Anti-CD25 for tumor-specific cell depletion

Abstract

The present invention provides antibody sequences found in antibodies that bind to human CD25, in particular, sequences of anti-CD25-a-634 antibodies that do not block CD25 binding to IL-2 or IL-2 signaling. Antibodies and antigen-binding portions thereof comprising such sequences can be used in pharmaceutical compositions and therapeutic methods, in particular for the treatment of cancer.

Description

Technical Field

[0001] The present invention relates to anti-CD25 antibodies, pharmaceutical compositions comprising anti-CD25 antibodies and therapeutic uses of these antibodies. Background Art

[0002] Cancer immunotherapy involves using the subject's own immune system to treat or prevent cancer. Immunotherapy utilizes the fact that cancer cells usually have slightly different molecules that can be detected by the immune system on their surface. These molecules or cancer antigens are the most common proteins, but also include molecules such as carbohydrates. Therefore, immunotherapy involves inducing the immune system to attack tumor cells via these target antigens. However, malignant tumors (particularly solid tumors) can escape immune surveillance through a variety of mechanisms mediated by tumor cells themselves and by components of the tumor microenvironment. In the latter, tumor infiltration by regulatory T cells (Treg cells or Treg), and more specifically, the unfavorable balance of effector T cells (Teff) versus Treg (i.e., a lower ratio of Teff to Treg) has been proposed as a key factor (Smyth M et al., 2014).

[0003] Since its discovery, Treg has been found to be crucial in mediating immune homeostasis and promoting the establishment and maintenance of peripheral tolerance. However, in the case of cancer, its role is more complicated. Since cancer cells express both self-associated antigens and tumor-associated antigens, the presence of Treg (which attempts to inhibit effector cell responses) can help tumor progression. Therefore, in general, the infiltration of Treg in established tumors represents one of the main obstacles to effective anti-tumor responses and the treatment of cancer. The inhibitory mechanism adopted by Treg is believed to significantly promote the limitation or even failure of current therapies, especially immunotherapies that rely on inducing or enhancing anti-tumor responses (Onishi H et al.; 2012).

[0004] Treg cells have been consistently shown to promote the establishment and progression of tumors in murine models and that their absence results in delayed tumor progression (Elpek et al., 2007; Golgher et al., 2002; Jones et al., 2002; Onizuka et al., 1999; Shimizu et al., 1999). In humans, high tumor infiltration by Treg cells and, more importantly, a low ratio of effector T (Teff) cells to Treg cells are associated with poor outcomes in a variety of cancers (Shang et al., 2015). Conversely, high Teff / Treg cell ratios are associated with favorable responses to immunotherapy in both humans and mice (Hodi et al., 2008; Quezada et al., 2006). Nevertheless, the elimination of Tregs in tumors is complex, and the results of preclinical and clinical studies in this field are inconsistent, primarily due to the difficulty in identifying targets that are specific for Tregs.

[0005] CD25 is one of the potential molecular targets for realizing Treg removal. In fact, CD25 (also known as interleukin-2 high affinity receptor α chain (IL-2Rα)) is constitutively expressed at a higher level on Treg cells, and it is not present on T effector cells or expressed at a lower level on T effector cells and is therefore a promising Treg removal target. IL-2 / CD25 interaction has been the target of several studies in murine models, most of which involve the use of rat anti-mouse CD25 antibody PC61 (Setiady Y et al., 2010.). The CD25 binding and functional activity of this antibody have been compared with the activity of a group of monoclonal antibodies produced by different authors (Lowenthal JW et al., 1985.; Moreau, J.-L et al.; Volk HD et al., 1989; Dantal J et al., 1991). While the original study demonstrated prophylactic rather than therapeutic activity of PC61, the current study showed that the Fc-optimized form of this anti-CD25 antibody caused intratumoral Treg depletion and provided significant therapeutic benefit in several murine tumor models (Vargas A et al., 2017).

[0006] Available anti-CD25 antibodies (such as PC61) block or inhibit the binding of IL-2 to CD25, as do many other anti-mouse CD25 antibodies and most antibodies disclosed as anti-human CD25 antibodies; see, for example, WO2004 / 045512, WO2006 / 108670, WO1993 / 011238, WO1990 / 007861, and WO2017 / 174331. For example, basiliximab and daclizumab are anti-human CD25 antibodies that inhibit the binding of IL-2 to CD25, and have been developed to reduce the activation of T effector cells (Queen C et al., 1989 and Bielekova B, 2013). Basiliximab is a chimeric mouse-human CD25 antibody currently approved for graft-versus-host disease, and daclizumab is a humanized CD25 antibody approved for the treatment of multiple sclerosis.

[0007] Some other anti-CD25 antibodies still allow IL-2 to bind to CD25, such as clone 7D4 (anti-mouse CD25), clone 2E4 (anti-mouse CD25), clone MA251 (anti-human CD25) or 7G7B6 (anti-human CD25) (i.e., non-blocking antibodies). Inolimomab / BT536, although allegedly not blocking IL-2 binding to CD25, does block IL-2 signaling via CD25. 7D4 is a rat IgM anti-mouse CD25 antibody widely used to detect CD25-positive cells in the presence of PC61 or an antibody with similar binding properties or after treatment with PC61 or an antibody with similar binding properties (Onizuka S et al., 1999). Very little literature discloses any functional properties of the 7D4-IgM antibody alone or in comparison to PC61 (Kohm A et al., 2006; Hallett W et al., 2008; Fecci P et al., 2006; McNeill A et al., 2007; Setiady Y et al., 2010; Couper K et al., 2007.). Indeed, the prior art does not teach the possibility of adapting or modifying to some extent the isotype or other structural features of 7D4 in order to obtain improved antibodies, particularly antibodies that can be used for cancer therapy. The ability of 7D4-IgM (either by itself or as an engineered antibody) or any anti-human CD25 designed or characterized to have CD25 binding characteristics similar to 7D4 to mouse CD25 has not been evaluated in detail for optimized Treg cell clearance.

[0008] As discussed above, the infiltration of Treg cells in tumors, and in particular the low ratio of effector Teff cells to Treg cells, can produce poor clinical outcomes. CD25 has been identified as a Treg marker and can therefore be a target of therapeutic antibodies intended to remove Treg. Importantly, CD25 is the α subunit of the receptor for IL-2 and IL-2 is a key cytokine for Teff reactions. Although the anti-CD25 antibodies that have been clinically tested so far remove Treg cells, they also block IL-2 signaling via CD25 (specifically CD25 / CD122 / CD132 complexes). The inventors have currently found that this kind of blocking of IL-2 signaling limits Teff reactions and that anti-CD25 antibodies that do not block IL2 signaling can effectively remove Treg cells while still allowing IL-2 to stimulate Teff cells, thereby providing antibodies that exhibit a stronger anti-cancer effect.

[0009] Therefore, there is a need in the art for improved anti-CD25 antibodies, particularly such antibodies that do not block CD25 binding to IL-2 or IL-2 signaling and that deplete Tregs, particularly in tumors, and that can be used in methods of treating cancer. Summary of the invention

[0010] The present invention provides new anti-CD25 antibody agents. In some embodiments, the anti-CD25 antibody agents provided are antibodies or antigen-binding fragments that specifically bind to CD25, particularly human CD25, without blocking interleukin 2 (IL-2) binding to CD25 or signaling of IL-2 via CD25, and effectively remove Tregs (particularly in tumors). Compared to the signaling level in the absence of the anti-CD25 antibody agent, the anti-CD25 antibody agent allows at least 50% of IL-2 signaling in response to the binding of IL-2 to CD25. The anti-CD25 antibody agent is therefore referred to as a non-blocking or non-IL-2 blocking anti-CD25 antibody agent.

[0011] In some embodiments, the anti-CD25 antibody agent or antigen binding fragment binds to CD25 without blocking the binding of IL-2 to CD25 or the signaling of IL-2 via CD25. That is, in some embodiments, the anti-CD25 antibody inhibits less than 50% of IL-2 signaling compared to IL-2 signaling in the absence of the antibody. The anti-CD25 antibody is characterized as a structural element that allows binding to CD25 without blocking the binding of IL-2 to CD25 or its signaling.

[0012] It was surprisingly found that anti-CD25 antibodies that do not block IL-2 binding or signaling via CD25 and eliminate Tregs have strong anti-tumor effects, particularly stronger anti-tumor effects than anti-CD25 antibodies that eliminate Tregs but block IL-2 signaling. The antibodies increase CD4+Teff / Treg and CD8+Teff / Treg ratios. Effective elimination of tumor-infiltrating Treg cells while retaining IL-2 signaling on Teff cells leads to therapeutic methods for treating cancer alone or in combination with other anticancer agents.

[0013] However, those skilled in the art will appreciate that the teachings of the present invention are not limited to the specific mechanism of action of the provided antibodies or antigen-binding fragments thereof. The relevant structural and / or functional features of the provided antibodies are described herein and are self-evident.

[0014] In some embodiments, provided anti-CD25 antibody agents are characterized by one or more features associated with binding to a specific epitope in the extracellular domain of human CD25 and / or render them particularly suitable for pharmaceutical use and / or manufacturing.

[0015] The provided technology, including the provided anti-CD25 antibody agents (e.g., provided antibodies or antigen-binding fragments thereof), compositions comprising the same, and / or uses thereof, are useful in medicine. In some embodiments, the provided technologies can be used for cancer treatment and / or prevention.

[0016] In some embodiments, the anti-CD25 antibody agent provided is exemplified as an antibody having the sequence of aCD25-a-634, and more generally exemplified as an antibody or agent that is or comprises one or more antigen-binding fragments or portions thereof, for example comprising an aCD25-a-634-HCDR3 amino acid sequence as the heavy chain variable complementary determining region 3, and / or, in some embodiments, comprising one or both of the aCD25-a-634 HCDR1 and HCDR2 sequences. References to anti-CD25 antibody agents herein include such aCD25-a-634 variants thereof, including affinity-matured variants thereof, unless the context otherwise indicates.

[0017] In one embodiment, an anti-CD25 antibody agent comprising the HCDR1, HCDR2 and HCDR3 amino acid sequences of aCD25-a-634 is provided. In another embodiment, an anti-CD25 antibody agent comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences of aCD25-a-634 is provided. In another embodiment, an anti-CD25 antibody agent comprising the heavy chain variable amino acid sequence of aCD25-a-634 is provided. In another embodiment, an anti-CD25 antibody agent comprising the heavy chain and light chain variable amino acid sequences of aCD25-a-634 or a variant thereof, including an affinity matured variant, is provided.

[0018] In some embodiments, an anti-CD25 antibody agent is provided that competes with aCD25-a-634 (or an antigen-binding fragment, derivative, or variant thereof) for binding to the extracellular domain of human CD25.

[0019] In some embodiments, the anti-CD25 antibody agents provided (e.g., the antibodies or antigen-binding fragments thereof provided, including variants and derivatives thereof, such as affinity matured variants) are administered at 10 -7 M or less (e.g. 10 -8 or 10 -9 or 10 -10 or 10 -11 or 10 -12 or 10 -13 In some embodiments, the anti-CD25 antibody agents provided (e.g., the antibodies or antigen-binding fragments thereof provided, including variants and derivatives thereof, such as affinity matured variants) bind to human CD25 with a Kd of 10 -7 Up to 10 -10 or 10 -7 Up to 10 -9 The Kd value for binding to human CD25.

[0020] In some embodiments, the anti-CD25 antibody agents provided (e.g., antibodies or antigen-binding fragments thereof, including variants and derivatives thereof) bind to an epitope on human CD25 that binds to aCD25-a-634 (or an antigen-binding fragment or derivative or variant thereof). In some embodiments, such anti-CD25 antibody agents provided may bind to the extracellular domain of human CD25. In some embodiments, the anti-CD25 antibody agents provided may bind to an epitope of CD25 (e.g., when assessed using one or more assays as described herein or otherwise known in the art). In some embodiments, the anti-CD25 antibody agents provided according to the present invention specifically bind to antibodies or antigen-binding fragments designated as aCD25ep-a and aCD25ep-b. In some embodiments, the antibodies or antigen-binding fragments provided may be expressed as 10 -7 M range or below, e.g. 10 -8 or 10 -9 or 10 -10 or 10 -11 or 10 -12 or 10 -13 In some embodiments, the anti-CD25 antibody agent (e.g., the provided antibody or antigen-binding fragment thereof, including variants and derivatives thereof, such as affinity matured variants) binds to human and non-human primate (e.g., cynomolgus monkey) CD25 (e.g., binds to an extracellular epitope on human and / or cynomolgus monkey CD25) with a Kd value in the range of 10 -7 Up to 10 -10 or 10 -7 Up to 10 -9 The Kd value for binding to human CD25.

[0021] Among other things, the present invention provides methods that can be used to identify and / or characterize particularly useful anti-CD25 antibody agents (e.g., anti-CD25 antibodies or antigen-binding fragments thereof) as described herein (e.g., anti-CD25 antibodies or antigen-binding fragments thereof characterized by particular structural and / or functional features such as specific binding to human CD25 (e.g., binding to an extracellular epitope thereof), comprising one or more CDR sequence elements as described herein (and in particular comprising a HCDR3 sequence element, optionally in combination with a HCDR1 and / or HCDR2 element), affecting IL-2 signaling activity as described herein, cytotoxic activity as described herein (e.g., with respect to CD25-positive cells such as immunomodulatory cells or cancer cells expressing CD25), and combinations thereof). In some embodiments, particularly useful anti-CD25 antibodies as described herein are characterized by a plurality of such features. In some embodiments, one or more antibodies described herein can be characterized as an anti-CD25 antibody agent.

[0022] Therefore, as exemplified herein, certain antibodies and / or antigen-binding fragments comprising aCD25-a-634 sequences (particularly aCD25-a-634-HCDR3 and / or aCD25-a-634-LCDR3) are characterized as such desirable structural and / or functional features; such antibodies and / or antigen-binding fragments thereof may be referred to herein as anti-CD25 antibody agents. In addition, antibodies and antigen-binding fragments thereof that compete with aCD25-a-634 may be particularly useful antibodies according to the present invention; such antibodies and / or antigen-binding fragments thereof may also be referred to herein as anti-CD25 antibody agents.

[0023] The antibodies (and / or antigen-binding fragments thereof) described herein are particularly useful in medicine (e.g., for treatment and / or prevention, e.g., for the treatment of cancer), and / or for methods requiring or involving targeting an epitope within the extracellular domain of human CD25. The provided antibodies or antigen-binding fragments thereof can be prepared to present the most suitable isotype, particularly a human isotype from the group consisting of IgG1, IgG2, IgG3 and IgG4 isotype antibodies, more particularly human IgG1 or human IgG2, preferably human IgG1.

[0024] In one aspect, the present invention provides aCD25-a-634-HCDR3 amino acid sequence and polypeptides comprising the same, such as, for example, an antibody or antigen-binding fragment comprising the aCD25-a-634-HCDR3 amino acid sequence (SEQ ID NO: 4) as the heavy chain variable complementary determining region 3. In some embodiments, such an antibody or antigen-binding fragment may be further characterized as comprising additional aCD25-a-amino acid sequence elements such as:

[0025] a) aCD25-a-634-HCDR1 amino acid sequence (SEQ ID NO: 2) as heavy chain variable complementarity determining region 1; and / or

[0026] b) aCD25-a-634-HCDR2 amino acid sequence (SEQ ID NO: 3) as heavy chain variable complementarity determining region 2.

[0027] In some embodiments, the antibodies or antigen-binding fragments thereof provided may further comprise the heavy chain variable complementary determining regions defined above (i.e., aCD25-a-634 amino acid sequence elements) in the correct order (particularly separated by the antibody backbone sequence), such as the heavy chain variable complementary determining regions contained in the aCD25-a-634-HCDR123 amino acid sequence (SEQ ID NO: 5), in particular for correctly exerting its binding and functional properties. For example, in some embodiments, the antibodies or antigen-binding fragments thereof provided may comprise the aCD25-a-634-HCDR123 amino acid sequence (SEQ ID NO: 5) and, optionally:

[0028] a) aCD25-a-634-LCDR1 amino acid sequence (SEQ ID NO: 6) as the light chain variable complementarity determining region 1;

[0029] b) aCD25-a-634-LCDR2 amino acid sequence (SEQ ID NO: 7) as the light chain variable complementarity determining region 2; and

[0030] c) aCD25-a-634-LCDR3 amino acid sequence (SEQ ID NO: 8) as the light chain variable complementarity determining region 3.

[0031] Therefore, in some embodiments, the present invention provides an antibody or antigen-binding fragment thereof comprising a variable heavy chain, the variable heavy chain comprising aCD25-a-634-HCDR123 amino acid sequence (SEQ ID NO: 5). Preferably, such an antibody or antigen-binding fragment thereof further comprises a variable light chain, the variable light chain comprising aCD25-a-634-LCDR123 amino acid sequence (SEQ ID NO: 9), as described in the embodiments. In addition, the aCD25-a-634 amino acid sequence also refers to an antibody sequence defined by several substitutions of the aCD25-a-634 amino acid sequence elements defined above. For example, such a sequence may be included in aCD25-a-634-HCDR3 (SEQ ID NO: 4) containing 1, 2, 3, 4 or more amino acid substitutions as a heavy chain variable complementary determining region 3 (HCDR3). In another embodiment, the aCD25-a-634 amino acid sequence also refers to an antibody sequence comprising as heavy chain variable complementarity determining regions 1, 2 and 3 (HCDR1, HCDR2 and HCDR3) sequences containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions in aCD25-a-634-HCDR1 (SEQ ID NO: 2), aCD25-a-634-HCDR2 (SEQ ID NO: 3) and aCD25-a-634-HCDR3 (SEQ ID NO: 4), and more preferably containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions in aCD25-a-634-HCDR123 (SEQ ID NO: 5). Antibodies exhibiting such aCD25-a-634 amino acid sequence elements and such substitutions can generally still exhibit the binding and / or functional properties of aCD25-a-634 and anti-CD25 antibody agents.

