histograms (C) of individual CD 8-positive, CD 4-positive, FoxP 3-negative and CD 4-positive, FoxP 3-positive T cell subpopulations within PBMCs and TILs isolated from other types of cancer were also obtained. Quantification (as percentage (%)) and Mean Fluorescence Intensity (MFI) (D) of CD25 expression for individual T cell subsets within each study patient group for melanoma (upper panel), NSCLC (middle panel) and RCC (lower panel) are also shown.

Figure 15-shows data on CD25 expression in patients treated with anti-PD-1. Multiple Immunohistochemistry (IHC) analysis of subcutaneous melanoma metastases before anti-PD-1 treatment ('baseline') and after two infusions ('week 6') was shown in parallel with quantification of CD8 and FoxP3 IHC staining at baseline and week 6 in two patients (one patient responded to treatment at week 6 and one patient did not respond to treatment) (B; showing mean counts/x 40 high power field). Percentages (%) of CD8 positive, CD25 positive and FoxP3 positive, CD25 positive, double-stained cells at baseline and at treatment (week 6) are shown for melanoma and RCC patients treated with anti-PD 1 (C).

FIG. 16-shows the structure and binding activity of the bispecific anti-IgG 1, anti-PD-L1-based Duobody (bs CD25/PD-L1), both of which have mutations in the human IgG1 isotype and specific amino acids (K409R for PC61-IgG1 and F405L for S70-IgG 1; A), generated by using the antigen-binding regions of anti-mouse CD25(PC61) and anti-mouse/human PD-L1 (clone S70). The specificity of bs CD25/PD-L1 for CD25 cell lines (SUP-T1 cells, human T lymphoblasts; SUP-T1[ VB ] have been used]CRL-1942^TM) Tests were performed in which the cell lines had been transfected with vectors expressing mouse CD25(CD25+ cell line) or mouse PD-L1(PD-L1+ cell line). The original cell line and the other two resulting cell lines have been used to compare the binding capacity of bsCD25/PD-L1 to the binding of the relevant monospecific antibody (aCD25, clone PC 61; aPD-L1, clone S70). The CD25+ cell line and the PD-L1+ cell line were mixed with each other in a 1: 1 ratio (or each separately with untransfected control cells) and then incubated with bsCD25/PD-L1, aCD25, aPD-L1 or without any antibody (no antibody) for 30 minutes. After incubation, three cell samples were analyzed in flow cytometry to calculate the percentage of double positive cells in the different cell samples (B). Has been respectively madeThe specificity of bs CD25/PD-L1(BsAb) was confirmed using the CD25+ cell line and the PD-L1+ cell line. Each cell line has been labeled with bsCD25/PDL1 or the corresponding monospecific Ab as primary antibody (MsAb, anti-mouse CD25IgG1 for CD25+ cells and anti-mouse PD-L1 for PD-L1+ cells) or with buffer only. Cells were then incubated with aHuman AF647(aHuman) as a secondary antibody and an immobilizable viability dye in FACS buffer for 30 minutes. Cells incubated with the secondary antibody alone (aHuman AF647) or cells not incubated with the primary and secondary antibodies (unstained) were used as negative controls. The cells were then analyzed by flow cytometry to calculate the percentage of positive cells obtained with BsAb compared to MsAb (indicated in the right side of each figure; C).

Figure 17-shows the effect of bispecific IgG 1-based Duobody (Bs CD25 PD-L1), anti-mouse CD25(aCD25) IgG1, and anti-mouse PD-L1(aPD-L1) IgG1 monospecific antibodies (alone or mixed together (aCD25 and aPD-L1)) or isotype IgG1 control (as described in figure 16) on LN and tumor in MCA205 tumor mouse model (established as described in figure 3; 4 or 5 mice per group) and modulating T cells. Samples were used to isolate Tumor Infiltrating Lymphocytes (TIL) or lymph node cells (LN) and analyzed for the presence of effector and regulatory T cells th in each treatment group based on FoxP3, CD3 and CD4 positive (a) or CD8 positive/Treg (FoxP3 positive, CD4 positive) ratios (B). The in vivo effect of each treatment on the ability of tumor-infiltrating CD 4-positive T cells to respond to stimulation was also evaluated. TIL was re-stimulated in vitro using PMA and ionomycin in the presence of golgi thrombospondin inhibitors, and then extracellular staining was performed for CD5 and CD4, and intracellular staining was performed for interferon-gamma (IFNg) after fixation. The percentage of CD 5-positive and CD 4-positive T cells that were also positive for IFNg was analyzed by flow cytometry (C).

Examples

Materials and methods

Mouse

C57BL/6 and BALB/C mice were obtained from Charles River Laboratories. Fcerlg^-/-And Fcgr3^-/-Mice were kindly provided by s.beers (University of Southampton, UK). Fcgr4^-/-And Fcgr2b^-/-Mice were a hefty gift from j.v. ravech (The rockcooker University, New York, USA). All animal studies were performed under ethical approval and regulations at the college of london university and the uk interior.

