Immunostimulatory monoclonal antibody against human interleukin-2

Abstract

The present invention relates to a monoclonal antibody (mAb) specific for human interleukin-2 (hIL-2), or an antigen-binding fragment thereof, which binds to hIL-2 to inhibit the binding of hIL-2 to CD25, and the antibody is characterized by any of the following parameters: the variable chain of the mAb comprises the amino acid sequence of SEQ ID NO 005 or SEQ ID NO 006; the binding to hIL-2 is characterized by a dissociation constant (K D )≤7,5nmol / L; binding to hIL-2 is characterized by the dissociation rate (K off )≤1x10 ‑4 s ‑1 And / or the antibody shows no measurable cross-reactivity with murine IL-2.

Description

Field of the Invention

[0001] The present invention relates to antibodies that bind to human interleukin-2 (hIL-2). The present invention more specifically relates to antibodies that specifically bind to a specific epitope of hIL-2 and are able to inhibit the binding of hIL-2 to CD25 when bound to the epitope. In addition, the present invention relates to in vitro and in vivo therapeutic applications of said antibodies. Background of the Invention

[0003] Malignant melanoma is a common type of cancer in men and women. Once melanoma becomes metastatic and spreads to distant sites, the 5-year survival rate is very poor, calculated at about 15%. Current available treatment strategies for metastatic melanoma do little to improve this survival rate.

[0004] Interleukin-2 (IL-2) is a cytokine that can effectively stimulate cytotoxic lymphocytes to fight metastatic tumors. However, IL-2 can also stimulate the so-called CD25 + CD4 + Regulatory T cells (Treg cells), the CD25 + CD4 + Regulatory T cells (Treg cells) are critical for the prevention of autoimmune diseases. Importantly, Treg cells can significantly inhibit the anti-tumor response of cytotoxic lymphocytes, and therefore, they somewhat antagonize the beneficial anti-tumor effects of IL-2. Moreover, at the doses required to achieve clinical anti-tumor responses, IL-2 may exert toxic side effects.

[0005] Standard IL-2 immunotherapy has been used for the immunotherapy of metastatic melanoma and metastatic renal cell carcinoma since the early 1980s, which resulted in FDA approval for these indications in 1996 and 1992, respectively. Although IL-2 given at high doses has shown objective response rates in approximately 17% of patients and complete regression in approximately 6-9% of patients with these deadly metastatic cancers, IL-2 given at these doses often results in toxic side effects such as hypotension, pulmonary edema, hepatocellular injury, gastrointestinal toxicity, and generalized edema. Moreover, as described above, IL-2 can stimulate Treg cells, which in turn can suppress anti-tumor CD8 + T cell and NK cell activity.

[0006] Combination of IL-2 with specific anti-IL-2 monoclonal antibodies (mAbs) has been shown to

[0007] (1) directing IL-2 preferentially to cytotoxic lymphocytes rather than Treg cells, and

[0008] (2) Rendering IL-2 more effective but less toxic (Boyman O, Kovar M, Rubinstein MP, Surh CD, and Sprent J. Selective stimulation of T cell subsets with antibody-cytokineimmune complexes. Science (2006) 311: 1924-1927; Krieg C, Letourneau S, Pantaleo G, and Boyman O. Improved IL-2 immunotherapy by selective stimulation of IL-2 receptors on lymphocytes and endothelial cells. Proceedings of the National Academy of Sciences USA (2010) 107: 11906-11911) Improving IL-2 therapy in experimental mouse models of cancer immunotherapy.

[0009] This approach has the following advantages: unmutated natural IL-2 is delivered to CD8 + T cells and NK cells, which then exert potent anti-tumor properties, while IL-2 complexed with this class of anti-IL-2 mAbs hardly activates Treg cells. Moreover, in mice, IL-2 complexed with this class of anti-IL-2 mAbs is less toxic than standard IL-2 immunotherapy. However, due to the lack of suitable anti-human IL-2 mAbs, this therapy has not been used in patients so far. SUMMARY OF THE INVENTION

[0011] The problem solved by the present invention is to provide an anti-human IL-2 monoclonal antibody capable of recognizing and binding to a specific epitope of human IL-2, thereby stimulating cytotoxic T cells and NK cells in favor of Treg cells, for use in in vitro and in vivo therapeutic applications. This problem is solved by the subject matter of the independent claims.

[0012] According to a first aspect of the present invention, a specific monoclonal antibody (mAb) or an antigen-binding fragment thereof for human interleukin-2 (hIL-2) is provided, wherein the antibody is capable of binding to a specific epitope in hIL-2, thereby inhibiting binding to CD25, thereby modulating the immunological effect of the interaction of hIL-2 / IL-2R. The antibody of the present invention is also characterized by at least one of the following parameters:

[0013] a) the variable chain of the mAb comprises an amino acid sequence having ≥85%, ≥90%, ≥95% or ≥99% identity compared to SEQ ID NO 005 or SEQ ID NO 006;

[0014] b) The binding of the antibody to hIL-2 is a reaction mAb*hIL-2 represents a binding complex of antibody and interleukin, characterized by a dissociation constant (K D )≤7.5nmol / L, ≤5nmol / L, ≤3nmol / L, ≤2nmol / L or ≤1.5nmol / L;

[0015] c) The binding of the antibody to hIL-2 is characterized by an off-rate (K off )≤1x10 -4 s -1 ,≤8x10 -5 s -1 ,≤6x10 -5 s -1 ,≤4x10 -5 s -1 ,≤3x10 -5 s -1 or ≤2,1x10 -5 s -1 ;

[0016] d) the resulting mAb*hIL-2 complex no longer effectively binds to human IL-2 receptor alpha (also known as CD25) based on the binding of the mAb to hIL-2, effectively returning the binding of human CD25 to the mAb*hIL-2 to background levels compared to the binding of human CD25 to free (uncomplexed) hIL-2, as measured by surface plasmon resonance; and / or

[0017] e) The antibodies showed no measurable cross-reactivity with murine IL-2.

[0018] The lack of cross-reactivity with mouse IL-2 is advantageous for preclinical studies, which often involve mouse models, such as the use of mAb*hIL-2 complexes to treat mouse tumor models, where the cross-reactive anti-IL-2 mAb may bind to endogenous mouse IL-2 and sequester it from endogenous mouse Treg cells, thereby enhancing the anti-tumor response.

[0019] The lack of cross-reactivity with murine IL-2 is also advantageous for preclinical safety and efficacy studies prior to development of candidate mAbs in human patients.

[0020] In certain embodiments, the hIL-2 mAb comprises at least one VH and / or V L A sequence having ≥80%, ≥85%, ≥90%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97% or ≥98% identity compared to SEQ ID NO 019 or SEQ ID NO 020.