[0032] Thus, in one embodiment, the present invention provides an anti-CD25 antibody agent (i.e., an antibody or an antigen-binding fragment thereof) comprising:

[0033] a. a heavy chain variable region sequence of aCD25-a-634 (SEQ ID NO: 5) (or a variant thereof, such as an affinity matured variant thereof) or a heavy chain variable region sequence having up to 5 amino acid substitutions compared to the heavy chain variable region sequence of aCD25-a-634 (or a variant thereof, such as an affinity matured variant thereof); and / or

[0034] b. a light chain variable region sequence of aCD25-a-634 (SEQ ID NO: 9) (or a variant thereof, such as an affinity matured variant thereof) or a light chain variable region sequence having up to 5 amino acid substitutions compared to the light chain variable region sequence of aCD25-a-634 (or a variant thereof, such as an affinity matured variant thereof).

[0035] In some embodiments, the present invention provides an anti-CD25 antibody agent (i.e., an antibody or an antigen-binding fragment thereof) comprising:

[0036] a. a heavy chain variable region sequence of aCD25-a-634 (SEQ ID NO: 5) (or a variant thereof, such as an affinity matured variant thereof), or a heavy chain variable region sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the heavy chain variable region sequence of aCD25-a-634 (or a variant thereof, such as an affinity matured variant thereof); and / or

[0037] b. a light chain variable region sequence of aCD25-a-634 (SEQ ID NO: 9) (or a variant thereof, such as an affinity matured variant thereof), or a light chain variable region sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the light chain variable region sequence of aCD25-a-634 (or a variant thereof, such as an affinity matured variant thereof).

[0038] The present invention also provides variant antibodies or antigen-binding fragments thereof of antibodies described herein. The present invention provides antibodies or antigen-binding fragments thereof, wherein any DG motif in the light chain or heavy chain of the antibody can be changed, for example, to reduce aspartic acid isomerization and / or any NG motif in the light chain or heavy chain of the antibody can be changed, for example, to reduce asparagine deamidation and / or any methionine in the light chain or heavy chain of the antibody can be changed, for example, to reduce methionine oxidation. For example, the DG motif can be changed to replace one or two amino acids in the motif with different amino acids. The NG motif can be changed to replace one or two amino acids in the motif with different amino acids. For example, such motifs can be mutated into EG, DQ or DA. The methionine residue can be changed to replace it with different amino acids, such as leucine or phenylalanine.

[0039] In this embodiment, the anti-CD25 antibody or antigen-binding fragment thereof can be, or can be derived from, for example, aCD25-a-634. Variant anti-CD25 antibodies provide additional antibodies having any and possibly all of the binding and functional properties of aCD25-a-634. For example, the variant anti-CD25 antibody is a non-IL-2 blocking antibody.

[0040] Thus, in some embodiments, the antibodies or fragments thereof provided herein can be mutated to remove or modify DG or NG motifs, particularly those present in CDR regions, to reduce sensitivity to chemical modification as is standard in the art. Such antibodies modified in this manner may require further modification (e.g., affinity maturation) before obtaining the final sequence.

[0041] In one embodiment of the invention, variant antibodies are provided that have the CDR1, CDR2 and CDR3 sequences of antibodies as disclosed herein (e.g., the CDR1, CDR2 and CDR3 sequences of aCD25-a-634), or the variable heavy chain and variable light chain of any antibody as disclosed herein (e.g., the variable heavy chain and variable light chain of any aCD25-a-634), but differ from the given sequence in that at least one or at least two DG or NG motifs (if present) in the CDRs have been changed to different motifs. The disclosed variants can be used and formulated as described for aCD25-a-634.

[0042] The invention also provides affinity matured antibodies, such as affinity matured variants derived from any of the antibodies disclosed herein.

[0043] In one embodiment, the affinity matured antibody is an affinity matured antibody with an altered DG motif and / or NG motif and / or altered to remove or mutate any methionine residues. The disclosed affinity matured variants can be used and formulated as described for aCD25-a-634.

[0044] In some embodiments, the present invention provides a method for preparing an anti-CD25 antibody, the method comprising providing an antibody as described herein (e.g., aCD25-a-634 or an antigen-binding fragment or variant thereof), and subjecting the antibody to affinity maturation, wherein the prepared antibody binds to CD25 with a greater affinity than the parent antibody. Preferably, the prepared antibody binds to CD25 with an affinity of at least 20%, at least 30%, at least 40%, and more preferably at least 50% greater than the parent antibody binding to CD25, for example as measured by Kd. Methods for measuring affinity are known in the art and are described in the following examples. Affinity-matured antibodies prepared by this method can be formulated and used as described herein for other anti-CD25 antibody agents.

[0045] Affinity maturation can be carried out according to any suitable method known to the technician.For example, in vitro antibody display systems are widely used to produce specific antibodies with high affinity.In these systems, phenotype (that is, antibody fragment) is combined with genotype (that is, antibody gene), thereby allowing to directly determine the sequence of antibody.Several systems have been developed to realize the display of antibody library to allow the selection of adhesive subsequently and to allow to select more and more high affinity variants by improving the stringency of selection.Antibody fragment can be expressed in yeast, ribosome, phage display particles or by directly being coupled to DNA.

[0046] Current antibody affinity maturation methods belong to two mutagenesis categories: random and non-random. Error-prone polymerase chain reaction (PCR), mutant strains and saturation mutagenesis are typical examples of random mutagenesis methods. Non-random techniques typically use alanine scanning or site-directed mutagenesis to produce a limited set of specific variants. In addition, recombinant methods for obtaining recombinant variants of parent antibodies can also be used to further improve antibody affinity.

[0047] Therefore, in one embodiment of the present invention, the method of affinity maturation is selected from the group consisting of random mutagenesis (e.g., error-prone polymerase chain reaction (PCR), mutant strains or saturation mutagenesis), non-random mutagenesis (e.g., alanine scanning or site-directed mutagenesis), recombination (e.g., DNA recombination, chain recombination or CDR recombination) and the use of the CRISPR-Cas9 system to introduce modifications.

[0048] Affinity maturation methods are described, for example, in Rajpal et al., Proc Natl Acad Sci USA, 2005, 102(24):8466-71, Steinwand et al., MAbs, 2014, 6(1):204-18, and Handbook of Therapeutic Antibodies, Wiley, 2014, Chapter 6, Antibody Affinity (pp. 115-140).

[0049] In some embodiments, a method for preparing a pharmaceutical composition is provided, the method comprising providing an antibody prepared according to the above method (i.e., for preparing an antibody by affinity maturation) and co-formulating the antibody with at least one or more pharmaceutically acceptable excipients. The antibody used to prepare the pharmaceutical composition can be an affinity-matured variant of aCD25-a-634. The pharmaceutical composition prepared by this method can be used in the treatment methods of the present invention, as described herein for other anti-CD25 antibody agents.

[0050] As described herein, antibodies and / or antigen-binding fragments that specifically bind to CD25 (e.g., may include one or more aCD25-a-634 amino acid sequence elements such as aCD25-a-634-HCDR3 or aCD25-a-634-HCDR123 and / or may compete with aCD25-a-634 for binding to human CD25 and non-human primate CD25, such as cynomolgus CD25, etc.) may be provided in any of a variety of forms. For example, in some embodiments, a suitable form may be or include: a monoclonal antibody, a domain antibody, a single-chain antibody, a Fab fragment, a F(ab')2 fragment, a single-chain variable fragment (scFv), a scFv-Fc fragment, a single-chain antibody (scAb), an aptamer or a nanobody. In some embodiments, the antibody or antigen-binding fragment thereof (particularly a monoclonal antibody) may be a rabbit, mouse, chimeric, humanized or fully human antibody or antigen-binding fragment thereof. In some embodiments, the provided antibodies or antigen-binding fragments thereof may be of IgG, IgA, IgE or IgM isotypes (preferably human isotypes), as may be best suited for a given use. In some embodiments, the provided antibodies or antigen-binding fragments thereof are of IgG isotypes, more particularly IgG1, IgG2, IgG3 or IgG4 isotypes. (In some embodiments, the antibodies or antigen-binding fragments thereof are from the human IgG1 subclass. In another embodiment, the antibodies or antigen-binding fragments thereof are from the human IgG2 subclass). In some embodiments, the provided antibodies or antigen-binding fragments thereof (e.g., provided as part of a multispecific binding agent, such as, for example, when it is desired to bind additional binding and / or functional portions to an anti-CD25 antibody agent such as aCD25-a-634 amino acid sequence, the separated antibodies or antigen-binding fragments may be included in bispecific antibodies, multispecific antibodies or other multispecific formats available in the art.

[0051] In some embodiments, the anti-CD25 antibody agent provided comprises a CD25 binding entity (e.g., an anti-CD25 antibody or an antigen-binding fragment thereof) and a conjugated payload such as a therapeutic agent or a diagnostic agent. In many such embodiments, the agent is considered and / or referred to as an "immunoconjugate". Examples of techniques and compounds that can be used to produce specific immunoconjugates such as antibody-drugs are disclosed in the literature (Beck A et al., 2017) and are described as being applicable to several known anti-CD25 antibodies (O'Mahony D et al., 2008; Oh et al., 2017; Kreitman RJ et al., 2016; Flynn MJ et al. 2016).

[0052] In some embodiments, the invention provides aCD25-a-634 amino acid sequence that identifies a provided antibody or antigen-binding fragment thereof (or variants thereof, including affinity matured variants). In some embodiments, such a sequence identifies a provided antibody or antigen-binding fragment thereof that binds to an epitope in human CD25 and optionally also binds to a corresponding epitope of non-human primate CD25 (e.g., cynomolgus monkey and / or murine CD25) (as an isolated protein or on a cell expressing CD25 (e.g., an immune cell or cell line, e.g., SU-DHL1 cell).

[0053] In some embodiments, the present invention provides methods for screening and / or characterizing antibodies or antigen-binding fragments that comprise an aCD25-a-634 amino acid sequence and / or exhibit comparable binding characteristics to antibodies or antigen-binding fragments thereof that comprise one or more aCD25-a-634 amino acid sequence elements (e.g., including an aCD25-a-634-HCDR3 amino acid sequence (SEQ ID NO: 4) and / or compete with aCD25-a-634), which amino acid sequence allows binding to the extracellular domain of CD25 (as an isolated protein and on the surface of cells expressing human CD25), thereby competing for the same epitope.

[0054] In addition, the present invention also provides a method for screening antibodies or antigen-binding fragments thereof, which exhibit functional characteristics comparable to antibodies or antigen-binding fragments thereof comprising one or more aCD25-a-634 amino acid sequence elements, such characteristics including lack of inhibition of the interaction between IL-2 and CD25, lack of inhibition of IL-2 signaling and cytotoxic activity, and acting as anti-CD25 antibody agents. Within these ranges, candidate antibodies can be tested in assays described in the Examples or other assays known in the art to determine the presence of any such characteristics, but the presence of all of the characteristics possible when evaluated in in vitro / ex vivo assays, cell-based assays, and / or animal models.

[0055] In some embodiments, the present invention provides nucleic acid molecules encoding isolated antibodies or antigen-binding fragments thereof, wherein the isolated antibodies or antigen-binding fragments thereof comprise an anti-CD25 antibody agent, such as aCD25-a-634 amino acid sequence (or variants thereof, including affinity-matured variants). In some embodiments, such provided nucleic acid molecules may contain codon-optimized nucleic acid sequences, and / or may be included in an expression cassette in an appropriate nucleic acid vector for expression in a host cell such as, for example, bacteria, yeast, insects, fish, mice, monkeys or human cells.

[0056] In some embodiments, the present invention provides a host cell comprising a heterologous nucleic acid molecule (e.g., a DNA vector) that expresses a provided anti-CD25 antibody agent (e.g., an antibody or an antigen-binding fragment thereof) having one or more properties of, e.g., an anti-CD25 antibody agent as described herein (e.g., comprising an aCD25-a-634 amino acid sequence). In some embodiments, the present invention provides a method for preparing an anti-CD25 antibody agent (e.g., an antibody or an antigen-binding fragment thereof) having one or more properties of, e.g., an anti-CD25 antibody agent as described herein (e.g., comprising an aCD25-a-634 amino acid sequence). In some embodiments, such a method may comprise culturing a host cell comprising a nucleic acid (e.g., a heterologous nucleic acid that may comprise and / or be delivered to the host cell via a vector). In some embodiments, the host cell (and / or heterologous nucleic acid sequence) is configured and constructed so that the anti-CD25 antibody agent (e.g., antibody or antigen-binding fragment thereof) is secreted from the host cell (e.g., so that it can be isolated from the cell culture supernatant) and / or is exposed on the cell surface (e.g., if the aCD25-a-634 amino acid sequence and sequence elements are intended for use in or with such cells, such as to graft the specificity of a monoclonal antibody onto T cells in an artificial T cell receptor).

[0057] In some embodiments, the antibody or antigen-binding fragment may be defucosylated. It is well known that antibody glycosylation may have an effect on the activity, pharmacokinetics and pharmacodynamics of antibodies (e.g., monoclonal antibodies, recombinant antibodies and / or antibodies otherwise modified or isolated) and Fc fusion proteins, and specific techniques may be used to obtain antibodies with desired glycosylation characteristics (Liu L, 2015). Methods for reducing antibody fucosylation levels may be used to enhance the effector function of cytotoxicity of antibodies (e.g., anti-CD25 antibodies as described herein, including antibodies that may be or are described as anti-CD25 antibody agents) used for use according to the present invention. Antibodies comprising specific aCD25-a-634 sequence elements exhibiting such properties can be produced, for example, by expressing the aCD25-a-634 sequence using techniques for cell lines that have been genetically engineered and have no or reduced fucosylation capacity (some of which are commercially available, such as Potelligent (Lonza), GlyMAXX (ProBiogen), those provided by Evitria), or by manipulating the manufacturing process, for example by controlling the molar osmotic pressure concentration and / or using enzyme inhibitors (see also, for example, the methods described in EP2480671).

[0058] In some embodiments, the present invention provides a composition (e.g., a pharmaceutical composition) comprising an antibody or antigen-binding fragment thereof provided having the desired properties as described herein (e.g., as described for the antibodies referred to herein as anti-CD25 antibody agents, particularly including, for example, aCD25-a-634 antibodies or antigen-binding fragments thereof or antibodies or antigen-binding fragments thereof that compete with aCD25-a-634 antibodies for binding to CD25). In some embodiments, such provided compositions are intended for and / or used for medical purposes, such as therapeutic, diagnostic or prophylactic uses. In some embodiments, such provided compositions may further comprise a pharmaceutically acceptable carrier or excipient and / or may be used to treat cancer. In some embodiments, pharmaceutical compositions may be formulated with one or more carriers, excipients, salts, buffers, etc., as known in the art. Those skilled in the art will recognize and be able to readily utilize a variety of formulation techniques, including those that may be particularly desirable and / or useful for a given method and / or site of administration, such as for parenteral (e.g., subcutaneous, intramuscular or intravenous injection), mucosal, intratumoral, peritumoral, oral or topical administration. In many embodiments, provided pharmaceutical compositions (comprising an anti-CD25 antibody agent as described herein (e.g., an anti-CD25 antibody or antigen-binding portion thereof)) are formulated for parenteral delivery (e.g., by injection and / or infusion). In some embodiments, such provided pharmaceutical compositions may be provided, for example, in the form of a prefilled syringe or bottle. In some embodiments, such provided pharmaceutical compositions may be provided and / or utilized, for example, in a dry (e.g., lyophilized) form; alternatively, in some embodiments, such provided pharmaceutical compositions may be provided and / or utilized in liquid form (e.g., as a solution, suspension, dispersion, emulsion, etc.), in gel form, etc.

[0059] In some embodiments, the present invention provides for use of an anti-CD25 antibody agent (e.g., an anti-CD25 antibody or antigen-binding fragment thereof) as described herein (e.g., comprising an aCD25-a-634 amino acid sequence element) and / or a composition comprising the same in treating cancer and / or preparing a medicament for treating cancer, such as a B-cell malignancy, a lymphoma (Hodgkin's lymphoma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, myeloma), a myeloproliferative disorder, a solid tumor (e.g., breast cancer, squamous cell carcinoma, colon cancer, head and neck cancer, lung cancer, genitourinary cancer, rectal cancer, gastric cancer, sarcoma, melanoma, esophageal cancer, liver cancer, testicular cancer, cervical cancer, mast cell tumor, hemangioma, eye cancer, laryngeal cancer, oral cancer, mesothelioma, skin cancer, rectal cancer, laryngeal cancer, bladder cancer, breast cancer, uterine cancer, prostate cancer, lung cancer, pancreatic cancer, kidney cancer, stomach cancer, Cancer can also be defined based on the presence of specific tumor-associated markers and antigens, such as CD20, HER2, PD-1, PD-L1, SLAM7F, CD47, CD137, CD134, TIM3, CD25, GITR, CD25, EGFR, etc., or cancers that have been identified as having biomarkers known as high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR). In addition, such conditions can also be considered when defining the precancerous, non-invasive state of the above-mentioned cancers, such as carcinoma in situ, congested myeloma, monoclonal gammopathy of undetermined significance, cervical intraepithelial neoplasia, MALTomas / GALTomes, and various lymphoproliferative disorders. Preferably, in some embodiments, the subject being treated has a solid tumor.