Cell lines and tissue culture

MC38, B16, CT26 and MCA205 tumor cells (3-methylcholanthrene-induced weakly immunogenic fibrosarcoma cells; from G.Kroemer, Gustave Roussy cancer institute) and 293T cells for retroviral production were cultured in Ducheng's modified eagle's medium (DMEM, Sigma) supplemented with 10% fetal calf serum (FCS, Sigma), 100U/mL penicillin, 100. mu.g/mL streptomycin and 2mM L-glutamine (all from Gibco). K562 cells for antibody production were cultured in phenol red free Iscove Modified Duchenne Medium (IMDM) supplemented with 10% IgG depleted fcs (life technologies). B16 (mouse skin melanoma cells) and CT26 (N-nitroso-N-methyl carbamate-induced, undifferentiated colon cancer cell line) cells and related culture conditions were obtained by ATCC.

Antibody production

The sequences of the heavy and light chain variable regions of anti-CD 25 were resolved from the PC-61.5.3 hybridoma by Rapid Amplification of CDNA Ends (RACE) and then cloned into the constant regions of murine IgG2a and kappa chains derived from pFUSs-CHIg-mG 2A and pFUSE2ss-CLIg-mk plasmid (Invivogen). Each antibody chain was then subcloned into a Murine Leukemia Virus (MLV) -derived retroviral vector. For preliminary experiments, antibodies were produced using K562 cells transduced with vectors encoding the heavy and light chains.

The re-cloned anti-CD 25 heavy chain variable DNA sequence from PC-61.5.3 antibody encodes the following protein sequence:

the re-cloned anti-CD 25 light chain variable DNA sequence from PC-61.5.3 antibody encodes the following protein sequence:

antibodies were purified from the supernatant using a protein G HiTrap MabSelect column (GE Healthcare), dialyzed against Phosphate Buffered Saline (PBS), concentrated and filter sterilized. For further experiments, antibody production was outsourced to Evitria AG. Commercial anti-CD 25 clone PC-61 was purchased from BioXcell.

The published anti-PDL 1(MPDL3280A/RG7446) variable heavy and light chain DNA sequences have been recloned and expressed as recombinant antibodies.

Physical tumor experiment

Cultured tumor cells were trypsinized, washed and resuspended in PBS and injected subcutaneously (s.c.) in flank (5 x10 for MCA205 and MC38 models in C57BL/6 mice⁵(ii) individual cells; 2.5X 10 model for B16 in C57BL/6 mice⁵Individual cells, 5x10 model for CT26 in BALB/c mice⁵Individual cell) cells) were injected intraperitoneally (i.p.) at the time points described in the legend, for functional experiments, tumors, draining lymph nodes and tissues were harvested after 10 days and processed for analysis by flow cytometry as described in Simpson et al (2013) J Exp Med210, 1695-a 710 for treatment experiments, tumors were measured twice weekly and volumes were calculated as the product of three orthogonal diameters when any diameter reached 150mm mice were euthanized, tumor-bearing mice were treated with 200 μ g of anti-CD 25-r1(α CD25-r1), anti-CD 25-m2a (α CD25-m2a) or anti-CTLA-4 (α CTLA-4) on days 5 and 7 and treated with 100 μ g of anti-PD-1 on days 6, 9 and 12 for treatment experiments, mice were treated only on days 5 for subtractive phenotype on days 5 and 7Tumor sizes were measured twice weekly, and mice were euthanized when any tumor size reached 150 mm. Peripheral Blood Mononuclear Cells (PBMC), Lymph Nodes (LN), and Tumors (TIL) were harvested on day 9, processed, and stained for flow cytometry analysis.

Flow cytometry

Collection was performed using BD LSR II Fortessa (BD Biosciences). The following directly conjugated antibodies were used: anti-CD 25(7D4) -FITC, CD4(RM4-5) -v500 (BDBiosciences); anti-IFN γ (XMG1.2) -AlexaFluor488, anti-PD-1 (J43) -PerCP-Cy5.5, anti-Foxp 3(FJK-16s) -PE, anti-CD 3(145-2C11) -PE-Cy7, anti-Ki 67(SolA15) -eFluor450, anti-CD 5(53-7.3) -eFluor450, fixable viability dye-eFluor 780 (eBioscience); anti-CD 8(53-6.7) -bright violet 650 (BioLegend); and anti-granzyme B (GB11) -apc (invitrogen). The following antibodies were used to stain human cells: anti-CD 25(BC96) -brilliant purple 650(Biolegend), anti-CD 4(OKT4) -AlexaFluor700(eBioscience), anti-CD 8(SK1) -V500, anti-Ki 67(B56) -fitc (bdbiosciences); anti-FoxP 3(PCH101) -PerCP-cy5.5 (eBioscience); anti-CD 3(OKT3) -brilliant violet 785 (Biolegend). Endonuclear staining of Foxp3 was performed using a Foxp3 transcription factor staining buffer set (eBioscience). For intracellular staining of cytokines, cells were restimulated with phorbol 12-myristate 13-acetate (PMA, 20ng/mL) and ionomycin (500ng/mL) (Sigma Aldrich) at 37C in the presence of GolgiPlug (BD Biosciences) for 4 hours and then stained using the Cytofix/Cytoperm buffer set (BD Biosciences). To quantify absolute cell numbers, a defined number of fluorescent beads (ThermoFisher beads for cell sorting by UV laser) were added to each sample prior to collection and used as a counting reference.

