Anti-PD1 antibody-attenuated IL2 immune complex and its use

Abstract

This specification discloses a modified human interleukin-2 (hIL2) protein, a human antibody molecule or an antigen-binding fragment thereof, and an immune complex containing the same, which specifically binds immunologically to human programmed cell death protein 1 (hPD1).

Description

Technical Field

[0001] Cross - References to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 352,842 (filing date: June 16, 2022), U.S. Provisional Patent Application No. 63 / 481,630 (filing date: January 26, 2023), and U.S. Provisional Patent Application No. 63 / 502,746 (filing date: May 17, 2023), the disclosures of which are hereby incorporated by reference in their entireties.

[0002] Sequence Listing This application includes a sequence listing that was electronically submitted in XML format, which is hereby incorporated by reference in its entirety. The name of the XML copy created on June 5, 2023 is 102085.021703_Sequence Listing.xml and the size is 696,000 bytes.

[0003] Technical Field This specification discloses a modified human interleukin - 2 (hIL2) protein, a human antibody molecule or an antigen - binding fragment thereof, and an immune complex containing them, which immunospecifically binds to human programmed cell death protein 1 (hPD1).

Background Art

[0004] Human IL2 (hIL2) is a glycosylated cytokine with four α -helix bundles of type 1 produced by CD4+ T cells and CD8+ T cells. Autocrine and paracrine IL2 signaling occurs through the involvement of either a high - affinity trimeric receptor complex containing IL2Rα (CD25), IL2Rβ (CD122), and IL2Rγ (CD132), or a medium - affinity dimeric receptor complex containing IL2Rβ (CD122) and IL2Rγ (CD132). IL2 has two opposing pleiotropic roles in that it not only stimulates the proliferation of T cells to produce effector T cells, memory T cells, and activated NK cells, but can also stimulate inhibitory regulatory T cells for the maintenance of immune homeostasis. Low doses of IL2 mainly stimulate regulatory T cells, some effector T cells, and NK cells, while high doses of IL2 widely stimulate cytotoxic T cells, effector T cells, NK cells, and regulatory T cells. However, the use of IL2 in the treatment of autoimmune diseases and cancer immunotherapy has been limited by off - target effects and toxicity associated with IL2 administration.

Summary of the Invention

[0005] This specification discloses a modified human interleukin 2 (hIL2) protein comprising substitutions at position 20 and position 38 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345, wherein the modified hIL2 protein has reduced potency against both the high - affinity hIL2 receptor and the medium - affinity hIL2 receptor as compared to the unmodified hIL2.

[0006] This specification also discloses a modified human interleukin 2 (hIL2) protein comprising D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitution at position 20 and R38E substitution at position 38 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345.

[0007] This specification also discloses a human antibody molecule or an antigen - binding fragment thereof that immunospecifically binds to human programmed cell death protein 1 (hPD1), and the human antibody molecule or its antigen - binding fragment a) A heavy chain complementarity determining region 1 (CDR1) comprising the amino acid sequence shown in SEQ ID NO: 418, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 419, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 420, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 421, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 422, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 423; b) A heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 386, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 387, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 388, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 389, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 390, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 391; c) A heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 396, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 397, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 398, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 399, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 400, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 401; or, d) A heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 406, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 407, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 408, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 409, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 410, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 411; comprising.

[0008] This specification also discloses (a) A modified hIL2 protein comprising substitutions at positions 20 and 38 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345; and, (b) A human antibody molecule or an antigen-binding fragment thereof that binds immunospecifically to hPD1, and discloses an immune complex comprising the same, wherein the human antibody molecule or an antigen-binding fragment thereof (i) A heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 418, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 419, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 420, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 421, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 422, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 423; (ii) A heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 386, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 387, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 388, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 389, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 390, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 391; (iii) A heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 396, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 397, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 398, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 399, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 400, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 401; or, (iv) A heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 406, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 407, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 408, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 409, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 410, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 411; comprising.

[0009] This specification also discloses a pharmaceutical composition comprising the modified hIL2 protein, human antibody molecule or antigen-binding fragment or immune complex thereof disclosed in this specification.

[0010] This specification also discloses a polynucleotide comprising a nucleic acid sequence encoding the modified hIL2 protein, human antibody molecule or antigen-binding fragment or immune complex thereof disclosed in this specification, as well as a vector comprising the polynucleotide and a transformed cell comprising the vector.

[0011] This specification discloses a method for treating a target disease or disorder, the method comprising administering to the target a therapeutically effective amount of an immune complex or pharmaceutical composition disclosed herein to treat the disease or disorder.

[0012] Also disclosed is the use of an immune complex or pharmaceutical composition disclosed herein in the manufacture of a medicament for treating a disease, and the use of an immune complex or pharmaceutical composition disclosed herein for treating a disease or disorder.

[0013] This summary and the following detailed description will be better understood when considered in conjunction with the drawings. To illustrate the disclosed modified hIL2 protein, anti-hPD1 antibody or antigen-binding fragment thereof, and immune complex, the drawings show representative embodiments of the modified hIL2 protein, anti-hPD1 antibody or antigen-binding fragment thereof, and immune complex, but the modified hIL2 protein, anti-hPD1 antibody or antigen-binding fragment thereof, and immune complex are not limited to the specific embodiments disclosed.

Brief Description of the Drawings

[0014]

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[0015] Detailed description of representative embodiments The disclosed modified hLI2 protein, human antibody molecule or antigen-binding fragment thereof, and immune complex can be more readily understood by reference to the following detailed description in conjunction with the drawings that form a part of this disclosure. The disclosed modified hLI2 protein, human antibody molecule or antigen-binding fragment thereof, and immune complex are not limited to the specific modified hLI2 protein, human antibody molecule or antigen-binding fragment thereof, and immune complex described and / or shown in the specification, and the terms used in the specification are for illustrative purposes only and are not intended to limit the modified hLI2 protein, human antibody molecule or antigen-binding fragment thereof, and immune complex of the claims.

[0016] Unless otherwise specifically mentioned, descriptions regarding possible mechanisms, modes of action, or reasons for improvement are for illustrative purposes only, and the disclosed modified hLI2 protein, human antibody molecule or antigen-binding fragment thereof, and immune complex are not restricted by the accuracy or inaccuracy of such suggested mechanisms, modes of action, or reasons for improvement.

[0017] Throughout this document, the specification describes the modified hLI2 protein, human antibody molecule or antigen-binding fragment thereof, and immune complex, as well as methods of using the modified hIL2 protein, human antibody molecule or antigen-binding fragment thereof, and immune complex. When the specification describes and claims features and aspects related to the modified hLI2 protein, human antibody molecule or antigen-binding fragment thereof, and immune complex, such features and aspects are equally applicable to methods of using the modified hIL2 protein, human antibody molecule or antigen-binding fragment thereof, and immune complex. Similarly, when the specification describes and claims features and aspects related to methods of using the modified hLI2 protein, human antibody molecule or antigen-binding fragment thereof, and immune complex, such features and aspects are equally applicable to the modified hIL2 protein, human antibody molecule or antigen-binding fragment thereof, and immune complex.

[0018] When a numerical range is set forth or otherwise defined in the specification, that range includes the endpoints and all the individual integers and fractions within that range, and also includes narrower ranges formed by all possible combinations of those endpoints with the integers and fractions within, which narrower ranges are to be construed as if expressly set forth. Subgroups of larger groups of values within the stated range are formed as if such narrower ranges were expressly set forth. Even when a numerical range is described in the specification as being greater than a recited value, the range is finite and its upper limit is restricted by values that are operable within the scope of the disclosure of the specification. Even when a numerical range is described in the specification as being less than a recited value, the lower limit of the range is restricted by values other than zero. The scope of modified hIL2 proteins, human antibody molecules or antigen-binding fragments thereof and immune complexes is not intended to be limited to the specific values indicated in defining the scope. All ranges include their endpoints and are combinable.

[0019] When a numerical value is expressed as an approximation by use of the term "about", it is understood that the particular numerical value forms another aspect. Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. The term "about" as used in a numerical range, cutoff value or particular numerical value is used to indicate that the indicated numerical value may vary by up to 10%. Thus, the term "about" is used to encompass variations of ± 10% or less, ± 5% or less, ± 1% or less, ± 0.5% or less or ± 0.1% or less from a particular value.

[0020] It is also to be understood that particular features of the disclosed modified hIL2 proteins, human antibody molecules or antigen-binding fragments thereof and immune complexes, which are described in separate aspects for clarity, can be provided in combination in a single aspect. Conversely, various features of the disclosed modified hIL2 proteins, human antibody molecules or antigen-binding fragments thereof and immune complexes, which are described in a single aspect for brevity, can be provided separately or in any sub-combination.

[0021] In this specification, the singular also includes the plural.

[0022] Throughout the specification and claims, various terms related to aspects of the description are used. Such terms, unless otherwise specified, have their ordinary meaning in the relevant technical field. Other specifically defined terms are interpreted in a manner consistent with the definitions provided in this specification.

[0023] The term "comprising" is intended to include examples subsumed by the terms "consisting essentially of" and "consisting of", and likewise, the term "consisting essentially of" is intended to include examples subsumed by the term "consisting of".

[0024] The term "antibody molecule" is used in a broad sense and includes full-length immunoglobulin molecules and their antigen-binding fragments.

[0025] Immunoglobulins can be classified into five major classes, namely IgA, IgD, IgE, IgG, and IgM, according to the amino acid sequence of their heavy-chain constant domains. IgA and IgG are further classified into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Vertebrate antibody light chains can be classified into one of two distinct types, namely κ or λ, based on the amino acid sequence of their constant domains.

[0026] "Antigen-binding fragment" refers to a portion of an immunoglobulin molecule (i.e., "said antigen-binding fragment") that retains the antigen-binding properties of the parent full-length antibody. Representative antigen-binding fragments may have heavy-chain complementarity-determining regions (CDRs) 1, 2, and / or 3; light-chain CDRs 1, 2, and / or 3; heavy-chain variable region (VH); light-chain variable region (VL); and combinations thereof. Antigen-binding fragments include a monovalent fragment consisting of the Fab fragment, VL, VH, constant light chain (CL), and heavy-chain constant 1 (CH1) domains; an F(ab)2 fragment, a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; and a domain antibody (dAb) fragment consisting of a VH domain or a VL domain (Ward et al., Nature 341:544-546, 1989). The VH domain and the VL domain can form various types of single-chain antibodies by ligation with recombinant and synthetic linkers, where the VH / VL domains pair intramolecularly or, when the VH domain and the VL domain are expressed as separate single-chain antibodies, intermolecularly to form a monovalent antigen-binding site such as a single-chain Fv or diabody described in International Publication Nos. WO 98 / 44001, WO 88 / 01649, WO 94 / 13804, and WO 92 / 01047. These antibody fragments are obtained by techniques well known to those skilled in the art and the fragments are screened for utility in the same manner as full-length antibodies.

[0027] The phrase "immunologically specifically binds" refers to the ability of the disclosed antibody molecule to preferentially bind to its target (hPD1 in the case of the anti-hPD1 antibody molecule) without preferentially binding to other molecules in a sample containing a mixed population of molecules. An antibody molecule that immunologically specifically binds to hPD1 substantially does not contain antibodies having different antigen specificities (e.g., an anti-hPD1 antibody substantially does not contain antibodies that specifically bind to antigens other than hPD1). However, an antibody molecule that immunologically specifically binds to hPD1 may cross-react with other antigens such as an ortholog of hPD1 including cynomolgus monkey (Macaca fascicularis) PD1. The antibody molecules disclosed in this specification can immunologically specifically bind to both naturally produced hPD1 and PD1 recombinantly prepared in mammals or prokaryotic cells.

[0028] An antibody variable region consists of four "framework" regions separated by three "antigen-binding sites". Antigen-binding sites are specified by various terms: (i) Complementary determining regions (CDRs) (three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3)) are based on sequence variability (Wu and Kabat J Exp Med 132:211-50, 1970; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991); and (ii) "hypervariable regions" ("HVR" or "HV", three in VH (H1, H2, H3) and three in VL (L1, L2, L3)) refer to regions of the antibody variable domain that are structurally hypervariable as defined by Chothia and Lesk (Chothia and Lesk Mol Biol 196:901-17, 1987). The AbM definition of CDRs is also widely used; this is a compromise between the Kabat and Chothia numbering schemes and is so called because it was used by Oxford Molecular's AbM antibody modeling software (Rees, A.R., Searle, S.M.J., Henry, A.H. and Pedersen, J.T. (1996) In Sternberg M.J.E. (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172). Other terms include "IMGT-CDR" (Lefranc et al., Dev Comparat Immunol 27:55-77, 2003) and "specificity determining residue usage (SDRU)" (Almagro Mol Recognit 17:132-43, 2004). The International ImMunoGeneTics (IMGT) database (http: / / www_imgt_org) provides standardized numbering and definitions of antigen-binding sites.The correspondence between the descriptions of CDR, HV, and IMGT is described in Lefranc et al., Dev Comparat Immunol 27:55-77, 2003.

[0029] "Framework" or "framework sequence" is the remaining sequence of the variable region other than the antigen-binding site. As noted above, since the antigen-binding site can be specified by various terms, the exact amino acid sequence of the framework will vary depending on how the antigen-binding site is specified.

[0030] The terms "human antibody", "fully human antibody" and similar terms refer to antibodies having heavy and light chain variable regions in which both the framework and the antigen-binding site are derived from sequences of human origin. Where the antibody includes a constant region, the constant region is also derived from a sequence of human origin. A human antibody contains heavy chain and / or light chain variable regions "derived from" sequences of human origin when the variable regions of the antibody are obtained from a system that uses immunoglobulin genes of the human germline or rearranged immunoglobulin genes. Such systems include human immunoglobulin gene libraries displayed on phage, and non-human transgenic animals such as mice or chickens having the human immunoglobulin loci described herein. A "human antibody" may contain amino acid differences compared to the amino acid sequences encoded by human germline or rearranged immunoglobulin genes, for example, due to naturally occurring somatic mutations or the intentional introduction of substitutions in the variable domain (framework and antigen-binding site) or constant domain. Typically, a "human antibody" has an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence encoded by human germline or rearranged immunoglobulin genes. In some cases, a "human antibody" may include, for example, a consensus framework sequence obtained from analysis of the sequences of human frameworks as described in Knappik et al., J Mol Biol 296:57-86, 2000, or a synthetic HCDR3 incorporated into a human immunoglobulin gene library displayed on phage as described in, for example, Shi et al., J Mol Biol 397:385-96, 2010 and WO 2009 / 085462. Antibodies in which the antigen-binding site is derived from a species other than human are not included in the definition of "human antibody".

[0031] Human antibodies are derived from human immunoglobulin sequences, but may be prepared using systems such as phage display incorporating synthetic CDRs and / or synthetic frameworks, or by inducing in vitro mutations to improve antibody properties in the variable region and / or constant region and / or both, resulting in antibodies in vivo that are not naturally present in the human antibody germline repertoire.

[0032] "Recombinant antibody" includes all antibodies prepared, expressed, produced or isolated by the following recombinant means: antibodies isolated from animals (e.g., mice) into which genes of human immunoglobulins have been introduced or chromosomes have been transferred, or hybridomas prepared therefrom (further described below); antibodies isolated from host cells transformed to express such antibodies; antibodies isolated from recombinant combinatorial antibody libraries; and antibodies prepared, expressed, produced or isolated by other means including splicing to other DNA sequences of human immunoglobulin gene sequences, or antibodies prepared in vitro by Fab arm exchange.

[0033] "Monoclonal antibody" refers to a population of antibody molecules of a single molecular composition. The composition of a monoclonal antibody exhibits a single binding specificity and affinity for a particular epitope, or in the case of a bispecific monoclonal antibody, dual binding specificities for two different epitopes. Thus, a monoclonal antibody refers to a population of antibodies with a single amino acid composition for each heavy chain and each light chain, except for well-known modifications such as deletion of lysine at the C-terminus of the antibody heavy chain. The glycosylation of a monoclonal antibody may not be uniform within the antibody population. A monoclonal antibody may be monospecific or multispecific, or monovalent, bivalent or multivalent. Bispecific antibodies are included within the term monoclonal antibody.

[0034] "Epitope" refers to a part of an antigen to which an antibody specifically binds. Epitopes usually consist of parts of the chemically active (e.g., polar, non-polar, hydrophobic) surface, such as amino acids or polysaccharide side chains, and may have specific three-dimensional structural characteristics and specific charge characteristics. Epitopes may be composed of contiguous and / or discontinuous amino acids that form a spatial unit of conformation. In discontinuous epitopes, amino acids of various parts of the linear sequence of the antigen are brought into proximity in three-dimensional space by the folding of the protein molecule.

[0035] "Variant" refers to a polypeptide or polynucleotide that is different from a reference polypeptide or reference polynucleotide by one or more modifications such as substitution, addition or deletion. In this specification, the term "mutation" is intended to mean one or more intentional substitutions to a polypeptide or polynucleotide.

[0036] "Treatment", "treating" and similar terms refer to both therapeutic treatment and prophylactic or preventive measures, including reduction in the severity and / or frequency of symptoms, elimination of symptoms and / or the cause underlying the symptoms, reduction in the frequency or likelihood of symptoms and / or the cause underlying them, and improvement or repair of damage caused directly or indirectly by a disease or disorder. Treatment also includes prolonging the survival period as compared to the predicted survival period of an untreated subject. Subjects to be treated / treated include not only those having a disease or disorder, but also those likely to have a disease or disorder and those in whom a disease or disorder should be prevented.

[0037] In this specification, "administering to a subject" and similar terms denote the procedure of injecting the disclosed modified hLI2 protein, immune complex or pharmaceutical composition into a subject to bring the target cells, tissues or body parts of the subject into contact with the disclosed modified hIL2 protein or an immune complex containing the same.

[0038] The term "therapeutically effective amount" refers to the amount of a modified hIL2 protein, immune complex or pharmaceutical composition described in the specification that is effective to achieve a particular biological or therapeutic result (e.g., but not limited to, a biological or therapeutic result disclosed, described or exemplified in the specification). The therapeutically effective amount may vary depending on factors such as the medical condition, age, gender, and weight of the individual, as well as the ability of the modified hIL2 protein, immune complex or pharmaceutical composition to elicit a desired response in the subject. Representative indicators of a therapeutically effective amount include, for example, improvement in the well-being of the patient, reduction of symptoms, arrest or delay of symptom progression, and / or disappearance of symptoms.