[0021] In certain embodiments, the variable chain of the hIL-2 mAb comprises an amino acid sequence having ≥85%, ≥90%, ≥95% or ≥99% identity compared to SEQ ID NO 003, 004, 005 or 006, and the hIL-2 mAb is characterized by a dissociation constant of ≤7.5 nmol / L, ≤5 nmol / L, ≤3 nmol / L, ≤2 nmol / L or ≤1.5 nmol / L.

[0022] In certain embodiments, the variable chain of the hIL-2 mAb comprises an amino acid sequence having ≥85%, ≥90%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% identity to SEQ ID NO 005 or 006, and the hIL-2 mAb is characterized by an off-rate of ≤1x10 -4 s -1 ,≤8x10 -5 s -1 ,≤6x10 -5 s -1 ,≤4x10 -5 s -1 ,≤3x10 -5 s -1 or ≤2,1x10 -5 s -1 .

[0023] In certain embodiments, the variable chain of the hIL-2 mAb comprises an amino acid sequence having ≥85%, ≥90%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99% identity to SEQ ID NO 005 or 006, and the hIL-2 mAb exhibits no measurable cross-reactivity with murine IL-2.

[0024] In certain embodiments, for administration to human patients, the sequence of the hIL-2 mAb is humanized to prevent adverse reactions.

[0025] In certain embodiments, the hIL-2 mAb is provided as an antigen binding fragment (Fab) or a single chain variable fragment (scFv).

[0026] In certain embodiments, the hIL-2 mAb comprises at least one complementarity determining region (CDR) sequence having ≥80%, ≥85%, ≥90%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97% or ≥98% identity to SEQ ID NO 007, 008, 009, 010, 011 or 012.

[0027] According to a second aspect of the present invention, a nucleic acid molecule encoding a monoclonal antibody or an antigen-binding fragment thereof capable of binding to human interleukin-2 according to the first aspect of the present invention is provided.

[0028] In certain embodiments, the nucleic acid molecule of the second aspect of the invention has a sequence identity of ≥60%, ≥70%, ≥80%, ≥90%, ≥95% or ≥99% compared to SEQ ID NO 003 to 004.

[0029] According to the third aspect of the present invention, a vector comprising the nucleic acid molecule of the present invention is provided.

[0030] According to a fourth aspect of the present invention, a cell comprising or expressing the nucleic acid molecule of the present invention is provided.

[0031] According to a fifth aspect of the present invention, a cell capable of producing the antibody according to the first aspect of the present invention is provided.

[0032] According to a sixth aspect of the present invention, there is provided a hybridoma cell line producing a monoclonal antibody, characterized in that the antibody produced is the antibody of the first aspect of the present invention.

[0033] According to a seventh aspect of the present invention, there is provided a therapeutic formulation for treating cancer or other diseases that benefit from immunostimulatory therapy (such as viral infection), comprising administering to a subject simultaneously or at different time points

[0034] i. the monoclonal antibody (mAb) of the first aspect of the present invention, and / or

[0035] ii. Human interleukin-2 or human IL-2 mutant.

[0036] According to an eighth aspect of the present invention, a fusion protein is provided. The fusion protein comprises:

[0037] a. hIL-2 binding peptide fragment, wherein the polypeptide is characterized by any of the following parameters:

[0038] i. the hIL-2 binding peptide fragment comprises an amino acid sequence having ≥85%, ≥90%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97% or ≥98% identity to SEQ ID NO 021 or SEQ ID NO 022;

[0039] ii. The binding of the polypeptide fragment to hIL-2 of hIL-2 is characterized by a dissociation constant (KD) of ≤7,5nmol / L, ≤5nmol / L, ≤3nmol / L, ≤2nmol / L or ≤1,5nmol / L;

[0040] iii. The hIL-2 binding peptide fragment binds to hIL-2 and is characterized by a dissociation rate (K off )≤1x10 -4 s -1 ,≤8x10 -5 s -1 ,≤6x10 -5 s -1 ,≤4x10 -5 s -1 ,≤3x10 -5 s -1 or ≤2,1x10 -5 s -1 ;

[0041] and / or

[0042] iv. The hIL-2 binding peptide fragment showed no measurable cross-reactivity with murine IL-2.

[0043] b. a human IL-2 polypeptide fragment having ≥85%, ≥90%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97% or ≥98% identity to SEQ ID NO 001, and, optionally,

[0044] c. an amino acid linker of 1 to 50 amino acids, specifically an amino acid linker of 5 to 40 amino acids, more specifically an amino acid linker of 10 to 30 amino acids, even more specifically an amino acid linker of about 15 to 25 amino acids, which connects the hIL-2 binding peptide fragment to the human IL-2 polypeptide fragment as a single polypeptide chain.

[0045] In other words, the fusion protein retains antibody binding and directs human interleukin-2 to stimulate selected immune cells such as CD8 + The capacity of T cells and NK cells.

[0046] The advantage of using such a fusion protein is that the human IL-2 will not be able to be separated from the antibody and the therapy will consist of one single product rather than two, which facilitates various aspects of manufacturing, dosing and regulatory compliance.

[0047] According to the ninth aspect of the present invention, there is provided an antibody or antigen binding fragment thereof that is separated by binding to a specific epitope. The epitope may be an epitope to which the antibody or antigen binding fragment thereof described in other aspects of the present invention binds. In one embodiment, the antibody or molecule that is separated binds to an epitope of human interleukin-2 (hIL-2), the epitope comprising amino acids K52, P54, K55, T57, R58, T61, F62, K63, Q94 and K96. In another embodiment, the antibody or molecule that is separated binds to an epitope that also comprises any one or more of amino acids N50, N53, N91, L92, A93 and N97. Also provided is an antibody or molecule that is separated, comprising an antigen recognition surface, the antigen recognition surface having an epitope recognition feature equivalent to the antibody or antigen binding fragment thereof described in other aspects.

[0048] Where alternatives to single separable features (such as, for example, coding sequences or binding epitopes) are arranged herein as "embodiments", it will be understood that such alternatives may be freely combined to form discrete embodiments of the invention disclosed herein.

[0049] The present invention is further illustrated by the following examples and figures, from which other embodiments and advantages can be derived. These examples are intended to illustrate the present invention but not to limit its scope.

[0050] definition

[0051] “Human interleukin-2” or “hIL-2” means the protein designated UniProt ID P60568 and reproduced as SEQ ID NO: 1.

[0052] Identity in the context of this specification is a single quantitative parameter representing the result of a position-by-position sequence comparison. Methods of sequence comparison are known in the art; the publicly available BLAST algorithm is one example. One such example of nucleic acid sequence comparison is the BLASTN algorithm, which uses the default settings: Expected threshold: 10; Word length: 28; Maximum match in query range: 0; Match / mismatch score: 1, -2; Gap loss: linear. In the absence of other measured variables, identity should be measured according to the above description.

[0053] In the context of this specification, the term antibody is used with its known meaning in the fields of cell biology and immunology; it refers to intact antibodies, any antigen-binding fragments or single chains thereof, and related or derived constructs. An intact antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (V H ) and the heavy chain constant region (C H ). The heavy chain constant region consists of three domains C H 1. C H 2 and C H Each light chain consists of a light chain variable region (abbreviated as V L ) and the light chain constant region (C L ). The light chain constant region consists of a domain C L Composition. H and V L The V region can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L It consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system.