Human tissue

Peripheral Blood (PBMC) and Tumor Infiltrating Lymphocytes (TIL) were studied in three independent groups with advanced melanoma (n ═ 10, 12 lesions), early non-small cell lung cancer (NSCLC) (n ═ 8), and Renal Cell Carcinoma (RCC) (n ═ 5). The presented human data were from three independent, ethically approved translation studies (melanoma REC number 11/LO/0003, NSCLC-REC number 13/LO/1546, RCC-REC number 11/LO/1996). Written informed consent was obtained in all cases.

Isolation of Tumor Infiltrating Lymphocytes (TILs)

The tumor is brought directly from the operating room to the pathology department, where the tumor representative area is isolated. The samples were subsequently minced under sterile conditions, then enzymatically digested (RPMI-1640 with Liberase TL research grade (Roche) and DNase I (Roche)) for 30 minutes at 37 deg.C, followed by mechanical dissociation using a mild MACS (Miltenyi Biotech). The resulting single cell suspension was filtered and the leukocytes were enriched by passage through a Ficoll-paque (GE healthcare) gradient. Viable cells were counted and frozen at-80 ℃ in human AB serum (Sigma) containing 10% dimethyl sulfoxide, then transferred to liquid nitrogen.

Phenotypic analysis of TILs and PBMCs by multiparameter flow cytometry

Tumor samples and PBMCs were thawed, washed in complete RPMI, resuspended in FACS buffer (500mL PBS, 2% FCS, 2nM EDTA) and placed in round bottom 96-well plates. Master mixtures of surface antibodies were prepared at the manufacturer's recommended dilutions: CD8-V500, SK1 clone (BD Biosciences), PD-1-BV605, EH12.2H7 clone (Biolegend), CD3-BV 785. Immobilizable viability dyes (eFlour780, eBioscience) are also included in the surface master mix. After permeabilization for 20 minutes using the intracellular fixation and permeabilization buffer set (eBioscience), an intracellular staining set consisting of the following antibodies used at the dilutions recommended by the manufacturer was applied: granzyme B-V450, GB11 clone (BDbiosciences), FoxP3-PerCP-Cy5.5, PCH101 clone (eBioscience), Ki67-FITC, clone B56(BD Biosciences), and CTLA-4-APC, L3D10 clone (Biolegend).

Multiple immunohistochemistry

Tumor samples were fixed in buffered formalin and embedded in paraffin. 2-5 μm tissue sections were cut and stained with the following antibodies for immunohistochemistry: anti-CD 8(SP239), anti-CD 4(SP35) (Spring biosciences inc.), anti-FoxP 3(236A/E7) (a gift from dr. g. roncador CNIO, Madrid, Spain), and anti-CD 25(4C9) (Leica Biosystems). For multiple staining, endogenous peroxidase was inactivated by using cell conditioning 1 reagent (Ventana medical systems, Inc.) and hydrogen peroxide, and paraffin-embedded tissue sections were incubated with primary antibody for 30 minutes after antigen retrieval. Detection was performed using peroxidase-based Detection reagents (OptiView DAB IHC Detection Kit Ventana Medical Systems, Inc.) and alkaline Phosphatase Detection reagents (UltraView Universal alkaline Phosphatase Red Detection Kit, Ventana Medical Systems, Inc.). Another immuno-alkaline phosphatase cycle is performed by using an alternative substrate (fast blue if fast red has been used previously, or vice versa). Immunohistochemistry and protein reactivity patterns were evaluated. Multiple immunostaining scores were also performed. Approval of this study was obtained from the national research ethics service, research ethics committee 4(REC reference number 09/H0715/64).

Construction and validation of bispecific Duobody based on anti-CD 25 and anti-PD-L1

Bs CD25 PD-L1Duobody (Labrijn AF et al, natprotoc.2014, 9: 2450-63) has been generated and produced from two parent IgG1 containing a single matched point mutation in the CH3 domain that allows Fab exchange according to techniques described in the literature. Briefly, an anti-mouse CD25(PC 61; mouse IgG1 isotype, as described above) and an anti-mouse/human PD-L1 (clone S70, also known as Attributab, MPDL3280A, RG7446 or clone YW243.55. S70; see WO2010077634 and Herbst R et al 2014, Nature 515: 563-7) were cloned into a mammalian expression vector (504865) having a K409R mutation (for PC61-IgG1) and a F405L mutation (for S70-IgG1) in the CH3 domainExpression vector-mouse 3.2kb Puro Set-Novagen) while the light chain remains the same and is produced as a separate recombinant protein in mammalian cells. These parent IgG1 were mixed in equimolar amounts in vitro under permissive redox conditions (e.g., 75mM 2-MEA; 5 hour incubation) to achieve half-molecule weightsAnd (4) grouping. After removal of the reducing agent to allow reoxidation of the interchain disulfide bonds, the resulting heteromeric proteins were analyzed for exchange efficiency using SDS-PAGE chromatography-based or mass spectrometry-based methods. In the case of Bs CD25PD-L1, mass spectrometry has confirmed that the molecular weight of the heterodimeric protein is 151Kd, corresponding to the addition of the molecular weights of the clone S70 single heavy and light chain (74Kd) and PD61-IgG1 single heavy and light chain (77Kd), and indicates that half of each parent IgG1 has been incorporated in a single molecule.