[0060] Thus, in some embodiments, the present invention provides a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of a composition comprising an anti-CD25 antibody agent (e.g., an anti-CD25 antibody or antigen-binding fragment thereof) as provided herein (e.g., comprising an aCD25-a-634 amino acid sequence) or an antibody or antigen-binding fragment thereof that competes with an antibody comprising an aCD25-a-634 amino acid sequence for binding to CD25. Preferably, in some embodiments, the subject suffers from a solid tumor.

[0061] Therefore, in some embodiments, the present invention provides a method for eliminating regulatory T cells of a subject, the method comprising the following steps: administering to the subject an effective amount of a composition comprising an anti-CD25 antibody agent (e.g., an anti-CD25 antibody or an antigen-binding fragment thereof) provided as described herein (e.g., comprising an aCD25-a-634 amino acid sequence) or an antibody or antigen-binding fragment that competes with an antibody comprising an aCD25-a-634 amino acid sequence for binding to CD25. In one embodiment, the subject suffers from a solid tumor. In one embodiment, the subject suffers from a blood cancer.

[0062] In some embodiments, the methods provided may further include administering to the subject at least one additional agent or therapy simultaneously or sequentially in any order (i.e., such that the subject receives a combination therapy). In some embodiments, this at least one additional agent or therapy can be or include an anti-cancer drug (e.g., a chemotherapeutic agent), radiation therapy (by applying radiation externally to the body or by administering a radioconjugated compound), an anti-tumor antigen or marker antibody (the antigen or marker is, for example, CD4, CD38, CA125, PSMA, c-MET, VEGF, CD137, VEGFR2, CD20, HER2, HER3, SLAMF7, CD326, CAIX, CD40, CD47, or EGF receptor), a checkpoint inhibitor or immunomodulatory antibody (e.g., targeting PD-1, PD-L1, TIM3, CD38, GITR, CD134, CD134L, CD137, CD137L, CD80, CD86, B7-H3, B7-H4, B7RP1, LAG3, ICOS, TIM3, GAL9, CD28, AP2M1, SHP-2, OX-40, VISTA, TIGIT, BTLA, HVEM, CD160, etc.), vaccines (specifically, cancer vaccines, such as GVAX), adjuvants, standard use regimens, one or more other compounds that target cancer cells or stimulate an immune response to cancer cells, or any combination thereof. Preferably, the additional agent is an immune checkpoint inhibitor, such as a PD-1 antagonist, such as an anti-PD-1 antibody or an anti-PD-L1 antibody. In certain specific embodiments, when the at least one additional agent or therapy is or comprises an antibody, the form of such antibody and / or the antigen targeted by such antibody can be selected from those listed in the literature and may be applicable to a given cancer (Sliwkowski M & Mellman I, 2013; Redman JM et al., 2015; Kijanka M et al., 2015). Such antigens and corresponding antibodies include, but are not limited to, CD22 (Blinatumomab), CD20 (Rituximab, Tositumomab), CD56 (Lorvotuzumab), CD66e / CEA (Labetuzumab), CD152 / CTLA-4 (Ipilimumab), CD221 / IGF1R (MK-0646), CD326 / Epcam (Edrecolomab), CD340 / HER2 (Trastuzumab, Pertuzumab), and EGFR (Cetuximab, Panitumumab).In an embodiment, when at least one additional agent or therapy is a chemotherapeutic agent, the chemotherapeutic agent may be those chemotherapeutic agents known in the art for cancer therapy. Such chemotherapeutic agents include, but are not limited to, alkylating agents, anthracyclines, epothilones, nitrosoureas, ethyleneimine / methylmelamine, alkyl sulfonates, alkylating agents, antimetabolites, pyrimidine analogs, epipodophyllotoxins, enzymes such as L-asparaginase; biological response modifiers such as IFNα,. IL-2, IL-12, G-CSF and GM-CSF; platinum coordination compounds such as cisplatin, oxaliplatin and carboplatin, anthracenediones, 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 steroid antagonists such as prednisone and equivalents, dexamethasone, and aminoglutethimide; progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate; estrogens such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogens such as tamoxifen; androgens including testosterone propionate and fluoxymesterone / equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs, and leuprolide; nonsteroidal antiandrogens such as flutamide; molecules targeting the TGFβ pathway, indoleamine dehydrogenase, and sedatives. deoxigenase (IDO), arginase and / or CSF1R; PARP inhibitors such as olaparib, veliparib, iniparib, rucaparib; and MET inhibitors such as tivantinib, foretinib, golvatinib, cabozantinib and crizotinib.

[0063] Further, the present invention provides a variety of kits or articles containing the anti-CD25 antibody agents (e.g., anti-CD25 antibodies or antigen-binding fragments thereof) provided as described herein (e.g., comprising aCD25-a-634 amino acid sequence) or related compositions that allow the administration, storage or other uses of such isolated antibodies or antigen-binding fragments. In some embodiments, the provided kits include containers, syringes, vials or other containers containing such compositions, optionally with one or more preparations, diluents, reagents, solid phases and / or instructions for the proper use of the kit.

[0064] In some embodiments, identification, characterization and / or validation of a specific anti-CD25 antibody agent (e.g., an anti-CD25 antibody or antigen-binding fragment thereof) as described herein (e.g., comprising an aCD25-a-634 amino acid sequence) for a particular use, such as a medical use, and in particular for the treatment of cancer, can be performed using one or more assays or systems as described herein. In some embodiments, such identification, characterization and / or validation can involve analyzing activity in one or more cell-based assays, for example, using different experimental settings and / or selected groups (e.g., cancer-derived cell lines). In some embodiments, particularly given certain desired anti-CD25 antibody agent activities as described herein associated with proposed immune mechanisms, the desired identification, characterization and / or validation can involve collecting relevant data generated in animal models in which cancer is induced or in which cancer cells are transplanted as xenografts or as isogenic / allogeneic cancer-derived cells. Alternatively or additionally, in some embodiments, animal models may be utilized that involve delivery of human cells such as PBMCs (i.e., humanized PBMC mouse model) or CD34+ hematopoietic stem cells alone (i.e., CD34+ humanized mice) or CD34+ hematopoietic stem cells together with liver and thymus (e.g., NSG-BLT mice) to allow evaluation of the activity of anti-CD25 antibody agents on human immune cells within the model system.

[0065] In some embodiments, sequences related to an anti-CD25 antibody agent (e.g., an anti-CD25 antibody or antigen-binding fragment thereof) as described herein (e.g., comprising an aCD25-a-634 amino acid sequence or otherwise including the structural and / or functional properties of an agent described herein as an anti-CD25 antibody agent) can be cloned into and / or expressed in the context of an antibody backbone that is more appropriate or desired for pharmaceutical and / or technical reasons. For example, such sequences (perhaps such as codon-optimized VH and VL coding sequences) can be cloned together with a human IgG1 constant region (hIgG1) and expressed using an appropriate antibody expression vector and cell line (e.g., a CHO-derived cell line, e.g., CHO-S). In some specific embodiments, expression and secretion of a provided antibody sequence in a human IgG1 format antibody can be analyzed after transfection under reducing conditions in a cell lysate and non-reducing conditions in a supernatant, which will later be used to purify the antibody (by affinity chromatography, gel filtration, and / or other appropriate techniques). The binding and / or other functional properties of the provided anti-CD25 antibody sequences (e.g., anti-CD25 antibody agents - hIgG1) in human IgG1 format can be analyzed, for example, by using one or more assays described in the Examples below. For example, such provided antibodies in hIgG1 format can be evaluated, for example, for binding to human and cynomolgus monkey PBMCs or purified Tregs or in vitro derived Tregs or CD25 positive cell lines (such as SU-DHL-1 or Karpas299) using flow cytometry.

[0066] In addition, the effects of one or more anti-CD25 antibody agents (e.g., anti-CD25 antibodies or antigen-binding fragments thereof) as described herein (e.g., comprising the aCD25-a-634 amino acid sequence or otherwise comprising the structural and / or functional properties of an agent described herein as an anti-CD25 antibody agent-such as an anti-CD25 antibody agent-hIgG1) on human primary tumor cells and / or immune cells (including regulatory T cells) isolated from healthy human donors and / or patients can be assessed. To study the potential effects of anti-CD25 antibody agents, the antibodies can be used to treat PBMCs and / or cells isolated from tumors (and / or organs, such as lymph nodes) and / or tumor explants or other 3D organoids, and / or purified or in vitro generated regulatory T cells or other CD25-positive cells, such as some NK cells or dendritic cells. Potential readouts include CD25 positive cell survival, elimination and / or apoptosis, tumor cell killing, effector immune cell proliferation and function (e.g., granzyme B production, cytotoxic activity, degranulation), antigen specific responses (as measured, for example, by proliferation, degranulation, or cytokine production in response to antigen). Alternatively or in addition, mice or non-human primates can be treated and cell status can be tracked in blood samples (analyzed as whole blood or isolated PBMCs) or after isolating various organs and / or cells from the animal by, for example, flow cytometry or immunohistochemistry.

[0067] Alternatively or in addition, one or more properties of an anti-CD25 antibody agent (e.g., an anti-CD25 antibody or antigen-binding fragment thereof) as described herein (e.g., comprising an aCD25-a-634 amino acid sequence or otherwise including the structural and / or functional properties of an agent described herein as an anti-CD25 antibody agent such as an anti-CD25 antibody agent-hIgG1) can be evaluated individually or in combination by studying the effects of such anti-CD25 antibody agents on cells expressing CD25 (e.g., Tregs or cancer cells expressing CD25). Readouts may include cell killing, apoptosis, intracellular signaling monitoring (e.g., Stat-5 phosphorylation), effects on IL-2 binding to CD25 and signaling (e.g., Stat-5 phosphorylation or other signaling downstream of the IL-2 receptor), and IL-2-dependent functional activities (e.g., proliferation and cytokine production). When an aCD25 antibody agent-hIgG1 antibody is administered to cynomolgus monkeys or related mouse models, the in vivo cellular effects of the antibody can then be tracked.

[0068] To obtain further understanding of the molecular interactions between the provided anti-CD25 antibody agents and human CD25, the crystal structures of the anti-CD25 antibody agents (e.g., to give a specific example, aCD25-a-634-hIgG1 antibody) and human CD25 protein can be determined. The solubility and / or stability of the provided anti-CD25 antibody agents (especially including, for example, aCD25-a-634-hIgG1 antibodies) can be evaluated by solubility studies, accelerated stress studies, freeze-thaw studies, and formal stability studies. The stability and / or stability of the provided anti-CD25 antibody agents can be evaluated by visual inspection, size exclusion chromatography, dynamic light scattering, and OD 280 / 320 Absorbance was used to track antibody aggregation. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 : Related protein sequences of aCD25-a-634. Each CDR of the heavy chain (aCD25-a-634-HCDR1 (SEQ ID NO: 2), aCD25-a-634-HCDR2 (SEQ ID NO: 3), and aCD25-a-634-HCDR3 (SEQ ID NO: 4)) and each CDR of the light chain (aCD25-a-634-LCDR1 (SEQ ID NO: 5), aCD25-a-634-LCDR2 (SEQ ID NO: 6), and aCD25-a-634-LCDR3 (SEQ ID NO: 7)) are indicated and underlined within the framework sequence of the heavy and light chain antibodies initially identified in the screening process (aCD25-a-634-HCDR123 (SEQ ID NO: 5) and aCD25-a-634-LCDR123 (SEQ ID NO: 9), respectively).

[0070] Figure 2 : Consensus sequence of human CD25 (Uniprot code P01589) (SEQ ID NO: 1). The extracellular domain of mature CD25 corresponding to amino acids 22-240 is underlined.

[0071] Figure 3 : Characterization of aCD25-a-634 binding to CD25 expressed on in vitro differentiated human Treg cells (A), SU-DHL-1 cells (B), or SR-786 cells (C) at increasing antibody concentrations and compared to a human IgG1 isotype control.

[0072] Figure 4 : Characterization of aCD25-a-634 binding to CD25 expressed on the HSC-F cynomolgus monkey T cell line at increasing antibody concentrations and compared to a human IgG1 isotype control.

[0073] Figure 5: aCD25-a-634 binding to his-tagged recombinant human CD25 measured by biolayer interferometry on an Octet Red 96 instrument. 20.83 nM of rhCD25-his was loaded onto the Ni NTA biosensor and followed by different concentrations of antibody (as indicated in the figure) and then allowed to dissociate in kinetic buffer.

[0074] Figure 6 : Non-competitive binding of aCD25-a-634 to IL-2 (A) and competitive binding of IL-2 competing antibodies to IL-2 (B) were shown by biolayer interference measurement on Octet Red 384 using a standard sandwich format competition (binning) assay. Anti-human CD25 antibody aCD25-a-634 was loaded onto the AHQ sensor. The sensor was then exposed to 100 nM human CD25 and subsequently to human IL-2. Additional binding of human IL2 after antigen association indicates an unoccupied epitope (non-competitor), while no binding indicates epitope blocking (competitor).

[0075] Figure 7 : Non-competitive binding of aCD25-a-634 and daclizumab to CD25 was shown by biolayer interference measurement on the Octet Red 384 system using a standard sandwich format competition assay. The reference monoclonal anti-human CD25 antibody daclizumab was loaded onto the AHQ sensor. The sensor was then exposed to 100 nM human CD25 antigen and then to anti-human CD25 antibody (aCD25-a-634). Additional binding by the second antibody after antigen association indicates an unoccupied epitope (non-competitor), while no binding indicates epitope blocking (competitor).

[0076] Figure 8 : Characterization of aCD25-a-634 blocking IL-2 signaling compared to human IgG1 isotype control, daclizumab, or the absence of primary antibody in a STAT5 phosphorylation assay using human PBMCs. Cells were incubated with 10 μg / ml antibody followed by increasing concentrations of IL-2 (as indicated in the accompanying figure). Analysis was limited to the percentage of CD3-positive cells phosphorylating STAT5.

[0077] Fig. 9 : Functional characterization of aCD25-a-634 compared to human IgG1 isotype control, daclizumab, or commercially available mouse anti-human IL-2 neutralizing antibody (clone: ​​AB12-3G4) as a positive control using PanT cells. Cells were incubated with 10ug / ml antibody and then activated with CD3 / CD28 beads for 72 hours before flow cytometry analysis. Results show the percentage of granzyme B-positive proliferating CD4 (A) or CD8 (B) T cells.

[0078] Fig.10 : Functional characterization of the killing of CD25-positive cell lines in ADCC assays compared to human IgG1 isotype controls by aCD25-a-634. In the presence of different concentrations of antibodies, CD25 high-expressing cells SU-DHL-1 cells (A) or CD25 low-expressing cells SR-786 cells (B) were co-cultured with purified NK cells (as shown in the accompanying drawings). Target cell lysis was measured by releasing calcein into the supernatant four hours after being added to NK cells. Data were normalized for saponin-treated controls.

[0079] Fig.11 : Functional characterization of aCD25-a-634 in causing ADCP in a reporter bioassay compared to a human IgG1 isotype control. SU-DHL-1 cells expressing CD25 were co-cultured with Jurkat T cells genetically engineered to express FcγRIIa and a NFAT response element (NFAT-RE-luc2) driving luciferase expression in the presence of varying concentrations of antibody (as indicated in the figure).

[0080] Fig.12 : Evaluation of the therapeutic activity of 7D4 mIgG2a (anti-mouse CD25 non-IL-2 blocking, Treg depleting antibody) (D) alone in combination with IL-2 neutralizing antibody (E) or IL-2 blocking antibody (F) in female BALB / c mice bearing CT26 syngeneic colon tumors. The activity of mouse IgG2a control (A), IL-2 neutralizing antibody only (B) and IL-2 blocking antibody only (C) were tested for comparison. DETAILED DESCRIPTION

[0081] Some definitions of terms, techniques, and embodiments used herein are provided below, most or most of which are generally understood by those skilled in the art.

[0082] Administration: As used herein, the term "administering" refers to administering a composition to a subject. It can be administered to an animal subject (e.g., a human) by any appropriate route. For example, in some embodiments, administration may be bronchial (including by bronchial instillation), buccal, enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, in a specific organ or tissue (e.g., intrahepatic, intratumoral, peritumoral, etc.), transmucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including intratracheal instillation), transdermal, vaginal, and vitreous. Administration may involve intermittent medication. Alternatively, administration may involve continuous medication (e.g., perfusion) for at least a selected period of time. As known in the art, antibody therapy is usually administered parenterally, such as by intravenous, subcutaneous, or intratumoral injection (e.g., especially when a higher dose is required in a tumor).

[0083] Agent: As used herein, the term "agent" may refer to a compound or entity of any chemical class, including, for example, polypeptides, nucleic acids, carbohydrates, small molecules, metals, or combinations thereof. Specific embodiments of agents that can be used according to the present invention include small molecules, drugs, hormones, antibodies, antibody fragments, aptamers, nucleic acids (e.g., siRNA, shRNA, antisense oligonucleotides, ribozymes), peptides, peptide mimetics, etc. The agent may be or include a polymer.