[0039] As used herein, the term "subject" is intended to mean an animal, particularly a mammal. Accordingly, the methods are applicable to humans and non-human animals, but are most preferably used in humans. As used herein, "subject" and "patient" are synonymous.

[0040] As used herein, immune complex and fusion protein are synonymous.

[0041] Modified human interleukin 2 (hIL2) protein As used herein, a modified hIL2 protein is disclosed that includes substitutions at position 20 and position 38 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345, and the modified hIL2 protein has reduced potency against both the high-affinity hIL2 receptor and the intermediate-affinity hIL2 receptor as compared to unmodified hIL2. The disclosed modified hIL2 protein is also referred to herein as "attenuated" IL2.

[0042] Suitable substitutions at position 20 are, for example, any of D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E.

[0043] Suitable substitutions at position 38 are, for example, any of R38E, R38N, R38G, R38H, R38I, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D or R38K.

[0044] In some embodiments, any of the D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E substitutions can be combined with the R38E substitution.

[0045] The modified hIL2 protein can comprise any of the amino acid sequences shown in SEQ ID NOs: 134 - 150, 307, 344, 607 - 611, 614, 617, or 620. The modified hIL2 protein can comprise any of the amino acid sequences shown in SEQ ID NOs: 134 - 150, 307, 344, 608, 611, 614, or 620. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 134. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 135. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 136. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 137. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 138. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 139. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 140. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 141. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 142. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 143. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 144. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 145. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 146. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 147. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 148. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 149. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 150. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 307. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 344. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 607. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 608.In some embodiments, the modified hIL2 protein comprises the amino acid sequence set forth in SEQ ID NO: 609. In some embodiments, the modified hIL2 protein comprises the amino acid sequence set forth in SEQ ID NO: 610. In some embodiments, the modified hIL2 protein comprises the amino acid sequence set forth in SEQ ID NO: 611. In some embodiments, the modified hIL2 protein comprises the amino acid sequence set forth in SEQ ID NO: 614. In some embodiments, the modified hIL2 protein comprises the amino acid sequence set forth in SEQ ID NO: 617. In some embodiments, the modified hIL2 protein comprises the amino acid sequence set forth in SEQ ID NO: 620. The modified IL2 protein having the amino acid sequence set forth in any of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617 or 620 can further comprise a T3A substitution and / or a C125A substitution. In some embodiments, the modified IL2 protein having the amino acid sequence set forth in any of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617 or 620 further comprises a T3A substitution. In some embodiments, the modified IL2 protein having the amino acid sequence set forth in any of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617 or 620 further comprises a C125A substitution. In some embodiments, the modified IL2 protein having the amino acid sequence set forth in any of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617 or 620 further comprises a T3A substitution and a C125A substitution.

[0046] The modified hIL2 protein may comprise a D20A substitution and an R38E substitution.

[0047] As used herein, the terms "potency reduction" and related terms such as "potency reduction" and "attenuation" of IL2 activity refer to the EC compared to unmodified hIL2 in an IL2-dependent assay 50Refers to the decrease in the potency of the modified hIL2 determined by the increase in value. As described in the specification, the decrease in the potency of the modified hIL2 occurs with both high-affinity and medium-affinity IL2 receptors. The IL2-dependent assay for determining potency may be the recombinant human erythroleukemia TF1 (TF1+IL2Rβ) or human natural killer NK-92 cell proliferation assay described in the specification. In one embodiment, the IL2-dependent assay for determining potency is the recombinant human erythroleukemia TF1 (TF1+IL2Rβ) cell proliferation assay. In another embodiment, the IL2-dependent assay for determining potency is the human natural killer NK-92 cell proliferation assay. Another IL2-dependent assay for determining potency may be the TF1+IL2Rβ or human natural killer NK-92 pSTAT5 assay described in the specification. Unmodified hIL2 may be hIL2 expressed by prokaryotes such as Proleukin® (having the amino acid sequence of natural human IL2 except for the C125S substitution that removes the unbound cysteine and having no normal human carbohydrate expression at residue T3), or unmodified hIL2 may be hIL2 having the amino acid sequence shown in SEQ ID NO: 345 expressed in mammalian cell lines such as CHO or HEK cell lines, or hIL2 having the amino acid sequence shown in SEQ ID NO: 345 with a C125S substitution.

[0048] The modified hIL2 protein can further include a substitution at position 3 of the amino acid sequence of the unmodified IL2 shown in SEQ ID NO: 345. Suitable substitutions include, for example, T3A. In some embodiments, the modified hIL2 protein includes the T3A substitution, the D20A substitution, and the R38E substitution. In some aspects, the modified hIL2 protein includes the amino acid sequence shown in SEQ ID NO: 216.

[0049] Alternatively, the modified hIL2 protein can further include a deletion at position 3 of the amino acid sequence of the unmodified IL2 shown in SEQ ID NO: 345. In some embodiments, the modified hIL2 protein includes the deletion of amino acids 1-3, the D20A substitution, and the R38E substitution. In some aspects, the modified hIL2 protein includes the amino acid sequence shown in SEQ ID NO: 218.

[0050] The modified hIL2 protein can further include a deletion or substitution at position 125 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345. The substitution at position 125 can be C125A. In some embodiments, the modified hIL2 protein includes D20A substitution, R38E substitution, and C125A substitution. In some embodiments, the modified hIL2 protein includes the amino acid sequence shown in SEQ ID NO: 215. In some embodiments, the modified hIL2 protein includes T3A substitution, D20A substitution, R38E substitution, and C125A substitution. In some embodiments, the modified hIL2 protein includes the amino acid sequence shown in SEQ ID NO: 217. In some embodiments, the modified hIL2 protein includes a deletion of amino acids 1-3, D20A substitution, R38E substitution, and C125A substitution. In some embodiments, the modified hIL2 protein includes the amino acid sequence shown in SEQ ID NO: 219.

[0051] The modified hIL2 protein, for example, in the hIL2-dependent cell proliferation assay described in the specification, in the comparison of EC 50 values, compared to unmodified hIL2, may show a decrease in potency for high-affinity IL2 receptor (hIL2Rαβγ) of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold. In some embodiments, the modified hIL2 protein may show a decrease in potency for high-affinity IL2 receptor (hIL2Rαβγ) of more than about 10,000-fold compared to unmodified hIL2. A greater decrease in hIL2 potency at the high-affinity hIL2 receptor is possible and may be acceptable in the modified hIL2 proteins described in the specification, but such a decrease may not be quantifiable by the methods described in the specification due to limitations in the cell proliferation assay conditions.

[0052] In addition, the modified hIL2 protein, for example, in the hIL2-dependent cell proliferation assay described in the specification, in the EC 50In the comparison of values, as compared to unmodified hIL2, there may be a decrease in potency against the intermediate affinity IL2 receptor (hIL2Rβγ) of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold or at least about 10,000-fold. In some embodiments, the modified hIL2 protein may show a decrease in potency against the intermediate affinity IL2 receptor (hIL2Rβγ) of more than about 10,000-fold as compared to unmodified hIL2.

[0053] The modified hIL2 protein, for example, has an EC in the hIL2-dependent cell proliferation assay described in the specification 50 In the comparison of values, as compared to unmodified hIL2, there may be a decrease in potency against the high affinity IL2 receptor (hIL2Rαβγ) of up to about 10,000-fold and a decrease in potency against the intermediate affinity IL2 receptor (hIL2Rβγ) of up to about 10,000-fold. The modified hIL2 protein may show a decrease in potency against the high affinity IL2 receptor (hIL2Rαβγ) of more than about 10,000-fold and a decrease in potency against the intermediate affinity IL2 receptor (hIL2Rβγ) of more than about 10,000-fold as compared to unmodified hIL2.

[0054] As exemplified in the specification, the modified hIL2 protein can be fused with an anti-PD1 antibody or an antigen-binding fragment thereof. The hIL2 protein can be fused with an anti-PD1 antibody or an antigen-binding fragment thereof at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment or the C-terminus of the antigen-binding fragment. In some embodiments, the modified hIL2 protein is directly fused with an anti-PD1 antibody or an antigen-binding fragment thereof by a peptide bond. The modified hIL2 protein can be directly fused, for example, by a peptide bond with the C-terminal amino acid residue of the anti-PD1 antibody heavy chain. In some embodiments, the modified hIL2 protein is fused with an anti-PD1 antibody or an antigen-binding fragment thereof via a linker.

[0055] The fusion of a modified hIL2 protein with an antibody or an antigen-binding fragment thereof can restore the ability of the modified hIL2 protein to bind to and activate the human intermediate-affinity IL2 receptor on PD1-expressing cells, particularly T cells such as tumor-infiltrating lymphocytes. In some embodiments, the hIL2 protein fused with an antibody or an antigen-binding fragment thereof exhibits an efficacy equivalent to that of wild-type hIL2 against the intermediate-affinity IL2 receptor on PD1-expressing cells.

[0056] The fusion of a modified hIL2 protein with an antibody or an antigen-binding fragment thereof can be used to selectively transmit the signal of IL2 to cells expressing PD1, which is the target of the antibody or an antigen-binding fragment thereof. Without being bound by theory, it is believed that by targeting a specific cell population with the modified hIL2 protein, the therapeutic effect of IL2 (e.g., anti-tumor immunity) can be dramatically amplified without causing off-target systemic toxicity.

[0057] This specification also discloses a modified human interleukin 2 (hIL2) protein comprising a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E substitution at position 20 and an R38E substitution at position 38 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345.

[0058] The modified hIL2 protein can comprise any of the amino acid sequences shown in SEQ ID NO: 307, 607 - 611, 614, 617 or 620. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 307. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 607. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 608. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 609. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 610. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 611. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 614. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 617. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 620. The modified IL2 protein having the amino acid sequence shown in any of SEQ ID NO: 307, 607 - 611, 614, 617 or 620 can further comprise a T3A substitution and / or a C125A substitution. In some embodiments, the modified IL2 protein having the amino acid sequence shown in any of SEQ ID NO: 307, 607 - 611, 614, 617 or 620 further comprises a T3A substitution. In some embodiments, the modified IL2 protein having the amino acid sequence shown in any of SEQ ID NO: 307, 607 - 611, 614, 617 or 620 further comprises a C125A substitution. In some embodiments, the modified IL2 protein having the amino acid sequence shown in any of SEQ ID NO: 307, 607 - 611, 614, 617 or 620 further comprises a T3A substitution and a C125A substitution.

[0059] The modified hIL2 protein may comprise a D20A substitution and an R38E substitution. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 149.

[0060] The modified hIL2 protein can further include a substitution at position 3 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345. Suitable substitutions include, for example, T3A. In some embodiments, the modified hIL2 protein includes a T3A substitution, a D20A substitution, and an R38E substitution. In some aspects, the modified hIL2 protein includes the amino acid sequence shown in SEQ ID NO: 216.

[0061] Alternatively, the modified hIL2 protein can further include a deletion at position 3 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345. In some embodiments, the modified hIL2 protein includes a deletion of amino acids 1-3, a D20A substitution, and an R38E substitution. In some aspects, the modified hIL2 protein includes the amino acid sequence shown in SEQ ID NO: 218.

[0062] The modified hIL2 protein can further include a deletion or substitution at position 125 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345. The substitution at position 125 can be C125A. In some embodiments, the modified hIL2 protein includes a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL2 protein includes the amino acid sequence shown in SEQ ID NO: 215. In some embodiments, the modified hIL2 protein includes a T3A substitution, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL2 protein includes the amino acid sequence shown in SEQ ID NO: 217. In some embodiments, the modified hIL2 protein includes a deletion of amino acids 1-3, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL2 protein includes the amino acid sequence shown in SEQ ID NO: 219.

[0063] The modified hIL2 protein, for example, has an EC in the hIL2-dependent cell proliferation assay described in the specification 50In the comparison of values, there may be a decrease in potency against the high-affinity IL2 receptor (hIL2Rαβγ) of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold or at least about 10,000-fold compared to unmodified hIL2. In some embodiments, the modified hIL2 protein may show a decrease in potency against the high-affinity IL2 receptor (hIL2Rαβγ) of more than about 10,000-fold compared to unmodified hIL2. A greater decrease in hIL2 potency at the high-affinity hIL2 receptor is possible and may be acceptable in the modified hIL2 proteins described herein, but such a decrease may not be quantifiable by the methods described herein due to limitations in cell proliferation assay conditions.

[0064] In addition, the modified hIL2 protein may have an EC 50 In the comparison of values, there may be a decrease in potency against the intermediate-affinity IL2 receptor (hIL2Rβγ) of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold or at least about 10,000-fold compared to unmodified hIL2. In some embodiments, the modified hIL2 protein may show a decrease in potency against the intermediate-affinity IL2 receptor (hIL2Rβγ) of more than about 10,000-fold compared to unmodified hIL2.

[0065] The modified hIL2 protein may have an EC 50In the comparison of values, there may be a decrease in efficacy of up to about 10,000-fold against the high-affinity IL2 receptor (hIL2Rαβγ) and a decrease in efficacy of up to about 10,000-fold against the medium-affinity IL2 receptor (hIL2Rβγ) compared to unmodified hIL2. The modified hIL2 protein may show a decrease in efficacy of more than about 10,000-fold against the high-affinity IL2 receptor (hIL2Rαβγ) and a decrease in efficacy of more than about 10,000-fold against the medium-affinity IL2 receptor (hIL2Rβγ) compared to unmodified hIL2.

[0066] As exemplified in the specification, the modified hIL2 protein can be fused with an anti-PD1 antibody or an antigen-binding fragment thereof. The hIL2 protein can be fused with an anti-PD1 antibody or an antigen-binding fragment thereof at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment. In some embodiments, the modified hIL2 protein is directly fused to an anti-PD1 antibody or an antigen-binding fragment thereof by a peptide bond. The modified hIL2 protein can be directly fused, for example, by a peptide bond to the C-terminal amino acid residue of the anti-PD1 antibody heavy chain. In some embodiments, the modified hIL2 protein is fused to an anti-PD1 antibody or an antigen-binding fragment thereof via a linker.

[0067] The fusion of the modified hIL2 protein with an antibody or an antigen-binding fragment thereof can restore the ability of the modified hIL2 protein to bind to and activate the human medium-affinity IL2 receptor on PD1-expressing cells, particularly T cells such as tumor-infiltrating lymphocytes. In some embodiments, the hIL2 protein fused to an antibody or an antigen-binding fragment thereof exhibits an efficacy equivalent to that of wild-type hIL2 against the medium-affinity IL2 receptor on PD1-expressing cells.

[0068] The fusion of a modified hIL2 protein with an antibody or an antigen-binding fragment thereof can be used to selectively transmit the signal of IL2 to cells expressing the PD1 target of the antibody or an antigen-binding fragment thereof. Without being bound by theory, it is believed that by targeting specific cell populations with the modified hIL2 protein, the therapeutic effect of IL2 (e.g., anti-tumor immunity) can be dramatically amplified without causing off-target systemic toxicity.

[0069] Human anti-human programmed cell death protein 1 (hPD1) antibody This specification discloses a human antibody molecule or an antigen-binding fragment thereof that immunospecifically binds to hPD1, and the human antibody molecule or an antigen-binding fragment thereof a) a heavy chain complementarity-determining region (CDR1) comprising the amino acid sequence shown in SEQ ID NO: 418, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 419, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 420, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 421, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 422, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 423; b) a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 386, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 387, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 388, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 389, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 390, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 391; c) a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 396, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 397, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 398, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 399, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 400, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 401; or, d) a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 406, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 407, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 408, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 409, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 410, and SEQ ID NO: 411; comprising.

[0070] In some embodiments, the human antibody molecule or antigen-binding fragment thereof comprises a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 418, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 419, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 420, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 421, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 422, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 423 (referred to herein as "H7-632").

[0071] In some embodiments, the human antibody molecule or antigen-binding fragment thereof comprises a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 386, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 387, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 388, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 389, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 390, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 391 (referred to herein as "2H7").

[0072] In some embodiments, the human antibody molecule or antigen-binding fragment thereof comprises a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 396, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 397, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 398, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 399, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 400, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 401 (referred to herein as "C51E6-5").

[0073] In one aspect, a human antibody molecule or an antigen-binding fragment thereof comprises a heavy-chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 406, a heavy-chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 407, a heavy-chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 408, a light-chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 409, a light-chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 410, and a light-chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 411 (referred to herein as "A2").

[0074] The disclosed human antibody molecule or an antigen-binding fragment thereof has the following activities: · Binding to PD1 that does not inhibit the binding of PDL1 to PD1; · Binding to PD1 in the presence of standard therapeutic anti-PD1 antibodies used clinically (e.g., KEYTRUDA® and OPDIVO®); · Highly selective for PD1 and does not immunospecifically bind to other related B7 family members; · Binding to PD1 on human activated T cells (EC by flow cytometry binding assay 50 is about 0.1 - 0.2 nM); and can exhibit one or more of the above.

[0075] A human antibody molecule or an antigen-binding fragment thereof can comprise a heavy-chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 416 and a light-chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 417 (referred to herein as "H7-632").

[0076] A human antibody molecule or an antigen-binding fragment thereof can comprise a heavy-chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 384 and a light-chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 385 (referred to herein as "2H7").

[0077] A human antibody molecule or an antigen-binding fragment thereof can comprise a heavy-chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 394 and a light-chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 395 (referred to herein as "C51E6-5").

[0078] A human antibody molecule or an antigen-binding fragment thereof can comprise a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 404 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 405 (referred to herein as "A2").

[0079] A human antibody molecule or an antigen-binding fragment thereof can comprise a heavy chain constant region of IgG1.

[0080] A human antibody molecule or an antigen-binding fragment thereof can have substitutions or deletions in the constant region to minimize Fc-mediated immune effector functions such as antibody-dependent cell cytotoxicity by FcγRIIIA, antibody-dependent cell phagocytosis dependent on FcγRI and FcγRIIa, and complement-dependent cell cytotoxicity by C1q binding. In some embodiments, the human antibody molecule comprises an L235A substitution according to EU numbering of the amino acids. In some embodiments, the human antibody molecule comprises a G237A substitution according to EU numbering of the amino acids. In some embodiments, the human antibody molecule comprises L235A and G237A substitutions according to EU numbering of the amino acids.