The specificity of Bs CD25PD-L1 has been further confirmed by flow cytometry as described in example 5, using the parental antibody as control and IgG1 recognition detection antibody (aHuman, Alexa) diluted in FACS buffer (PBS + 2% FCS +2mM EDTA) used according to the literature and manufacturer's instructions (aHuman, Alexa)647, AffiniPure goat anti-human IgG, Fc gamma fragment specificity; jackson Labs 109-. Additional flow cytometry and cytobiological materials were the immobilizable viability dyes, eFluor780(Ebioscience65086514), PMA (50 ng/ml; Santa Cruz Biotechnology, sc-3576) and ionomycin (400 ng/ml; Sigma 10634) and Golgi-embolic protein inhibitors (BD Bioscience, 512301 KZ).

Validation of Bs CD25PD-L1 in the MCA205 model was performed by using the same method as shown in the previous examples, isotype control, monospecific antibodies (100 μ g each) or bispecific Duobody (200 μ g each) were administered on day 7 after MCA205 injection, and mouse tissues were obtained and prepared on day 10.

Example 1-high expression of CD25 in tregs makes it a suitable target for their depletion

The interleukin-2 high affinity receptor α (IL2R α) CD25 has historically been used as a true surface marker of tregs and is therefore a target for antibody-mediated Treg depletion because there is controversy as to whether anti-CD 25(aCD25) can also lead to elimination of activated effector T cells, the expression of CD25 in a subpopulation of lymphocytes in tumors and peripheral lymphoid organs was analyzed.

Mice were injected subcutaneously (s.c.) in the flank with MCA205(5 x 10)⁵Individual cells, C57BL/6 mice), B16(2.5X 10)⁵Individual cells, C7BL/6 mice) or CT26(5 x 10)⁵Individual cells, BALB/c mice) cells, and 10 days later Tumors (TILs) and draining lymph nodes were harvested and processed for analysis by flow cytometry.

An attempt was made to assess the relative expression of CD25 in tumors, draining lymph nodes, and individual subpopulations of T lymphocytes within the blood of tumor-bearing mice 10 days after tumor challenge. The results are shown in figure 1. In different models of transplantable tumor cell lines, including MCA205 sarcoma, MC38 colon adenocarcinoma, B16 melanoma, and CT26 colorectal cancer, CD25 expression was consistently higher in CD4 positive, Foxp3 positive T cells (Treg) and minimal in CD4+ Foxp 3-and CD8+ T cells (FIG. 1(A)), as previously described (Sakaguchi et al 1995. JImmunol; Shimizu et al 1999.J Immunol). The effect on Treg depletion in the MCA205 tumor model was studied in more detail due to its immunogenicity and higher T cell infiltration (fig. 1 (B-C)). In contrast to in vitro studies, CD25 was observed in the effector compartment in vivo (CD 4)⁺FoxP3^-And CD8⁺T cells). Although CD25 was found to be tumor-infiltrating CD8⁺And CD4⁺FoxP3^-T effector cells (Teff) were slightly upregulated.The percentage of CD25 positive cells (3.08% -8.35% CD8+, 14.11% -26.87% CD4 positive, Foxp3 negative cells) and the expression level on a per cell basis (mean fluorescence intensity (MFI) 166.6 in CD8 positive cells and 134 in CD4 positive, Foxp3 negative cells) were significantly lower than in tregs (83.66% -90.23%, MFI 1051.9; p < 0.001). Finally, CD25 is also expressed on tregs present in draining lymph nodes and blood, but the expression level based on Mean Fluorescence Intensity (MFI) is higher in tumor infiltrating tregs. The significantly lower expression of CD25 on Teff cells compared to Treg cells indicates that CD25 is a significant level of expression on tregsSuitable and attractive targets for Treg depletion in higher tumors.

Example 2-isotype switching is essential for efficient and safe intratumoral Treg depletion with anti-CD 25

Traditionally, anti-CD 25 antibody (α CD25) clone PC-61 (rat IgG1, k) (α CD25-R1) has been used for Treg depletion in mouse models where it has been repeatedly shown to result in the elimination of tregs in peripheral lymphoid organs to avoid interspecies differences in Fc γ R junctions, PC-61 constant regions were exchanged with murine IgG2a, κ (α CD25-m2a) -the classical mouse depletion isotype-exchange, and Treg numbers in the periphery and in the tumor were quantified and compared with the effect of anti-CTLA 4(α CTLA4, clone 9H10), which is known to result in the depletion of tumor-infiltrating tregs.