[0084] Antibody: As used herein, the term "antibody" refers to a polypeptide comprising typical immunoglobulin sequence elements sufficient to confer specific binding to a specific target antigen such as CD25, particularly human CD25 and human CD25 extracellular domain. As known in the art, naturally occurring intact antibodies are tetrameric agents of approximately 150 kD, consisting of two identical heavy chain polypeptides (each about 50 kD) and two identical light chain polypeptides (each about 25 kD) associated with each other in a structure often referred to as a "Y-shaped" structure. Each heavy chain comprises at least four domains (each about 110 amino acids long): an amino-terminal variable (VH) domain (located at the top of the Y structure), followed by three constant domains: CH1, CH2, and carboxyl-terminal CH3 (located at the bottom of the trunk of the Y). A short region called a "switch" connects the heavy chain variable region and the constant region. A "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides in the intact antibody to each other. Each light chain comprises two domains, an amino-terminal variable (VL) domain, followed by a carboxyl-terminal constant (CL) domain, which are separated from each other by another "switch". A complete antibody tetramer comprises two heavy chain-light chain dimers, wherein the heavy and light chains are linked to each other by a single disulfide bond; two additional disulfide bonds link the heavy chain hinge regions to each other, so that the dimers are linked to each other and form a tetramer. Naturally produced antibodies are also glycosylated, typically on the CH2 domain, and each domain has a structure characterized as an "immunoglobulin fold", which is formed by two β sheets (e.g., 3, 4, or 5 sheets) stacked relative to each other in a compressed antiparallel β cylinder. Each variable domain contains three hypervariable loops called "complementarity determining regions" (CDR1, CDR2, and CDR3; as understood in the art, for example, determined according to the Kabat numbering scheme) and four somewhat invariant "framework" regions (FR1, FR2, FR3, and FR4). When a natural antibody is folded, the FR region forms a β sheet that provides a structural skeleton for the domain, and the CDR loop regions from both the heavy chain and the light chain are combined together in three-dimensional space so that they produce a single hypervariable antigen binding site located at the top of the Y structure. The Fc region of a naturally occurring antibody binds to elements of the complement system and also binds to effector cells, including, for example, receptors on effector cells that mediate cytotoxicity. As known in the art, the affinity and / or other binding properties of the Fc region of an Fc receptor can be regulated by glycosylation or other modifications that can improve the developability of the antibody (Jarasch A et al., 2015).

[0085] In some embodiments, antibodies produced and / or utilized in accordance with the present invention include a glycosylated Fc domain, including Fc domains with modified or engineered such glycosylation. For purposes of the present invention, in certain embodiments, any polypeptide or polypeptide complex comprising sufficient immunoglobulin domain sequences as found in natural antibodies may be referred to and / or used as an "antibody," whether such polypeptide is produced naturally (e.g., by an organism in response to an antigen) or produced by recombinant engineering, chemical synthesis, or other artificial systems or methods. In some embodiments, antibodies are polyclonal or oligoclonal, produced as a group of antibodies, each associated with a single antibody sequence and binding to a more or less unique epitope within an antigen (e.g., different epitopes within the extracellular domain of human CD25 associated with different reference anti-CD25 antibodies).

[0086] Polyclonal or oligoclonal antibodies can be provided in a single formulation for medical use as described in the literature (Kearns JD et al., 2015). In some embodiments, the antibody is monoclonal. In some embodiments, the antibody has a constant region sequence that has the characteristics of a mouse, rabbit, primate or human antibody. In some embodiments, as known in the art, antibody sequence elements are humanized, primatized, chimeric, etc. In addition, the term "antibody" as used herein in appropriate embodiments (unless otherwise specified, it is obvious from the context) may refer to any construct or form known or developed in the art for utilizing antibody structural and functional characteristics in alternative displays, for example, as an antigen-binding fragment as defined below. For example, the antibody utilized according to the present invention is in a form selected from (but not limited to) the following: complete IgG, IgE and IgM, bispecific or multispecific antibodies (e.g., etc.), single chain variable domain (scFv), polypeptide-Fc fusion, Fab, camelid-like antibodies, heavy chain shark antibodies (IgNAR), shielded antibodies (e.g., ) or a fusion protein with a polypeptide that allows expression and exposure on the cell surface (as a scFv in a construct for obtaining an artificial T cell receptor for the specificity of a monoclonal antibody to be transplanted to a T cell). The shielded antibody may include a blocking or "shielding" peptide that specifically binds to the antigen binding surface of the antibody and interferes with the antigen binding of the antibody. The shielding peptide is connected to the antibody by a cleavable linker (e.g., by a protease). Under the desired environment, such as in a tumor environment, the selective cleavage of the linker allows the shielding / blocking peptide to dissociate, so that antigen binding can occur in the tumor and thus limit potential toxicity issues. In some embodiments, the antibody may lack a covalent modification (e.g., attachment of a polysaccharide) that it would have if it were naturally produced. Alternatively, the antibody may contain a covalent modification (e.g., attachment of a polysaccharide, a load [e.g., a detectable portion, a therapeutic portion, a catalytic portion, etc.] or other side groups [e.g., polyethylene glycol, etc.]).

[0087] Non-IL-2 blocking antibody agents: The anti-CD25 antibody agents and antigen-binding fragments of the present invention are non-IL-2 blocking antibody agents. The term "non-IL-2 blocking antibody agent" as used herein refers to those anti-CD25 antibody agents (e.g., anti-CD25 non-IL-2 blocking antibodies) that are capable of specifically binding to the CD25 subunit of the IL-2 receptor without blocking the binding of IL-2 to CD25 or IL-2 signaling via CD25. Compared to the signaling level in the absence of the anti-CD25 antibody agent, the anti-CD25 antibody agent allows at least 50% of IL-2 signaling in response to IL-2 binding to CD25. Preferably, the anti-CD25 antibody agent allows at least 75% of IL-2 signaling in response to CD25, compared to the signaling level in the absence of the anti-CD25 antibody agent.

[0088] The CD25 subunit of the IL-2 receptor is also known as the α subunit of the IL-2 receptor and is found on activated T cells, regulatory T cells, activated B cells, some thymocytes, bone marrow precursor cells, and oligodendrocytes. CD25 associates with CD122 and CD132 to form a heterotrimeric complex that serves as a high affinity receptor for IL-2. The consensus sequence of human CD25 is shown in Figure 2 middle.

[0089] "Specific binding" is understood to mean that the antibody or antigen-binding fragment has less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 In a preferred embodiment, the dissociation constant is less than 10-8 M, for example, in 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 Within the range of M.

[0090] As used herein and with reference to "non-blocking", "not blocking", etc., "non-IL-2 blocking" (with respect to non-blocking of IL-2 binding to CD25 in the presence of an anti-CD25 antibody) includes embodiments in which the anti-CD25 antibody or antigen-binding fragment inhibits less than 50% of IL-2 signaling, compared to IL-2 signaling in the absence of the antibody. In specific embodiments of the invention as described herein, the anti-CD25 antibody or antigen-binding fragment inhibits less than about 40%, 35%, 30%, preferably less than about 25% of IL-2 signaling, compared to IL-2 signaling in the absence of the antibody. An anti-CD25 non-IL-2 blocking antibody allows binding to CD25 without interfering with IL-2 binding to CD25, or does not substantially interfere with IL-2 binding to CD25. Reference herein to a non-IL-2 blocking antibody is alternatively expressed as an anti-CD25 antibody that "does not inhibit interleukin 2 binding to CD25" or an anti-CD25 antibody that "does not inhibit signaling of IL-2".

[0091] Some anti-CD25 antibody agents may allow IL-2 to bind to CD25, but still block signaling through the CD25 receptor. Such antibody agents are not within the scope of the present invention. Alternatively, non-IL-2 blocking anti-CD25 antibody agents allow IL-2 to bind to CD25 to promote at least 50% of the signaling level through the CD25 receptor compared to signaling in the absence of the anti-CD25 antibody agent.

[0092] IL-2 signaling through CD25 can be measured by methods as discussed in the Examples and as known in the art. Comparison of IL-2 signaling can be performed in the presence and absence of an anti-CD25 antibody agent under the same or substantially the same conditions.

[0093] In some embodiments, IL-2 signaling can be measured by measuring the level of phosphorylated STAT5 protein in cells using standard Stat-5 phosphorylation assay.For example, the Stat-5 phosphorylation assay for measuring IL-2 signaling can be related to the presence of anti-CD25 antibodies with a concentration of 10ug / ml to cultivate PMBC cells for 30 minutes and then add different concentrations of IL-2 (0.1U / ml, 1U / ml or 10U / ml) for 10 minutes.Alternatively, for example, the Stat-5 phosphorylation assay for measuring IL-2 signaling can be related to the presence of anti-CD25 antibodies at 10ug / ml concentrations to cultivate PMBC cells for 30 minutes and then add different concentrations of IL-2 (for example, 10U / ml or 0.25U / ml, 0.74U / ml, 2.22U / ml, 6.66U / ml or 20U / ml of different concentrations) for 10 minutes.Then the cells can be permeabilized, and then the level of STAT5 protein can be measured by the fluorescently labeled antibody of flow cytometry analysis phosphorylated STAT5 peptides. The percentage of blockade of IL-2 signaling was calculated as follows: Blockade % = 100 × [(Stat5 + Cells without antibody group%-Stat5 + Cells 10ug / ml antibody group %) / (Stat5 + cells without Ab group %)].

[0094] In some embodiments, the anti-CD25 antibody agents described herein are characterized in that they are highly effective in depleting tumor-infiltrating regulatory T cells, particularly within tumors.

[0095] In some embodiments, the anti-CD25 antibody agent is characterized in that it binds to an Fcγ receptor with high affinity, preferably binds to at least one activated Fcγ receptor with high affinity. Preferably, the antibody binds to at least one activated Fcγ receptor with an affinity of less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 The anti-CD25 antibody agent binds to at least one activated Fcγ receptor with a dissociation constant of M. In some embodiments, the anti-CD25 antibody agent is characterized by other characteristics with respect to the Fcγ receptor, in particular:

[0096] (a) binds to an Fcγ receptor with an activation inhibition ratio (A / I) greater than 1; and / or

[0097] (b) binds to at least one of FcγRI, FcγRIIc and FcγRIIIa with higher affinity than it binds to FcγRIIb.

[0098] In some embodiments, the CD25 antibody agent is an IgG1 antibody, preferably a human IgG1 antibody, which is 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 FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the antibody is capable of binding to FcγRIIIa. In some embodiments, the antibody is capable of binding to FcγRIIIa and FcγRIIa and optionally FcγRI. In some embodiments, the antibody is capable of binding to FcγRIIIa and FcγRIIa with high affinity, for example, with a binding affinity of less than about 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 In some embodiments, the antibody binds to the inhibitory receptor FcγRIIb with a dissociation constant of greater than 10 M. -7 M, higher than 10 -6 M or above 10 -5 The dissociation constant of M binds to FcγRIIb.

[0099] In some embodiments, the desired anti-CD25 antibody agent as described herein is characterized in that it is cytotoxic or induces phagocytosis of cells expressing CD25 (e.g., expressing higher levels of CD25), such as immunosuppressive cells or tumor cells (e.g., in each case, expressing CD25 on its surface). In some embodiments, the anti-CD25 antibody agent is characterized in that: for immune cells (e.g., when in contact with immune cells, and in particular with immune cells expressing CD25) and tumor cells, the activity (e.g., level and / or type) is reasonably comparable to the activity of aCD25-a-634. In some embodiments, the relevant activity is or includes: clearing Treg cells (e.g., in solid tumors) or certain cells expressing CD25 (e.g., high-expressing cells) by ADCP, ADCC or CDC, promoting T cell, B cell or NK cell expansion, skewing the T cell pool, etc., and combinations thereof. In some embodiments, the increased level and / or activity, or the decreased level and / or the increased or unchanged level or activity, is assessed or determined relative to the level and / or activity observed under otherwise comparable conditions in the absence of the entity or moiety. Alternatively or in addition, in some embodiments, the increased level and / or activity is equivalent to or greater than the level and / or activity observed under comparable conditions in the presence of a reference anti-CD25 antibody agent (e.g., a suitable reference anti-CD25 antibody, which in various embodiments is a CD25 antibody that blocks IL-2 binding to CD25, such as daclizumab or basiliximab). In various embodiments, the anti-CD25 antibody agent used in accordance with the present invention is or comprises an entity or moiety that directly or indirectly binds to CD25 (typically to its extracellular domain). In some embodiments, the anti-CD25 antibody agent is, comprises, or competes with an anti-CD25 antibody as exemplified herein, an antigen-binding fragment thereof (e.g., comprising one or more CDRs, all heavy chain CDRs, all light chain CDRs, all CDRs, heavy chain variable regions, light chain variable regions, or both heavy chain variable regions and light chain variable regions), an affinity-matured variant thereof (or an antigen-binding fragment thereof), or any alternative form of any of the foregoing (e.g., chimeric, humanized, multispecific, alternative isotypes, etc.). Alternatively or additionally, in some embodiments, an anti-CD25 antibody agent as described herein may be characterized by one or more features, which may be features that facilitate: screening; manufacturing; preclinical testing; and / or identification of relevant epitopes within human CD25); and / or formulation, administration, and / or effectiveness in a particular context (e.g., cancer therapy), as disclosed herein.

[0100] Antigen: As used herein, the term "antigen" refers to an agent that elicits an immune response and / or binds to a T cell receptor (e.g., when presented by an MHC molecule) and / or a B cell receptor. Antigens that elicit a humoral response are involved in the production of antigen-specific antibodies or, as demonstrated in the example against the extracellular domain of CD25, can be used to screen antibody libraries and identify candidate antibody sequences for further characterization.

[0101] Antigen binding fragment: As used herein, the term "antigen binding fragment" encompasses an agent that includes or comprises one or more portions of an antibody as described herein sufficient to confer on the antigen binding fragment the ability to specifically bind to an antigen targeted by the antibody. For example, in some embodiments, the term encompasses any polypeptide or polypeptide complex that includes an immunoglobulin structural element sufficient to confer specific binding. Exemplary antigen binding fragments include, but are not limited to, small modular immunopharmaceuticals (Small Modular ImmunoPharmaceuticals, "SMIPsTM"), single-chain antibodies, camelid-like antibodies, single-domain antibodies (e.g., shark single-domain antibodies), single-chain or tandem diabodies VHH, Microantibodies, Ankyrin repeat protein or DART, TCR-like antibodies, MicroProtein, CoVX antibodies, bicyclic peptides, Kunitz domain derived antibody constructs, or any other antibody fragments as long as the desired biological activity is exhibited. In some embodiments, the term encompasses other protein structures, such as stapled peptides, antibody-like binding peptide mimetics, antibody-like binding scaffold proteins, monofunctional antibodies, and / or other non-antibody protein scaffolds, such as those reviewed in the literature (Vazquez-Lombardi R et al., 2015). In some embodiments, the antigen binding fragment is or comprises a polypeptide, the amino acid sequence of which includes one or more structural elements identified as complementary determining regions (CDRs) by those skilled in the art. In some embodiments, the antigen binding fragment is or comprises a polypeptide, the amino acid sequence of which includes at least one reference CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) substantially identical to the CDR found in the anti-CD25 antibody as described herein (e.g., in the aCD25-a-634 amino acid sequence element), and in particular at least one heavy chain CDR, such as HCDR3 (e.g., aCD25-a-634-HCDR3 sequence). In some embodiments, an antigen-binding fragment is or comprises a polypeptide having an amino acid sequence that includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is identical to or contains a small number (e.g., 1, 2, 3, or 4) of amino acid changes (e.g., substitutions, additions, or deletions; in most cases substitutions) of the reference CDR sequence or more than the reference CDR, while maintaining binding to the target of the antibody from which the reference CDR is derived (e.g., aCD25-a-634). In some embodiments, an antigen-binding fragment is or comprises a polypeptide or a complex thereof that includes all three CDRs of a heavy chain or light chain from a reference antibody (e.g., from aCD25-a-634) (or, in some embodiments, sequences substantially identical thereto); in some embodiments, an antigen-binding fragment is or comprises a polypeptide or a complex thereof that includes all six CDRs from a reference antibody (e.g., from aCD25-a-634) (or, in some embodiments, sequences substantially identical thereto). In some embodiments, the antigen binding fragment is or comprises a polypeptide or a complex thereof, which includes the heavy chain and / or light chain variable domains of a reference antibody (e.g., aCD25-a-634) (or, in some embodiments, a sequence substantially identical thereto). In some embodiments, the term "antigen binding fragment" encompasses non-peptide and non-protein structures, such as nucleic acid aptamers, such as RNA aptamers and DNA aptamers. Aptamers are oligonucleotides (e.g., DNA, RNA, or analogs or derivatives thereof) that bind to specific targets such as polypeptides. Aptamers are short, single-stranded oligonucleotides that specifically bind to various molecular targets such as small molecules, proteins, nucleic acids, and even synthetic cells and tissues.These small nucleic acid molecules can form secondary and tertiary structures that can specifically bind to proteins or other cellular targets and are essentially chemical equivalents of antibodies. Aptamers are highly specific, relatively small in size and non-immunogenic. Aptamers are usually selected from a bio-panning method known as SELEX (Systematic Evolution of Ligands by Exponential enrichment) (see, for example, Ellington et al., 1990; Tuerk et al., 1990; Ni et al., 2011). Methods for producing aptamers for any given target are known in the art. Peptide aptamers including affimers are also contemplated. Affimers are small, highly stable proteins that are engineered to display peptide loops that provide high-affinity binding surfaces for specific target proteins. It is a low molecular weight (12-14 kDa) protein derived from the cysteine ​​protease inhibitor family of cystatin. Affimer proteins are composed of a scaffold, which is a stable protein based on cystin protein folding. It was shown that two peptide loops and N-terminal sequences can be randomly bound to different target proteins with high affinity and specificity similar to antibodies. The peptides are stabilized after the protein scaffold restricts the possible conformations that the peptides can adopt, thus increasing the binding affinity and specificity compared to the free peptide library.