[0054] In the context of this specification, the term antigen binding portion or antigen binding fragment is used with its known meaning in the field of cell biology and immunology; it refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen (e.g., interleukin-2). The antigen binding function of an antibody can be performed by a fragment of an intact antibody. Examples of binding fragments encompassed within the term antigen binding portion or antigen binding fragment of an antibody include Fab fragments, consisting of V L , V H , C L and C H A monovalent fragment consisting of a V domain; a F(ab)2 fragment, a bivalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region; and a Fd fragment, a fragment consisting of a V H and C H Domain composition; Fv fragment, composed of the V domain of a single arm of an antibody L and V H Domain composition; single domain antibody (dAb) fragment, which consists of V H Domain or V LDomain composition; and isolated complementarity determining regions (CDRs). HCDR means the CDR of the heavy chain, and LCDR means the CDR of the light chain.

[0055] In the context of this specification, the term chimeric antibody is used with its known meaning in the field of cell biology and immunology; it refers to an antibody molecule in which the constant region or part thereof is changed, replaced or exchanged so that the antigen binding site (variable region) is connected to a different or changed type of constant region, effector function and / or species, or a completely different molecule that confers new properties to the chimeric antibody, such as an enzyme, cytokine, toxin, hormone, growth factor, drug, etc. For example, the antibody can be modified by replacing the constant region of the antibody with a cytokine. Due to being replaced by a cytokine, the chimeric antibody can retain its specificity for recognizing an antigen while also having the function of the original cytokine molecule or part of its function.

[0056] In the context of this specification, the term hybridoma is used with its known meaning in the field of cell biology and biochemistry; it refers to a hybrid cell produced by the fusion of a B-cell producing a specific antibody with a myeloma (B cell cancer) cell. Hybridoma cells can grow in tissue culture medium and produce monospecific antibodies (monoclonal antibodies).

[0057] In the context of the present specification, the term single-chain variable fragment (scFv) is used with its known meaning in the fields of cell biology and biochemistry; it refers to the variable region of the heavy chain (V H ) and light chain variable region (V L ) are fusion proteins connected by a short linker peptide of 10 to about 25 amino acids. ScFv retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker.

[0058] In the context of this specification, the term antigen-binding fragment (Fab) is used in its known sense in the fields of cell biology and immunology; it refers to the region of an antibody that binds an antigen. It consists of the heavy chain (V H ) and light chain (V L ) are composed of a constant region and a variable domain, respectively. These domains form the antigen binding site at the amino terminus of the monomer.

[0059] In the context of this specification, the term dissociation constant (K D ) is used in its known sense in the fields of chemistry and physics; it refers to the equilibrium constant that measures the tendency of a larger object to reversibly dissociate into smaller components, such as when a complex dissociates into its component molecules. DIt is expressed in molar units [M] and corresponds to the concentration of [Ab] when half of the binding sites of [Ag] are bound. In other words, the concentration of unbound [Ab] is equal to the concentration of [AbAg] complex. The dissociation constant can be calculated according to the following formula:

[0060]

[0061] [Ab]: antibody concentration; [Ag]: antigen concentration; [AbAg]: antibody-antigen complex concentration

[0062] In the context of this specification, the terms off-rate (Koff; [1 / sec]) and on-rate (Kon; [1 / sec*M]) are used with their known meanings in the fields of chemistry and physics; they refer to the rate constants measuring the dissociation (Koff) or association (Kon) of an antibody with its target antigen. Koff and Kon can be determined experimentally using methods well established in the art. Methods for determining Koff and Kon of an antibody employ surface plasmon resonance. This is, for example, or The principle behind the biosensor system of the system. They can also be used to determine the dissociation constant KD by using the following formula:

[0063]

[0064] In the context of this specification, the term humanized antibody is used with its known meaning in the fields of cell biology and biochemistry; it refers to an antibody originally produced by immune cells of a non-human species, the sequence of which has been modified to increase its similarity to antibody variants naturally produced in humans.

[0065] In the context of this specification, the term no measurable cross-reactivity refers to the lack of ability of an antibody to recognize and bind to orthologous proteins from other species. For example, if, under appropriate conditions, binding of an antibody to human interleukin-2 to mouse interleukin-2 cannot be detected by a sufficiently sensitive method (such as surface plasmon resonance), then the antibody has no measurable cross-reactivity with mouse interleukin-2. An example of no measurable cross-reactivity is shown in Fig. 9 Antibody in the lower panel (NARA1).

[0066] As used herein, an antibody or protein that "specifically binds to hIL-2" is intended to mean an antibody or protein that binds to human IL-2 polypeptide with a K of 100 nM or less, 10 nM or less, 1 nM or less, 100 pM or less, or 10 pM or less. D An antibody or protein that "cross-reacts with an antigen other than human IL-2" is intended to mean an antibody that cross-reacts with an antigen other than human IL-2 with a K of 10 nM or less, 1 nM or less, or 100 pM or less. DAn antibody that binds to the antigen. An antibody that "does not cross-react with a specific antigen" is intended to mean an antibody that binds to the antigen with a K of 100 nM or higher. D , or K of 1 μM or higher D , or K of 10 μM or higher D Antibodies that bind to the antigen. In certain embodiments, such antibodies that do not cross-react with the antigen exhibit essentially undetectable binding to these proteins in standard binding assays.

[0067] The term "epitope" means a protein determining region capable of specific binding to an antibody. An epitope is usually composed of chemically active surface groups of molecules such as amino acids or sugar side chains and usually has specific three-dimensional structural characteristics, as well as specific charge characteristics. The difference between conformational epitopes and non-conformational epitopes is that the binding to the former is lost in the presence of denaturing solvents, but the binding to the latter is not lost.

[0068] The term "epitope binding domain" or "EBD" refers to a portion of a binding molecule (e.g., an antibody or an epitope binding fragment or derivative thereof) that specifically interacts with a binding site on a target epitope (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). EBD also refers to one or more fragments of an antibody that retain the ability to specifically interact with an IL-2 epitope (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution) and inhibit signal transduction. Examples of antibody fragments include, but are not limited to, scFv, Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; F(ab)2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragments, consisting of VH and CH1 domains; Fv fragments, consisting of the VL and VH domains of a single arm of an antibody; dAb fragments (Ward et al., (1989) Nature 341:544-546), which consist of a VH domain; and isolated complementary determining regions (CDRs).