Based on previous evidence demonstrating the importance of intratumoral Treg depletion in co-defining the activity of immunomodulatory antibodies, an attempt was made to compare the effects of α CD25-r1 on Teff and Treg frequency in blood, draining Lymph Nodes (LN), and Tumor Infiltrating Lymphocytes (TILs) in the MCA205 mouse model due to their higher immunogenicity and to evaluate any potential negative effects of anti-CD 25 on intratumoral activated Teff.

Subcutaneous inoculation of 5X10⁵Tumor bearing mice were injected with 200 μ g of anti-CD 25-r1(α CD25-r1), anti-CD 25-m2a (α CD25-m2a), or anti-CTLA-4 (α CTLA-4) on days 5 and 7 after MCA205 cells, Peripheral Blood Mononuclear Cells (PBMC), Lymph Nodes (LN), and Tumors (TIL) were harvested on day 9, processed and stained for flow cytometry analysis the results are shown in fig. 2 and 3.

In vivo administration α CD25 reduced the number of CD25+ cells in lymph nodes and in particular in the blood, independent of antibody isotype, when quantifying the number of tregs by their expression of the characteristic transcription factor Foxp3, both isotypes were again equally effective in the periphery, but surprisingly only the mouse IgG2a isotype resulted in a significant reduction in the frequency and absolute number of tumor-infiltrating tregs to levels comparable to those observed in the case of α CTLA4, although CD25 expression was upregulated in a fraction of tumor-infiltrating effector T cells (see example 1), no significant reduction in the number of CD8+ and CD4+ Foxp 3-in the periphery or tumor was observed, as a result, both α CD25 isotypes resulted in an increase in the Teff/Treg ratio in the periphery, however, only CD25-m2a was increased in a similar manner to that of anti-CTLA 5 (which is known to prefer the tumor site rather than the peripheral one) to increase in the number of treff/Treg ratio, as a reduction in the number of the previously determined and the number of extracellular accumulation of tumor as a reduction in the serum-alanine aminotransferase in mice, thus the mice treated mice, the number of the mice was not observed in the mice, the serum-mediated reduction of the primary tumor-mediated by the various tumor-mediated effects of the earlier studies (CD a, the rat-mediated by the rat, the rat-mediated clinical effects of the rat, thus the rat-mediated clinical effects of the rat, the decrease of the rat, the decrease in the rat, the decrease of the rat, the rat.

The expression levels of activating and inhibitory Fc γ rs on different leukocyte subpopulations in blood, spleen, LN and tumors of mice bearing subcutaneous MCA205 tumor were also determined (figure 4). Relative to all other organs studied, Fc γ R appeared to be more expressed on tumor-infiltrating myeloid cells (granulocytes, conventional dendritic cells and monocytes/macrophages). The binding affinity of two Fc variants against CD25 to Fc γ R was also determined by surface plasmon resonance (table 1).

TABLE 1

	rlgG1	mlgG2a
			FcγRI	n.b.	1.1×10-8
FcγRIIb	2.6×10-6	4.2×10-6
			FcγRIII	2.5×10-6	4.5×10-6
FcγRIV	n.b.	2.2×10-7

These data demonstrate that the mIgG2a isotype binds all Fc γ R subtypes with high activation to inhibition ratio (a/I). In contrast, the rgig 1 isotype binds with similar affinity to single activating Fc γ R, Fc γ RIII and inhibitory Fc γ RIIb, resulting in low a/I ratio (< 1).

The number of tumor-infiltrating tregs in mice lacking different Fc γ R expression was established in different mouse models to distinguish which specific Fc γ rs are involved in anti-CD 25-mediated Treg depletion (fig. 5). C57BL/6 control mice and fcerrlg-/-mice were injected subcutaneously with MCA205 cells and tumors, draining lymph nodes and blood were harvested, processed and stained for flow cytometry analysis. Regulatory T cells were identified by CD4 and FoxP3 expression. The percentage of Foxp3 positivity from total CD4 positive cells shows how the anti-CD 25 effect is due to expression of the Fcerlg gene. For Fcer1g^-/-Analysis of mice that do not express either of activated Fc γ rs (I, III and IV) demonstrated complete absence of Treg depletion α CD25-R1 in the periphery and peripheryAnd α CD25-m2a Treg depletion in tumors thus results from Fc γ R mediated ADCC and does not block IL-2 binding to CD25 depletion by α CD25-m2a is not dependent on any separate activation of Fc γ R, wherein Treg depletion is in Fcgr3^-/-And Fcgr4^-/-Thus, the depletion of peripheral tregs by α CD25-r1, despite the high intratumoral expression of this receptor, was not reduced in tumors, however, in mice lacking the inhibitory receptor fcyriib expression, intratumoral Treg depletion was effectively restored in this case, the intratumoral Treg depletion between α CD25-r1 and α CD25-m2a was comparable.

Example 3 anti-CD 25 therapy in synergy with anti-PD-1 to eradicate established tumors and increase survival of tumor-bearing mice

It was hypothesized that α CD25-m2a had better therapeutic outcomes in the treatment of established tumors due to its better efficiency in intratumor Treg depletion, when tumors were established, α CD25-m2a and α CD25-r1 were evaluated for anti-tumor activity against established tumors by administering a single dose of α CD25 5 days after subcutaneous implantation of MCA205 cells, results are provided in figure 6.