[0102] Biological sample. As used herein, the term "biological sample" or "sample" generally refers to a sample obtained or derived from a biological source of interest (e.g., a tissue or an organism or a cell culture), as described herein. The source of interest can be an organism, such as an animal or a human. A biological sample can comprise biological tissue or a fluid.

[0103] Cancer: The terms "cancer," "malignancy," "neoplasm," "tumor," and "neoplasm" are used interchangeably herein to refer to cells that exhibit relatively abnormal, uncontrolled, and / or spontaneous growth, such that they exhibit an abnormal growth phenotype characterized by an apparent loss of control over cell proliferation. In general, cells of interest for detection or treatment in the present application include precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and non-metastatic cells. The teachings of the present invention may be relevant to any and all cancers. To provide a few non-limiting examples, in some embodiments, the teachings of the present invention are applied to one or more cancers, such as, for example: hematopoietic cancers, including leukemias, lymphomas (Hodgkin's and non-Hodgkin's), myelomas and myeloproliferative diseases; sarcomas, melanomas, adenomas, solid tissue tumors, squamous cell carcinomas of the mouth, throat, larynx and lung, liver cancer, genitourinary cancers such as prostate, cervical, bladder, uterine and endometrial cancers and renal cell carcinomas, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancers of the endocrine system, thyroid cancer, parathyroid cancer, head and neck cancer, breast cancer, gastrointestinal cancer and cancers of the nervous system, benign lesions such as papilloma, etc. The antibodies of the present invention can be used to treat tumors expressing CD25+. Cancers involving the treatment of tumors expressing CD25 may include, but are not limited to, lymphomas such as Hodgkin's lymphoma, and lymphocytic leukemias such as chronic lymphocytic leukemia (CLL).

[0104] Combination therapy: As used herein, the term "combination therapy" refers to those situations in which a subject is exposed to two or more treatment regimens (e.g., two or more therapeutic agents) simultaneously. In some embodiments, two or more agents may be administered simultaneously. Alternatively, such agents may be administered sequentially; or, such agents may be administered in overlapping dosing regimens.

[0105] Comparable: As used herein, the term "comparable" refers to two or more agents, entities, situations, effects, groups of conditions, etc. that may not be identical to one another but are sufficiently similar to permit comparisons therebetween (e.g., comparison of levels and / or activities) so that conclusions can be reasonably drawn based on the observed differences or similarities. Such comparable groups of conditions, effects, situations, individuals, or populations are characterized by a plurality of substantially identical features and one or a few different features. In the context, one skilled in the art will understand what degree of identity is necessary in any given case for two or more such agents, entities, situations, groups of conditions, effects, or populations, etc. to be considered comparable.

[0106] Comprising: Compositions or methods described herein as "comprising" one or more specified elements or steps are open-ended, meaning that the specified elements or steps are required, but other elements or steps may be added within the scope of the composition or method. It is also understood that any composition or method described as "comprising" (or its "comprising") one or more specified elements or steps also describes a corresponding, more limited composition or method "consisting essentially of" (or its "consisting essentially of") the same specified elements or steps, meaning that the composition or method includes the specified required elements or steps, and may also include additional elements or steps that do not materially affect the basic and novel characteristics of the composition or method.

[0107] Clearance: As used herein, reference to "clearance" (with respect to clearing regulatory T cells by an anti-CD25 antibody agent) means that the number, ratio or percentage of Tregs is reduced relative to when an anti-CD25 antibody that does not inhibit the binding of interleukin 2 to CD25 is not administered. In specific embodiments of the invention as described herein, more than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of tumor-infiltrating regulatory T cells are cleared.

[0108] Dosage form: As used herein, the term "dosage form" refers to a physically discrete unit of an active agent (e.g., a therapeutic or diagnostic agent) for administration to a subject. Each unit contains a predetermined amount of an active agent. In some embodiments, such an amount is a unit dose (or a complete portion thereof) that is suitable for administration according to a regimen determined to be associated with a desired or beneficial outcome when administered to a relevant population (i.e., using a therapeutic regimen). It is understood by those skilled in the art that the total amount of a therapeutic composition or therapeutic agent administered to a particular subject is determined by one or more attending physicians and may involve the administration of multiple dosage forms.

[0109] Dosage regimen: As used herein, the term "dosage regimen" refers to a group of unit doses (usually more than one) administered independently to a subject, usually at intervals of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, the dosing regimen comprises multiple doses, each of which is spaced apart by a time period of the same length. Alternatively, the dosing regimen comprises multiple doses and at least two different time periods separating separate doses. In some embodiments, all doses within the dosing regimen have the same amount of unit dose. Alternatively, different doses within the dosing regimen have different amounts. In some embodiments, the dosing regimen comprises a first dose in a first dosage, followed by one or more additional doses of a second dosage that is different from the first dosage. The dosing regimen may comprise a first dose in a first dosage, followed by one or more additional doses of a second dosage that is the same as the first dosage. In some embodiments, the dosing regimen is associated with a desired or beneficial outcome when administered across a related population (i.e., a therapeutic dosing regimen).

[0110] Epitope: As used herein, the term "epitope" refers to the portion of an antigen that binds to an antibody or antigen-binding fragment. In some embodiments where the antigen is a polypeptide, when the antigen is in a relevant conformation, the epitope is a conformation consisting of portions of the antigen that are not covalently continuous in the antigen but close to each other in three-dimensional space. For example, for CD25, conformational epitopes are those consisting of amino acid residues that are not continuous in the CD25 extracellular domain, and linear epitopes are those consisting of amino acid residues that are continuous in the CD25 extracellular domain. In some embodiments, the epitopes utilized according to the present invention are provided by reference to epitopes that bind to anti-CD25 antibody agents provided herein (e.g., those that bind to aCD25-a-634). Means for determining the exact sequence and / or specific amino acid residues of the epitope of aCD25-a-634 are known in the literature and in the Examples and include competition with peptides, from antigen sequences, binding to CD25 sequences from different species, truncation and / or mutagenesis (e.g., by alanine scanning or other site-directed mutagenesis), phage display-based screening, yeast display technology or (co) crystallization techniques.

[0111] Identity: Percent identity (%) as known in the art is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences as determined by comparing the sequences. In the art, identity also means, as the case may be, the degree of sequence relatedness between polypeptide or polynucleotide sequences as determined by the match between such sequence strings. Although there are a variety of methods for measuring the identity between two polypeptide or two polynucleotide sequences, the methods commonly used to determine identity are encoded by computer programs. Preferred computer programs for determining the identity between two sequences include, but are not limited to, the GCG program package (Devereux, et al., Nucleic Acids Research, 12, 387 (1984), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec. Biol. 215, 403 (1990)). The percent identity of two amino acid sequences or two nucleic acid sequences is determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced into the first sequence for optimal alignment with the sequence) and comparing the amino acid residues or nucleotides at corresponding positions. The "optimal alignment" is the alignment of the two sequences that yields the highest percent identity. The percent identity is determined by comparing the number of identical amino acid residues or nucleotides in the sequences (i.e., % identity = number of identical positions / total number of positions × 100). In general, unless otherwise specified or implied by the context, reference to % identity herein refers to % identity along the entire length of the molecule.

[0112] Immune effector cells: Immune effector cells refer to immune cells that participate 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 lytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils.

[0113] The immune effector cells participating in the effector phase of the immune response can express specific Fc receptors and perform specific immune functions. Effector cells can induce antibody-dependent cell-mediated cytotoxicity (ADCC), such as neutrophils that can induce ADCC. Effector cells can also induce antibody-dependent cell-mediated phagocytosis (ADCP), which is present in the phagocytosis of target antigens, target cells or microorganisms (such as macrophages capable of ADCP). For example, monocytes, macrophages, neutrophils, eosinophils and lymphocytes expressing FcγR participate in specific killing of target cells and present antigens to other components of the immune system, or combine cells presenting antigens. As discussed, antibodies according to the present invention can be optimized for the ability to induce ADCC and / or ADCP.

[0114] Regulatory T cells: As used herein, "regulatory T cells" ("Treg" or "Treg cells") refer to a CD4+T lymphocyte line dedicated to controlling autoimmunity, allergies, and infections. Typically, it regulates the activity of T cell populations, but it can also affect certain innate immune system cell types. Tregs are usually identified by the expression of the biomarkers CD4, CD25, and Foxp3, as well as low expression of CD127. Naturally occurring Treg cells typically constitute approximately 5%-10% of peripheral CD4+T lymphocytes. However, within the tumor microenvironment (i.e., tumor-infiltrating Treg cells), they can constitute up to 20%-30% of the total CD4+T lymphocyte population.

[0115] 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 (TGFβ) in vivo, and thus directly inhibit T cell activation and inhibit APC function by inhibiting the expression of MHC molecules, CD80, CD86 and IL-12. Treg cells can also suppress immunity by expressing high levels of CTLA4, which can bind to CD80 and CD86 on antigen-presenting cells and prevent the proper activation of effector T cells.

[0116] Patient: As used herein, the term "patient" or "subject" refers to any organism to which a provided composition may be administered, e.g., for experimental, diagnostic, prophylactic, aesthetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). In some embodiments, the patient is human. In some embodiments, the patient suffers from or is susceptible to one or more disorders or conditions. The patient may display one or more symptoms of a disorder or condition, or be diagnosed with one or more disorders or conditions (e.g., cancer, or the presence of one or more tumors). In some embodiments, the patient is receiving or has received certain therapies to diagnose and / or treat such diseases, disorders, or conditions.

[0117] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" as applied to a carrier, diluent or excipient used to formulate a composition as disclosed herein means that the carrier, diluent or excipient must be compatible with the other ingredients of the composition and not poisonous to the recipient thereof.

[0118] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a treatment regimen in an amount of a unit dose suitable for administration, which exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. The pharmaceutical composition can be formulated for administration in solid or liquid form, including forms suitable for: oral administration, such as drench (aqueous or non-aqueous solution or suspension), lozenge (e.g., lozenges targeting buccal, sublingual and systemic absorption), bolus, powder, granule, paste for application to the tongue; parenteral administration, such as by subcutaneous, intramuscular, intravenous, intratumoral or epidural injection as a sterile solution or suspension or sustained release preparation; topical administration, such as application to the skin, lungs or oral cavity as an ointment, ointment or controlled release patch or spray; intravaginal, intrarectal, sublingual, ocular, transdermal, nasal, pulmonary and to other mucosal surfaces.

[0119] Solid tumor: As used herein, the term "solid tumor" refers to an abnormal tissue mass that does not usually contain a cyst or liquid area. Solid tumors can be benign or malignant. Different types of solid tumors are named for the cell types that form them. Examples of solid tumors are sarcomas (including cancers caused by transformed cells of mesenchymal cell sources such as cancellous bone, cartilage, fat, muscle, blood vessels, hematopoiesis or fibrous connective tissue), tumors (including tumors caused by epithelial cells), melanomas, lymphomas, mesotheliomas, neuroblastomas, retinoblastomas, etc. Cancers related to solid tumors include but are not limited to brain cancer, lung cancer, gastric cancer, duodenal cancer, esophageal cancer, breast cancer, colorectal cancer, renal cancer (renal cancer), bladder cancer, kidney cancer (kidney cancer), pancreatic cancer, prostate cancer, ovarian cancer, melanoma, oral cancer, sarcomas, eye cancer, thyroid cancer, urethral cancer, vaginal cancer, cervical cancer, lymphoma, etc.

[0120] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" means an amount (e.g., an amount of a pharmaceutical agent or pharmaceutical composition) sufficient to treat a disease and / or condition when administered according to a therapeutic dosing regimen to a population suffering from or susceptible to such disease and / or condition. A therapeutically effective amount is an amount that reduces the incidence and / or severity of a disease, disorder, and / or condition, stabilizes the disease, disorder, and / or condition, and / or delays the onset of one or more symptoms of the disease, disorder, and / or condition. One skilled in the art will appreciate that the term "therapeutically effective amount" is not actually required to achieve successful treatment in a particular individual.

[0121] Treatment: As used herein, the term "treatment" refers to any administration of a substance (e.g., a provided anti-CD25 antibody agent, such as aCD25-a-634 or any other agent) that partially or completely relieves, ameliorates, alleviates, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms. In some embodiments, treatment may involve direct administration of an anti-CD25 antibody agent (e.g., aCD25-a-634) (e.g., as an injectable aqueous composition, optionally comprising a pharmaceutically acceptable carrier, excipient, and / or adjuvant for intravenous, intratumoral, or peritumoral injection) or administration using a regimen that includes obtaining cells from a subject (e.g., from blood, tissue, or tumor, with or without selection based on the presence or absence of marker expression), contacting the cells with an anti-CD25 antibody agent (e.g., aCD25-a-634) in vitro, and administering such cells to a subject (with or without selection based on the presence or absence of marker expression).

[0122] Dosage and administration. Pharmaceutical compositions comprising an anti-CD25 antibody agent as described herein (e.g., anti-CD25 or an antigen-binding fragment thereof, e.g., comprising aCD25-a-634-HCDR3 amino acid sequence) used in accordance with the present invention can be prepared for storage and / or delivery using any of a variety of techniques and / or techniques known and / or available to those skilled in the art. In some embodiments, the provided anti-CD25 antibody agent is administered according to a dosing regimen approved by a regulatory agency such as the U.S. Food and Drug Administration (FDA) and / or the European Medicines Agency (EMEA), e.g., for a related indication. In some embodiments, the provided anti-CD25 antibody agent is administered in combination with one or more other agents or therapies, e.g., for a related indication, which one or more other agents or therapies themselves can be administered according to a dosing regimen approved by a regulatory agency such as the U.S. Food and Drug Administration (FDA) and / or the European Medicines Agency (EMEA). However, in some embodiments, use of provided anti-CD25 antibody agents may allow for reduced dosing of approved agents or therapies used in combination with anti-CD25 antibody agent therapies (e.g., lower amounts of active in one or more doses, fewer dosing times, and / or reduced dosing frequency). In some embodiments, dosing and / or administration may be adapted to other drugs also being administered, patient conditions, and / or the form of the anti-CD25 antibody agent (e.g., modified as an immunoconjugate, single domain antibody, or bispecific antibody).

[0123] Furthermore, in some embodiments, it may be desirable to adjust dosing regimens based on timing and / or threshold expression levels of CD25, and in particular to design continuous dosing regimens, whether for a particular cell type, a particular tumor or type thereof, or a particular patient population (e.g., carrying a genetic marker). In some such embodiments, the therapeutic dosing regimen may be combined with or adjusted based on detection methods or other criteria that assess the expression of one or more inducible markers prior to and / or during treatment.

[0124] In some embodiments, the medication and administration according to the present invention utilize an active agent having the desired purity in combination with any or multiple forms of one or more physiologically acceptable carriers, excipients or stabilizers. These include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form may depend on the intended mode of administration and / or therapeutic application, typically in the form of an injectable or infusible solution, such as a composition similar to that used to treat human subjects with antibodies.

[0125] In some embodiments, the composition can be prepared with a carrier that protects the agent from rapid release and / or degradation, such as a controlled release formulation, including implants, transdermal patches, and microcapsule delivery systems. Biodegradable biocompatible polymers can be used, such as polyanhydrides, polyglycolic acid, polyorthoesters, and polylactic acid. Typically, pharmaceutical compositions and dosing regimens consistent with good medical practice and suitable for related agents (e.g., for agents such as antibodies) are used to prepare, administer, and administer each active agent in a therapeutically effective amount. The pharmaceutical composition containing the active agent can be administered by any appropriate method known in the art, including but not limited to oral, mucosal, by inhalation, topical, buccal, nasal, rectal, or parenteral (e.g., intravenous, infusion, intratumoral, intranodular, subcutaneous, intraperitoneal, intramuscular, intradermal, transdermal, or other types of administration involving physical ruptures in the tissue of the object and administration of the pharmaceutical composition through such ruptures).

[0126] In some embodiments, the dosing regimen of a particular active agent may involve intermittent or continuous (e.g., by perfusion or slow release system) administration, for example to achieve a specific desired pharmacokinetic profile or other exposure pattern in one or more tissues or body fluids of interest of the subject. In some embodiments, different agents administered in combination may be administered via different delivery routes and / or according to different time schedules. Alternatively or in addition, 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, one or more doses of the first active agent are administered via a common route and / or as part of a single composition with one or more other active agents.

[0127] Factors to consider in optimizing the route and / or dosing schedule for a given treatment regimen may include, for example, the specific cancer being treated (e.g., type, stage, location, etc.), the clinical condition of the subject (e.g., age, general health, weight, etc.), the site of delivery of the agent, the nature of the agent (e.g., an antibody or other protein-based compound), the mode and / or route of administration of the agent, the presence or absence of combination therapy, and other factors known to medical practitioners.