[0081] A human antibody molecule or an antigen-binding fragment thereof can comprise a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 414 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 415 (referred to herein as "H7-632-hIgG1-LAGA").

[0082] A human antibody molecule or an antigen-binding fragment thereof can comprise a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 424 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 425 (referred to herein as "2H7-hIgG4").

[0083] A human antibody molecule or an antigen-binding fragment thereof can comprise a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 426 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 427 (referred to herein as "C51E6-5-hIgG4").

[0084] A human antibody molecule or an antigen-binding fragment thereof can comprise a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 428 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 429 (referred to herein as "A2-hIgG4").

[0085] A human antibody molecule or an antigen-binding fragment thereof can be fused to a modified hIL2 protein comprising substitutions at positions 20 and 38 of the amino acid sequence of unmodified IL2 set forth in SEQ ID NO: 345. A human antibody molecule or an antigen-binding fragment thereof can be fused to a modified hIL2 protein disclosed herein.

[0086] When not fused to an antibody molecule or an antigen-binding fragment thereof, the modified hIL2 protein may exhibit a decrease in potency against both the high-affinity hIL2 receptor and the intermediate-affinity hIL2 receptor as compared to unmodified hIL2.

[0087] Suitable substitutions at position 20 of the modified hIL2 are, for example, any of D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E.

[0088] Suitable substitutions at position 38 of the modified hIL2 protein are, for example, any of R38E, R38N, R38G, R38H, R38I, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D or R38K.

[0089] In some embodiments, any of the D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E substitutions can be combined with the R38E substitution.

[0090] The modified hIL2 protein can comprise any of the amino acid sequences shown in SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617 or 620. The modified hIL2 protein can comprise any of the amino acid sequences shown in SEQ ID NOs: 134-150, 307, 344, 608, 611, 614 or 620. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 134. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 135. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 136. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 137. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 138. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 139. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 140. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 141. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 142. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 143. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 144. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 145. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 146. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 147. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 148. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 149. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 150. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 307. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 344. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 607. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 608.In some embodiments, the modified hIL2 protein comprises the amino acid sequence set forth in SEQ ID NO: 609. In some embodiments, the modified hIL2 protein comprises the amino acid sequence set forth in SEQ ID NO: 610. In some embodiments, the modified hIL2 protein comprises the amino acid sequence set forth in SEQ ID NO: 611. In some embodiments, the modified hIL2 protein comprises the amino acid sequence set forth in SEQ ID NO: 614. In some embodiments, the modified hIL2 protein comprises the amino acid sequence set forth in SEQ ID NO: 617. In some embodiments, the modified hIL2 protein comprises the amino acid sequence set forth in SEQ ID NO: 620. The modified IL2 protein having the amino acid sequence set forth in any of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617 or 620 can further comprise a T3A substitution and / or a C125A substitution. In some embodiments, the modified IL2 protein having the amino acid sequence set forth in any of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617 or 620 further comprises a T3A substitution. In some embodiments, the modified IL2 protein having the amino acid sequence set forth in any of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617 or 620 further comprises a C125A substitution. In some embodiments, the modified IL2 protein having the amino acid sequence set forth in any of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617 or 620 further comprises a T3A substitution and a C125A substitution.

[0091] The modified hIL2 protein can comprise a D20A substitution and an R38E substitution.

[0092] The modified hIL2 protein can further comprise a substitution at position 3 of the amino acid sequence of unmodified IL2 set forth in SEQ ID NO: 345. Suitable substitutions include, for example, T3A. In some embodiments, the modified hIL2 protein comprises a T3A substitution, a D20A substitution and an R38E substitution. In some aspects, the modified hIL2 protein comprises the amino acid sequence set forth in SEQ ID NO: 216.

[0093] Alternatively, the modified hIL2 protein can further include a deletion at position 3 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345. In some embodiments, the modified hIL2 protein includes a deletion of amino acids 1-3, a D20A substitution, and an R38E substitution. In some aspects, the modified hIL2 protein includes the amino acid sequence shown in SEQ ID NO: 218.

[0094] The modified hIL2 protein can further include a deletion or substitution at position 125 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345. The substitution at position 125 can be C125A. In some embodiments, the modified hIL2 protein includes a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL2 protein includes the amino acid sequence shown in SEQ ID NO: 215. In some embodiments, the modified hIL2 protein includes a T3A substitution, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL2 protein includes the amino acid sequence shown in SEQ ID NO: 217. In some embodiments, the modified hIL2 protein includes a deletion of amino acids 1-3, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL2 protein includes the amino acid sequence shown in SEQ ID NO: 219.

[0095] When the modified hIL2 protein does not fuse with a human antibody molecule or an antigen-binding fragment thereof, for example, the EC in the hIL2-dependent cell proliferation assay described in the specification 50In value comparisons, when compared to unmodified hIL2, there may be a decrease in potency against high-affinity IL2 receptor (hIL2Rαβγ) of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold. In some embodiments, when the modified hIL2 protein is not fused to a human antibody molecule or an antigen-binding fragment thereof, there may be a decrease in potency against high-affinity IL2 receptor (hIL2Rαβγ) of more than about 10,000-fold compared to unmodified hIL2. A greater decrease in hIL2 potency at the high-affinity hIL2 receptor is possible and may be acceptable in the modified hIL2 proteins described herein, but such a decrease may not be quantifiable by the methods described herein due to limitations in cell proliferation assay conditions.

[0096] In addition, when the modified hIL2 protein is not fused to a human antibody molecule or an antigen-binding fragment thereof, for example, the EC in the hIL2-dependent cell proliferation assay described herein 50 In value comparisons, when compared to unmodified hIL2, there may be a decrease in potency against intermediate-affinity IL2 receptor (hIL2Rβγ) of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold. In some embodiments, when the modified hIL2 is not fused to a human antibody molecule or an antigen-binding fragment thereof, there may be a decrease in potency against intermediate-affinity IL2 receptor (hIL2Rβγ) of more than about 10,000-fold compared to unmodified hIL2.

[0097] When the modified hIL2 protein is not fused to a human antibody molecule or an antigen-binding fragment thereof, for example, the EC in the hIL2-dependent cell proliferation assay described herein 50In value comparison, compared with unmodified hIL2, there may be a reduction in potency against the high-affinity IL2 receptor (hIL2Rαβγ) by up to about 10,000-fold and a reduction in potency against the medium-affinity IL2 receptor (hIL2Rβγ) by up to about 10,000-fold. When the modified hIL2 protein is not fused with a human antibody molecule or its antigen-binding fragment, compared with unmodified hIL2, there may be a reduction in potency against the high-affinity IL2 receptor (hIL2Rαβγ) exceeding about 10,000-fold, and a reduction in potency against the medium-affinity IL2 receptor (hIL2Rβγ) exceeding about 10,000-fold.

[0098] The fusion of the modified hIL2 protein with an antibody or its antigen-binding fragment can restore the ability of the modified hIL2 protein to bind to and activate the human medium-affinity IL2 receptor on PD1-expressing cells, especially T cells such as tumor-infiltrating lymphocytes. In some embodiments, the hIL2 protein fused with an antibody or its antigen-binding fragment exhibits potency equivalent to that of wild-type hIL2 against the medium-affinity IL2 receptor on PD1-expressing cells.

[0099] The modified hIL2 protein can be fused with a human antibody molecule or its antigen-binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment. In some embodiments, the hIL2 protein is directly fused to the antibody or its antigen-binding fragment by a peptide bond. hIL2 can be directly fused, for example, to the C-terminal amino acid residue of the antibody heavy chain by a peptide bond. In some embodiments, the hIL2 protein is fused to the antibody or its antigen-binding fragment via a linker.

[0100] The fusion of a human antibody molecule or its antigen-binding fragment with the modified hIL2 protein can be used to selectively transmit the signal of IL2 to cells expressing PD1. Without being bound by theory, it is believed that targeting a specific cell population expressing PD1 with the modified hIL2 protein can dramatically amplify the therapeutic effect of IL2 (e.g., anti-tumor immunity) while reducing or minimizing off-target systemic toxicity.

[0101] Immune complex Disclosed herein is an immune complex comprising the modified hIL2 protein disclosed in this specification and the human antibody molecule or antigen-binding fragment thereof disclosed in this specification. The immune complex is (a) a modified hIL2 protein comprising substitutions at position 20 and position 38 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345; and (b) a human antibody molecule or antigen-binding fragment thereof that binds immunospecifically to hPD1, wherein the human antibody molecule or antigen-binding fragment thereof (i) a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 418, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 419, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 420, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 421, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 422, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 423; (ii) a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 386, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 387, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 388, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 389, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 390, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 391; (iii) a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 396, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 397, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 398, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 399, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 400, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 401; or (iv) a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 406, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 407, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 408, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 409, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 410, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 411; It can include.

[0102] A suitable substitution at position 20 of the modified hIL2 portion of the immune complex is, for example, any of D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E.

[0103] A suitable substitution at position 38 of the modified hIL2 portion of the immune complex is, for example, any of R38E, R38N, R38G, R38H, R38I, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D or R38K.

[0104] In some embodiments, any of the D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E substitutions can be combined with the R38E substitution.

[0105] The modified hIL2 protein portion of the immune complex can comprise any of the amino acid sequences shown in SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620. The modified hIL2 protein portion of the immune complex can comprise any of the amino acid sequences shown in SEQ ID NOs: 134-150, 307, 344, 608, 611, 614, or 620. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 134. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 135. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 136. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 137. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 138. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 139. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 140. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 141. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 142. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 143. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 144. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 145. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 146. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 147. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 148. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 149. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 150. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 307. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 344. In some embodiments, the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 607.In some embodiments, the modified hIL2 protein comprises the amino acid sequence set forth in SEQ ID NO: 608. In some embodiments, the modified hIL2 protein comprises the amino acid sequence set forth in SEQ ID NO: 609. In some embodiments, the modified hIL2 protein comprises the amino acid sequence set forth in SEQ ID NO: 610. In some embodiments, the modified hIL2 protein comprises the amino acid sequence set forth in SEQ ID NO: 611. In some embodiments, the modified hIL2 protein comprises the amino acid sequence set forth in SEQ ID NO: 614. In some embodiments, the modified hIL2 protein comprises the amino acid sequence set forth in SEQ ID NO: 617. In some embodiments, the modified hIL2 protein comprises the amino acid sequence set forth in SEQ ID NO: 620. The modified IL2 protein having the amino acid sequence set forth in any of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617 or 620 can further comprise a T3A substitution and / or a C125A substitution. In some embodiments, the modified IL2 protein having the amino acid sequence set forth in any of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617 or 620 further comprises a T3A substitution. In some embodiments, the modified IL2 protein having the amino acid sequence set forth in any of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617 or 620 further comprises a C125A substitution. In some embodiments, the modified IL2 protein having the amino acid sequence set forth in any of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617 or 620 further comprises a T3A substitution and a C125A substitution.

[0106] The modified hIL2 protein portion of the immune complex can comprise a D20A substitution and an R38E substitution.

[0107] The modified hIL2 protein portion of the immune complex can further comprise a substitution at position 3 of the amino acid sequence of unmodified IL2 set forth in SEQ ID NO: 345. Suitable substitutions include, for example, T3A. In some embodiments, the modified hIL2 protein portion of the immune complex comprises a T3A substitution, a D20A substitution and an R38E substitution. In some aspects, the modified hIL2 protein portion of the immune complex comprises the amino acid sequence set forth in SEQ ID NO: 216.

[0108] Alternatively, the modified hIL2 protein moiety of the immune complex can further include a deletion at position 3 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345. In some embodiments, the modified hIL2 protein moiety of the immune complex includes a deletion of amino acids 1-3, a D20A substitution, and an R38E substitution. In some aspects, the modified hIL2 protein moiety of the immune complex includes the amino acid sequence shown in SEQ ID NO: 218.

[0109] The modified hIL2 protein moiety of the immune complex can further include a deletion or substitution at position 125 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345. The substitution at position 125 can be C125A. In some embodiments, the modified hIL2 protein moiety of the immune complex includes a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL2 protein moiety of the immune complex includes the amino acid sequence shown in SEQ ID NO: 215. In some embodiments, the modified hIL2 protein moiety of the immune complex includes a T3A substitution, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL2 protein moiety of the immune complex includes the amino acid sequence shown in SEQ ID NO: 217. In some embodiments, the modified hIL2 protein moiety of the immune complex includes a deletion of amino acids 1-3, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL2 protein moiety of the immune complex includes the amino acid sequence shown in SEQ ID NO: 219.

[0110] The modified hIL2 protein moiety of the immune complex, for example, has an EC in the hIL2-dependent cell proliferation assay described in the specification 50In the comparison of values, as compared to unmodified hIL2, there may be a decrease in potency against the high-affinity IL2 receptor (hIL2Rαβγ) by at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold. In some embodiments, the modified hIL2 protein moiety of the immune complex may show a decrease in potency against the high-affinity IL2 receptor (hIL2Rαβγ) that is greater than about 10,000-fold as compared to unmodified hIL2. A greater decrease in hIL2 potency at the high-affinity hIL2 receptor is possible and may be acceptable in the modified hIL2 proteins described in the specification, but such a decrease may not be quantifiable by the methods described in the specification due to limitations in the cell proliferation assay conditions.

[0111] In addition, the modified hIL2 protein moiety of the immune complex, for example, the EC in the hIL2-dependent cell proliferation assay described in the specification 50 In the comparison of values, as compared to unmodified hIL2, there may be a decrease in potency against the intermediate-affinity IL2 receptor (hIL2Rβγ) by at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold. In some embodiments, the modified hIL2 protein moiety of the immune complex may show a decrease in potency against the intermediate-affinity IL2 receptor (hIL2Rβγ) that is greater than about 10,000-fold as compared to unmodified hIL2.

[0112] The modified hIL2 protein moiety of the immune complex, for example, the EC in the hIL2-dependent cell proliferation assay described in the specification 50In the comparison of values, compared with unmodified hIL2, there may be a decrease in potency against the high-affinity IL2 receptor (hIL2Rαβγ) up to about 10,000-fold and a decrease in potency against the medium-affinity IL2 receptor (hIL2Rβγ) up to about 10,000-fold. The modified hIL2 protein moiety of the immune complex may show a decrease in potency against the high-affinity IL2 receptor (hIL2Rαβγ) exceeding about 10,000-fold and a decrease in potency against the medium-affinity IL2 receptor (hIL2Rβγ) exceeding about 10,000-fold compared with unmodified hIL2.

[0113] The hIL2 protein moiety of the immune complex can be fused with an antibody or its antigen-binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment. In some embodiments, the hIL2 protein moiety of the immune complex is directly fused with a human antibody molecule or its antigen-binding fragment by a peptide bond. The hIL2 protein moiety of the immune complex can be directly fused, for example, by a peptide bond with the C-terminal amino acid residue of the antibody heavy chain. In some embodiments, the hIL2 protein moiety of the immune complex is fused with a human antibody molecule or its antigen-binding fragment via a linker.

[0114] The fusion of the modified hIL2 protein with a human antibody molecule or its antigen-binding fragment can restore the ability of the modified hLI2 protein to activate the medium-affinity IL2 receptor. In some embodiments, the immune complex can activate the medium-affinity IL2 receptor to an extent equivalent to the activation of the medium-affinity IL2 receptor by wild-type hIL2.

[0115] In some embodiments, a portion of the human antibody molecule or its antigen-binding fragment, the immune complex, comprises a heavy-chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 418, a heavy-chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 419, a heavy-chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 420, a light-chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 421, a light-chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 422, and a light-chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 423.

[0116] In some embodiments, a human antibody molecule or an antigen-binding fragment thereof, a portion of an immune complex, comprises a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 386, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 387, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 388, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 389, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 390, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 391.

[0117] In some embodiments, a human antibody molecule or an antigen-binding fragment thereof, a portion of an immune complex, comprises a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 396, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 397, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 398, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 399, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 400, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 401.

[0118] In some embodiments, a human antibody molecule or an antigen-binding fragment thereof, a portion of an immune complex, comprises a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 406, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 407, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 408, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 409, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 410, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 411.

[0119] A human antibody molecule or an antigen-binding fragment thereof, a portion of an immune complex, can comprise a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 416 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 417.

[0120] A human antibody molecule or an antigen-binding fragment thereof, a portion of an immune complex, can comprise a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 384 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 385.

[0121] A human antibody molecule or an antigen-binding fragment thereof, a part of an immune complex, can include a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 394 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 395.

[0122] A human antibody molecule or an antigen-binding fragment thereof, a part of an immune complex, can include a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 404 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 405.

[0123] A human antibody molecule or an antigen-binding fragment thereof, a part of an immune complex, can include the heavy chain constant region of IgG1.

[0124] A human antibody molecule or an antigen-binding fragment thereof, a part of an immune complex, can have substitutions or deletions in the constant region to minimize Fc-mediated immune effector functions such as antibody-dependent cell cytotoxicity by FcγRIIIA, antibody-dependent cell phagocytosis dependent on FcγRI- and FcγRIIa, and complement-dependent cell cytotoxicity by C1q binding. In some embodiments, the human antibody molecule part of the immune complex includes an L235A substitution with EU numbering of amino acids. In some embodiments, the human antibody molecule part of the immune complex includes a G237A substitution with EU numbering of amino acids. In some embodiments, the human antibody molecule part of the immune complex includes L235A and G237A substitutions with EU numbering of amino acids.

[0125] A human antibody molecule or an antigen-binding fragment thereof, a part of an immune complex, can include a heavy chain containing the amino acid sequence shown in SEQ ID NO: 414 and a light chain containing the amino acid sequence shown in SEQ ID NO: 415.

[0126] A human antibody molecule or an antigen-binding fragment thereof, a part of an immune complex, can include a heavy chain containing the amino acid sequence shown in SEQ ID NO: 424 and a light chain containing the amino acid sequence shown in SEQ ID NO: 425.

[0127] A human antibody molecule or an antigen-binding fragment thereof, a part of the immune complex, can comprise a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 426 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 427.

[0128] A human antibody molecule or an antigen-binding fragment thereof, a part of the immune complex, can comprise a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 428 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 429.