[0069] EBDs also include single domain antibodies, large antibodies, single chain antibodies, minibodies, three chain antibodies, four chain antibodies, v-NARs and bi-scFvs (as known in the art (see, e.g., Hollinger and Hudson, (2005) Nature Biotechnology 23: 1126-1136)), bispecific single chain diabodies, or single chain diabodies designed to bind two different epitopes. EBDs also include antibody-like molecules or antibody mimetics, including but not limited to minibodies, large antibodies, Fn3-based protein backbones, ankyrin repeats (also known as DARpins), VASP polypeptides, avian pancreatic polypeptide (aPP), tetranectin, Affililin, Knottin, SH3 domains, PDZ domains, amylase inhibitors, neocarcinomastatins, protein A domains, lipocalins, transferrins, and Kunitz domains that specifically bind epitopes, which are within the scope of the present invention. Antibody fragments can be grafted into scaffolds based on polypeptides such as fibronectin type III (Fn3) (see US Pat. No. 6,703,199, which describes fibronectin polypeptide monomers).

[0070] The present invention also includes antibodies against human IL-2, which are isolated antibodies.

[0071] As used herein, the phrase "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds hIL-2 is substantially free of antibodies that specifically bind antigens other than hIL-2). However, an isolated antibody that specifically binds hIL-2 may have cross-reactivity with other antigens (e.g., IL-2 molecules from other species). Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0072] The terms "nucleic acid" and "polynucleotide" or "nucleotide coding sequence" are used interchangeably and refer to a polymeric form of nucleotides of any length, deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, DNA of any sequence isolated, RNA of any sequence isolated, nucleic acid probes, siRNA, shRNA, RNAi substances and primers. Polynucleotides can be modified or replaced at one or more bases, sugars and / or phosphates, with any of the various modifications or replacements described herein or known in the art. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the nucleotide structure can be modified before or after assembling the polymer. The sequence of nucleotides can be spaced apart by non-nucleotide components. After polymerization, the polynucleotides can be further modified, such as by conjugation with a labeling component. The term also refers to double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of a polynucleotide of the invention includes a double-stranded form and each of the two complementary single-stranded forms known or predicted to comprise the double-stranded form.

[0073] The term "polypeptide" is used interchangeably with the term "protein" and refers in its broadest sense to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In another embodiment, the subunits may be linked by other bonds, e.g., ester bonds, ether bonds, etc.

[0074] In one embodiment, the terms "treating" or "treatment" of any disease or condition (e.g., cancer) as used herein refers to alleviation of the disease or condition (e.g., slowing or arresting or reducing the development of the disease or at least one of its clinical symptoms). In another embodiment, "treating" or "treatment" refers to alleviation or reduction of at least one physical parameter, including those that are not discernible by the patient. In yet another embodiment, "treating" or "treatment" refers to regulating the disease or condition physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of a physical parameter), or both. Unless specifically described herein below, methods for assessing the treatment and / or prevention of a disease are generally known in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1Anti-human IL-2 binders were shown. Supernatants of B cell clones obtained after B cell hybridoma fusion were added to plates previously coated with human IL-2. Anti-human IL-2 mAbs were detected using biotinylated anti-mouse IgG antibodies.

[0077] Figure 2 Screening for anti-human IL-2 mAbs binding to putative specific human IL-2 epitopes. The plate was coated and blocked with 5344 (hIL-2 mAb without super-antagonistic behavior targeted herein), followed by the addition of human IL-2 to allow binding of cytokines to 5344, thereby covering the specific epitope of IL-2. Then, supernatants that produced a positive signal in the first screening (see Figure 1 ). After allowing the mAb in the supernatant to bind to the IL-2-5344 complex, a biotinylated MAB602 antibody was added to the plate in order to assess whether the test mAb in the supernatant binds to the same region as MAB602 (the so-called "competitor") or to a different region than MAB602. The competitor mAb resulted in an absorbance value (OD450) that was two-fold less than that obtained with MAB602 alone (in this case, OD = 1.1, as shown in H11).

[0078] Figure 3 Concentration-dependent competition of B cell hybridomas was demonstrated. Supernatants from eight competitor B cell hybridoma clones from the first screen (see Figure 2 ) and concentrated before use. Supernatants of these eight competitor B cell hybridoma clones (labeled 1 to 8) were added in increasing amounts. Competing competitor B cell hybridoma clones reduced OD450 as much or even more than MAB602, which was evident for clones 1 and 2. Different concentrations of MAB602 (green circles) served as controls.

[0079] Figure 4 In vivo proliferation of CD8+T cells is shown. Carboxyfluorescein succinimidyl ester (CFSE)-labeled CD8+T cells of CD45.1-congenic IL-7 transgenic mice were transferred to CD45.2-congenic wild-type recipient mice, followed by daily injections of phosphate buffered saline (PBS), IL-2, IL-2 plus MAB602 (IL-2 / MAB602), IL-2 plus 5344 (IL-2 / 5344), IL-2 plus hybridoma 1 (IL-2 / Hyb#1), or IL-2 plus hybridoma 2 (IL-2 / Hyb#2) for 4 days. On the 5th day, lymph nodes and spleens were analyzed for the CFSE characteristics of donor CD45.1+CD8+T cells. Results obtained with lymph nodes are shown, and similar results were obtained in the spleen.

[0080] Figure 5 shows the phenotypic characteristics of endogenous CD8+ T cells and NK cells after in vivo treatment with IL-2 plus hybridoma 1 and hybridoma 2. Figure 4 Process mice, then evaluate endogenous CD8+T cell subsets and NK cells in lymph nodes and spleen by flow cytometry.(A) CD8 and CD3 characteristics (left figure) of total lymph node cells and CD44 (activated T cells or memory T cells) and CD122 (IL-2 receptor β-subunit, present on activated T cells or memory T cells) characteristics of CD3+CD8+ lymph node cells, or (B) NK1.1 and CD3 characteristics of mice receiving indication treatment.Activation / memory CD8+T cells are high for CD44, and are middle high for CD122.NK cells are CD3 negative, and NK1.1 is positive.Similar results are obtained using splenocytes.

[0081] Figure 6 shows the total number of activated / memory CD8+ T cells and NK cells in lymph nodes and spleen. Animals were treated and analyzed as in Figure 5. Lymph nodes ( Fig. 6A ) and spleen ( Figure 6B ) were analyzed by measuring the absolute number of CD44-high CD8+ T cells (so-called memory phenotype, MP CD8+) and the absolute number of CD3-negative NK1.1+ NK cells.

[0082] Figure 7 The surface plasmon resonance binding curves of commercially available monoclonal antibody MAB602 (left figure) and monoclonal antibody NARA1 (right figure) (which is the object of the present invention) and human IL-2 are shown. For this experiment, an amine-coupled GLM chip was used. The carboxylic acid groups on the chip were activated at 30 ml / min using a mixture of 1-ethyl-3-3-dimethylaminopropylcarbodiimide chloride (0.2M EDC) and thio-N-hydroxythiosuccinimide (0.05M s-NHS) for 420 seconds (s). Antibodies NARA1 and MAB602 were coated on the chip at 100 mg / ml (in sodium acetate buffer (10 mM pH 4.5)). After adding ethanolamine HCl at 30 ml / min for 300 s, inactivation was performed. Finally, different concentrations of human IL-2 (starting from 100 nM, followed by three-fold dilution) were added at 100 ml / min, 600 s binding, and 240 s dissociation.