[0128] Those skilled in the art will appreciate that, for example, a specific route of delivery may affect the dosage and / or the required dosage may affect the route of delivery. For example, when the purpose is a specific high concentration of a drug in a specific site or position (e.g., in a tissue or organ), concentrated delivery (e.g., intratumoral delivery) may be desirable and / or useful. In some embodiments, one or more features of a specific pharmaceutical composition and / or a used dosage regimen may be modified over time (e.g., increasing or decreasing the amount of active substance in any single dose, increasing or decreasing the time interval between doses, etc.), for example, to optimize the desired therapeutic effect or response (e.g., a treatment or biological response associated with the functional characteristics of an anti-CD25 antibody agent as described herein). Typically, the type of administration, dosage, and frequency of administration of an active agent according to the present invention are controlled by the safety and efficacy requirements of the application when one or more related agents are administered to a mammal (preferably a human). Typically, such a dosage feature is selected to provide a specific and generally detectable therapeutic response compared to that observed in the absence of treatment. In the context of the present invention, exemplary desired therapeutic responses may involve, but are not limited to, inhibition and / or reduction of tumor growth, tumor size, metastasis, one or more of symptoms and side effects associated with tumors, and increased apoptosis of cancer cells, treatment-related reduction or increase of one or more cell markers or circulating markers, etc. Such criteria can be readily assessed by any of a variety of immunological, cytological and other methods disclosed in the literature. For example, a therapeutically effective amount of an anti-CD25 antibody agent alone or in combination with another agent can be determined to be sufficient to enhance the killing of cancer cells as described in the Examples.

[0129] A therapeutically effective amount of an anti-CD25 antibody agent as an active agent, or a composition comprising such an agent, can be readily determined using techniques available in the art including, for example, consideration of one or more factors such as the disease or condition being treated, the stage of the disease, the age and health and physical condition of the mammal being treated, the severity of the disease, the specific compound being administered, and the like.

[0130] In some embodiments, the therapeutically effective amount is an effective dose (and / or unit dose) of the active agent, which can be at least about 0.01 mg / kg body weight, at least about 0.05 mg / kg body weight, at least about 0.1 mg / kg body weight, at least about 1 mg / kg body weight, at least about 5 mg / kg body weight, at least about 10 mg / kg body weight, or greater (e.g., 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 body weight). One skilled in the art will appreciate that in some embodiments such guidelines may be adjusted for the molecular weight of the active agent. The dose may also vary for route of administration, treatment cycle, or thus a dose escalation schedule that can be used to determine the maximum tolerated dose and dose-limiting toxicity (if any) associated with administering increasing doses of an isolated antibody or antigen-binding fragment thereof comprising the aCD25-a-634-HCDR3 amino acid sequence.

[0131] The therapeutic composition should generally be sterile and stable under manufacturing and storage conditions. The composition can be formulated into a solution, microemulsion, dispersion, liposome or other ordered structure suitable for high drug concentration. A sterile injectable solution can be prepared by incorporating the desired amount of antibody in a suitable solvent having one or a combination of the above-listed ingredients, followed by filtration sterilization. Typically, a dispersion is prepared by incorporating the active compound into a sterile carrier containing an alkaline dispersion medium and other required ingredients from the above-listed ingredients. In the case of a powder for preparing a sterile injectable solution, a preferred preparation method is vacuum drying and freeze drying, which produces a powder of the active ingredient plus any other required ingredients from a previously aseptically filtered solution. For example, by using a coating, by maintaining the desired particle size in the case of a dispersion and by using a surfactant to maintain the appropriate fluidity of the solution. The extended absorption of the injectable composition can be achieved by including an agent (e.g., monostearate and gelatin) that delays absorption in the composition.

[0132] The preparation of each agent should ideally be sterile, such as can be achieved by filtering through a sterile filtration membrane, and then packaged or sold in a form suitable for bolus administration or continuous administration. Injectable preparations can be prepared, packaged or sold in unit dosage form, such as in ampoules or multi-dose containers containing preservatives. Preparations for parenteral administration include, but are not limited to: suspensions, solutions, emulsions in oily or aqueous vehicles; pastes; and implantable sustained release or biodegradable preparations, as described below. Non-toxic parenterally acceptable diluents or solvents (such as water or 1,3 butanediol) can be used to prepare sterile injectable preparations. Other useful parenterally administrable preparations include those containing active ingredients in microcrystalline form, in liposomal preparations, or as components of biodegradable polymer systems. Compositions for sustained release or implantation may include pharmaceutically acceptable polymeric or hydrophobic materials such as emulsions, ion exchange resins, slightly soluble polymers or salts.

[0133] Each pharmaceutical composition used according to the present invention may contain a pharmaceutically acceptable dispersant, wetting agent, suspending agent, isotonic agent, coating, antibacterial and antifungal agent, carrier, excipient, salt or stabilizer, which is non-toxic to the subject at the dosages and concentrations employed. A non-exhaustive list of such additional pharmaceutically acceptable compounds includes: buffers, such as phosphate, citric acid and other organic acids; antioxidants, including ascorbic acid and methionine; salts containing pharmacologically acceptable anions (such as acetate, benzoate, bicarbonate, bisulfate, isethionate, lactate, lactobionate, laurate, malate, maleate, salicylate, stearate, subacetate, succinate, tannate, tartrate, theochlorate, toluenesulfonate, thiethiodode and valerate); preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexahydroxyquaternium chloride; benzalkonium chloride; benzethonium chloride; sodium chloride; phenol, butyl alcohol or benzyl alcohol ; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, glutamic acid, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, fucose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn protein complexes); and / or nonionic surfactants, such as TWEEN®. TM 、PLURONICS TM or polyethylene glycol (PEG).

[0134] In some embodiments, when two or more active agents are utilized according to the present invention, such agents may be administered simultaneously or sequentially. In some embodiments, another agent is administered at a specific time relative to the administration of one agent. In some embodiments, the desired relative dosing regimen of the agents administered in combination may be evaluated or empirically determined, for example, using ex vivo, in vivo, and / or in vitro models; in some embodiments, such evaluations or empirical determinations are performed in vivo, in a specific patient or patient population (e.g., to generate correlations).

[0135] In some embodiments, one or more active agents used in the implementation of the present invention are administered according to an intermittent dosing regimen comprising at least two cycles. When two or more agents are combined and each is administered by this intermittent cyclic regimen, the individual doses of different agents can cross each other. In some embodiments, one or more doses of the second agent are administered after a period of time of the dose of an anti-CD25 antibody agent as described herein. In some embodiments, the second agent of each dose is administered after a period of time of the dose of an anti-CD25 antibody agent as described herein. In some embodiments, one or more doses of the second agent are administered before a period of time of the dose of an anti-CD25 antibody agent. In some embodiments, the anti-CD25 antibody agent as described herein can also be involved in subsequent administration not only by the same route but also by alternate administration routes (such as by subcutaneous (or intramuscular) administration and intratumoral administration), in one or more treatment cycles of one week, two weeks, four weeks or more, and the same regimen (or by extending the interval between administrations) is repeated for such a cycle, depending on the patient's response. Also, in some embodiments, the precise schedule (e.g., number of doses, intervals between doses (e.g., relative to each other or relative to another event, such as administration of another therapy)), amounts of doses, etc.) of one or more subsequent cycles may be different compared to one or more other cycles.

[0136] By using any of the routes of administration, doses and / or regimens as described herein, the anti-CD25 antibody agents as described herein can be identified, characterized and / or validated, for example, taking into account one or more criteria measured in patients using biopsies, blood samples and / or other clinical criteria. In some embodiments, as an alternative or in addition to directly assessing tumor size and / or cancer metastasis, the therapeutic efficacy of the anti-CD25 antibody agents as described herein can be determined in a method that assesses one or more of the following different general criteria: a reduction in regulatory T cells in circulation, tumors and / or lymphoid organs; direct cytotoxicity to cancer cells (apoptosis and necrosis of cancer cells); an increase in tumor-infiltrating immune cells (such as CD4-positive and / or CD8-positive tumor-infiltrating T cells); an increase in circulating immune cells in the blood (total or specific subpopulations of lymphocytes, NK cells, monocytes, dendritic cells, macrophages, B cells, etc.); and / or a certain degree of differential expression before treatment relative to after treatment in only responding or non-responding patients (as determined by RNA sequencing, bulk flow cytometry and / or other bulk sequencing methods). Alternatively or additionally, in some embodiments, such identification, characterization and / or validation may involve tracking at the molecular level by screening for mRNA and / or protein expression of one or more specific proteins or sets of proteins. In some embodiments, one or more such techniques may allow identification or relevant information for assessing responses to anti-CD25 antibody agents as described herein, for example, which may be related to tissue distribution and / or markers of specific cell populations within (or near) a tumor and / or circulating in the blood.

[0137] Such methods and immunobiological data can allow not only the determination of one or more efficacy and / or safety parameters or characteristics, but also in some embodiments can provide a rationale for selecting a particular dose, route or dosing regimen, for example, which can be used in one or more clinical trials for a given indication alone and / or in combination with other drugs, standard of care regimens, or immunotherapies that may provide other therapeutic benefits. Thus, in a series of other embodiments of the invention, the anti-CD25 antibody agents as described herein are used in a method of treating a patient suffering from a disease (such as cancer) or preventing a disease (such as cancer) after determining the presence (and / or absence) of a combination of expression of one or more genes at the RNA and / or protein level in a patient's cells or tissues (such as tumors, blood samples, blood isolates) prior to or after treatment with such an agent. Thus, such methods can allow for the definition of one or more biomarkers or more complex gene expression signatures (or cell population profiles) associated with a therapeutically effective amount of a desired anti-CD25 antibody agent, a therapeutically relevant biomarker that predicts that a subject may have an anti-tumor or anti-infective response following treatment with an anti-CD25 antibody agent as described herein, or a therapeutically relevant biomarker that predicts that a subject may respond to compound treatment following treatment with an anti-CD25 antibody agent.

[0138] Alternatively or additionally, in some embodiments, CD25 expression in human cancers and / or other human tissues may be considered, for example, by aggregating data on CD25 distribution in stroma and / or immune subsets in various cancers, tissues, and / or patients to predetermine and / or later evaluate the dosing and administration of a specific anti-CD25 antibody agent as disclosed herein. Such data can be generated using common techniques (such as flow cytometry, mass cytometry, immunohistochemistry, or mRNA expression libraries) in common cancer types and / or tissues (central nervous system, esophagus, stomach, liver, colon, rectum, lung, bladder, heart, kidney, thyroid, pancreas, uterus, skin, breast, ovary, prostate, and testis) for identifying the relationship between CD25 expression in various immune and non-immune subsets and / or its relationship to cell infiltration measurements and / or cancer-associated markers (such as Foxp3 and PD-1 / PD-L1) associated with subsets of cancer cells or immune cells. CD25 expression may be restricted (or not restricted) to immune subsets in tumor tissues (such as NK cells and other effector or regulatory immune cells), and if positive, the correlation between CD25 expression and immune checkpoint inhibitors can be determined, so it is recommended to appropriately use anti-CD25 antibody agents in combination with compounds that target such immune checkpoint inhibitors (e.g., PD-1 antagonists, such as anti-PD-1 antibodies or anti-PD-L1 antibodies).

[0139] Articles and kits; In some embodiments of the present invention, the anti-CD25 antibody agent as described herein is provided as a separate article. In some embodiments of the present invention, the article containing the anti-CD25 antibody agent is provided in a container with a label or by a container with a label. Suitable containers may include, for example, bottles, vials, syringes, and test tubes. In some embodiments, the container may be made of various materials (such as glass or plastic). In some embodiments, the container holds a composition that effectively treats a specific disease, disorder, or condition, or a stage or type thereof. In some embodiments, the container may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle). For example, in some embodiments, a composition comprising an anti-CD25 antibody agent as described herein is packaged in a transparent glass vial with a rubber stopper and an aluminum seal. The label on or associated with the container indicates that the composition is used to treat the selected condition.

[0140] In some embodiments, the article of manufacture may further comprise a separate container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and dextrose solution, and / or may further comprise other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. For example, in some embodiments, the article of manufacture may allow for the provision of an intravenous formulation of each agent in the form of a sterile aqueous solution containing a total of 2 mg, 5 mg, 10 mg, 20 mg, 50 mg, or more, formulated with appropriate diluents and buffers at a final concentration of 0.1 mg / ml, 1 mg / ml, 10 mg / ml, or at a higher concentration.

[0141] In some embodiments, the anti-CD25 antibody agent as described herein can be provided in a test kit in a lyophilized form or other types of dosage units using any compatible pharmaceutical carrier, and the lyophilized form is reconstituted using any suitable aqueous solution provided or not provided with the test kit. The anti-CD25 antibody agent in one or more unit dosage forms can be provided in a packaging or dispensing device. For example, such packaging or device can include metal or plastic foil, such as blister packaging. For the correct use of this multi-component kit, it can further include a buffer, a diluent, a filter, a needle, a syringe, and a package insert with instructions for treating cancer.

[0142] In some embodiments, instructions associated with an article or kit as described herein may be in the form of a label, brochure, publication, recording, chart, or any other means that may be used to inform the proper use of the agent, preparation, and other materials in the article and / or kit and / or to monitor the possible effects of the agent, preparation, and other materials in the article and / or kit. Instructions may be provided with the article and / or kit.

[0143] Example

[0144] Example 1: In vitro production of antibodies that bind CD25

[0145] Materials and methods

[0146] CD25 antigen preparation

[0147] Mouse CD25-HIS, human CD25-Fc, and untagged recombinant proteins were purchased from R&D Systems Biotechne. Cynomolgus monkey CD25-Fc and CD25-HIS recombinant proteins were purchased from Sino Biological. Protein reagent biotinylation was performed using the EZ-Link Sulfo-NHS-Biotinylation Kit (Thermo Scientific, catalog number 21425). The CD25 antigen was concentrated to approximately 1 mg / mL and the buffer was exchanged into PBS, followed by the addition of a 1:7.5 molar ratio biotinylation reagent (EZ-Link Sulfo-NHS-Biotinylation Kit, Thermo Scientific, catalog number 21425). The mixture was kept at 4°C overnight, followed by another buffer exchange to remove free biotin in the solution. Biotinylation was confirmed by binding of the streptavidin sensor to the labeled protein on the ForteBio.

[0148] Library interrogation and selection methods for isolating anti-CD25 antibodies:

[0149] Design, production and amplification of eight native synthetic yeast libraries (each with approximately 10 9 Species diversity), as described previously (see, e.g., Xu et al., 2013; WO2009036379; WO2010105256; WO2012009568). Eight parallel selections were performed using eight native libraries selected for monomeric human CD25.

[0150] For the first two rounds of selection, magnetic bead sorting was performed using the Miltenyi MACs system essentially as described (Siegel et al., 2004). Briefly, yeast cells (approximately 10 cells / mL) were plated at 30°C in FACS wash buffer (PBS with 0.1% BSA). 10Cells / library) were incubated with 3ml 10nM biotinylated dimer human CD25 antigen for 15min. After washing once with 50ml ice-cold washing buffer, the cell pellet was resuspended in 40mL washing buffer, and 500μl streptavidin microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany. Catalog number 130-048-101) were added to the yeast and incubated at 4°C for 15min. Then, the yeast was precipitated, resuspended in 5mL washing buffer and loaded onto a MACS LS column (Miltenyi Biotec, Bergisch Gladbach, Germany. Catalog number 130-042-401). After loading 5mL, the column was washed 3 times with 3ml FACS washing buffer. The column was then removed from the magnetic field, and the yeast was eluted with 5mL growth medium and then cultured overnight.

[0151] After two rounds of MACS, four rounds of sorting using flow cytometry (FACS) were performed, which are described in the following three paragraphs.

[0152] For the first round of FACS selection, approximately 4 × 10 7 Yeast pellets were washed three times with wash buffer and incubated with 10 nM each of biotinylated dimeric human CD25 antigen or 200 nM biotinylated monomeric mouse CD25 antigen for 15 min at 30° C. Yeast were then washed twice and stained with 1:100 dilution of goat anti-human F(ab')2κ-FITC (SouthernBiotech, Birmingham, Alabama, catalog number 2062-02) and secondary reagents of 1:500 dilution of streptavidin-Alexa Fluor 633 (Life Technologies, Grand Island, NY, catalog number S21375) or 1:50 dilution of Extravidin-phycoerythrin (Sigma-Aldrich, St Louis, catalog number E4011) at 4° C. for 15 min. After washing twice with ice-cold wash buffer, the cell pellets were resuspended in 0.4 mL of wash buffer and transferred to filter-capped sorting tubes. Sorting was performed using a FACS ARIA sorter (BD Biosciences) and the sorting gate was determined to select only CD25 binding. The selected murine populations from the first round of FACS were combined into one group. This group was then sorted for human CD25 binding to increase or identify cross-reactive binders in the third round of FACS (described below).

[0153] The second and third rounds of FACS for the selected human population involved positive or negative sorting of CD25 binders to reduce reagent multispecific binders (Xu et al., 2013). Depending on the amount of multispecific binding or target binding of the particular selection output, positive sorting was followed by negative sorting or vice versa to enrich for the fully bound population with limited amounts of multispecific binding. Samples from the output of the three rounds of selection were plated and sequenced.

[0154] The fourth round of FACS consisted mainly of positive selection using 10 nM biotinylated dimeric cynomolgus CD25 as antigen. These selected samples were then plated and sequenced similarly to what was done with the third round of selection output.