[0129] The immune complex has the following characteristics: · Binding to PD1 that does not inhibit the binding of PDL1 to PD1; · Binding to PD1 in the presence of standard therapeutic anti-PD1 antibodies used clinically (e.g., Keytruda® and Opdivo®); · Highly selective for PD1 and does not bind immunospecifically to other related members of the B7 family; · Binding to PD1 on human activated T cells (EC by flow cytometry binding assay 50 is about 0.1 - 0.2 nM); · In the comparison of EC 50 values in, for example, the hIL2-dependent cell proliferation assay described in the specification, at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold reduction in potency against the high-affinity IL2 receptor (hIL2Rαβγ) compared to unmodified hIL2; can have one or more of the above. In some embodiments, the modified hIL2 protein portion of the immune complex may exhibit a reduction in potency against the high-affinity IL2 receptor (hIL2Rαβγ) of more than about 10,000-fold compared to unmodified hIL2. A greater reduction in hIL2 potency at the high-affinity hIL2 receptor is possible and may be acceptable in the modified hIL2 proteins described in the specification, but such a decrease is subject to the limitations of the cell proliferation assay conditions; · For example, EC in the hIL2-dependent cell proliferation assay described in the specification 50In the comparison of values, there is a decrease in potency against the intermediate affinity IL2 receptor (hIL2Rβγ) of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold or at least about 10,000-fold as compared to unmodified hIL2. Therefore, it may not be quantifiable by the methods described in the specification. In some embodiments, the modified hIL2 protein moiety of the immune complex may exhibit a decrease in potency against the intermediate affinity IL2 receptor (hIL2Rβγ) of more than about 10,000-fold as compared to unmodified hIL2; · Recovery and increase of the subset of human memory T cells expressing PD1 in GvHD model animals · Minimal or no effect on body weight, blood chemistry or hematological parameters after single administration of 1 mg / kg and 10 mg / kg to cynomolgus monkeys.

[0130] In some embodiments, the immune complex comprises a modified hLI2 protein comprising T3A substitution, R38E substitution, D20A substitution and C125A substitution, fused to the C-terminus of the antibody heavy chain of a human anti-hPD1 antibody comprising a human IgG1 framework with L235A substitution and G237A substitution. In some embodiments, the immune complex comprises a light chain comprising the amino acid sequence shown in SEQ ID NO: 415 and a heavy chain-hIL2 protein fusion comprising the amino acid sequence shown in SEQ ID NO: 532.

[0131] The disclosed immune complex can selectively transmit the signal of IL2 to PD1-expressing T cells. A human antibody molecule or an antigen-binding fragment thereof, which is a part of the immune complex, is simply used to deliver the modified hIL2 to cells expressing PD1, and does not block the function of the PD1 receptor like conventional anti-PD1 inhibitory antibodies such as Opdivo (registered trademark) and Keytruda (registered trademark). The main mechanism of action of the immune complex disclosed in the specification is due to the T cell-selective activity of IL2. The human PD1 receptor is mainly expressed in a small subset of T cells with strong tumor reactivity. Without being bound by theory, targeting this T cell population with the modified hIL2 protein portion of the immune complex is thought to be able to dramatically amplify anti-tumor immunity while reducing or minimizing off-target systemic IL2 toxicity by the cell population lacking PD1 expression.

[0132] Pharmaceutical Compositions, Polynucleotides, Vectors, and Cells Disclosed herein are pharmaceutical compositions comprising the modified hIL2 protein disclosed in this specification, a human antibody molecule or an antigen-binding fragment thereof disclosed in this specification, or the immune complex disclosed in this specification. In some embodiments, the pharmaceutical composition comprises the modified hIL2 protein disclosed in this specification. In some embodiments, the pharmaceutical composition comprises a human antibody molecule or an antigen-binding fragment thereof disclosed in this specification. In some embodiments, the pharmaceutical composition comprises the immune complex disclosed in this specification.

[0133] Disclosed herein are polynucleotides comprising nucleic acid sequences encoding the modified hIL2 protein disclosed in this specification, a human antibody molecule or an antigen-binding fragment thereof or the immune complex disclosed in this specification. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding the modified hIL2 protein disclosed in this specification. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a human antibody molecule or an antigen-binding fragment thereof disclosed in this specification. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding the immune complex disclosed in this specification.

[0134] Disclosed herein is a vector comprising a polynucleotide comprising a nucleic acid sequence encoding a modified hIL2 protein disclosed herein, a human antibody molecule or an antigen-binding fragment thereof disclosed herein, or an immune complex disclosed herein. In some embodiments, the vector comprises a polynucleotide comprising a nucleic acid sequence encoding a modified hIL2 protein disclosed herein. In some embodiments, the vector comprises a polynucleotide comprising a nucleic acid sequence encoding a human antibody molecule or an antigen-binding fragment thereof disclosed herein. In some embodiments, the vector comprises a polynucleotide comprising a nucleic acid sequence encoding an immune complex disclosed herein.

[0135] Disclosed herein are also transformed cells comprising the vector disclosed herein.

[0136] Therapeutic Methods and Uses Disclosed herein is a method for treating a disease or disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of an immune complex or pharmaceutical composition disclosed herein to treat the disease.

[0137] Also disclosed is the use of an immune complex or pharmaceutical composition disclosed herein in the manufacture of a medicament for treating a disease. Also disclosed is the use of an immune complex or pharmaceutical composition disclosed herein for treating a disease or disorder.

[0138] The disclosed immune complexes and pharmaceutical compositions can be used to treat diseases or disorders in which it is beneficial to stimulate the immune system of a subject. In some embodiments, the immune response of the subject is insufficient, and the disclosed immune complexes and pharmaceutical compositions stimulate the immune response of the subject. The antibody portion of the immune complex can serve to direct the modified hIL2 protein to the subject's immune cells, for example, by binding to an antigen expressed on the surface of immune cells. In the disclosed immune complex of modified hLI2 protein-human anti-hPD1 antibody, for example, the anti-PD1 antibody (or antigen-binding fragment thereof) portion of the immune complex can bind to PD1 expressed on T cells, thereby delivering the modified hIL2 protein to the T cells. By targeting specific cells with the modified IL2 protein, the therapeutic effect of the IL2 protein can be dramatically amplified without causing off-target systemic toxicity by cell populations lacking antigen expression. The disclosed methods and uses can be used, for example, to treat cancer, autoimmune and inflammatory diseases, and chronic infectious and contagious diseases. Representative cancers include bladder cancer, brain tumors, head and neck cancers, pancreatic cancer, lung cancer, non-small cell lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, stomach cancer, prostate cancer, blood cancers, skin cancer, melanoma, squamous cell carcinoma, bone cancer, and kidney cancer. Representative autoimmune and inflammatory diseases include systemic lupus erythematosus (SLE), type 1 diabetes, rheumatoid arthritis, ankylosing spondylitis, psoriasis, Behcet's disease, granulomatosis with polyangiitis, Takayasu's disease, Crohn's disease, ulcerative colitis, autoimmune hepatitis, sclerosing cholangitis, Sjogren's syndrome, alopecia areata, and inflammatory myopathy. Representative infectious diseases include HIV and hepatitis B.

[0139] In some embodiments, the disease is cancer. The methods and uses can include administering to the subject a therapeutically effective amount of the modified hLI2 protein-antibody complex disclosed herein to treat the cancer. In some aspects, the cancer is melanoma. In some aspects, the cancer is non-small cell lung cancer.

Examples

[0140] The following examples are provided to further illustrate some of the aspects disclosed in the specification. The examples are intended to exemplify the disclosed aspects and are not intended to be limiting.

[0141] General method Protocol A Flow cytometry screening of the binding of human PD1 to an anti-hPD1 antibody or an anti-hPD1 antibody-attenuated hIL2 fusion For antibodies and antibody-attenuated hIL2 fusion proteins, they were characterized with a full titration curve to test their binding to hPD1. A mammalian vector encoding amino acids 1-185 (SEQ ID NO: 346) of human PD1 was transfected into the Jurkat cell line to stably express a portion of the transmembrane domain and the extracellular domain of human PD1, and this transfected cell line was used to determine the binding of anti-hPD1 antibodies. Jurkat+hPD1 cells were washed and added to a 96-well plate at 100,000 cells / well in FACS buffer (PBS, 0.2% heat-inactivated fetal bovine serum). The cells were blocked for 10 minutes at 4°C with a 1:50 dilution of a human FcR blocking reagent (Miltenyi) and washed with FACS buffer.

[0142] Antibodies or antibody-attenuated hIL2 immune complexes (fusion proteins) were serially diluted 6-fold in FACS buffer for an 8-point curve and added at a volume of 100 μL for 1 hour to Jurkat cells expressing human PD1 on ice. The cells were washed and resuspended in FACS buffer containing a 1:40 dilution of an allophycocyanin-conjugated anti-human IgG Fc monoclonal antibody. The cells were washed again and resuspended in FACS buffer containing a 1:1000 dilution of Sytox Green (Thermo Fisher), and flow cytometry analysis was performed using a BD FACS Canto II, BD Celesta or BD Fortessa (BD Biosciences). The geometric mean fluorescence intensity (gMFI) was calculated with FlowJo software version 10. The half maximal effective concentration (EC 50) The value of was calculated from the geometric mean fluorescence intensity (gMFI) of allophycocyanin over the titrated concentrations using GraphPad Prism 7 software.

[0143] Protocol B Flow Cytometry Competitive Screening of the Binding of Human PD1 with Anti-hPD1 Antibody or Anti-hPD1 Antibody-Attenuated hIL2 Fusion Antibodies and antibody-attenuated hIL2 fusion proteins were tested for their ability to bind to human PD1 in the presence of a saturating concentration of anti-hPD1 #1-mIgG2b-N297A (clone 5C4, formatted on a mouse IgG2b-N297A background, containing the sequences of the variable regions of the heavy and light chains of nivolumab, described in US Patent Application Publication No. 2009 / 0217401) (SEQ ID NOs: 348 and 349) or anti-hPD1 #2-mIgG2b-N297A (clone 109A-H / K09A-L-11, formatted on a mouse IgG2b-N297A background, containing the sequences of the variable regions of the heavy and light chains of pembrolizumab, described in International Publication No. 2008 / 156712) (SEQ ID NOs: 350 and 351).

[0144] Antibodies or antibody-attenuated hIL2 fusion proteins were serially diluted six-fold for 8-point titration curves in the presence and absence of saturating amounts (10 μM) of anti-hPD1 #1-mIgG2b-N297A or anti-hPD1 #2-mIgG2b-N297A. Briefly, Jurkat cells stably expressing hPD1 (described in Protocol A above) were washed and resuspended in FACS buffer containing a 1:50 dilution of the hooking reagent. Cells were incubated at 4°C for 10 minutes and washed. Cells were resuspended in 100 μL of anti-hPD1 #1-mIgG2b-N297A or anti-hPD1 #2-mIgG2b-N287A diluted to 10 μM in FACS buffer and incubated at 4°C for 1 hour. Cells were washed and incubated for 1 hour at 4°C with 100 μL of the antibody or antibody-attenuated hIL2 fusion protein serially diluted six-fold for the 8-point curve. To detect bound anti-hPD1 antibody or anti-hPD1-attenuated hIL2 fusion protein, cells were washed again and incubated on ice for 45 minutes with a 1:40 dilution of allophycocyanin-conjugated anti-human IgG Fc monoclonal antibody. Cells were washed and resuspended in FACS buffer containing a 1:1000 dilution of Sytox Green (Thermo Fisher). For comparison, Jurkat cells stably expressing human PD1 were incubated with only the titrated antibody or antibody-attenuated hIL2 fusion protein (without anti-hPD1 #1-mIgG2b-N297A or anti-hPD1 #2-mIgG2b-N297A), followed by incubation with a 1:40 dilution of allophycocyanin-conjugated anti-human IgG Fc secondary antibody. As controls, the variable regions of anti-hPD1 #1 and anti-hPD1 #2 were cloned into the hIgG4 framework and evaluated with and without the addition of anti-hPD1 #1-mIgG2b-N297A or anti-hPD1 #2-mIgG2b-N297A. Flow cytometry was performed on a BD Canto II, BD Celesta or BD Fortessa (BD Biosciences) flow cytometer, and gMFI was calculated with FlowJo software version 10. EC 50It was calculated from the geometric mean fluorescence intensity (gMFI) of allophycocyanin over the titrated concentrations using GraphPad Prism 7 software.

[0145] Protocol C Cell-based screening to evaluate the properties of an anti-hPD1 non-antagonist antibody or an anti-hPD1 antibody-attenuated hIL2 fusion The ability of human PD1 antibodies and anti-hPD1-attenuated hIL2 fusion proteins to block the binding of hPD1 to its ligand hPDL1 (SEQ ID NO: 584) was revealed. Anti-hPD1 antibodies and anti-hPD1-attenuated hIL2 fusion proteins were characterized as antagonists or non-antagonists using an in vitro cell-based human PD1 / PDL1 inhibition bioassay (Promega, Cat# J1255). In this co-culture assay, two cell lines were utilized: FcγRIIb artificial antigen-presenting cells / Chinese hamster ovary K1 (aAPC / CHO-K1) and Jurkat effector cells. aAPC / CHO-K1 cells stably express both the human PDL1 ligand and a cell surface protein, activate the cognate T cell receptor (TCR), and Jurkat effector cells express hPD1 and a luciferase reporter under the control of the nuclear factor of activated T cells response element (NFAT-RE). When these cells were co-cultured in the presence of a non-antagonist antibody, TCR signaling was inhibited by the hPD1 / hPDL1 interaction and luminescence was not detected. In the presence of an antibody that antagonizes the interaction between hPDL1 (SEQ ID NO: 584) and hPD1, the inhibitory signal was disrupted and luminescence was detected.

[0146] In accordance with the manufacturer's instructions, thaw-and-use assays were performed. Briefly, first, aAPC / CHO-K1 cells were thawed and seeded at 30,000 cells per well in a flat-bottom 96-well plate and cultured in an incubator at 37 °C and 5% CO2 for 18 hours. After the cells adhered, the medium was removed, and antibodies or antibody-attenuated hIL2 fusion proteins at 200 nM or 1000 nM were diluted in 40 μL of assay buffer (RPMI1640 medium + 1% FBS) and added to the aAPC / CHO-K1 cells. As a negative control, a human IgG4 isotype monoclonal antibody targeting keyhole limpet hemocyanin (KLH) clone C3 (SEQ ID NOs: 585 and 586) was used. Jurkat effector cells expressing hPD1 were added at 24,000 cells (40 μL) per well. The final concentration of the immobilized antibody tested was 100 nM or 500 nM. In some examples, this co-culture assay was used to test various concentrations of anti-hPD11 or anti-hPD1-attenuated hIL2 fusion protein, and the highest concentration in the 5-fold titration series was 500 nM (Figure 7).

[0147] Incubate for an additional 18 - 20 hours in a 37 °C, 5% CO2 incubator to continue the co-culture assay. To read the luminescence signal, bring the plate to room temperature and add 80 μL of Bio-Glo™ reagent to each well. Incubate the plate in the dark at room temperature for 15 minutes and read the luminescence with a Victor X illuminometer (Perkin Elmer). Average the relative light units (RLU) of three measurements and graph them using GraphPad Prism 7 software.

[0148] Protocol D In vitro phosphorylated STAT5 assay for testing attenuation of hIL2 variants The attenuation level of the hIL2 receptor activation activity of the antibody-attenuated hIL2 fusion protein was characterized using a phosphorylation STAT5 assay. The variants were tested in both hIL2-responsive human natural killer NK-92 cells and recombinant human erythroleukemia TF1 cells. The NK-92 cell line naturally expresses physiological levels of the high-affinity hIL2 receptor (IL2Rαβγ), whereas the TF1 cell line, which naturally expresses IL2Rγ (SEQ ID NO: 352), was engineered to stably express human CD122 (IL2Rβ) (SEQ ID NO: 353) as well for expression of the intermediate-affinity hIL2 receptor complex (IL2Rβγ). This TF1+IL2Rβ stable cell line does not express IL2Rα (SEQ ID NO: 354). Using both the NK-92 and TF1+IL2Rβ cell lines, the attenuation levels of IL2 potency in these cell-based potency assays were evaluated as fixed-concentration screening and as a complete titration curve.

[0149] To perform fixed-concentration screening, 100,000 NK-92 cells or TF1+IL2Rβ cells were seeded in 96 wells in 50 μL of fresh growth medium without human IL2 cytokine and incubated overnight at 37 °C in a CO2 incubator. After 15 - 16 hours, the human IL2-starved cells were treated with 25.7 nM of recombinant hIL2 (designated rhIL2) (SEQ ID NO: 345) or antibody-attenuated hIL2 fusion protein in the NK-92 cell assay and with 33.3 nM of hIL2 or hIL2 variant in the TF1+IL2Rβ cell assay. The cells were incubated at 37 °C, 5% CO2 for 10 minutes. The cells were fixed with Cytofix buffer (BD Biosciences) at 37 °C for 10 minutes, then treated with Perm buffer III (BD Biosciences) on ice for 30 minutes and permeabilized. The fixed and permeabilized cells were stained with 0.5 μL of Alexa Fluor-647-conjugated anti-Stat5 antibody (BD Biosciences) per sample in the dark at room temperature for 45 minutes, after which hIL2-dependent Stat5 phosphorylation was detected. The cells were washed and the reagents were diluted with BD Pharmingen buffer (BD Biosciences). The stained cells were acquired on a FACS-Celesta cytometer (BD Biosciences) and analyzed with FlowJo software version 10.7.2. The assay was performed in cohorts and normalized per plate using rhIL2. The degree of attenuation of the selected antibody-attenuated hIL2 fusion protein was evaluated for both the NK-92 and TF1+IL2Rβ cell lines with an 8-point, 6-fold serial titration curve from 1200 nM to 7 pM. The pStat5 curve was performed in the same manner as the above method. EC 50 values were calculated from the geometric mean fluorescence intensity (gMFI) across the titrated concentrations using GraphPad Prism 7 software. The fold change in activity from rhIL2 was calculated by dividing the EC 50 value of the variant by the EC 50 of hIL2.