[0083] Figure 8 Surface plasmon resonance binding curves showing human IL-2 bound to monoclonal antibody NARA1 and IL-2 receptor subunit CD25 (used herein as Fc fusion of CD25-Fc), CD122, monoclonal antibody MAB602, or anti-hIL-2 antibodies that bind to different human IL-2 epitopes than NARA1 and MAB602. Figure 7 A chip coated with NARA1 and MAB602 as described in . The chip was regenerated using 10 mM glycine, pH 2.5, 30 ml / min, 60 s. A saturating concentration (1 mM) of human IL-2 was added at 100 ml / min, 120 s for binding, and 0 s for dissociation. Immediately after IL-2 binding to the antibody, a second analyte was added at 100 ml / min, 120 s for binding, and 240 s for dissociation. The concentrations used for cross-binding were: MAB602: 50 nM; NARA1: 50 nM; positive control: 50 nM; CD25-Fc: 500 nM; CD122: 138 nM. As expected, an anti-hIL-2 antibody recognizing a different hIL-2 epitope (herein referred to as the 'positive control') bound strongly to the hIL-2 / NARA1 complex when hIL-2 was bound to NARA1 ( Figure 8 Alternatively, when hIL-2 is already bound to NARA1, IL-2Ra (in the form of CD25-Fc) cannot bind to hIL-2 (pink line, Figure 8 ), however, when hIL-2 has bound to NARA1, IL-2Rβ (CD122) still binds to hIL-2 (orange line, Figure 8 ).

[0084] Fig. 9 Surface plasmon resonance binding curves of monoclonal antibodies MAB602 (upper panel) and NARA1 (lower panel) to murine IL-2 are shown. Figure 7 and Figure 8 The same chip used for the data in . Regeneration of the chip was performed with 10 mM glycine, pH 2.5, 30 ml / min, 60 s. Mouse IL-2 (mIL-2) or human IL-2 (hIL-2) was injected at 100 ml / min starting at 10 nM and then diluted three times, with 120 s for binding, 5 s and 240 s for dissociation. In the upper figure, MAB602 shows cross-reactivity by binding to mouse IL-2. In particular, for higher concentrations of mouse interleukin-2 (>1 nM), the binding curve is significantly different from the background level, with response units 10 (RU) greatly exceeding 10. However, at all tested concentrations, NARA1 (lower figure) showed no measurable cross-reactivity with mouse IL-2.

[0085] Fig.10 An overview of the three-dimensional structure of the Proleukin / Fab-NARA1 complex as obtained in Example 1 is provided.

[0086] Fig.11Further analysis of epitope residues is provided. The X-axis lists the amino acid sequence and number of SEQ ID No 1. The upper part of the Y-axis shows the corresponding residues from Proleukin in The total number of atoms of NARA1-Fab within the molecule, and the lower part of the Y-axis shows the reduced solvent accessible area of ​​the corresponding residues from Proleukin due to binding to NARA1-Fab.

[0087] Fig.12 The most critical epitope residues recognized by NARA1-Fab are indicated.

[0088] Fig.13 Overlay of the Proleukin / NARA1-Fab complex with the IL-2 / CD25 / CD122 / CD132 quaternary complex is shown.

[0089] Fig.14 Overlays of the C helices from IL-2_C145A (PDB: 3INK), Superkine (PDB: 3QB1), IL-2 / CD25 / CD122 / CD132 (PDB: 2B5I) and Proleukin / NARA1-Fab are shown. DETAILED DESCRIPTION OF THE INVENTION

[0091] Until now, there have been no monoclonal antibodies applicable to the disclosed invention. The inventors disclose their anti-human IL-2 mAb, which allows the following key steps for the use and commercialization of this technology in clinical applications:

[0092] • Anti-human IL-2 mAb was further sequenced and well characterized.

[0093] • Humanization of anti-human IL-2 mAbs, which is crucial to avoid (or minimize) immunogenicity in patients.

[0094] Different formats of anti-human IL-2 mAbs were generated, such as IgG, IgG1, IgG4, Fab, and single-chain Fv (scFv).

[0095] • Generation of a fusion protein consisting of human IL-2 and anti-human IL-2 mAb (or a fragment of anti-human IL-2 mAb): Such a construct has the advantage of consisting of only one component instead of two components, as in IL-2 bound to anti-human IL-2 mAb.

[0096] The inventors have generated and characterized a novel IL-2-binding protein that binds human IL-2 and, when tested in mice, exerts its ability to inhibit cytotoxic lymphocytes, including CD8 +To achieve these goals, certain difficulties had to be overcome.

[0097] Human IL-2 shows a high degree of similarity to mouse and rat IL-2, so human IL-2 is able to stimulate mouse lymphocytes in vitro and in vivo. Moreover, IL-2 is present in high concentrations in primary immune organs (such as bone marrow), which is why IL-2 is sometimes a "forbidden" antigen, meaning that it is very difficult to generate a B cell response that leads to neutral antibodies against IL-2. However, after immunizing C57BL / 6 mice with purified recombinant human IL-2 plus adjuvant, the inventors were able to obtain a response of polyclonal anti-human IL-2 antibodies.

[0098] • Of the antibody responses generated, only some mAbs effectively bound IL-2 (the so-called "binders"), and of those only about 0.35% interacted with the putative active site of IL-2.

[0099] • Finally, among these anti-human IL-2 mAbs, some showed the expected specific and potent in vivo activity as assessed by specific in vivo assays performed in mice, which cannot be replaced by in vitro experiments.

[0100] The inventors have developed a specific screening assay that allows the detection of specific anti-human IL-2 antibodies (so-called "binders") in the sera of immunized animals and in the supernatants of B-cell clones obtained after B-cell hybridoma fusion. In a second step, standard binders and those binders targeting putative specific epitopes of the human IL-2 molecule can be distinguished. An example of such an in vitro enzyme-linked immunosorbent assay (ELISA) performed with different B-cell clones is shown in Figures 1 to 3 middle.

[0101] After in vitro screening of anti-human IL-2 mAbs, these mAbs were characterized in vivo. For this purpose and in order to obtain sufficient amounts of mAbs, mAbs were concentrated from the supernatants of hybridomas, the amounts were estimated using ELISA, and finally the anti-human IL-2 mAbs were tested in mice. CD8 + The results obtained for proliferation and expansion of T cells and NK cells are shown in Figure 4 To Figure 6.

[0102] To characterize the binding properties of the anti-human IL-2 mAbs, surface plasmon resonance binding assays were used to test binding to human interleukin-2. The commercially available anti-human IL-2 mAb MAB602 was measured as a comparison. Figure 7, the binding curves of MAB602 (left) and NARA1 (the antibody of the present invention; right) with different concentrations of human interleukin-2 are shown. The dissociation constants (K D ) and the rate constant K on and K off Shown in Table 1.