[0155] Affinity maturation of clones identified in initial selection

[0156] Heavy chains from the multispecific binder negative sorting output were used to prepare light chain diversified libraries for four additional rounds of selection. The first of these rounds of selection utilized Miltenyi MAC beads conjugated to either 10 nM biotinylated dimeric human CD25 as antigen or 100 nM biotinylated monomeric murine CD25 as antigen.

[0157] After MAC bead selection, three rounds of FACS sorting were performed. The first of these rounds used 100 nM monomeric human CD25, 10 nM dimeric cynomolgus monkey CD25, or 200 nM monomeric mouse CD25. The second round of FACS was a negative selection using a multispecific reagent to enrich for a complete binding population with a limited amount of multispecific binding. The third round of FACS included titration of human CD25 antigen down to 1 nM and parallel selection using 100 nM or 10 nM mouse CD25.

[0158] In parallel with the third round of FACS described in the immediately preceding paragraph, competitive selection was performed. For competitive selection, 200 nM competitor IgG (IgG generated from the initial selection with known groupings on human CD25 (described above) to select antibodies that did or did not compete with the groupings) was incubated with 10 nM biotinylated dimeric human CD25 for 30 minutes, followed by incubation with yeast for 30 minutes.

[0159] Individual colonies from each round of FACS selection described above were plated and picked for sequencing characterization.

[0160] IgG and Fab production and purification:

[0161] Yeast clones were grown to saturation and then induced at 30°C with shaking for 48h. After induction, yeast cells were precipitated and supernatant was harvested for purification. IgG was purified using a protein A column and eluted with acetic acid pH 2.0. Fab fragments were produced by papain digestion and purified on CaptureSelect IgG-CH1 affinity matrix (Life Technologies, catalog number 1943200250).

[0162] Preparation of α-fucosylated aCD25-a-634 human IgG1 expressed in mammalian cells:

[0163] The synthesis of the codon-optimized VH and VL coding sequences of the antibody is carried out and the cDNA of the variable region is cloned into an antibody expression vector (Evitria, Switzerland) using conventional (non-PCR-based) cloning techniques. The cDNA of the oxidoreductase GDP-6-deoxy-d-lyxose-4-hexulose reductase (RMD) is cloned into an expression vector (Evitria, Switzerland). Plasmid DNA is prepared under low endotoxin conditions based on anion exchange chromatography. Evitria uses the CHO K1 cells (originally derived from ATCC, and adapted to serum-free growth in suspension culture in Evitria) adapted to suspension for preparation. Seeds are grown in eviGrow culture medium, which is a chemically determined, animal-free, serum-free medium. Evitria proprietary transfection reagent (customized Evitria) is used to transfect cells with the expression vector of IgG1 and RMD enzyme. After transfection, cells are grown in eviMake2, which is animal-free, serum-free medium. The supernatant was collected by centrifugation and subsequent filtration (0.2 μm filter membrane). TM SuRe TM Purification. The glycosylation pattern of the antibody was characterized using LC / MS, showing >99% a-fucosylation.

[0164] Affinity measurement of anti-CD25 antibodies:

[0165] The affinity of CD25 antibody was determined by measuring its Kd using ForteBio. ForteBio affinity measurements were performed as previously described (Estep et al., 2013). In brief, ForteBio affinity measurements were performed by loading IgG online onto an AHQ sensor. The sensor was balanced offline for 30 minutes in the assay buffer and then monitored online for 60 seconds to determine the baseline. Monovalent binding measurements were obtained by loading CD25 dimers onto an AHC sensor and then exposing to a solution containing 100 nM antibody Fab. For affinity binding measurements, 20.83 nM antigen (recombinant human CD25 with a HIS tag) was loaded onto the Ni-NTA sensor for 5 min, then transferred to the assay buffer for 0.5 min for washing and again in the assay buffer for 1 min for baseline determination. Then, the antibody was allowed to stand at different concentrations (see Figure 5 ) for 20 min. It was then transferred to assay buffer for 26.6 min for dissociation rate measurement. Kinetic data were fitted using a 1:1 binding model in the data analysis software provided by ForteBio.

[0166] Ligand Binding:

[0167] The ligand binding of antibodies was performed using a standard sandwich binning assay on a Forte Bio Octet Red384 system (Pall Forte Bio Corp., USA). Anti-human CD25 antibody (aCD25-a-634) was loaded onto the AHQ sensor and unoccupied Fc binding sites on the sensor were blocked using an unrelated human IgG1 antibody. The sensor was exposed to 100 nM human CD25 and subsequently to 100 nM human IL-2. The data was processed using Forte Bio data analysis software 7.0. Additional binding of human IL2 after antigen association indicates an unoccupied epitope (non-competitor), while no binding indicates epitope blocking (competitor).

[0168] Epitope Grouping:

[0169] Epitope grouping of antibodies was performed using a standard sandwich format grouping assay on a Forte Bio Octet Red384 system (Pall Forte Bio Corporation, Menlo Park, CA). Anti-human CD25 antibody IgG was loaded onto the AHQ sensor and unoccupied Fc binding sites on the sensor were blocked using an irrelevant human IgG1 antibody. The sensor was then exposed to 100 nM target antigen followed by daclizumab or basiliximab. Data were processed using ForteBio's Data Analysis Software 7.0. Additional binding of the second antibody after antigen association indicates an unoccupied epitope (non-competitor), while no binding indicates epitope blocking (competitor).

[0170] In addition, different formats were used for determination, with human CD25 antigen fixed first. In brief, competition was carried out using standard sequential binding assays on the ForteBioOctet Red96 system (Pall Forte Bio Corp., USA). 26.8nM of recombinant human CD25 with HIS tag was loaded onto the Ni-NTA biosensor for 200s. After the baseline step using kinetic buffer, the sensor was exposed to the first antibody of 66.6nM for 600s and then exposed to the second anti-CD25 antibody (also 66.6nM for 600s). ForteBio data analysis software 9.0 was used to process data. The additional binding of the second antibody indicated an unoccupied epitope (non-competing epitope), and no binding indicated epitope blocking (competing epitope).

[0171] Binding of anti-CD25 antibodies to cells expressing CD25:

[0172] Candidate hits were evaluated by binding to lymphoma human cell lines SU-DHL-1 and SR-786, Karpas299, in vitro differentiated Treg cells and HSC-F cynomolgus monkey T cell lines. Cells were first blocked with Trustain (Biolegend) and then incubated with anti-CD25 antibodies titrated in half-log serial dilutions of the highest concentration of 20 μg / ml at 4 ° C for 30 minutes and then incubated with PE-conjugated anti-human IgG Fc antibodies (Biolegend) to detect binding to human cell lines (SU-DHL-1 and SR-786) expressing CD25. The cells were washed again and resuspended in FACS buffer containing DAPI and obtained on Intellicyt iQue. Live cells were gated using FSC vs. SSC parameters by flow cytometry during sample acquisition. The geometric mean intensity of the stained cells was plotted on an XY graph, and the geometric mean intensity was plotted relative to the logarithm of the concentration, and the data was fitted to a nonlinear regression curve, from which EC50 was calculated.

[0173] The test article (anti-CD25 primary antibody) was stained with 30 μg / ml antibody, followed by half-log serial dilution (7 points) on ice for 30 minutes to detect the binding of in vitro differentiated Treg expressing CD25. This was followed by staining with a secondary antibody (Alexa Fluor647-AffiniPure Fab fragment goat anti-human IgG (H+L)-(JacksonImmunoResearch)) at a concentration of 1 μg / ml on ice for 30 minutes. All samples were stained in duplicate. Live cells were gated using FSC vs. SSC parameters by flow cytometry during sample acquisition. The mean fluorescence intensity (MFI) of the stained cells was plotted on an XY graph, and the MFI was plotted against the logarithm of the concentration, and the data was fitted into a nonlinear regression curve, from which EC50 was calculated.

[0174] Binding to the HSC-F cynomolgus monkey T cell line expressing CD25 was detected by staining the test article (anti-CD25 primary antibody) with 20 μg / ml antibody followed by half-log serial dilutions (7 points) on ice for 30 minutes. This was followed by staining with a secondary antibody (rabbit anti-human Fcg F(ab')2- (Jackson ImmunoResearch)) at a concentration of 5 μg / ml on ice for 30 minutes. All samples were stained in triplicate. To minimize cross-linking-induced cell death mediated by binding of the secondary antibody, cell lines were tested in 4 test article staining groups simultaneously. Live lymphocytes were gated by flow cytometry using FSC vs. SSC parameters during sample acquisition. The mean fluorescence intensity (MFI) of live cells was plotted on an XY graph, the MFI was plotted against the logarithm of the concentration, and the data were fitted to a nonlinear regression curve from which the EC50 was calculated.

[0175] Binding to CD25-expressing Karpas299 cells was detected by staining the test article (anti-CD25 primary antibody) with 20 μg / ml antibody followed by half-log serial dilutions (7 points) on ice for 30 minutes. This was followed by staining with a secondary antibody (Alexa Fluor 647-AffiniPure F(ab')2 fragment rabbit anti-human IgG Fcγ fragment-(Jackson ImmunoResearch)) at a concentration of 1 μg / ml on ice for 30 minutes. All samples were stained in duplicate. Live cells were gated using FSC vs. SSC parameters by flow cytometry during sample acquisition. The mean fluorescence intensity (MFI) of the stained cells was plotted on an XY graph, the concentration logarithm versus MFI was plotted, and the data was fitted to a nonlinear regression curve from which the EC50 was calculated.

[0176] Recloning, production and characterization of aCD25-a-634 as human IgG1 expressed in mammalian cells: Synthesis of codon-optimized VH and VL coding sequences of antibodies was performed by Genewiz. The cDNA of the variable region was cloned into an antibody expression vector (Icosagen, EST) containing human IgG1 heavy chain and kappa light chain constant regions (P01857 and P01834, respectively). The full-length heavy chain and light chain cDNAs were verified by sequencing in the final vector and then recloned using QMCF technology (Icosagen) for expression, which is a stable episomal expression system using CHO-based cells (CHOEBNALT85) and appropriate vectors for the production of recombinant proteins and antibodies. CHOEBNALT85 cells were transfected with 1 μg of expression plasmid for antibody production. 48 h after transfection, 700 μg / ml G418 was added to select cell populations containing plasmids. For production, the temperature was changed to 30°C and the culture was additionally fed. At the end of production, the culture supernatant was clarified by centrifugation (1000g, 30min, 15°C), PMSF was added and the supernatant was treated or frozen until purification. The hIgG1 antibody was purified into PBS or PBS 100mM L-Arg by MabSelect SuRe affinity chromatography and followed by Superdex 200 gel filtration. The human IgG1 antibody produced in CHOEBNALT85 cells was characterized for affinity to recombinant human CD25, cross-reactivity to mouse and cynomolgus monkey CD25, and epitope grouping compared to selected CD25 binding antibodies.

[0177] aCD25-a-634 epitope mapping:

[0178] Different sets of linear, monocyclic, β-turn mimetic, disulfide bridge mimetic, non-continuous disulfide bridge, non-continuous epitope mimetic peptides representing the human CD25 sequence (Uniprot record number P01589) were synthesized using solid phase Fmoc synthesis (Pepscan BV, The Netherlands; Timmermann P et al., 2007; Langedijk JP et al., 2011). Binding of the a-634 antibody to each of the synthetic peptides was tested in ELISA (Pepscan, The Netherlands). The peptide array was incubated with the primary antibody solution (4°C overnight). After washing, the peptide array was incubated with a 1 / 1000 dilution of the appropriate antibody peroxidase conjugate (2010-05; Southern Biotech) at 25°C for one hour. After washing, the peroxidase substrate 2,2'-azo-di-3-ethylbenzthiazoline sulfonate (ABTS) and 20 μl / ml of 3% H2O2 were added. After one hour, the color development was measured. A charge coupled device (CCD) camera and image processing system were used to quantify the color development. The values ​​obtained from the CCD camera ranged from 0 to 3000 mAU, similar to a standard 96-well plate ELISA reader. To verify the quality of the synthetic peptides, separate sets of positive and negative control peptides were synthesized in parallel and screened with irrelevant control antibodies.

[0179] Anti-human CD25 Ab competition assay:

[0180] Antibody competition was performed using a standard continuous binding assay on a Forte Bio Octet Red96 system (Pall Forte Bio Corp., USA). 26.8 nM recombinant human CD25 with his tag was loaded onto a Ni-NTA biosensor for 200 s. After a baseline step using a dynamic buffer, the sensor was exposed to a 66.6 nM first antibody for 600 s, followed by exposure to a second anti-CD25 antibody (also at 66.6 nM for 600 s). Forte Bio data analysis software 9.0 was used to process the data. The additional binding of the second antibody indicated an unoccupied epitope (non-competing epitope), while no binding indicated epitope blocking (competing epitope).

[0181] result

[0182] A yeast-based antibody display library as described in Materials and Methods was used to isolate monoclonal antibodies (mAbs) that bind to recombinant human CD25 extracellular protein sequences (rhCD25). These antibodies were sequenced and unique clones were generated in yeast cells (Barnard GC et al., 2010). Cell culture supernatants of each yeast clone expressing the unique antibody sequence were screened for rhCD25 binding.

[0183] A panel of mAbs was identified based on binding to rhCD25, sequence uniqueness, and expression levels. These antibodies were further characterized for binding to recombinant cynomolgus monkey and mouse CD25 extracellular domain protein sequences. Clones were characterized showing IgG binding values ​​within the 10-fold range to monovalent rhCD25 and / or recombinant cynomolgus monkey CD25 extracellular protein sequences. -8 M and 10 -10 In addition, each antibody was characterized as competing or not competing with IL-2 ligand or reference anti-CD25 antibody daclizumab. Antibody clones were also evaluated by flow cytometry for binding to human cells expressing different levels of CD25, such as lymphoma cell lines, SU-DHL-1, and in vitro differentiated Treg cells.

[0184] The results of the rhCD25, cross-grouping and IL-2 competitive binding assays are shown in Table 1 ( Figure 3-7 ).

[0185] Table 1:

[0186]

[0187] Binding of anti-CD25 antibodies was shown in Figure 3-7 middle.

[0188] Finally, to eliminate antibody sequences that would be prone to aggregation and non-specific interactions, antibodies were screened in a polyspecific reagent (PolySpecific Reagent; PSR) assay and affinity capture self-interaction nanoparticle spectroscopy (Affinity-Capture Self-Interaction Nanoparticle Spectroscopy; AC-SINS), which is a method that allows high-throughput screening of early antibody development (Liu Y et al., 2014). None of the selected antibodies were evaluated as positive in the subsequent assay and were therefore not removed from the group.

[0189] Among the selected hits sequenced and characterized as described above, clone aCD25-a-634 was the first to present novel complementarity determining regions (CDRs; Figure 1 ) that does not compete with daclizumab or basiliximab for binding to human CD25.-8 M to 10 -10 The Kd value of M binds to the human CD25 extracellular protein sequence.

[0190] Therefore, the aCD25-a-634 sequence ( Figure 1 ) Antibodies that specifically bind to CD25 are identified and their activities associated with defining the functional characteristics of an anti-CD25 antibody agent (as that term is used herein) can be functionally assessed by cell-based assays or animal models.

[0191] Example 2: Cell-based Model for Validation of CD25 Modulating Antibody Agents

[0192] Materials and methods

[0193] In vitro IL-2 signaling measured by STAT5 phosphorylation

[0194] IL-2 blockade was characterized using a STAT5 phosphorylation assay to detect IL-2 signaling. Previously frozen PBMCs (Stemcell Technologies) were cultured in 96-well U-bottom plates in the presence of 10 μg / ml anti-CD25 antibody for 30 minutes, followed by the addition of IL-2 (Peprotech) at different concentrations of 0.1 U / ml, 1 U / ml, or 10 U / ml in RPMI 1640 (Life Technologies) containing 10% FBS (Sigma), 2 mM L-glutamine (Life Technologies), and 10,000 U / ml Pen-Strep (Sigma) for 10 minutes. When the cells were fixed and analyzed using eBioscience TMIL-2-induced STAT5 phosphorylation was terminated when the cells were permeabilized with Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen) and treated with BD Phosflow Perm Buffer III (BD Biosciences). The cells were then stained with antibodies labeled with surface and intracellular fluorescent dyes (STAT5-Alexa Fluor 647 clone 47 / stat5 / pY694 BD Bioscience, CD3-PerCP-Cy5.5 clone UCHT1 Biolegend, CD4-BV510 clone SK3 BD Bioscience, CD8-Alexa Fluor 700 clone RPA-T8 Invitrogen, CD45RA-PE-Cy7 clone HI100 Invitrogen, FoxP3-Alexa Fluor 488 clone 236A / E7 Invitrogen) and samples were acquired on a Fortessa LSR X20 flow cytometer (BD Bioscience) and analyzed using BD FACSDIVA software. Use FCS-H vs. FCS-A to exclude doublets and use SSC-A vs. FCS-A parameters to qualify lymphocytes. Use CD3 PerCP-Cy5.5-A vs. FCS-A plots to qualify CD3 + T cells and gated on a histogram showing counts versus STAT5 Alexa Fluor 647-A to determine STAT5 + CD3 + T cell population. The percentage of blockade of IL-2 signaling was calculated as follows: Blockade % = 100 × [(Stat5 + Cells without Ab group%-Stat5 + Cells 10ug / mlAb group %) / (Stat5 + Different T cell subsets (CD4 + 、CD8 + 、CD4 + FoxP3 + Further analysis of STAT5 phosphorylation in naive and memory T cells (Figure 5—figure supplement 1A). Graphical and statistical analyses were performed using GraphPad Prism v7 (results not shown).