[0150] Protocol E In vitro cell-based proliferation assay to test the attenuation of antibody-attenuated hIL2 fusion protein Antibody-attenuated hIL2 fusion proteins were also tested in an IL2-dependent cell proliferation assay for attenuated hIL2 activity. 10,000 NK-92 cells (expressing the high-affinity receptor hIL2Rαβγ) or TF1+IL2Rβ cells (expressing the intermediate-affinity receptor hIL2Rβγ) suspended in 50 μL of fresh growth medium without hIL2 cytokine were seeded into 96-well U-bottom cell culture plates. An 8-point, 6-fold serial titration of the antibody-attenuated hIL2 fusion protein with a maximum concentration of 996 nM was diluted in fresh medium and overlaid on the cells in the wells. The cells were incubated in a 5% CO2 incubator at 37 °C for 3 days for TF1+IL2Rβ cells and 4 days for NK-92 cells. To measure proliferation, Cell-Titer-Glo (Promega) was added to the wells and incubated at room temperature for 10 minutes, and luminescence was read for 0.1 second per well using a VictorX Multilabel Plate Reader (Perkin Elmer). EC 50 50 was calculated from the relative light units (RLU) over the titrated concentrations using GraphPad Prism 7 software. The fold change in activity from rhIL2 was the EC 50 value of the variant divided by the EC 50 of hIL2. The assay was performed in cohorts but was normalized per plate using the EC 50 of rhIL2

[0151] Example 1 Optimization of antibody-attenuated hIL2 fusion protein variants and determination of hIL2 activity against intermediate- and high-affinity hIL2 receptor complexes To determine the optimal structure of the antibody-attenuated hIL2 fusion protein, unattenuated hIL2 was fused to an anti-DNase I antibody (clone 1H3) (SEQ ID NO: 379, SEQ ID NO: 374), designated 1H3-hIgG, in various ways shown in Figure 1 in the variable region of the antibody. Variants were designated hIL-2 Nterm light chain df (SEQ ID NO: 379, SEQ ID NO: 356) and hIL-2 Nterm heavy chain df (SEQ ID NO: 358, SEQ ID NO: 374), which were hIL2 directly fused (df) to the N-terminus of the heavy or κ light chain of the human anti-DNase I antibody (clone 1H3) immunoglobulin hIgG1, or were designated hIL-2 Nterm light chain L6 fusion (SEQ ID NO: 379, SEQ ID NO: 357) and hIL-2 Nterm heavy chain L6 fusion (SEQ ID NO: 359, SEQ ID NO: 374), which were hIL2 fused via a 6-amino acid linker (L6) (SEQ ID NO: 355). Variants in which the hIL2 portion was fused to the C-terminus of the heavy or light chain via df or L6 were also produced and designated hIL-2 Cterm heavy chain df (SEQ ID NO: 360, SEQ ID NO: 374), hIL-2 Cterm heavy chain L6 fusion (SEQ ID NO: 361, SEQ ID NO: 374), hIL-2 Cterm light chain df (SEQ ID NO: 379, SEQ ID NO: 362), hIL-2 Cterm light chain L6 fusion (SEQ ID NO: 379, SEQ ID NO: 363). Furthermore, variants were produced in which the extracellular domain of CD25 / IL2Rα (amino acids 1-164) (SEQ ID NO: 126) was fused to the N-terminus or C-terminus of the heavy or κ light chain to inhibit the binding of CD25, an IL2 receptor, to IL2 (Figure 2).In these constructs, the human CD25 extracellular domain (amino acids 1-164) (SEQ ID NO: 126) was fused to human IL2 via a 20 amino acid linker (L20) (SEQ ID NO: 364), and then fused directly or via an L6 linker (SEQ ID NO: 355) to the heavy or light chain of 1H3-hIgG1 at the N-terminus: hCD25-L20-hIL-2 Nterm heavy chain df (SEQ ID NO: 365, SEQ ID NO: 374), hCD25-L20-hIL-2 Nterm heavy chain L6 fusion (SEQ ID NO: 366, SEQ ID NO: 374), hCD25-L20-hIL-2 Nterm light chain df (SEQ ID NO: 379, SEQ ID NO: 367), hCD25-L20-hIL-2 Nterm light chain L6 fusion (SEQ ID NO: 379, SEQ ID NO: 368). Finally, a final set of variants was produced with the CD25 / IL2Rα extracellular domain portion (SEQ ID NO: 126) fused to the C-terminus of the heavy chain and κ light chain: hCD25-L20-hIL-2 Cterm heavy chain df (SEQ ID NO: 369, SEQ ID NO: 374), hCD25-L20-hIL-2 Cterm heavy chain L6 fusion (SEQ ID NO: 370, SEQ ID NO: 374), hCD25-L20-hIL-2 Cterm light chain df (SEQ ID NO: 379, SEQ ID NO: 371), hCD25-L20-hIL-2 Cterm light chain L6 fusion (SEQ ID NO: 379, SEQ ID NO: 372). These antibody-hIL2 fusion proteins were produced, expressed, and purified by Protein A using standard techniques. The 16 N- or C-terminal and linker variants were evaluated in an in vitro cell-based phosphorylated STAT5 assay using an 8-point 6-fold serial titration as described in Protocol D.

[0152] EC calculated from an 8-point, 6-fold serial titration curve of the geometric mean fluorescence intensity (gMFI) calculated with FlowJo version 10 software 50 are summarized in Table 1. Also, for each variant, the fold change from rhIL2 was calculated as a measure of the attenuation level compared to the activity of the rhIL2 positive control. Some EC50 The value could not be calculated with GraphPad Prism 7 software and was displayed as "not calculated (NC)"; however, from the dose titration curve, there was no attenuation in these variants.

[0153] When compared with rhIL2 in cell lines expressing high-affinity hIL2 receptor (NK-92) or medium-affinity hIL2 receptor (TF1 + IL2Rβ), the fusion of the hIL2 moiety with the N-terminus or C-terminus of the immunoglobulin heavy chain did not cause a decrease in IL2 activity. When compared with the fusion using a 6-amino acid linker (L6) between IL2 and the antibody component, the direct fusion (df) of hIL2 with the antibody component of the fusion protein did not cause a change in IL2 activity. Similarly, when compared with rhIL2, the fusion of the IL2 component with the heavy or light chain of the antibody component did not cause a change in IL2 activity. All fusion protein variants of the N- or C-terminus and linker with hIL2 and hCD25 / hIL2Rα moiety were predicted to have a decreased binding of the fusion protein to CD25, the hIL2 receptor on the cell surface. In experiments, the hIL2 activity of these constructs was significantly attenuated (at least 45-fold) against the high-affinity IL2 receptor (NK-92) and showed an 18-fold attenuation against the medium-affinity hIL2 receptor (TF1 + IL2Rβ).

[0154] [Table 1]

[0155] Example 2 Production of antibody-attenuated hIL2 fusion protein variants and determination of the binding rate constants to recombinant human CD25 and / or human CD122 Since a decrease in hIL2 activity was not observed in various N-terminal or C-terminal immunoglobulin heavy chain fusion proteins, the hIL-2 Cterm heavy chain L6 fusion (SEQ ID NOs: 361, 374), named "1H3-hIgG1-L6-hIL2", was used as the basic construct for antibody-attenuated hIL2 fusion protein variants with substitutions in the hIL2 portion. To examine the role of residues in the recognition of any of human CD25 / IL2Rα and / or human CD122 / IL2Rβ, or CD132 / IL2Rγ (human IL2R subunits), one and / or more amino acids of human IL2 residues were substituted. Over 300 variants of antibody-attenuated hIL2 fusion proteins with substitutions in the hIL2 portion were produced and evaluated six times. These variants were first screened at a fixed concentration and in a dose titration curve against IL2-dependent cell lines (NK-92 and TF1 + IL2Rβ) by a flow-based phosphorylated STAT5 (pSTAT5) assay. Phosphorylated STAT5 is a signal downstream of IL2 activity and was used as a snapshot measurement of IL2 potency. An IL2-dependent cell proliferation assay was also performed to measure IL2 activity for 3 - 4 days. The criteria for selection of attenuated hIL2 included: (1) a decrease in IL2 potency by more than 50% agonist activity in the NK-92 cell line and the TF1 + IL2Rβ cell line; and (2) moderate to high production yields.

[0156] The human anti-DNase I antibody-hIL2 fusion protein was produced by fusing the C-terminus of the heavy chain of human IL2 or a human IL2 variant (SEQ ID NOs: 1 to 344, 377, 378 and 575) with the human anti-DNase I antibody (clone 1H3, human IgG1 isotype) via an L6 linker, and combining it with the hIgG1 light chain (1H3-hκLC; SEQ ID NO: 374) to produce the 1H3-hIgG1-L6-hIL2 fusion protein (shown in Table 28). Also, the mouse anti-yellow fever virus antibody-hIL2 fusion protein was produced by fusing the C-terminus of the heavy chain of a human IL2 variant with a mouse anti-yellow fever virus antibody (clone 2D12, mouse IgG1 isotype) having a D265A substitution for reducing immune effector function via an L6 linker, and combining it with the 2D12-mIgG1 light chain (2D12-mκLC; SEQ ID NO: 376) to produce the 2D12-mIgG1-D265A-L6-hIL2 fusion protein (shown in Table 28). A part of these mouse anti-yellow fever virus antibody-hIL2 fusion proteins was formatted on the human IgG1 constant region and combined with the 2D12-hκ light chain (2D12-hκLC; SEQ ID NO: 573), and was produced by the same method as described above. The repetition of IL2 amino acid substitution was performed 6 times and named groups 1 to 6. The 1H3-hIgG1-L6-hIL2, 2D12-mIgG1-D265A-L6-hIL2 and 2D12-hIgG1-L6-hIL2 fusion proteins were produced, expressed and purified by protein A using standard techniques.

[0157] Group 1 included only a series of 2D12-mIgG1-D265A-L6-hIL2 or 2D12-hIgG1-L6-hIL2 fusion proteins having substitutions or combinations of substitutions in human IL2 predicted to be involved in binding only to any one of the IL2 receptor subunits CD25 / IL2Rα, CD122 / IL2Rβ, or CD132 / IL2Rγ. The fusion proteins of this group contained the following substitutions in IL2 predicted to modulate binding to CD25 / IL2Rα: F42K (SEQ ID NO: 1), V69A (SEQ ID NO: 2), V69E (SEQ ID NO: 3), V69F (SEQ ID NO: 4), V69G (SEQ ID NO: 5), V69H (SEQ ID NO: 6), V69I (SEQ ID NO: 7), V69K (SEQ ID NO: 8), V69L (SEQ ID NO: 9), V69M (SEQ ID NO: 10), V69Q (SEQ ID NO: 11), V69S (SEQ ID NO: 12), V69T (SEQ ID NO: 13), V69W (SEQ ID NO: 14), V69Y (SEQ ID NO: 15), V69R (SEQ ID NO: 581), (F42K / F44K) (SEQ ID NO: 16), (F44K / Y45R) (SEQ ID NO: 17), (F42K / V69R) (SEQ ID NO: 18), (Y45R / V69R) (SEQ ID NO: 19), (F42K / F44K / Y45R) (SEQ ID NO: 20), (F42A / Y45A / L72G) (SEQ ID NO: 574), (R38A / F42K / Y45R) (SEQ ID NO: 21), (R38E / F42K / Y45R) (SEQ ID NO: 22), (K43E / F42K / Y45R) (SEQ ID NO: 23), (K43T / F42K / Y45R) (SEQ ID NO: 24), (F42K / Y45R / E62A) (SEQ ID NO: 25), (P65R / F42K / Y45R) (SEQ ID NO: 26), (P65S / F42K / Y45R) (SEQ ID NO: 27), (V69A / F42K / Y45R) (SEQ ID NO: 28), (V69D / F42K / Y45R) (SEQ ID NO: 29), or (V69R / F42K / Y45R) (SEQ ID NO: 30).This group of substitutions included the following substitutions predicted to modulate binding to CD122 / IL2Rβ: D20A (SEQ ID NO: 31), D20N (SEQ ID NO: 32), D20K (SEQ ID NO: 33), N88A (SEQ ID NO: 34), N88G (SEQ ID NO: 35), N88H (SEQ ID NO: 36), N88K (SEQ ID NO: 37), (D20A / D84A) (SEQ ID NO: 38), (D20A / E15A) (SEQ ID NO: 39), (D20A / E95A) (SEQ ID NO: 40), (D20A / N88A) (SEQ ID NO: 41), (D20A / S87A) (SEQ ID NO: 42), (D84A / N88A) (SEQ ID NO: 43), (E15A / N88A) (SEQ ID NO: 44) or (S87A / N88A) (SEQ ID NO: 45). Group 1 also included the following substitutions of IL2 predicted to modulate binding of IL2 to CD132 / IL2-Rγ: Q126L (SEQ ID NO: 377) or Q126E (SEQ ID NO: 378). The IL2 substitutions examined in Group 1 were not predicted to modulate binding to multiple IL2 receptor subunits.

[0158] Group 2 included a series of 1H3-hIgG1-L6-hIL2 fusion proteins having one or more substitutions in human IL2 predicted to be involved only in CD25 / IL2Rα binding. The fusion proteins of this group included the following substitutions in IL2 predicted to modulate binding to CD25 / IL2Rα: R38A (SEQ ID NO: 46), R38D (SEQ ID NO: 47), R38E (SEQ ID NO: 48), R38Q (SEQ ID NO: 49), F42R (SEQ ID NO: 50), F42A (SEQ ID NO: 51), F42D (SEQ ID NO: 52), F42H (SEQ ID NO: 53), K43A (SEQ ID NO: 54), K43E (SEQ ID NO: 55), K43Q (SEQ ID NO: 56), Y45A (SEQ ID NO: 57), Y45K (SEQ ID NO: 58), Y45S (SEQ ID NO: 59), Y45R (SEQ ID NO: 60), E61A (SEQ ID NO: 61), E61R (SEQ ID NO: 62), E61K (SEQ ID NO: 63), E62A (SEQ ID NO: 64), E62R (SEQ ID NO: 65), E62K (SEQ ID NO: 66), E62Y (SEQ ID NO: 67), E68Y (SEQ ID NO: 68), E68A (SEQ ID NO: 69), E68K (SEQ ID NO: 70), E68R (SEQ ID NO: 71), E68L (SEQ ID NO: 72), L72Y (SEQ ID NO: 73), L72R (SEQ ID NO: 74), L72A (SEQ ID NO: 75), L72D (SEQ ID NO: 76), L72H (SEQ ID NO: 77), L72F (SEQ ID NO: 78), (R38D / E61R) (SEQ ID NO: 79), (R38D / E61R / K43E) (SEQ ID NO: 80) or (T3A / F42A / Y45A / L72G / C125A) (SEQ ID NO: 81). The T3A substitution was introduced into the IL2 amino acid sequence to remove the predicted O-linked glycosylation site of human IL2 (see, for example, WO 2012 / 107417), and the C125A substitution was introduced into the IL2 amino acid sequence to remove the unpaired cysteine residue (see, for example, WO 2018 / 184964). The IL2 substitutions examined in Group 2 were predicted not to modulate binding of IL2 to CD132 / IL2Rγ and were also not predicted to modulate binding to multiple IL2 receptor subunits.

[0159] Group 3 included a series of 1H3-hIgG1-L6-hIL2 fusion proteins having one or more substitutions in human IL2 predicted to be involved only in CD122 / IL2Rβ binding. The fusion proteins of this group included the following substitutions in IL2 predicted to modulate binding to CD122 / IL2Rβ: E15A (SEQ ID NO:82), E15R (SEQ ID NO:83), E15K (SEQ ID NO:84), H16A (SEQ ID NO:85), H16Y (SEQ ID NO:86), H16E (SEQ ID NO:87), L19A (SEQ ID NO:88), D20I (SEQ ID NO:89), D20S (SEQ ID NO:90), D20H (SEQ ID NO:91), D20T (SEQ ID NO:92), D20W (SEQ ID NO:93), D20Y (SEQ ID NO:94), D20R (SEQ ID NO:95), D20F (SEQ ID NO:96), R81A (SEQ ID NO:97), D84A (SEQ ID NO:98), D84R (SEQ ID NO:99), D84K (SEQ ID NO:100), S87A (SEQ ID NO:101), N88Y (SEQ ID NO:102), N88D (SEQ ID NO:103), N88R (SEQ ID NO:104), N88E (SEQ ID NO:105), N88F (SEQ ID NO:106), N88I (SEQ ID NO:107), I92A (SEQ ID NO:108), I92Y (SEQ ID NO:109), I92S (SEQ ID NO:110), I92F (SEQ ID NO:111), I92R (SEQ ID NO:112), I92D (SEQ ID NO:113), I92E (SEQ ID NO:114), E95A (SEQ ID NO:115), E95R (SEQ ID NO:116), E95K (SEQ ID NO:117), (D20Y / H16E) (SEQ ID NO:118), (D20Y / H16A) (SEQ ID NO:119), (D20Y / H16Y) (SEQ ID NO:120), (D20Y / I92A) (SEQ ID NO:121), (D20Y / I92S) (SEQ ID NO:122), (D20Y / I92R) (SEQ ID NO:123), (D20Y / E95R) (SEQ ID NO:124) or (D20Y / E95A) (SEQ ID NO:125).

[0160] Group 4 included a series of fusion proteins comprising the CD25 / IL2Rα extracellular domain portion (SEQ ID NO: 126), the 20 amino acid linker (L20) (SEQ ID NO: 364), and 1H3-hIgG1-L6-hIL2 HC fused to a human IL2 variant, having one or more substitutions to residues predicted to be involved in binding to CD122 / IL2Rβ. The fusion proteins of this group contained the following substitutions in IL2 predicted to modulate binding to CD122 / IL2Rβ: E15A (SEQ ID NO: 82), D20I (SEQ ID NO: 89), D20S (SEQ ID NO: 90), D20H (SEQ ID NO: 91), D20W (SEQ ID NO: 93), D20Y (SEQ ID NO: 94), D20R (SEQ ID NO: 95), D20F (SEQ ID NO: 96), D84K (SEQ ID NO: 100), S87A (SEQ ID NO: 101), N88Y (SEQ ID NO: 102), N88D (SEQ ID NO: 103), N88R (SEQ ID NO: 104), N88E (SEQ ID NO: 105), N88F (SEQ ID NO: 106), N88I (SEQ ID NO: 107), I92A (SEQ ID NO: 108), E95A (SEQ ID NO: 115) or E95K (SEQ ID NO: 117). This group of antibody-attenuated hIL2 fusion proteins is designated 1H3-hIgG1-L6-hCD25(1-164)-L20-hIL2.