[0103] Table 1

[0104]

[0105] Table 1: Binding properties of anti-human IL-2 mAbs to human IL-2 Example

[0106] Antibodies of the invention include antibody NARA1, which is deduced, isolated and structurally characterized by its full-length heavy chain amino acid sequence set forth in SEQ ID NO:5 and its full-length light chain amino acid sequence set forth in SEQ ID NO:6.

[0107] The corresponding variable region V of NARA1 H and V L The amino acid sequences of are SEQ ID NO: 19 (variable heavy chain) and SEQ ID NO: 20 (variable light chain).

[0108] The nucleotide coding sequences of the full-length light and heavy chains of NARA1 are SEQ ID NO: 3 (heavy chain coding sequence, including leader sequence) and SEQ ID NO: 4 (light chain coding sequence, including leader sequence).

[0109] The variable light and heavy chain nucleotide coding sequences of NARA1 are SEQ ID NO: 21 (variable heavy chain coding sequence) and SEQ ID NO: 22 (variable light chain coding sequence).

[0110] The CDR regions of NARA1 are described using the Kabat system (Kabat, EA et al. 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, see also Zhao & Lu 2009, Molecular Immunology 47: 694-700). For ease of reading, when the CDR regions are described according to the Kabat definition, they are referred to as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 thereafter, respectively. The CDR regions of NARA1 are: HCDR1 described in SEQ ID NO: 7, HCDR2 described in SEQ ID NO: 8, HCDR3 described in SEQ ID NO: 9, LCDR1 described in SEQ ID NO: 10, LCDR2 described in SEQ ID NO: 11, LCDR3 described in SEQ ID NO: 12.

[0111] The nucleotide coding sequence of the CDR region of NARA1 is: the HCDR1 coding sequence described in SEQ ID NO:13, the HCDR2 coding sequence described in SEQ ID NO:14, the HCDR3 coding sequence described in SEQ ID NO:15, the LCDR1 coding sequence described in SEQ ID NO:16, the LCDR2 coding sequence described in SEQ ID NO:17, and the LCDR3 coding sequence described in SEQ ID NO:18.

[0112] Also provided are fusion proteins described in SEQ ID NO: 23 and SEQ ID NO: 24. SEQ ID NO: 23 is a fusion protein comprising the variable heavy chain of NARA1, the N-terminus of which is fused to the C-terminus of hIL-2 via a GxS linker. SEQ ID NO: 24 is a fusion protein comprising the variable light chain of NARA1, the N-terminus of which is fused to the C-terminus of hIL-2 via a GxS linker.

[0113] (a) Example 1. Crystal structure of NARA1

[0114] (i) Materials and methods

[0115] The complex structure of a human interleukin 2 mutant (SEQ ID NO: 2) (called "Proleukin") bound to the Fab fragment of the antibody "NARA1" (SEQ ID NO: 5 and 6) was determined. The residue numbers of the resulting Proleukin are given according to the numbering of wild-type IL-2.

[0116] As discussed in detail below, the sequence differences between Proleukin and wild-type hIL-2 are irrelevant, and Proleukin is a valid model for structural analysis of hIL-2.

[0117] To define the epitope, X-ray crystallography was used to solve the atomic resolution structure of the above complex. X-ray crystallography is a technique that has been routinely and widely used to generate structural data for biomolecules, including antibodies and their complexes with antigens (Adms et al., (2013) Annual Review Biophysics 42: 265-287; Garman, (2014) Science 343: 1102-1108; Joachimiak, (2009) Current Opinio Structural Biology 19: 573-584.)

[0118] The antigen Proleukin is commercially available as a lyophilized powder containing excipients (approximately 50 mg of mannitol, 0.18 mg of sodium dodecyl sulfate, 0.173 mg of sodium dihydrogen phosphate and 0.89 mg of sodium dihydrogen phosphate are mixed per 1 mg of Proleukin). Before use in complex formation, Proleukin was purified by reverse phase HPLC to remove excipients.

[0119] The Fab fragment (NARA1-Fab) of NARA1 is produced by protein A chromatography followed by papain cleavage of full-length antibody. In brief, 6.5ml full-length NARA1 (9mg / ml, in 50mM citric acid buffer containing 90mM sodium chloride at pH 7.0) is mixed with 5mM DTT and 590ug papain (Roche). The cleavage reaction is kept at room temperature for 16 hours and terminated (Roche) by adding 15ul 56mM E64 solution. Then with 25mM Tris, 25mM NaCl, pH 8.0, the cleavage solution is diluted 10 times, and it is loaded on 5ml protein A columns (GE Healthcare) balanced with 5 times the volume of 25mM Tris, 25mM NaCl, pH 8.0, and the Fab fragment is in the fraction loaded through, and the Fc fragment is combined with the protein A column.

[0120] To form the complex, the Proleukin powder after HPLC was dissolved in H2O at a concentration of 5.5 mg / ml. An excess of 6.6 mg Proleukin was added dropwise to the 11.5 mg NARA1 Fab fragment solution. Excess Proleukin that precipitated under the current conditions was removed by centrifugation. The complex was then purified by gel filtration on Superdex 200 10x300 (GE Healthcare) using a running buffer of 25 mM Tris, 25 mM NaCl, pH 7.4.

[0121] The Proleukin / NARA1-Fab complex after gel filtration was concentrated to 14 mg / ml and screened by vapor diffusion methods such as the hanging drop method. The protein solution was mixed 1:1 with the reservoir solution to a total size of 0.4 ul. The experiment was set up using a Phoenix mechanical system (Art Robbins Instruments), stored in a RockImager chamber (Formulatrix) at 19°C, and automatically imaged. Crystals were collected 4 days after screening under conditions of 20% w / v polyethylene glycol 3350 and 0.2 M sodium nitrate. Crystals were cryopreserved with a reservoir solution containing 10% glycerol and flash frozen in liquid nitrogen prior to data collection. Using a Pilatus pixel detector, use Diffraction data were collected at beamline PX-II at the Swiss Light Source (Villigen, Switzerland) with an x-ray radiation wavelength of 100 Å.

[0122] Data sets were processed with XDS and XSCALE (version December 6, 2010), and structures were solved by molecular replacement using the program PHASER using the protein database entry "3INK" as a search model for IL-2 and the protein database entry "3TTI" as a search model for Fab fragments. Iterative model building and refinement were performed using the programs Coot (Crystallographic Object-Oriented Toolkit) and AUTOBUSTER (Bricogne et al., 2011). All figures were generated using the program PyMOL (Molecular Graphics System; DeLano Scientific: Palo Alto, CA; http: / / www.pymol.org).

[0123] The epitope residues were defined as those within 100 μg / mL from any atom in the Fab fragment of NARA1. Those residues from Proleukin that were within 1.000 Å and further confirmed by the CCP4 programs CONTACT and AREAIMOL (Collaborative Computational Project, Number 4, version 6.4.0). Similar paratope residues were defined as those with a distance of 1.000 Å to any atom in Proleukin. Those residues from NARA1-Fab within.