Consistent with the observed lack of ability to deplete intratumoral tregs, a single dose of α CD25 (day 5) administered to mice with established tumors resulted in no protection in the case of α CD25-r1 on the other hand, a growth delay and long-term survival (15.4%) was observed in mice administered α CD25-m2a, due to the clinical relevance of agents targeting the co-inhibitory receptor PD-1 as a target for immunotherapy and the critical role of PD-1 in controlling T cell regulation within the tumor microenvironment, assuming CD25⁺Treg cell depletion and PD-1 blockade can have a synergistic effect in the combination in the same model, the use of anti-PD-1 (α PD-1, clone RMP 1-14; dose of 100. mu.g every three days) α CD25 in combination with PD-1 blockade was tested as monotherapyα PD-1 in the treatment of established MCA205 tumor models and the combination with α CD25-r1 did not improve its effect, however, a single dose of aCD25-m2a followed by α PD-1 therapy eradicated established tumors in 78.5% of mice, resulting in long-term survival over 100 days similar results were observed in MC38 and CT26 tumor models, where α CD25-m2a had a partial therapeutic effect synergistic with α PD-1 therapy, as opposed to the combination with α CD25-r1 (which failed to deplete tumor-infiltrating tregs in these tumors).

To understand the mechanism of action of the synergistic effect of the α CD25-m2a and α PD-1 combination, the phenotype and function of Tumor Infiltrating Lymphocytes (TILs) present in the MCA205 tumor microenvironment at the end of the treatment regimen (24 hours after the third dose of α PD-1) was evaluated (fig. 7.) monotherapy with α PD-1 had no effect on both Teff proliferation and the degree of Teff infiltration in tumors, where there was also observed a high frequency of persistent tregs (data not shown), and a low Teff/Treg ratio consistent with a lack of therapeutic activity, in contrast, intratumor tregs with α CD25-m2a abrogated a higher proportion of proliferation in tumors and interferon- γ (IFN- γ) producing CD4 24⁺FoxP3^-T cells, which correspond to high Teff/Treg ratios and anti-tumor responses. This effect was further enhanced in the combination of anti-PD-1, which resulted in even higher proliferation and a 1.6-fold increase in the number of CD 4-positive, FoxP 3-negative T cells producing IFN- γ, compared to monotherapy with anti-CD 25-m2 a. In contrast, the lack of Treg depletion observed with anti-CD 25-rl resulted in no change in Teff proliferation or IFN- γ production when used as monotherapy or in combination with anti-PD-1.

Data generated with PC61 of the original mouse IgG1 isotype or the mouse IgG2a isotype that allows efficient Treg depletion indicate that such anti-CD 25 alone or in combination with anti-cancer antibodies can effectively reject established tumors, particularly for those tumors that require efficient intratumoral Treg depletion.

As shown above, administration of a single dose of α CD25-m2a followed by α PD-1 treatment had a positive effect on tumor size and mouse survival in the MCA205 murine model this therapeutic effect due to anti-CD 25-m2 a/anti-PD 1 was dependent on the activity of CD8 positive T cells, as further administration of anti-CD 8 antibody brought tumor size and mouse survival to levels observed in untreated animals (fig. 8), thus, MCA205 tumor elimination was dependent on the effect of α PD-1/α CD25 synergy on both CD8 positive and Treg cell populations, and the overall effector T cell response was not negatively affected by anti-CD 25 antibody depletion.

This synergy was also observed against the less immunogenic B16 melanoma tumor model when α CD25-m2a and α PD-1 was combined with the GM-CSF expressing whole tumor cell vaccine Gvax (fig. 9) — in this system Gvax treatment alone and in combination with α PD-1 or α CD25-r1 failed to prevent tumor growth or prolong survival of tumor bearing mice in this setting Gvax alone in combination with α CD25-m2a (alone or with α PD-1) alone in this setting, no such improvement was observed in any of the treatment groups administered α CD25-r 1.

Similar results were observed for the synergistic effect of immune checkpoint inhibitors with α CD25-m2a in the MC38 tumor model when either α PD-1 (fig. 10) or α PD-L1 (fig. 11) was administered CT26 tumor model also demonstrates the therapeutic effect of these combinations (fig. 12 and 13) thus, this advantageous property provides the possibility of unexpectedly improving the response to therapy based on immune checkpoint inhibitors when α CD25-m2a already has partial therapeutic effect due to Treg depletion.

Example 4-CD25 is highly expressed in Treg-infiltrating human tumors and anti-PD-1 therapy induces infiltration of Tregs expressing CD25 in human tumors

To validate CD25 as a possible target for Treg depletion in humans, its expression levels in peripheral blood and tumor-infiltrating lymphocytes were compared by flow cytometry and Immunohistochemistry (IHC) using biological samples obtained from ovarian, bladder, melanoma, non-small cell carcinoma (NSCS), and Renal Cell Carcinoma (RCC) patients.