[0161] Group 5 included a series of 1H3-hIgG1-L6-hIL2 having combinations of substitutions in IL2 predicted to be involved in the binding of IL2 to CD25 / IL2Rα and CD122 / IL2Rβ or CD132 / IL2Rγ. Further, in some variants, the first 3 amino acids at the N-terminus of the hIL2 portion were deleted (Δ1-3APT). The fusion proteins of Group 5 included the following substitutions in IL2 predicted to modulate the binding of IL2 to CD25 / IL2Rα and CD122 / IL2Rβ: (F42D / D20A) (SEQ ID NO: 127), (F42R / D20A) (SEQ ID NO: 128), (F42K / D20A) (SEQ ID NO: 129), (F42A / D20A) (SEQ ID NO: 130), (F42H / D20A) (SEQ ID NO: 131), (Y45R / D20A) (SEQ ID NO: 132), (Y45K / D20A) (SEQ ID NO: 133), (R38N / D20A) (SEQ ID NO: 134), (R38G / D20A) (SEQ ID NO: 135), (R38H / D20A) (SEQ ID NO: 136), (R38I / D20A) (SEQ ID NO: 137), (R38L / D20A) (SEQ ID NO: 138), (R38M / D20A) (SEQ ID NO: 139), (R38F / D20A) (SEQ ID NO: 140), (R38P / D20A) (SEQ ID NO: 141), (R38S / D20A) (SEQ ID NO: 142), (R38T / D20A) (SEQ ID NO: 143), (R38W / D20A) (SEQ ID NO: 144), (R38Y / D20A) (SEQ ID NO: 145), (R38V / D20A) (SEQ ID NO: 146), (R38A / D20A) (SEQ ID NO: 147), (R38Q / D20A) (SEQ ID NO: 148), (D20A / R38E) (SEQ ID NO: 149), (R38D / D20A) (SEQ ID NO: 150), (K43E / D20A) (SEQ ID NO: 151), (E61A / D20A) (SEQ ID NO: 152), (E62A / D20A) (SEQ ID NO: 153), (E62Y / D20A) (SEQ ID NO: 154), (L72D / D20A) (SEQ ID NO: 155), (L72H / D20A) (SEQ ID NO: 156), (L72R / D20A) (SEQ ID NO: 157), (F42D / I92D) (SEQ ID NO: 158), (F42R / I92D) (SEQ ID NO: 159), (F42H / I92D) (SEQ ID NO: 160), (F42A / I92D) (SEQ ID NO: 161), (H16A / F42A) (SEQ ID NO: 575), (K43E / I92D) (SEQ ID NO: 162),(Y45R / I92D) (Accession No. 163), (Y45K / I92D) (Accession No. 164), (E62A / I92D) (Accession No. 165), (E62Y / I92D) (Accession No. 166), (L72D / I92D) (Accession No. 167), (L72H / I92D) (Accession No. 168), (L72R / I92D) (Accession No. 169), (R38D / I92D) (Accession No. 170), (R38E / I92D) (Accession No. 171), (R38Q / I92D) (Accession No. 172), (R38A / I92D) (Accession No. 173), (R38E / N88R) (Accession No. 174), (R38E / D84R) (Accession No. 175), (R38E / D84K) (Accession No. 176), (F42A / Y45R / D20A) (Accession No. 177), (F42H / Y45R / D20A) (Accession No. 178), (R38D / E61R / D20A) (Accession No. 179), (R38E / E61R / D20A) (Accession No. 180), (R38Q / E61R / D20A) (Accession No. 181), (R38A / E61R / D20A) (Accession No. 182), (R38A / D20A / E95A) (Accession No. 183), (D20A / E95A / R38D) (Accession No. 184), (D20A / E95A / R38E) (Accession No. 185), (D20A / E95A / R38Q) (Accession No. 186), (D20A / E95A / F42R) (Accession No. 187), (D20A / E95A / F42A) (Accession No. 188), (D20A / E95A / F42D) (Accession No. 189), (D20A / E95A / F42H) (Accession No. 190), (D20A / E95A / F42K) (Accession No. 191), (D20A / E95A / K43A) (Accession No. 192), (D20A / E95A / K43E) (Accession No. 193), (D20A / E95A / K43Q) (Accession No. 194), (D20A / E95A / Y45A) (Accession No. 195), (D20A / E95A / Y45K) (Accession No. 196), (D20A / E95A / Y45S) (Accession No. 197), (D20A / E95A / Y45R) (Accession No. 198), (D20A / E95A / E61A) (Accession No. 199), (D20A / E95A / E62A) (Accession No. 200), (D20A / E95A / E62R) (Accession No. 201), (D20A / E95A / E62K) (Accession No. 202), (D20A / E95A / E62Y) (Accession No. 203),(D20A / E95A / E68Y) (Accession No. 204), (D20A / E95A / E68A) (Accession No. 205), (D20A / E95A / E68L) (Accession No. 206), (D20A / E95A / L72Y) (Accession No. 207), (D20A / E95A / L72R) (Accession No. 208), (D20A / E95A / L72A) (Accession No. 209), (D20A / E95A / L72D) (Accession No. 210), (D20A / E95A / L72H) (Accession No. 211), (D20A / E95A / L72F) (Accession No. 212), (F42K / Y45R / D20A / S87A) (Accession No. 213), (F42K / Y45R / D20A / E95A) (Accession No. 214), (D20A / R38E / C125A) (Accession No. 215), (T3A / D20A / R38E) (Accession No. 216), (T3A / D20A / R38E / C125A) (Accession No. 217), (Δ1-3APT / D20A / R38E) (Accession No. 218) or (Δ1-3APT / D20A / R38E / C125A) (Accession No. 219). The fusion proteins of Group 5 contained the following substitutions of IL2 predicted to modulate the binding to CD25 / IL2Rα and CD132 / IL2R: (R38E / Q22A) (Accession No. 220), (R38E / T123A) (Accession No. 221), (R38E / I129A) (Accession No. 222), (R38E / S130A) (Accession No. 223), (R38E / Q126A) (Accession No. 224), (R38E / Q126D) (Accession No. 225), (R38E / Q126V) (Accession No. 226), (R38E / Q22A / S130A) (Accession No. 227), (F42K / Y45R / Q126D) (Accession No. 228) or (D20A / E95A / Q126D) (Accession No. 229). The mutations in the hIL2 sequence of the antibody-attenuated hIL2 fusion proteins of Group 5 (numbering according to the IL2 sequence) are listed in Accession Nos. 127 to 229 and 575.,

[0162] Group 6 included a series of 1H3-hIgG1-L6-hIL2 having combinations of substitutions in human IL2 that are predicted to be involved in the binding of IL2 to CD25 / IL2Rα and CD122 / IL2Rβ but not in the binding of IL2 to CD132 / IL2Rγ. The fusion proteins of Group 6 included the following combinations of substitutions in IL2 that are predicted to modulate the binding of IL2 to CD25 / IL2Rα and CD122 / IL2Rβ: (D20A / E61R) (SEQ ID NO: 230), (D20A / E61N) (SEQ ID NO: 231), (D20A / E61D) (SEQ ID NO: 232), (D20A / E61Q) (SEQ ID NO: 233), (D20A / E61G) (SEQ ID NO: 234), (D20A / E61H) (SEQ ID NO: 235), (D20A / E61I) (SEQ ID NO: 236), (D20A / E61L) (SEQ ID NO: 237), (D20A / E61K) (SEQ ID NO: 238), (D20A / E61M) (SEQ ID NO: 239), (D20A / E61F) (SEQ ID NO: 240), (D20A / E61P) (SEQ ID NO: 241), (D20A / E61S) (SEQ ID NO: 242), (D20A / E61T) (SEQ ID NO: 243), (D20A / E61W) (SEQ ID NO: 244), (D20A / E61Y) (SEQ ID NO: 245), (D20A / E61V) (SEQ ID NO: 246), (D20A / F42N) (SEQ ID NO: 247), (D20A / F42Q) (SEQ ID NO: 248), (D20A / F42E) (SEQ ID NO: 249), (D20A / F42G) (SEQ ID NO: 250), (D20A / F42I) (SEQ ID NO: 251), (D20A / F42L) (SEQ ID NO: 252), (D20A / F42M) (SEQ ID NO: 253), (D20A / F42P) (SEQ ID NO: 254), (D20A / F42S) (SEQ ID NO: 255), (D20A / F42T) (SEQ ID NO: 256), (D20A / F42W) (SEQ ID NO: 257), (D20A / F42Y) (SEQ ID NO: 258), (D20A / F42V) (SEQ ID NO: 259), (D20A / Y45A) (SEQ ID NO: 260), (D20A / Y45N) (SEQ ID NO: 261), (D20A / Y45D) (SEQ ID NO: 262), (D20A / Y45Q) (SEQ ID NO: 263), (D20A / Y45E) (SEQ ID NO: 264), (D20A / Y45G) (SEQ ID NO: 265), (D20A / Y45H) (SEQ ID NO: 266), (D20A / Y45I) (SEQ ID NO: 267).(D20A / Y45L)(SEQ ID NO: 268), (D20A / Y45M)(SEQ ID NO: 269), (D20A / Y45F)(SEQ ID NO: 270), (D20A / Y45P)(SEQ ID NO: 271), (D20A / Y45S)(SEQ ID NO: 272), (D20A / Y45T)(SEQ ID NO: 273), (D20A / Y45W)(SEQ ID NO: 274), (D20A / Y45V)(SEQ ID NO: 275), (I92D / F42N)(SEQ ID NO: 276), (I92D / F42Q)(SEQ ID NO: 277), (I92D / F42E)(SEQ ID NO: 278), (I92D / F42G)(SEQ ID NO: 279), (I92D / F42I)(SEQ ID NO: 280), (I92D / F42L)(SEQ ID NO: 281), (I92D / F42K)(SEQ ID NO: 282), (I92D / F42M)(SEQ ID NO: 283), (I92D / F42P)(SEQ ID NO: 284), (I92D / F42S)(SEQ ID NO: 285), (I92D / F42T)(SEQ ID NO: 286), (I92D / F42W)(SEQ ID NO: 287), (I92D / F42Y)(SEQ ID NO: 288), (I92D / F42V)(SEQ ID NO: 289), (I92D / Y45A)(SEQ ID NO: 290), (I92D / Y45N)(SEQ ID NO: 291), (I92D / Y45D)(SEQ ID NO: 292), (I92D / Y45Q)(SEQ ID NO: 293), (I92D / Y45E)(SEQ ID NO: 294), (I92D / Y45G)(SEQ ID NO: 295), (I92D / Y45H)(SEQ ID NO: 296), (I92D / Y45I)(SEQ ID NO: 297), (I92D / Y45L)(SEQ ID NO: 298), (I92D / Y45M)(SEQ ID NO: 299), (I92D / Y45F)(SEQ ID NO: 300), (I92D / Y45P)(SEQ ID NO: 301), (I92D / Y45S)(SEQ ID NO: 302), (I92D / Y45T)(SEQ ID NO: 303), (I92D / Y45W)(SEQ ID NO: 304), (I92D / Y45V)(SEQ ID NO: 305), (R38E / D20H)(SEQ ID NO: 306), (R38E / D20S)(SEQ ID NO: 307), (F42A / N88R)(SEQ ID NO: 308), (F42A / N88D)(SEQ ID NO: 309), (R38E / D84A)(SEQ ID NO: 310), (R38E / D84N)(SEQ ID NO: 311), (R38E / D84Q)(SEQ ID NO: 312), (R38E / D84E)(SEQ ID NO: 313), (R38E / D84G)(SEQ ID NO: 314),(R38E / D84H) (Accession No. 315), (R38E / D84I) (Accession No. 316), (R38E / D84L) (Accession No. 317), (R38E / D84M) (Accession No. 318), (R38E / D84F) (Accession No. 319), (R38E / D84P) (Accession No. 320), (R38E / D84S) (Accession No. 321), (R38E / D84T) (Accession No. 322), (R38E / D84W) (Accession No. 323), (R38E / D84Y) (Accession No. 324), (R38E / D84V) (Accession No. 325), (R38E / I92A) (Accession No. 326), (R38E / I92R) (Accession No. 327), (R38E / I92N) (Accession No. 328), (R38E / I92Q) (Accession No. 329), (R38E / I92E) (Accession No. 330), (R38E / I92G) (Accession No. 331), (R38E / I92H) (Accession No. 332), (R38E / I92L) (Accession No. 333), (R38E / I92K) (Accession No. 334), (R38E / I92M) (Accession No. 335), (R38E / I92F) (Accession No. 336), (R38E / I92P) (Accession No. 337), (R38E / I92S) (Accession No. 338), (R38E / I92T) (Accession No. 339), (R38E / I92W) (Accession No. 340), (R38E / I92Y) (Accession No. 341), (R38E / I92V) (Accession No. 342), (R38E / H16E) (Accession No. 343) or (R38K / D20A) (Accession No. 344). Mutations in the hIL2 sequence of the antibody-attenuated hIL2 fusion proteins of Group 6 (numbering according to the IL2 sequence) are listed in SEQ ID NOs: 230 to 344.,

[0163] The association rate constants of the purified 1H3-hIgG1-L6-hIL2 variant protein with recombinant human CD25 and human CD122 were determined by biolayer interferometry (BLI). Briefly, binding experiments were performed at 25 °C using an Octet® Red96 instrument (Pall ForteBio). The extracellular domains of human CD25 and human CD122 with a polyhistidine tag at the C-terminus were captured on an anti-His2 sensor (Pall ForteBio). The sensors loaded with the receptor were immersed in seven-point serial three-fold dilutions of each 1H3-hIgG-L6-hIL2 variant starting from a maximum concentration of 300 nM. The 1H3-hIgG1-L6-hIL2 fusion protein was diluted in an assay buffer (pH 7.2) consisting of phosphate-buffered saline (PBMS) supplemented with 0.1% BSA and 0.02% Tween® 20. The loaded sensors were regenerated with 10 mM glycine buffer (pH 1.7). The rate constants were calculated using a monovalent binding model.

[0164] The binding constants (k on ) of 74 immunoglobulin-hIL2 fusion protein variants that bind to recombinant human CD25 and recombinant human CD122 off ), dissociation constants (k D ) and equilibrium constants (K

[0165]

Table 2-1

[0166]

Table 2-2

[0167]

Table 2-3

[0168] The binding (k on ) constants, dissociation (koff )Constants and equilibrium constant (K D ) are shown in Table 3.

[0169]

Table 3-1

[0170]

Table 3-2

[0171]

Table 3-3

[0172] Example 3 Attenuation test for high- and medium-affinity hIL2 receptors by cell-based potency pSTAT5 screening at fixed concentration The attenuation of the antibody-attenuated hIL2 fusion protein described in Example 2 was tested in NK-92 (expressing high-affinity hIL2 receptor) and TF1+IL2Rβ (expressing medium-affinity hIL2 receptor) cell lines using protocol D in a fixed-concentration pSTAT5 screening. The fold change in geometric mean fluorescence intensity (gMFI) of the antibody-attenuated hIL2 fusion protein from free cytokine wild-type rhIL2, a measure of the decrease in IL2 activity, is shown in Tables 4 to 8. In the fixed-concentration screening, the fold change was calculated by dividing the gMFI of rhIL2 by the gMFI of the variant. In experiments with a complete titration curve, the fold change from rhIL2 was calculated by dividing the EC 50 value of rhIL2 by the EC 50 of the variant. The fold change was rounded to an integer. The decrease in gMFI in the NK-92 and TF1+IL2Rβ cell lines compared to the gMFI of rhIL2 indicated attenuation of IL2 activity in both high- and medium-affinity receptors. The fixed-concentration cell-based potency pSTAT5 screening of the variants of group 1 described in Example 2 was not performed.

[0173] The IL2 agonist activity of each variant tested was also evaluated to clarify its characteristics as a full or partial IL2 agonist or as having no (inactive) IL2 activity. The dose titration curve of the 1H3-hIgG1-L6-hIL2 fusion protein that reached the maximum gMFI level shown by the rhIL2 positive control was considered an antibody-attenuated hIL2 fusion protein with full agonist activity. The partial agonist activity was calculated as a percentage of the maximum gMFI of rhIL2 as 100% relative to full activity. Antibody-attenuated hIL2 fusion proteins with a maximum gMFI at the highest concentration of 1200 nM less than 10% of the rhIL2 maximum gMFI were considered to have no (inactive) agonist activity. Some EC 50 values and attenuation levels were estimated values because the activity did not reach a maximum and could not be accurately calculated with GraphPad Prism 7 software.

[0174] The results of the pSTAT5 fixed concentration demonstrated that some single residue substitutions attenuated the IL2 activity against the high-affinity cell line (NK-92), and a combination of substitutions that modulated the binding to the α and β chains or the α and γ chains was required to significantly attenuate the IL2 activity of the high-affinity IL2 receptor (more than 20-fold attenuation from recombinant hIL2).

[0175]

Table 4

[0176]

Table 5-1

[0177]

Table 5-2

[0178]

Table 6

[0179]

Table 7-1

[0180]

Table 7-2

[0181]

Table 7-3

[0182]

Table 8-1

[0183]

Table 8-2

[0184]

Table 8-3

[0185] Example 4 Attenuation Test of IL2 Fusion Proteins against High-Affinity and Medium-Affinity hIL2 Receptors by Cell-Based Potency pSTAT5 Dose Titration Screening The attenuation of the antibody-attenuated hIL2 fusion proteins (1H3-hIgG1-L6-hIL2 fusion proteins in Groups 2 to 6) described in Example 2 was tested with the pSTAT5 titration curve using the NK-92 and TF1 + IL2Rβ cell lines described in Protocol D.

[0186] Using the geometric mean fluorescence intensity (gMFI) of the Alexa Fluor 647 pSTAT5 positive signal, a four-parameter logistic curve was generated, and then the EC 50Values were calculated. These values were compared with the rhIL2 control as the measured values of attenuation. The fold change in activity from rhIL2 calculated by the gMFI of Alexa Fluor 647 is summarized in Tables 9 to 13.