[0124] (ii) Results

[0125] The Proleukin / NARA1-Fab complex was resolved in space group C1 2 1 The unit cell size is α = 90°, β = 102.9°, γ = 90°. For detailed structural analysis please refer to Table 2. In each asymmetric unit, there is one complex molecule.

[0126] Table 2. Analysis of Proleukin / NARA1-Fab complex results

[0127]

[0128] 1) Epitope and paratope analysis

[0129] Fig.10 An overview of the three-dimensional structure of the Proleukin / Fab-NARA1 complex as obtained in Example 1 is provided. The light chain of the Fab fragment of NARA1 is designated A, the heavy chain of the Fab fragment of NARA1 is shown as B, the epitope residues recognized by NARA1-Fab are designated D, and Proleukin is designated C, with the mutation C145S highlighted.

[0130] Fig.11 Provides further analysis of epitope residues. The X-axis lists the amino acid sequence and number of SEQ ID No 1. The upper part of the Y-axis represents the residues corresponding to those from Proleukin. The total number of atoms in NARA1-Fab, and the lower part of the Y-axis represents the reducing solvent accessible area after binding with NARA1-Fab

[0131] The Proleukin used in Example 1 contained a C145S mutation. Fig.10As shown in , C145S is far from the epitope region. In addition, the superposition of the Cα atoms between the Proleukins in Example 1 and the Cα atoms from wild-type hIL-2 complexed with CD25, CD122 and CD132 (PDB: 2B5I) shows The rmsd of 1.3 fold was 0.1, indicating that the mutation did not perturb the overall structure. Therefore, the C145S mutant Proleukin is a valid model for structural analysis of wild-type hIL-2.

[0132] hIL-2 is a four-helix bundle protein, and the four helices are named A, B, C, and D from the N-terminus to the C-terminus, respectively. Fig.10 As shown, the epitope recognized by NARA1-Fab is a conformational epitope and as Fig.11 Shown is a span of two regions: one region (N50-K63) contains a loop and a short helix and connects helix A and helix B, and the other region (N91-N97) contains a loop and connects helix B and helix C.

[0133] The epitope residues and the interacting paratope residues from NARA1-Fab are summarized in Table 3. Among all the epitope residues, Fig.11 Arg58 shown in is the most critical epitope residue for binding to NARA1-Fab, as this residue alone has 42 interacting atoms from NARA1-Fab and accounts for 17.7% of the total reducing solvent accessible surface area due to binding to NARA1-Fab. Fig.12 As shown in , Arg58 forms two strong salt bridges with Glu35 in HCDR1 and Asp100 from LCDR3, respectively. Arg58 also forms a π-interaction with the aromatic ring of Try100 from LCDR3. Residues K52, P54, K55, T57, T61, F62, K63, Q94, and K96 are also considered important for binding to NARA1-Fab, as they all show equal to / greater than 5 interacting atoms from NARA1-Fab, and as shown in Fig.11 Shown is greater than Reduction solvent accessible area.

[0134] Table 3. Summary of epitopes and paratopes

[0135]

[0136]

[0137] Fig.12 Arg58 is shown as the most critical epitope residue recognized by NARA1-Fab. A represents Proleukin, B represents the heavy chain, and C represents the light chain. The involved residues are shown as bars.

[0138] 2) NARA1-Fab binding properties

[0139] Fig.13 Overlay of the Proleukin / NARA1-Fab complex with the IL-2 / CD25 / CD122 / CD132 quaternary complex is shown.

[0140] like Fig.13 The structural overlay of the two complexes shown in clearly demonstrates that NARA1-Fab forms a direct competition with CD25, but not with CD122 / CD132, which is consistent with the observation that the IL-2 / NARA1 complex primarily exhibits pro-T effector cell activity but not pro-Treg activity.

[0141] 3) Helix C of Proleukin in complex with NARA1-Fab adopts a conformation similar to helix C in the quaternary complex

[0142] Fig.14 Overlays of the C helices from IL-2_C145A (PDB: 3INK), Superkine (PDB: 3QB1), IL-2 / CD25 / CD122 / CD132 (PDB: 2B5I) and Proleukin / NARA1-Fab are shown.

[0143] The polar interface between helix C in IL-2 and CD122 plays an important role in the binding between the two parts (Wang et al. (2005) Science 310: 1159-1163). In 2012, Levin et al. have demonstrated that superkine (IL-2 mutant) has helix C alone, which adopts a conformation similar to that of helix C in the quaternary complex, and superkine shows a binding affinity to CD122 that is ~215 times higher than that of wild-type IL-2 (Levin et al, (2012) Nature 484: 529-533). It has been observed that such conformational changes in helix C are associated with conformational stability, which reduces the energy penalty for binding to CD122. As Fig.14 As shown in , the conformation of helix C from Proleukin complexed with NARA1-Fab is also similar to the conformation of helix C observed in Superkine and IL-2 / CD25 / CD122 / CD132 quaternary complexes, therefore, it is likely that the Proleukin / NARA1-Fab complex may exhibit higher binding affinity to CD122 than wild-type hIL-2.

[0144] (b) Example 2. Linear peptide maps of NARA1 and MAB602

[0145] In order to locate the epitope of NARA1 and MAB602 antibodies, a first library of 15-mer peptides was generated based on the sequence of human IL2. A second library of selected 15-mer peptides was also generated based on mutations of 3 specific residues F(62), Y(65) and L(92). The latter mutations were performed based on Roche / Glycart IL2 mutant proteins, as disclosed in WO2012 / 107417A1 with such 3 mutations. Previous work (unpublished) performed in the Boyman laboratory showed that commercially available mouse anti-human IL2 mAb 602 with similar functions to A1 strongly reduced binding to the F42A mutant of IL2 (one of the IL2 docking sites for CD25).

[0146] (i) Materials and methods

[0147] Thus, each peptide in the first library has 15 amino acids and is obtained by scanning the target sequence (see Table 4, reference peptides 1 to 41) in steps of 3 residues starting from the N-terminus. Thus, a ladder diagram is generated and each peptide contains 12 overlapping residues of the preceding peptide in the ladder diagram and 12 overlapping residues of the following peptide. In summary, 41 peptides are generated from the expressed human IL2 sequence.

[0148] A second library of peptides was generated by mutating F(62), Y(65) and L(92) to alanine in all corresponding peptides in the first library generated as described above (see Table 4, reference peptide numbers 42 to 60).

[0149] For both libraries, the parental cysteine ​​has been replaced with serine (underlined residue) to avoid nonspecific binding.