Independent of anatomical location, tumor type or stage, CD25 expression was observed to be significantly higher (50% -85%) in tregs than CD25 expression in CD4+ Foxp3- (10% -15%) and CD8+ (< 5%) T cells. Similar to the murine model, the level of CD25 expression as assessed by MFI was significantly higher on CD4+ FoxP3+ tregs relative to CD4+ FoxP 3-and CD8+ Teff in all studied tumor subtypes.

Multiple Immunohistochemistry (IHC) further supported these observations. Analysis of melanoma, NSCLC and RCC tumors from the same patient cohort showed that CD25 expression was limited to FoxP3 positive cells even in dense CD8 positive, T cell infiltrated regions. Remarkably, this expression profile remained consistent regardless of tumor subtype, stage, site of resection, current or previous treatment, and they were consistent with the data obtained in the mouse model.

Furthermore, RCC samples showed a rare number of tregs in untreated tumors compared to the high proportion of tregs observed in subcutaneous murine tumors. However, anti-PD-1 therapy resulted in significant infiltration by CD8+ T cells and tregs (Foxp3 positive cells). In addition, data generated from the melanoma and RCC samples confirmed that CD25 is highly expressed by Foxp3 positive cells, while its expression was minimal among Foxp3 negative, CD8 positive cells.

When CD25 expression is assessed in the context of therapeutic immunomodulation. A core biopsy was performed on the same lesion at baseline and 4 cycles of nivolumab or 2 cycles of pembrolizumab) in patients with advanced renal cancer and melanoma, respectively. Despite systemic immune modulation, CD25 expression was still limited to FoxP3 positive tregs, even in areas of dense CD8 positive T cell infiltration assessed by multiple IHC.

These findings confirm the translational value of the described preclinical data and further support the concept of selective therapeutic targeting of tregs by CD25 in human cancers. Furthermore, the CD25 expression profile associated with anti-PD 1 therapy in human solid cancers provides a theoretical basis for therapeutic combinations of anti-human CD25 antibodies with CD25 binding and Fc γ receptor specificity comparable to those shown for anti-mouse CD25PC61(IgG2a) and immune checkpoint inhibitors such as PD-1 antagonists.

Example 5-combination of anti-CD 25 and anti-PD-L1-based bispecific antibodies and antibodies provides potent Treg-depleting and cytokine-inducing properties

The previous examples have shown that the Treg-depleting, CD 25-binding properties of PC 61-based antibodies with the appropriate isotype can be used in combination with other anti-cancer compounds, such as antibodies targeting immune checkpoint proteins, e.g., PD-1 antagonists (being anti-PD-1 or anti-PD-L1 antibodies). These findings suggest the construction of bispecific antibodies that combine two antigen binding properties and a related isotype (e.g., IgG 1).

This approach has been validated by using the Duobody technique, termed Bs CD25PD-L1, within a single heteromeric protein, which allows for the efficient association of a single heavy and light chain with two different monospecific antibodies produced separately (fig. 16A). The binding specificity of this antibody has been verified using two genetically modified human cell lines, each expressing mouse CD25 or mouse PD-L1; and the binding specificity was compared to that of the original monospecific antibody (fig. 16B and C). These cell lines have been tested by flow cytometry (alone or mixed in equal amounts), indicating that BsCD25 PD-L1 retains its dual CD25, PD-L1 specificity, even allowing detection of the complex of double positive cells formed by the simultaneous binding of Bs CD25PD-L1 to CD25 positive and PD-L1 positive cells.

The functional properties of BsAb CD25PD-L1 have been evaluated in vivo by using the cell interaction and depletion model used in the previous examples to validate PC 61. The MCA205 model was used to assess the effect of BsAb on effector and regulatory T cells in tumors and LNs. In this model, BsAb CD25PD-L1 recognized and depleted CD 4-positive, Foxp 3-positive regulatory T cells, and increased the CD 8-positive, Foxp 3-positive regulatory T cell ratio in tumors and LNs with equivalent efficacy to the combination of anti-CD 25(PC61-m2a) or monospecific anti-CD 25 and anti-PD-L1 antibodies (fig. 17 AB). Furthermore, BsAb CD25PD-L1 increased the number of CD 4-positive, CD 5-positive cells expressing interferon gamma at a level at least similar to the combination of monospecific anti-CD 25 and anti-PD-L1, and possibly better than the combination of anti-CD 25m2a antibody alone (fig. 17C).

The data indicate how treatment of cancer with PC 61-based Treg-depleted anti-human CD25 antibodies can be improved not only by selecting the appropriate isotype, but also in effective combination with other anti-cancer drugs, in particular with anti-cancer antibodies that bind to different cell surface antigens. This approach can be achieved by generating and administering the two products as a novel mixture of monospecific antibodies or as a novel bispecific antibody that associates and is generated to maintain the Treg depletion, CD25 binding and other binding properties of the parent monoclonal antibody.

All documents mentioned herein are incorporated by reference in their entirety, with particular attention to the subject matter to which they are cited. Various modifications and variations of the described methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology, cellular immunology or related fields are intended to be within the scope of the following claims.