[0187] The increase in fold change from rhIL2 indicated the degree of attenuation of hIL2 activity. Antibody-attenuated hIL2 fusion proteins that were tested for pSTAT5 titration curves were also evaluated for agonist activity and classified as fully active, partially active, or no activity (inactive). Antibody-attenuated hIL2 fusion proteins whose dose titration curves reached the maximum gMFI level as rhIL2 were considered variants with full agonist activity. Partial agonist activity was calculated as described in Example 3. Antibody-attenuated hIL2 fusion proteins with less than 10% activity compared to rhIL2 were classified as inactive. Since some fold changes from rhIL2 did not generate a complete four-parameter logistic curve and could not be accurately calculated with GraphPad Prism 7 software (not calculated or denoted as "NC"), these values are estimated values (annotated as such in Tables 9 to 13). However, these variants showed more than 10,000-fold attenuation relative to rhIL2 on the graph (data not shown). This is denoted as ">10,000, NC on the graph" in Tables 9 to 13. a The complete titration curve of pSTAT5 showed results similar to those shown in Example 3, where substitutions that regulate binding to both the α-chain and β-chain significantly attenuated IL2 activity for the high-affinity IL2 receptor compared to single substitutions for binding to only the α-chain or β-chain. The complete titration assay of pSTAT5 was further able to distinguish variants with substitutions that cause inactivity from highly attenuated variants. Finally, the comparison of dose titration curves was more accurate in terms of the level of attenuation than the fixed-concentration assay.

[0188] The complete titration curve of pSTAT5 showed results similar to those shown in Example 3, where substitutions that regulate binding to both the α-chain and β-chain significantly attenuated IL2 activity for the high-affinity IL2 receptor compared to single substitutions for binding to only the α-chain or β-chain. The complete titration assay of pSTAT5 was further able to distinguish variants with substitutions that cause inactivity from highly attenuated variants. Finally, the comparison of dose titration curves was more accurate in terms of the level of attenuation than the fixed-concentration assay.

[0189] [Table 9]

[0190]

Table 10-1

[0191]

Table 10-2

[0192]

Table 11

[0193]

Table 12-1

[0194]

Table 12-2

[0195]

Table 12-3

[0196]

Table 13

[0197] Example 5 Attenuation Test for High-Affinity and Medium-Affinity hIL2 Receptors by Cell-Based Proliferation Assay The antibody-attenuated hIL2 fusion proteins (2D12-mIgG1-D265A-L6-hIL2, 2D12-hIgG1-L6-hIL2, and 1H3-hIgG1-L6-hIL2 fusion proteins) of Groups 1 to 6 in Example 2 were selected, and the attenuation of IL2 activity was tested in the proliferation assays of the NK-92 cell line and the TF1 + IL2Rβ cell line as described in Protocol E. The results of the assays are shown in Tables 14 to 19.

[0198] The 1H3-hIgG1-L6-hIL2 fusion protein that showed significant attenuation in the pSTAT5 titration curve of Example 4 was tested in this cell-based proliferation assay. pSTAT5 requires a mere 10-minute stimulation, which may be a small snapshot of IL2-dependent activity, in the readout downstream of IL2 activity. In the proliferation assay, cells were incubated with 2D12-mIgG1-D265A-L6-hIL2, 2D12-hIgG1-L6-hIL2, 1H3-hIgG1-L6-hIL2 fusion protein, or recombinant hIL2 control for 3 to 4 days, and a more physiologically appropriate readout of IL2-dependent activity in vivo was obtained. In this proliferation assay, the IL2-dependent activities of the other 2D12-mIgG1-D265A-L6-hIL2 and 2D12-hIgG1-L6-hIL2 fusion proteins that were produced but not assayed for pSTAT5 were assayed.

[0199] Similar to the cell-based pSTAT5 dose titration experiment, the EC calculated from relative light units (RLU) instead of gMFI 50 and the analysis of the results were performed after the calculation of EC 50 in the same manner as in Example 4. Similar to the results confirmed in Example 4, the growth curves showed that some substitutions that regulate binding to both the α-chain and the β-chain significantly attenuated the IL2 activity against the high-affinity receptor compared to a single substitution for binding to only the α-chain or the β-chain. These selected 1H3-hIgG1-L6-hIL2 fusion proteins were also tested for growth in the TF1+IL2Rβ cell line, and some of the same substitutions were shown to significantly attenuate the IL2 activity against the intermediate-affinity receptor.

[0200] [Table 14-1]

[0201] [Table 14-2]

[0202] [Table 15]

[0203]

Table 16

[0204]

Table 17

[0205]

Table 18-1

[0206]

Table 18-2

[0207]

Table 18-3

[0208]

Table 19-1

[0209]

Table 19-2

[0210] Example 6 Production of Anti-hPD1 Antibody Various anti-hPD1 antibodies with desired properties were produced by several methods.

[0211] In one method, transgenic chickens (OmniChicken®) expressing human antibody genes (human light chains (VLCL or VKCK) and human VH) and chicken heavy chain constant regions were used to produce anti-hPD1 human monoclonal antibodies (Ching et al., mAbs 2018). The transgenic chickens were immunized every 14 days for 14 weeks with 100 μg of Fc-tagged human PD1 protein (huPD1-Fc, SEQ ID NO: 380). In another method, transgenic chickens were genetically immunized 6 times with DNA encoding human PD1 (SEQ ID NO: 347), followed by a final boost with 100 μg of huPD1-Fc (SEQ ID NO: 380). The serum immune response of each animal was monitored by ELISA against biotinylated human PD1 on streptavidin-coated plates.

[0212] Spleen cells were isolated from immunized animals and tested for positive antibody clones by the gel-encapsulated microenvironment (GEM) assay (described in Mettler Izquierdo, S., Varela, S., Park, M., Collarini, E. J., Lu, D., Pramanick, S., Rucker, J., Lopalco, L., Etches, R., & Harriman, W. (2016)). High-efficiency antibody discovery was achieved by multiplex microscopy. Microscopic observation (Oxford, England), 65(4), 341-352) was performed and human PD1-labeled beads were screened. The sequences of the positive clones were determined, the variable regions of the heavy and light chains were cloned, assembled into single-chain variable fragments, and fused to the hinge and Fc regions of immunoglobulins (ScFv-Fc). These unique scFv-Fc fusion proteins were transiently expressed in Expi293 cells and the binding activity of the supernatant was tested by ELISA on plates coated with huPD1-Fc (SEQ ID NO: 380) or cynomolgus PD1-Fc (SEQ ID NO: 381). A total of 102 unique pairs of anti-human PD1 variable heavy and variable light chains were identified by this method. 2H7-hIgG4 (SEQ ID NOs: 382-391, 424 and 425) and A2-hIgG4 (SEQ ID NOs: 402-411, 428 and 429) were the antibodies identified by this method.

[0213] By other methods, an anti-hPD1 antibody designated C51E6-hIgG4 was identified, which became an antibody designated C51E6-5-hIgG4 (SEQ ID NOs: 392-401, 426, 427) by germline optimization, was humanized, and became an antibody designated Abz1mod-hIgG4 (SEQ ID NOs: 449, 450) by sequence optimization.

[0214] The variable region sequences of anti-PD1 were expressed as human IgG4κ antibodies, and their binding ability to PD1-expressing cells was evaluated using flow cytometry as described in Protocol A of the general method. The binding of the antibodies to human PD1 was first screened using a Jurkat cell line expressing recombinant human PD1 (Jurkat+hPD1 cell line). The antibodies were serially diluted from a maximum concentration of 280 nM, and then an allophycocyanin-conjugated anti-human IgG secondary antibody was added to the cells for detection. Among the 92 hits, the EC 50 of the binding of 79 anti-PD1 antibodies was less than 30 nM (by flow cytometry). 2H7-hIgG4 (SEQ ID NOs: 382-391, 424, and 425), C51E6-5-hIgG4 (SEQ ID NOs: 392-401, 426, and 427), A2-hIgG4 (SEQ ID NOs: 402-411, 428, and 429), OMC.1.B6-hIgG4 (SEQ ID NOs: 438 and 439), OMC.1.D6-hIgG4 (SEQ ID NOs: 442 and 443), OMC.2.C6-hIgG4 (SEQ ID NOs: 440 and 441), 1H9-hIgG4 (SEQ ID NOs: 576 and 525), 1D5-hIgG4 (SEQ ID NOs: 577 and 527), and 2A3.H7-hIgG4 (SEQ ID NOs: 424 and 523) were included in the antibodies identified as antibodies that bind to hPD1 with medium to high affinity in a Jurkat cell line expressing human PD1 (SEQ ID NO: 346). The calculated values of the EC 50 of the binding to Jurkat cells recombinantly expressing hPD1 by flow cytometry in multiple experiments were 0.1-0.3 nM for 2H7-hIgG4, 1H9-hIgG4, 1D5-hIgG4, and 2A3.H7-hIgG4. The EC 50The calculated values were 2 - 4 nM for C51E6-5-hIgG4 and 3 - 16 nM for A2-hIgG4, OMC.1.B6-hIgG4, OMC.1.D6-hIgG4, and OMC.2.C6-hIgG4. In the antibody titrations of 2H7-hIgG4, C51E6-5-hIgG4, A2-hIgG4, 1H9-hIgG4, 1D5-hIgG4, 2A3.H7-hIgG4, OMC.1.B6-hIgG4, OMC.1.D6-hIgG4, and OMC.2.C6-hIgG4, binding was specific for hPD1 as they did not bind to the parental Jurkat cell line that does not express hPD1 (data not shown).

[0215] Example 7 Evaluation of the characteristics of anti-hPD1 antibody binding in the presence of anti-hPD1 #1-mIgG2b-N297A and anti-hPD1 #2-mIgG2b-N297A antibodies 2H7-hIgG4, C51E6-5-hIgG4, and A2-hIgG4 were evaluated for binding competition with hPD1 in the presence of anti-hPD1 #1-mIgG2b-N297A and anti-hPD1 #2-mIgG2b-N297A as described in Protocol B of the general method.

[0216] As a control, Opdivo® (nivolumab) was titrated in the presence of anti-hPD1 #1-mIgG2b-N297A at a saturating concentration of 10 μM (Figure 4A). The dose titration curve in the presence of the competitor anti-hPD1 #1-mIgG2b-N297A was significantly reduced (a 100- to 1000-fold shift to the right of the dose titration curve graph) compared to the dose titration curve of Opdivo® in the absence of the competitor anti-hPD1 #1-mIgG2b-N297A. Addition of anti-hPD1 #1-mIgG2b-N297A or anti-hPD1 #2-mIgG2b-N297A at a saturating concentration (10 μM) before exposure to 2H7-hIgG4, C51E6-5-hIgG4 or A2-hIgG4 did not interfere with the binding of hPD1 to 2H7-hIgG4, C51E6-5-hIgG4 or A2-hIgG4, as indicated by a shift of less than 10-fold in Figures 4B-4D, suggesting that 2H7-hIgG4, C51E6-5-hIgG4 and A2-hIgG4 did not compete for binding to PD1 in the presence of anti-hPD1 #1-mIgG2b-N297A or anti-hPD1 #2-mIgG2b-N297A.

[0217] Example 8 Characterization of hPD1 Non-Antagonist Antibodies The anti-hPD1 antibodies 2H7-hIgG4, C51E6-5-hIgG4 and A2-hIgG were tested for PD1 antagonist activity using an in vitro cell-based human PD1 / PDL1 inhibition bioassay as described in Protocol C of the general method. All antibodies except A2-hIgG4 were tested at a final concentration of 200 nM. A2-hIgG4 was tested at a final concentration of 500 nM.

[0218] Anti-hPD1 antibodies 2H7-hIgG4, C51E6-5-hIgG4, A2-hIgG4, OMC.1.B6-hIgG4, OMC.1.D6-hIgG4, OMC.2.C6-hIgG4, 1H9-hIgG4, 1D5-hIgG4, and 2A3.H7-hIgG4 all emitted light at an average of 3000 relative light unit (RLU) levels, showing an RLU similar to that of KLH-C3-hIgG4, which was the negative control (data not shown). Therefore, none of these anti-hPD1 antibodies showed hPD1 antagonist activity. In contrast, anti-hPD1 #1, a known hPD1 antagonist that blocks the binding of hPDL1 (SEQ ID NO: 584) to hPD1, showed light emission exceeding 14,000 RLU (data not shown).

[0219] Example 9 Anti-hPD1-attenuated hIL2 fusion proteins bind to Jurkat cells expressing human PD1 To construct vectors expressing various antibodies and antibody-attenuated hIL2 fusion proteins, corresponding polynucleotides encoding the sequences of antibodies, cytokines, cytokine receptors, and linkers were prepared and cloned into expression vectors. Antibodies or antibody fusion proteins were transiently expressed in human embryonic kidney (HEK) 293 cells and then purified by affinity chromatography using protein A or protein G-sepharose. The purified proteins were concentrated by ultrafiltration and buffer-exchanged into phosphate-buffered saline or phosphate-buffered saline containing 100 mM L-arginine and 10 mM L-histidine, and then the protein concentration was determined.

[0220] In some methods, 2H7-hIgG4, C51E6-5-hIgG4, and A2-hIgG4 having the S228P hinge stabilization mutation were fused directly (df) to hIL2 or fused to hIL2 using an L6 linker at the C-terminus of the immunoglobulin heavy chain. Illustrations of these hIL2 fusion proteins attenuated by these anti-PD1s are summarized in FIG. 5. As described in Example 2, various constructs with substitutions of hIL2 that attenuate hIL2 activity were produced. The binding of the anti-hPD1-attenuated hIL2 fusion proteins listed in Table 20 to hPD1 was tested in a Jurkat cell line expressing hPD1 as described in Protocol A of the general method. The variable regions of 2H7-hIgG4 (SEQ ID NOs: 384 and 385) were further optimized, and the isotype was changed to human IgG1 having the effector function null substitution L235A / G237A (LAGA, described in WO 1998 / 006248) to H7-632-hIgG1-LAGA (SEQ ID NOs: 414 and 415). The optimized H7-632-hIgG1-LAGA was also fused directly (df) to a variant of hIL2 with attenuated hIL2 activity (hIL2 T3A / D20A / R38E / C125A; SEQ ID NO: 217) to give H7-767 (SEQ ID NOs: 412-413, 415-423, 532), and the binding of both H7-632-hIgG1-LAGA and H7-767 to hPD1 was tested (Table 20). EC 50 values were calculated from the geometric mean fluorescence intensity (gMFI) over the titration concentrations using GraphPad Prism 7 software.

[0221] By producing anti-hPD1-attenuated hIL2 fusion proteins, binding to hPD1 was not reduced, and the anti-hPD1-attenuated hIL2 fusion proteins were still able to bind to Jurkat cells expressing human PD1. The calculated EC 50 values of the anti-hPD1-attenuated hIL2 fusion proteins are summarized in Table 20.

[0222]

Table 20

[0223] Addition of the attenuated hIL2 moiety to the anti-hPD1 antibody did not interfere with binding to human PD1, as indicated by an increase in the EC 50 of less than 2-fold in the binding of the anti-hPD1-hIL2 fusion protein to Jurkat+hPD1 cells compared to the anti-hPD1 antibody without the attenuated hIL2 moiety.

[0224] Example 10 Anti-hPD1-attenuated hIL2 fusion protein binds to hPD1 in the presence of anti-hPD1 #1 and anti-hPD1 #2 antibodies The binding of anti-hPD1-attenuated hIL2 fusion proteins to the hPD1 receptor in the presence of anti-hPD1 #1 and anti-hPD1 #2 was tested as described in Protocol B of the general method and Example 7. A reverse experiment was also conducted to examine the binding of anti-hPD1 #1-mIgG2b-N297A or anti-hPD1 #2-mIgG2b-N297A to hPD1 in the presence of a saturating concentration of the antibody-attenuated hIL2 fusion protein. In this format, Jurkat cells expressing hPD1 were seeded at 100,000 cells per well in FACS buffer, blocked with an anti-human FcγR blocking reagent (Miltenyi) at 4°C for 10 minutes, and washed. Antibody-attenuated hIL2 fusion proteins 2H7-hIgG4-df-hIL2 (D20A / R38E), C51E6-5-hIgG4-L6-hIL2 (D20A / R38E), A2-hIgG4-df-hIL2 (D20A / R38E), H7-767, and isotype control anti-DNase 1H3-hIgG4-df-hIL2 (D20A / R38E) were diluted to a final concentration of 280 nM in 100 μL of FACS buffer and incubated with Jurkat cells expressing hPD1 on ice for 1 hour. The cells were washed and resuspended in FACS buffer containing 6-fold serial titrations of anti-hPD1 #1-mIgG2b-N297A or anti-hPD1 #2-mIgG2b-N297A starting at a maximum concentration of 50 nM on ice for 1 hour. The cells were washed and resuspended in a 1:100 dilution of a phycoerythrin-conjugated anti-mouse IgG light chain κ monoclonal antibody on ice for 45 minutes. The cells were washed again and resuspended in FACS buffer containing a 1:1000 dilution of Sytox Green (Thermo Fisher). Flow cytometry analysis was performed using a BD FACS Canto II (BD Biosciences), and the gMFI was calculated using FlowJo software version 10. EC 50 50 was calculated from the gMFI of phycoerythrin over the titrated concentrations using GraphPad Prism 7 software.

[0225] Addition of attenuated hIL2 to anti-hPD1 antibodies 2H7-hIgG4, C51E6-5-hIgG4, and A2-hIgG4, similar to the results described in Example 7, did not reduce the ability of the anti-hPD1 protein to bind to human PD1 in the presence of anti-hPD1 #1-mIgG2b-N297A or anti-hPD1 #2-mIgG2b-N297A (Figures 16B - 16D). In this competition assay, H7-767 was also tested, and Figure 13B shows that H7-767 continues to bind to the hPD1 receptor in the presence of anti-hPD1 #1-mIgG2b-N297A or anti-hPD1 #2-mIgG2b-N297A. In contrast, the binding of the positive control anti-hPD1 #1 was significantly reduced in the presence of anti-hPD1 #1-mIgG2b-N297A or anti-hPD1 #2-mIgG2b-N297A (Figure 16A, Figure 13A).