[0150] Table 4. Library of reference peptides

[0151]

[0152]

[0153] Two groups of peptides were printed on the microarray sheet in triplicate and incubated with the target antibody (MAb602 and NARA1) and the control antibody. Additional incubation was performed with an unrelated antibody (mouse control IgG2a / λ and mouse control IgG2a / κ) from the same isotype, and a secondary antibody (anti-mouse IgG (Thermo84545, labeled DL650) or anti-mouse IgG (JIR 115-175-072, labeled Cy5)) to assess nonspecific binding due to the detection antibody. Experiments were performed as described in Maksimov P et al. 2012, PLoS One 7: e34212. doi: 10.1371 / journal.pone.0034212.

[0154] The determination of peptide-antibody binding was performed by RepliTope analysis, wherein the microarray of peptides (triplicate) was incubated with the first antibody, followed by incubation with a fluorescently labeled second antibody for the Fc portion of the first antibody. All steps were performed on a TECAN microarray processing station, enabling highly reliable and repeatable washing and incubation steps. After the incubation step and subsequent final washing step (to remove unbound second antibody), the microarray was dried using a nitrogen stream and scanned with an appropriate wavelength setting in a high-resolution microarray scanning system. Control incubations were performed to exclude false positive signals with unrelated antibodies of the same isotype.

[0155] Use point recognition software GenePix (Molecular Devices) to analyze and quantitatively obtain the image.For each point, extract the mean signal intensity (0 to 65535 arbitrary units).For further data evaluation, measure the MMC2 value.MMC2 equals the mean value of all three examples on the microarray.Except that the coefficient of variation (CV)-standard deviation is divided by mean value-is greater than 0.5, in this case, the mean value of the two closest numerical values ​​(MC2) is assigned to MMC2.

[0156] (ii) Results

[0157] The data are summarized in Table 5.

[0158] The anti-IL2(NARA1) antibody did not show any significant reactivity against the immobilized peptides. Only peptide 10 presented a weak response, however, this peptide was also weakly recognized by the mouse control antibody.

[0159] The commercially available antibody MAB602 (mIgG2a) provided some weak signals for peptides 22 to 26 and some strong signals for peptides 10 to 13.

[0160] Table 5. Results of linear epitope profiling

[0161]

[0162]

[0163] The overlapping sequences of the two sets of peptides were considered to contain amino acids that bind to the target antibody (Table 5). One stretch was a strong binder of MAB602, while the other stretch corresponded to a weak binder of MAB602:

[0164] Strong: (57)TRMLTF (62)

[0165] Weaker: (96) KNF (98)

[0166] Ala mutations of specific residues F42 (62), Y45 (65), L72 (92) showed that residue F42 (62) is a significant important residue for binding to antibody MAB602 (Table 6).

[0167] Table 6. Mutation characteristics

[0168]

[0169] Sequence Listing

[0170] Useful amino acid and nucleotide sequences for practicing the present invention are shown in Table 7.

[0171] Table 7. Sequence Listing

[0172]

[0173]

[0174]

[0175]

Claims

1. A monoclonal antibody (mAb) or an antigen-binding fragment thereof specific for human interleukin-2 (hIL-2), wherein the binding of the antibody to hIL-2 inhibits the binding of hIL-2 to CD25, and the antibody or antigen-binding fragment thereof is characterized by the following parameters: a. Binding to hIL-2 is characterized by the dissociation constant (K D )≤7,5nmol / L、≤5 nmol / L, ≤3nmol / L, ≤2nmol / L or ≤1,5nmol / L; b. Binding to hIL-2 is characterized by the off-rate (K off )≤1x10 -4 s -1 ,≤8x10 -5 s -1 ,≤6x10 -5 s -1 ,≤4x10 -5 s -1 ,≤3x10 -5 s -1 or ≤2,1x10 -5 s -1 ,by and c. comprising HCDR1 as shown in SEQ ID NO: 7, HCDR2 as shown in SEQ ID NO: 8, HCDR3 as shown in SEQ ID NO: 9, HCDR4 as shown in SEQ ID NO: LCDR1 as shown in SEQ ID NO: 10, LCDR2 as shown in SEQ ID NO: 11, and LCDR3 as shown in SEQ ID NO:

12.

2. A monoclonal antibody or antigen-binding fragment thereof specific for human interleukin-2 (hIL-2) as claimed in claim 1, wherein the antibody or antigen-binding fragment thereof shows no measurable cross-reactivity to mouse IL-2.

3. A nucleic acid molecule encoding the human interleukin-2 (hIL-2)-specific monoclonal antibody or an antigen-binding fragment thereof according to claim 1 or 2. A vector comprising the nucleic acid molecule according to claim 3.

5. A cell comprising the nucleic acid molecule of claim 3, or expressing the nucleic acid molecule of claim 3.

6. A cell capable of producing the human interleukin-2 (hIL-2)-specific monoclonal antibody (mAb) or an antigen-binding fragment thereof according to claim 1 or 2.

7. A therapeutic preparation for use as a medicament for treating cancer or viral infection, comprising: a. the hIL-2 monoclonal antibody (mAb) according to claim 1 or 2, or b. The hIL-2 monoclonal antibody (mAb) according to claim 1 or 2 and human interleukin-2.

8. Use of the antibody or molecule of claim 1 or 2 in the preparation of a medicament for treating cancer or viral infection.

9. A therapeutic preparation for use as a medicament for treating cancer or viral infection, comprising a fusion protein comprising: a. A monoclonal antibody (mAb) or an antigen-binding fragment thereof specific for human interleukin-2 (hIL-2) according to claim 1 or 2, b. Human IL-2, and c. An amino acid linker of 1 to 50 amino acids, which connects the human interleukin-2 (hIL-2)-specific monoclonal antibody (mAb) or its antigen-binding fragment to the human IL-2 into a single polypeptide chain.

10. The therapeutic formulation for use as a medicament for treating cancer or viral infection according to claim 9, wherein the amino acid linker is 5 to 40 amino acids.

11. The therapeutic formulation for use as a medicament for treating cancer or viral infection as claimed in claim 9, wherein the amino acid linker is 10 to 30 amino acids.

12. The therapeutic formulation for use as a medicament for treating cancer or viral infection as claimed in claim 9, wherein the amino acid linker is 15 to 25 amino acids.

13. Use of the therapeutic formulation for use as a medicament for treating cancer or viral infection as claimed in any one of claims 9 to 12 in the preparation of a medicament for treating cancer or viral infection.

14. A monoclonal antibody (mAb) or an antigen-binding fragment thereof specific for human interleukin-2 (hIL-2) as described in claim 1 or 2, which binds to an epitope of human interleukin-2 (hIL-2), wherein the epitope comprises amino acids K52, P54, K55, T57, R58, T61, F62, K63, Q94 and K96.

15. The human interleukin-2 (hIL-2)-specific monoclonal antibody (mAb) or an antigen-binding fragment thereof as claimed in claim 14, wherein the epitope further comprises any one or more of amino acids N50, N53, N91, L92, A93, and N97.

16. The human interleukin-2 (hIL-2)-specific monoclonal antibody (mAb) or an antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody is humanized.

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