[0195] In vitro T cell activation assay:

[0196] The effect of IL-2 signaling on Teff responses was characterized in a T cell activation assay that detects upregulation of intracellular granzyme B (GrB) and proliferation. Previously frozen primary human Pan T cells (Stemcell Technologies) were labeled with eFluor450 cell proliferation dye (Invitrogen) according to the manufacturer's recommendations and cultured at 1×10 5 Cells / well were added to 96 U-bottom well plates in RPMI 1640 (Life Technologies) containing 10% FBS (Sigma), 2mM L-glutamine (Life Technologies) and 10,000U / ml Pen-Strep (Sigma). Cells were then treated with 10μg / ml anti-CD25 antibody or control antibody followed by human T-activator CD3 / CD28 (20:1 cell to bead ratio; Gibco) and cultured in a humidified incubator at 37°C, 5% CO2 for 72 hours. To assess T cell activation, cells were stained with eBioscience FixableViability Dye efluor780 (Invitrogen) followed by fluorochrome-labeled antibodies for surface T cell markers (CD3-PerCP-Cy5.5 clone UCHT1 Biolegend, CD4-BV510 clone SK3 BD Bioscience, CD8-Alexa Fluor 700 clone RPA-T8 Invitrogen, CD45RA-PE-Cy7 clone HI100 Invitrogen, CD25-BUV737 clone 2A3 BD Bioscience) and then fixed and stained with eBioscience TM Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen) was used for permeabilization, followed by staining for intracellular GrB and nuclear FoxP3 (Granzyme B-PE clone GB11 BD Bioscience, FoxP3-APC clone 236A / E7). Samples were obtained on a Fortessa LSR X20 flow cytometer (BD Bioscience) and analyzed using BD FACSDIVA software. Doublets were excluded using FCS-H versus FCS-A, and lymphocytes were defined using SSC-A versus FCS-A parameters. GrB-PE-A versus proliferation eFluor450-A plots were used to assess the expression of live CD3 + CD4 lymphocyte gating + and CD8 + T cell subsets. Results are expressed as a percentage of total CD4+ or CD8 + The percentage of proliferating GrB-positive cells in the T cell population. Graphical and statistical analyses were performed using GraphPad Prism v7.

[0197] In vitro ADCC assay:

[0198] Antibody-dependent cell-mediated cytotoxicity assays (ADCC assays) were performed to characterize anti-human CD25 antibodies using SU-DHL-1 or SR-786 (CD25 positive) human cell lines as target cells and human NK cells as effector cell sources. NK cells were isolated from PBMCs of healthy donors using a NK cell negative separation kit (Stemcell Technologies). NK cells were cultured overnight in the presence of 2ng / mL IL-2 (Peprotech). SU-DHL-1 or SR-786 target cells were loaded with calcein-AM (Thermofisher) and plated at 37°C 5% CO2 for 30 minutes in the presence of anti-CD25 or isotype antibodies, in quadruplicate for each case. After culture, NK cells were added to the wells with a target: effector (T:E) ratio of 1:10 (10,000 target cells and 100,000 effector cells) and cultured for 4 hours at 37°C 5% CO2. Calcein fluorescence in the supernatant was read out on a BMG Fluostar plate reader. Calculate the specific lysis percentage relative to individual target cells (0% lysis) and target cells treated with 0.1% saponin (100% lysis). Graphpad Prism v7 was used to generate a graph of raw data to generate a dose response curve. Target cell lysis percentages were plotted on an XY graph, and the normalized Calcein AM release percentage was plotted against the logarithm of the concentration, and the data were fitted to a nonlinear regression curve, from which EC50 was calculated.

[0199] In vitro ADCP assay: using FcyRlla-H reporter assay:

[0200] ADCP bioassay effector cells (FcyRlla-H) were obtained from Promega (Catalog No. G9881 / 5; Lot No. 0000261099). 5,000 cells / well of SUDHL-1 target cells were plated (25 μl / well) using 96-well white polystyrene plates (Costar; Catalog No. 3917). The test antibody was serially diluted using 3-fold dilution and 25 μl was added to the cells. 50,000 effector cells were added to each well in a volume of 25 μl to obtain a 10:1 effector to target cell ratio. All target cells, antibodies, and effector cells were plated using cell culture medium. The plates were incubated at 37°C for 18 hours. The plates were then removed from the incubator and kept at room temperature for 20 minutes. 60 μl of Bio-Glo luciferase assay substrate buffer was added to each well, followed by a 30-minute incubation and luminescence was measured using the GloMax Multi Detection System (Promega).

[0201] In vitro ADCP assay using in vitro differentiated macrophages and Treg cells:

[0202] Antibody-dependent cell-mediated phagocytosis (ADCP) assays were also performed using in vitro differentiated Treg as target cells and monocyte-derived macrophages as effector cells. PBMCs were separated from leukocyte cones by Ficoll gradient centrifugation. CD14 microbeads (Miltenyi Biotec) were used to separate monocytes (CD14+ cells). Monocytes were cultured in RPMI 1640 (Life Technologies) containing 10% FBS (Sigma), 2mM L-glutamine (Life Technologies) and 10,000U / ml Pen-Strep (Sigma) in the presence of 50ng / ml M-CSF for 5 days, and fresh culture medium containing M-CSF was added after 3 days. Regulatory T cells (Treg) were separated using a human Treg cell differentiation kit (R&D Systems). According to the manufacturer's recommendations, these cells were cultured in a humid incubator at 37°C, 5% CO2 for 5 days and labeled with eFluor450 dye (Invitrogen). On day 5, macrophages and eFluor450-dye labeled Tregs were co-cultured at an effector to target ratio of 10 to 1 in the presence of anti-CD25 antibody or control for 4 hours, as described below. 4 Target cells (Treg) were added at 1×10 5Effector cells (macrophages) were added at 10 cells / well to achieve an effector to target ratio of 10 to 1. Anti-CD25 antibody was then added in duplicate at the highest concentration of 1 μg / ml and followed by a logarithmic series (7 points). Cells and antibodies were incubated at 37°C, 5% CO2 for 4 hours. To evaluate ADCP, cells were placed on ice, stained with cell surface marker CD14 (CD14-PerCP-Cy5.5 clone MfP9 BD Biosciences) and fixed with eBioscience fixation buffer. Two-color flow cytometric analysis was performed using a Fortessa LSR X20. Residual target cells were defined as eFluor450-dyed + / CD14 - Macrophages are defined as CD14 + Double labeled cells (eFluor450-dye + / CD14 + ) is considered to represent the phagocytosis of the target by macrophages. The phagocytosis of the target cells was calculated by the following formula: % phagocytosis = 100 x [(double positive %) / (double positive % + residual target %)].

[0203] statistics:

[0204] Prism software (GraphPad) was used to perform curve fitting to determine EC50 values ​​and maximal activity.

[0205] result

[0206] The aCD25-a-634 antibody (as well as other CD25 antibodies identified and characterized in Example 1) was further evaluated for its ability not to interfere with IL-2 signaling and its ability to kill CD25 expressing target cells. Ligand binding assays using Octet showed that aCD25-a-634 did not affect IL-2 binding to CD25 ( Figure 6 This was confirmed in the STAT5 assay, where aCD25-a-634 did not block IL-2 signaling, regardless of the IL-2 concentration tested, whereas IL-2 signaling was completely blocked by the reference antibody daclizumab ( Figure 8 Daclizumab, which was shown to block the interaction of CD25 with IL-2 via the so-called “Tac” epitope (Queen C et al., 1989 and Bielekova B, 2013), binds to a different epitope from aCD25-a-634 ( Figure 7 ), which may explain why daclizumab blocks IL-2 signaling while aCD25-a-634 does not in the STAT5 phosphorylation assay ( Figure 8In addition, daclizumab reduced the effector response of activated T cells (most likely due to its blockade of IL-2 signaling), whereas aCD25-a-634, which does not block IL-2 signaling, had no negative impact on T cell responses ( Fig. 9 Finally, when compared to IgG1 isotype antibodies, aCD25-a-634 showed a stronger ability to bind to IgG1 via ADCC ( Fig.10 ) and ADCP( Fig.11 ) Kill cells, tumor cells or regulatory T cells expressing CD25.

[0207] In summary, aCD25-a-634 has been characterized and exhibits potent killing of CD25-positive cells (Tregs or cancer cell lines) without interfering with IL-2 signaling and thus suppressing T effector cell responses. Therefore, aCD25-a-634 is a Treg-depleting antibody that can be used as a monotherapy or in combination to treat cancer.

[0208] Example 3: Preparation of variants of CD25 modulating antibody agents

[0209] aCD25-a-634 was subjected to further affinity maturation. Optimization was performed by introducing diversity into the heavy chain variable region. The CDRH3 of the antibody was recombined into a pre-made 8 The library of CDRH1 and CDRH2 variants with diversity was selected by one round of MACS and four rounds of FACS as described in the initial discovery. In the FACS round, the library was checked for PSR binding, species cross-reactivity, antigen cross-reactivity and affinity pressure, and sorted to obtain a population with the desired characteristics. For these selections, affinity pressure was applied by titrating down the biotinylated monomeric antigen or by pre-incubating the biotinylated antigen with the parent Fab or IgG for 30 min and then applying the pre-complexed mixture to the yeast library for a period of time that allowed the selection to reach equilibrium. Then higher affinity antibodies can be sorted.

[0210] Variants showing the best affinity are selected for mammalian production and can be further characterized using assays as described in Examples 1 and 2 above.

[0211] Example 4: Mouse model experiment

[0212] Materials and methods

[0213] Therapeutic activity of non-IL-2 blocking antibodies: Female BALB / c mice obtained from Charles River were injected subcutaneously in the flank with 3×10 5CT26 tumor cells, n=15 / group. Animals were randomized into treatment groups based on body weight on day 1. Treatment started on day 6 and mice were treated by injecting each antibody (mouse IgG2a isotype, IL-2 neutralizing antibody, PC61 mIgG1 (anti-mouse CD25 blocking IL-2 signaling antibody of mouse IgG1 isotype), and 7D4 mIgG2a (anti-mouse CD25 non-blocking IL-2 signaling antibody of mouse IgG2a isotype) once at 200 μg / animal. Animals received monotherapy treatment (one group for each antibody) or combination treatment of 7D4 mIgG2a and IL-2 neutralizing antibody or 7D4mIgG2a and PC61 mIgG1 antibody. When tumor volume reached 2000mm 3 Mice were sacrificed on or day 50 (whichever came first).

[0214] result

[0215] When compared to isotype control mouse IgG2a, anti-CD25 abolished tumor rejection induced by the non-IL-2 blocking antibody 7D4 mIgG2a in treated mice, whereas the other antibodies showed no effect as monotherapy. In combination with IL2-blocking antibodies, PC61 mIgG1 or IL2nAb abolished the therapeutic activity of the non-IL-2 blocking antibody 7D4 mIgG2a ( Fig.12 ). This demonstrates that the non-IL-2 blocking feature of 7D4 mIgG2a is key to therapeutic activity. It also suggests that the therapeutic activity of this antibody is dependent on an anti-tumor immune response mediated by T effector cells, which depends on IL-2 signaling for optimal activity. These results show that the absence of IL-2 / CD25 blocking activity is required for optimal therapeutic activity of CD25-targeted antibodies and support the use of anti-CD25 non-IL-2 blocking antibodies as described herein in cancer therapy.

[0216] sequence

[0217] A summary of the sequences included in this application is provided below:

[0218]

[0219] Equivalents and categories

[0220] Those skilled in the art will appreciate that the present invention is defined by the appended claims rather than by the examples or other description of certain embodiments contained herein.

[0221] Similarly, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0222] Unless otherwise specified above, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. Methods and materials similar or equivalent to the methods and materials described herein can be used to implement or test the present invention. In general, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry described herein are known and commonly used in the art, or are according to the manufacturer's instructions.

[0223] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

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[0271] Steinwand et al.,MAbs,2014,6(1):204-18 Sequence Listing <110> Tasker Therapeutics Ltd. Cancer Research Technologies Ltd. <120> Anti-CD25 for tumor-specific cell depletion <130> P115237WO <150> US62642218 <151> 2018-03-13 <160> 9 <170> PatentIn version 3.5 <210> 1 <211> 272 <212> PRT <213> Homo sapiens <400> 1 Met Asp Ser Tyr Leu Leu Met Trp Gly Leu Leu Thr Phe Ile Met Val 1 5 10 15 Pro Gly Cys Gln Ala Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro 20 25 30 His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn 35 40 45 Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr 50 55 60 Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys 65 70 75 80 Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro 85 90 95 Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro 100 105 110 Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro 115 120 125 Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val 130 135 140 Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His 145 150 155 160 Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His Gly Lys Thr Arg 165 170 175 Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly Glu Met Glu Thr Ser Gln 180 185 190 Phe Pro Gly Glu Glu Lys Pro Gln Ala Ser Pro Glu Gly Arg Pro Glu 195 200 205 Ser Glu Thr Ser Cys Leu Val Thr Thr Thr Asp Phe Gln Ile Gln Thr 210 215 220 Glu Met Ala Ala Thr Met Glu Thr Ser Ile Phe Thr Thr Glu Tyr Gln 225 230 235 240 Val Ala Val Ala Gly Cys Val Phe Leu Leu Ile Ser Val Leu Leu Leu 245 250 255 Ser Gly Leu Thr Trp Gln Arg Arg Gln Arg Lys Ser Arg Arg Thr Ile 260 265 270 <210> 2 <211> 9 <212> PRT <213> Homo sapiens <400> 2 Tyr Thr Phe Thr Gly Tyr Tyr Met His 1 5 <210> 3 <211> 17 <212> PRT <213> Homo sapiens <400> 3 Ser Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 4 <211> 8 <212> PRT <213> Homo sapiens <400> 4 Ala Arg Asp Gly Leu Met Asp Val 1 5 <210> 5 <211> 115 <212> PRT <213> Homo sapiens <400> 5 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ser Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Leu Met Asp Val Trp Gly Gln Gly Thr Ala Val Thr 100 105 110 Val Ser Ser 115 <210> 6 <211> 11 <212> PRT <213> Homo sapiens <400> 6 Arg Ala Ser Gln Ser Ile Ser Ser Trp Leu Ala 1 5 10 <210> 7 <211> 7 <212> PRT <213> Homo sapiens <400> 7 Asp Ala Ser Ser Leu Glu Ser 1 5 <210> 8 <211> 10 <212> PRT <213> Homo sapiens <400> 8 Gln Gln Tyr Asp Thr Tyr Ser Pro Phe Thr 1 5 10 <210> 9 <211> 108 <212> PRT <213> Homo sapiens <400> 9 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Thr Tyr Ser Pro 85 90 95 Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105

Claims

1. An anti-CD25 antibody or an antigen-binding fragment thereof, comprising: a) the amino acid sequence of SEQ ID NO: 2 as the heavy chain variable complementarity determining region 1; b) the amino acid sequence of SEQ ID NO: 3 as the heavy chain variable complementarity determining region 2; c) the amino acid sequence of SEQ ID NO: 4 as the heavy chain variable complementarity determining region 3; d) the amino acid sequence of SEQ ID NO: 6 as the light chain variable complementarity determining region 1; e) the amino acid sequence of SEQ ID NO: 7 as the light chain variable complementarity determining region 2; and f) the amino acid sequence of SEQ ID NO: 8 as the light chain variable complementarity determining region 3.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

5.

3. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:

9.

4. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof is: a monoclonal antibody, a Fab fragment, a F(ab')2 fragment, a single-chain variable fragment, a scFv-Fc fragment or a single-chain antibody.

5. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof is: rabbit, mouse, chimeric, humanized or fully human antigen-binding antibody.

6. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4 isotype antibodies.

7. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof binds to the extracellular domain of human CD25.

8. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof binds to cells expressing human CD25 on their surface and is an anti-CD25 antibody agent.

9. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment does not inhibit the binding of interleukin-2 to CD25.

10. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment does not inhibit interleukin-2 signaling through CD25.

11. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof is defucosylated.

12. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.

13. A nucleic acid vector comprising the nucleic acid molecule according to claim 12.

14. A host cell comprising the nucleic acid vector according to claim 13.

15. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the method comprising culturing the host cell according to claim 14.

16. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.

17. The composition according to claim 16, characterized in that The composition further comprises a pharmaceutically acceptable carrier or excipient.

18. The composition according to claim 17, characterized in that The composition is used for treating cancer.

19. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 or the composition according to claim 16 for the preparation of a medicament for treating cancer.

20. The use according to claim 19, characterized in that The antibody is administered to a subject together with a second agent.

21. The use according to claim 20, characterized in that The second agent is an immune checkpoint inhibitor or a cancer vaccine.

22. The use according to claim 20, characterized in that The second agent is an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is a PD-1 antagonist.

23. The use according to claim 22, characterized in that The PD-1 antagonist is an anti-PD-1 antibody or an anti-PD-L1 antibody.

24. The use according to claim 19, characterized in that The cancer is a solid tumor.

25. The use according to claim 19, characterized in that The cancer is a blood cancer.

26. A kit comprising the composition according to claim 16 in a container.

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