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Claims (29)

1. A method of treating a human subject having cancer, the method comprising the step of administering to the subject an anti-CD 25 antibody, wherein the subject has a solid tumor, and wherein the anti-CD 25 antibody is an IgG1 antibody that binds with high affinity to at least one activated fcgamma receptor selected from the group consisting of fcyri, fcyriic, and fcyriiia and depletes tumor-infiltrating regulatory T cells.

2. The method of claim 1, wherein the anti-CD 25 antibody has a dissociation constant (K) for CD25_d) Is less than 10^-８M, and/or a dissociation constant for at least one activated Fc gamma receptor of less than about 10^-6M。

3. The method of claim 1 or claim 2, wherein the anti-CD 25 antibody:

(a) binds to Fc γ receptors with an activation to inhibition ratio (a/I) higher than 1; and/or

(b) Binds at least one of Fc γ RI, Fc γ RIIc, and Fc γ RIIIa with a higher affinity than that of Fc γ RIIb.

4. The method of any one of claims 1 to 3, wherein the anti-CD 25 antibody is a monoclonal antibody.

5. The method of any one of claims 1-4, wherein the anti-CD 25 antibody is a human antibody, a chimeric antibody, or a humanized antibody.

6. The method according to any one of claims 1 to 5, wherein the anti-CD 25 antibody elicits an enhanced CDC, ADCC and/or ADCP response, preferably an increased ADCC and/or ADCP response, more preferably an increased ADCC response.

7. The method of any one of claims 1 to 6, wherein the anti-CD 25 antibody is administered to a subject having a defined tumor.

8. The method of any one of claims 1 to 7, wherein the method further comprises the step of identifying a subject having a solid tumor.

9. The method of any one of claims 1 to 8, wherein the method further comprises administering an immune checkpoint inhibitor to the subject.

10. The method of claim 9, wherein the immune checkpoint inhibitor is a PD-1 antagonist.

11. The method of claim 10, wherein the PD-1 antagonist is an anti-PD-1 antibody or an anti-PD-L1 antibody.

12. An anti-CD 25 antibody as defined in any one of claims 1 to 6.

13. An anti-CD 25 antibody as defined in any one of claims 1 to 6 for use in the treatment of cancer in a human subject, wherein the subject has a solid tumor.

14. Use of an anti-CD 25 antibody as defined in any one of claims 1 to 6 for the manufacture of a medicament for the treatment of cancer in a human subject, wherein the subject has a solid tumor.

15. An anti-CD 25 antibody for use according to claim 13 or for use according to claim 13, wherein the antibody is for administration in combination with an immune checkpoint inhibitor.

16. The anti-CD 25 antibody for the use according to claim 15 or the use according to claim 14, wherein the immune checkpoint inhibitor is a PD-1 antagonist.

17. A combination of an anti-CD 25 antibody as defined in any one of claims 1 to 6 and an immune checkpoint inhibitor as defined in any one of claims 9 to 11 for use in the treatment of cancer in a human subject, wherein the subject has a solid tumor and the anti-CD 25 antibody and the PD-1 antagonist are administered simultaneously, separately or sequentially.

18. A kit for use in the treatment of cancer, the kit comprising an anti-CD 25 antibody as defined in any one of claims 1 to 6 and an immune checkpoint inhibitor as defined in any one of claims 9 to 11.

19. A pharmaceutical composition comprising an anti-CD 25 antibody and an immune checkpoint inhibitor in a pharmaceutically acceptable medium.

20. A bispecific antibody comprising:

(a) a first antigen binding portion that binds CD 25; and

(b) a second antigen-binding moiety that binds to an immune checkpoint protein;

wherein the bispecific antibody is an IgG1 antibody that binds with high affinity to at least one Fc γ receptor activating and depleting tumor infiltrating regulatory T cells selected from the group consisting of Fc γ RI, Fc γ RIic, and Fc γ RIIIa.

21. The bispecific antibody of claim 20, wherein the immune checkpoint protein is selected from the group consisting of: PD-1, CTLA-4, BTLA, KIR, LAG3, VISTA, TIGIT, TIM3, PD-L1, B7H3, B7H4, PD-L2, CD80, CD86, HVEM, LLT1, GAL9, GITR, OX40, CD137, and ICOS.

22. The bispecific antibody of claim 21, wherein the immune checkpoint protein is expressed on a tumor cell.

23. The bispecific antibody of claim 21 or 22, wherein the immune checkpoint protein is PD-L1.

24. The bispecific antibody of claim 23, wherein the second antigen-binding moiety that binds PD-L1 is comprised in atezumab.

25. A method of treating cancer comprising the step of administering to a subject a bispecific antibody as defined in any one of claims 20 to 24.

26. The method of claim 25, wherein the subject has a solid tumor.

27. A bispecific antibody as defined in any one of claims 19 to 24 for use in the treatment of cancer in a subject.

28. The bispecific antibody for use according to claim 27, wherein the subject has a solid tumor.

29. A method of depleting regulatory T cells in a solid tumor in a subject, the method comprising the step of administering to the subject an anti-CD 25 antibody, wherein the antibody is as defined in any one of claims 1 to 6.