[0226] In the reverse competition assay, Figures 6A and 6B show that anti-hPD1 #1-mIgG2b-N297A (Figure 6A) and anti-hPD1 #2-mIgG2b-N297A (Figure 6B) were able to bind to hPD1 on Jurkat cells even in the presence of saturating concentrations (280 nM) of anti-hPD1-attenuated hIL2 fusion proteins 2H7-hIgG4-df-hIL2 (D20A / R38E), C51E6-5-hIgG4-df-hIL2 (D20A / R38E), or A2-hIgG4-df-hIL2 (D20A / R38E). The binding curves of the anti-hPD1-attenuated hIL2 fusion proteins at saturating concentrations before exposure to the anti-hPD1 fusion protein overlapped with the binding curves of anti-hPD1 #1-mIgG2b-N297A (no competition) or anti-hPD1 #2-mIgG2b-N297A (no competition). The binding curves also overlapped with that of the negative control fusion protein 1H3-hIgG4-df-hIL2 (D20A / R38E) at saturating concentrations that did not bind to hPD1.

[0227] Example 11 Anti-hPD1-attenuated hIL2 fusion proteins bind to recombinantly expressed cynomolgus PD1 The binding of the anti-hPD1-attenuated hIL2 fusion protein to cynomolgus PD1 was tested by flow cytometry using human embryonic kidney 293 cell line (HEK-293T) expressing SV40 large T cell antigen transiently transfected to express cynomolgus PD1. In each transfection reaction, 2 million HEK-293T cells were transfected with 2 μg of pCMV6-hygro-HA-cyno-PD1(1-185) (SEQ ID NO: 448), a mammalian vector containing the extracellular domain of cynomolgus PD1 tagged with human influenza hemagglutinin and a sequence encoding hygromycin resistance. Transfection was performed by electroporation. The transfected cells were blocked with human FcγR blocking reagent and stained with titrated amounts of the anti-hPD1-attenuated hIL2 fusion protein. Further, the cells were stained by adding phycoerythrin-conjugated anti-hemagglutinin clone 15B12 to stain the transfected cells, and allophycocyanin-conjugated anti-human IgG Fc secondary clone HP6017 (BioLegend Cat# 409306) was added to the cells to stain the bound antibody. The cells were analyzed on a BD Canto II, and live transfected (hemagglutinin-positive) cells were gated using FlowJo software version 10, and the geometric mean fluorescence intensity (gMFI) of allophycocyanin was calculated. The EC 50 50 values were calculated from the gMFI over the titrated concentrations using GraphPad Prism 7 software.

[0228] The anti-hPD1-attenuated hIL2 fusion protein bound to HEK-293T cells expressing cynomolgus PD1 in a manner similar to the binding profile seen in Jurkat T cells expressing human PD1 (Figure 17). The EC 50was 5 nM for 2H7-hIgG4-df-hIL2(D20A / R38E), 6 nM for C51E6-5-hIgG4-df-hIL2(D20A / R38E), and 11 nM for A2-hIgG4-df-hIL2(D20A / R38E). Anti-hPD1 #1 and anti-hPD1 #2 formatted as anti-hPD1-attenuated hIL2 fusion protein, which were comparators, also bound to cynomolgus PD1 at EC 50 values of 9 nM and 2 nM, respectively, suggesting that addition of the hIL2 portion attenuated to an anti-hPD1 antibody did not prevent binding to cynomolgus PD1.

[0229] Example 12 Anti-hPD1-attenuated hIL2 fusion proteins bind to activated primary human and cynomolgus PD1 Binding of anti-hPD1 antibodies and anti-hPD1-attenuated hIL2 fusion proteins to activated primary T cells expressing hPD1 was examined by flow cytometry. To test whether 2H7-hIgG4, C51E6-5-hIgG4 or A2-hIgG4 binds to native hPD1, cryopreserved human peripheral blood mononuclear cells (PBMCs) were thawed and activated with 50 ng / mL phorbol 12-myristate 13-acetate (PMA) and 1 μg / mL ionomycin to upregulate the hPD1 receptor. Activated PBMCs were collected, blocked with a 1:50 dilution of human FcγR blocking reagent (Miltenyi) for 10 min at 4°C, and stained with titrated concentrations of the anti-hPD1 antibodies 2H7-hIgG4, C51E6-5-hIgG4, A2-hIgG4, anti-hPD1 #1 and isotype control. Cells were then stained with a 1:20 dilution of allophycocyanin-conjugated anti-human IgG Fc to detect bound antibody. A cocktail of surface markers including anti-human CD3, anti-CD4 and anti-CD8 antibodies was used to distinguish immune subsets. Additionally, samples were tested for the expression of hPD1, hCD25, hCD122 and hCD132. Cells were analyzed on a BD Fortessa (BD Biosciences), T cell subsets were gated using FlowJo software version 10, and then the geometric mean fluorescence intensity (gMFI) of allophycocyanin was calculated. EC50 Values were calculated from the gMFI over the titrated concentrations using GraphPad Prism 7 software. To test the binding of the anti-hPD1-hIL2 fusion protein, cryopreserved CD3+ T cells were activated with PMA / ionomycin, and flow cytometry binding was performed as previously described.

[0230] The binding of the human PD1 antibody-attenuated hIL2 fusion protein to cynomolgus activated T cells was also tested by flow cytometry. Cynomolgus PBMC were activated with a mixture of 0.081 μM PMA and 1.34 μM ionomycin. After 24 hours, the cells were stained in the same procedure as the binding to the human PD1 primary cells, except that cynomolgus cross-reactive markers were used. Using FlowJo software version 10, live CD3 + CD4 + T cells or CD3 + CD8 + T cells were gated, and then the gMFI of allophycocyanin was calculated. EC 50 Values were calculated from the gMFI of the anti-hPD1 antibody or hPD1 antibody-attenuated hIL2 fusion protein over the titrated concentrations using GraphPad Prism 7 software.

[0231] In some variants tested, the attenuated hIL2 also included a T3A substitution that removes an O-linked glycosylation site and a C125A substitution that replaces a free cysteine residue.

[0232] After activation with PMA and ionomycin, 40 - 50% of CD4 + T cells were PD1 + and 30 - 40% of CD8 + T cells were PD1 + (data not shown). The EC of the binding of the human activated CD3 + CD4 + T cells by flow cytometry 50The calculated values were 0.1 - 0.7 nM for 2H7-hIgG4, 12 nM for C51E6-5-hIgG4, 30 nM for A2-hIgG4, and 0.04 nM for 2H7-hIgG4-df-hIL2(T3A / D20A / R38E / C125A) with human activated CD3 + CD8 + The EC of binding to 50 T cells was 0.1 - 0.8 nM for 2H7-hIgG4, 16 nM for C51E6-5-hIgG4, 22 nM for A2-hIgG4, and 0.03 nM for 2H7-hIgG4-df-hIL2(T3A / D20A / R38E / C125A) with H7-767 and human activated CD3 + CD4 + The EC of binding to 50 T cells was 0.19 nM with human activated CD3 + CD8 + and 0.12 nM with cynomolgus activated CD3 + CD4 + The EC of binding to 50 T cells was 0.09 nM for 2H7-hIgG4 and 0.04 nM for 2H7-hIgG4-df-hIL2(T3A / D20A / R38E / C125A) with cynomolgus activated CD3 + CD8 + The EC of binding to 50 T cells was 0.08 nM for 2H7-hIgG4 and 0.03 nM for 2H7-hIgG4-df-hIL2(T3A / D20A / R38E / C125A) with H7-767 and cynomolgus activated CD3 + CD4 + The EC of binding to 50 T cells was 0.26 nM with cynomolgus activated CD3 + CD8 + and 0.24 nM with cynomolgus activated CD3. This data indicates that when the hPD1 antibody was converted to an anti-hPD1-attenuated hIL2 fusion protein, the calculated EC 50 of binding to activated hPD1 was comparable to the calculated EC 50 of the naked hPD1 antibody that binds to hPD1.

[0233] Binding of H7-767 and H7-632-hIgG1-LAGA anti-PD1 naked antibodies to primary naive human CD4 + and CD8 + T cells was tested by flow cytometry. Frozen human CD3 + T cells were thawed and flow cytometry was performed as previously described. Both H7-767 and H7-632-hIgG1-LAGA anti-PD1 naked antibodies did not bind to human naive CD4 + and CD8 + T cells (data not shown).

[0234] Example 13 Quantitative determination of the binding of anti-hPD1 antibodies and anti-hPD1-attenuated hIL2 fusion proteins to recombinant human or cynomolgus PD1 by surface plasmon resonance (SPR) Surface plasmon resonance binding assays were performed using a high-throughput SPR Carterra® LSA™ to determine the binding affinities of anti-hPD1 antibodies and anti-hPD1-attenuated hIL2 fusion proteins. Proteins were diluted to 2 μg / mL or 10 μg / mL in 10 mM sodium acetate (pH 4.5) containing 0.01% Tween® 20 and coupled to a HC30M (Carterra Bio) chip using sulfo-N-hydroxysuccinimide / 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (sulfo-NHS / EDC) coupling chemistry and blocked with ethanolamine. Association rate constants for binding to commercially available recombinant His-tagged human PD1 and His-tagged cynomolgus PD1 (Acro Biosystems) were determined by a non-regenerative kinetic coupling method.

[0235] Anti-hPD1 antibodies and anti-hPD1-attenuated hIL2 fusion proteins were expressed in either a modified human IgG1 or modified IgG4 isotype having a κ light chain framework. L235E or L235A / G237A (LAGA, described in WO 1998 / 006248) (EU numbering) substitutions were introduced into the Fc region to abrogate effector functions of the immunoglobulin component.

[0236] Binding constants (k a ), dissociation constants (k d ), and equilibrium constants (K D ) of various anti-hPD1 antibodies and anti-hPD1 antibody-attenuated hIL2 fusion proteins with recombinant human or cynomolgus PD1 proteins were determined from titration curves and Carterra Kinetics software. The maximum possible SPR (R max ) generated and the residual standard deviation (Res SD) were also calculated. The results of the kinetic constant screening are summarized in Table 21, indicating that the addition of the attenuated hIL2 moiety to the anti-hPD1 antibody did not modulate the binding of the human PD1 or cynomolgus PD1 antigen to the PD1 antibody. In another experiment, the steady-state equilibrium dissociation constant (K D ) measured by SPR was 1.23×10 -9 M for H7-632-hIgG1-LAGA (SEQ ID NOs: 414 and 415) and 1.93×10 -9 M for H7-767.

[0237]

Table 21-1

[0238]

Table 21-2

[0239] Example 14 Determination by surface plasmon resonance (SPR) of whether anti-hPD1 antibodies and anti-hPD1-attenuated hIL2 fusion proteins compete with anti-hPD1 #1 and anti-hPD1 #2 for binding to PD1 Using the sandwich method, the competition between anti-hPD1 and anti-hPD1-attenuated hIL2 fusion protein was assayed. The antibody and the corresponding antibody-IL2 cytokine fusion protein were immobilized on the HC30M chip using the amine coupling chemistry described in Example 13. Following the analysis of the rate constants described in Example 13, 80 nM of human PD1 (Acro Biosystems, Cat # PD-1-H5221-100ug) was injected across the array. The competing anti-hPD1 and anti-hPD1-attenuated hIL2 fusion proteins (analyte species) were diluted to 30 μg / mL and subsequently injected into the array, and the binding parameters were evaluated by SPR. The evaluation of all anti-hPD1 and anti-hPD1-hIL2 fusion proteins was performed twice. Some of the variants tested had a modified human IgG1 or IgG4κ light chain framework with L235E or L235A / G237A (LAGA) substitutions that interfere with the effector function of immunoglobulins.

[0240] By screening pairs of anti-hPD1 or anti-hPD1-attenuated hIL2 fusion proteins, as shown in Table 22, two bins could be distinguished. The antibodies and fusion proteins in Group 1 could bind to hPD1 in the presence of all the antibodies and fusion proteins in Group 2, but competed with all members of the same group. The antibodies and fusion proteins in Group 2 could bind to hPD1 in the presence of all the antibodies and fusion proteins in Group 1, but competed with all members of the same group. None of the anti-hPD1 listed in Group 1 of Table 22 competed with Keytruda® and Opdivo®.

[0241]

Table 22

[0242] Example 15 Antagonistic effect of anti-hPD1-attenuated hIL2 fusion protein on hPD1 in the presence of anti-hPD1 #1 and anti-hPD1 #2 The antagonistic effect of the anti-hPD1-attenuated hIL2 fusion protein against hPD1 was tested. The characterization of the anti-hPD1-attenuated hIL2 fusion protein was performed according to Protocol C of the general method. The results are shown in Figure 7. Compared with the PD1 antagonists Keytruda (registered trademark) or Opdivo (registered trademark), 2H7-hIgG4-df-hIL2 (D20A / R38E), C51E6-5-hIgG4-L6-hIL2 (D20A / R38E) and A2-hIgG4-df-hIL2 (D20A / R38E) are non-antagonists against human PD1 because of their low luminescence levels. The antagonist activities of H7-632-hIgG1-LAGA and H7-767 were also tested as described in Protocol C of the general method. Figure 15 shows that H7-632-hIgG1-LAGA and H7-767 do not block the interaction between hPDL1 (SEQ ID NO: 584) and the hPD1 receptor.

[0243] For the competition assay by the cell-based co-culture assay described in Protocol C of the general method, several modifications were made. Samples of the anti-hPD1-attenuated hIL2 fusion protein were diluted to a fixed concentration of 400 nM, and 20 μL of this was added to 20 μL of titrated anti-hPD1 #1 or anti-hPD1 #2. 40 μL of the mixture was added to CHO cells. 40 μL of Jurkat PD1 effector cells was overlaid on the mixture of CHO cells and the anti-hPD1-attenuated hIL2 fusion protein. In this competition assay, a saturated anti-hPD1-attenuated hIL2 fusion protein at a final concentration of 100 nM was tested in combination with titrated anti-hPD1 #1 or anti-hPD1 #2. The rest of the assay was performed as described in Protocol C of the general method. Figures 18A and 18B show that the addition of 100 nM of the anti-hPD1-attenuated hIL2 fusion protein did not block or compete with the binding of hPDL1 (SEQ ID NO: 584) to titrated anti-hPD1 #1. The dose titration curve of anti-hPD1 #1 did not change from the curve in the absence of the competing antibody, suggesting that the presence of the anti-hPD1-attenuated hIL2 fusion protein did not compete with the function of anti-hPD1 #1 even at high concentrations. In the presence of 100 nM of 2H7-hIgG4-df-hIL2 (D20A / R38E) and 100 nM of C51E6-5-hIgG4-L6-hIL2 (D20A / R38E), the luminescence (RLU) of anti-hPD1 #2 decreased by 35% with a high concentration of anti-hPD1 #2 (Figure 18B), but from the standard deviation values, it is unclear whether this decrease is significant.

[0244] In the reverse experiment, anti-hPD1 #1 or anti-hPD1 #2 was diluted to a concentration of 400 nM, and 20 μL thereof was combined with 20 μL of the titrated anti-hPD1-attenuated hIL2 fusion protein. The anti-hPD1-attenuated hIL2 fusion protein was titrated continuously, and 40 μL of the mixture was added to CHO cells and then overlaid on 40 μL of Jurkat PD1 effector cells. The remainder of the assay was performed as described in protocol C of the general method. Figures 18C and 18D show that the addition of 100 nM of anti-hPD1 #1 (Figure 18C) or 100 nM of anti-hPD1 #2 (Figure 18D) does not impair the ability of the anti-hPD1-attenuated hIL2 fusion protein as an antagonist. A flat curve exceeding 18,000 relative light units (RLU) indicates no competition for antagonist activity, and the anti-hPD1-attenuated hIL2 fusion protein tested was able to exert antagonist function even in the presence of anti-hPD1 #1 or anti-hPD1 #2.

[0245] Example 16 Testing of attenuation of anti-hPD1-attenuated hIL2 fusion protein against high-affinity and medium-affinity hIL2 receptors using a cell-based proliferation assay Using the cell proliferation assay in NK-92 and TF1+IL2Rβ cell lines described in Protocol E of the general method, the level of attenuation of hIL2 activity of the anti-hPD1-attenuated hIL2 fusion protein was evaluated. Control fusion proteins included those having a human IgG4 or human IgG1 backbone directly fused to hIL2, or fusion proteins incorporating an anti-DNase I antibody (designated 1H3) having a linker (SEQ ID NO: 355) to show the effect of non-target attenuated hIL2 fusion proteins. The hIL2 sequences of these constructs contained the substitutions of attenuated hIL2 activity described in Example 2. Full, partial or non-agonist IL2 activity (inactivity) was also evaluated as in Example 3. Some of the variants tested were expressed in modified human IgG1 or IgG4 isotypes having a κ light chain and involved additional L235E or L235A / G237A (LAGA) substitutions in the Fc region to abrogate the effector function of the immunoglobulin. In some antibody-cytokine fusion proteins, the hIL2 cytokine was fused at the C-terminus of the light chain (LC fusion).

[0246] The EC of each antibody-cytokine fusion protein from relative light units (RLU) 50 was determined and the fold change in EC compared to recombinant human IL2 (rhIL2) 50 was calculated. The fold change from rhIL2 and agonist activity are summarized in Table 23. Agonist activity was determined as full, partial or inactive by comparison of the maximum luminescence of the antibody-attenuated hIL2 fusion protein and rhIL2. The dose titration curve of the antibody-attenuated hIL2 fusion protein that reached the maximum luminescence as rhIL2 was considered a variant with full activity. Partial activity was calculated as a percentage of full activity with the maximum luminescence of rhIL2 as 100%. Antibody-attenuated hIL2 fusion proteins with a maximum RLU at the highest concentration of 1200 nM less than 10% of the rhIL2 maximum RLU were considered to have no agonist activity or be inactive. For some variants, as noted in a Table 23, the EC 50 values were only estimated because the maximum luminescence was not reached.

[0247]

Table 23-1

[0248]

Table 23-2

[0249]

Table 23-3

[0250]

Table 23-4

[0251] Example 17 Recovery of IL2 Activity of Anti-hPD1-Attenuated hIL2 Fusion Protein in a Cell Line Expressing Medium-Affinity hIL2 Receptor and hPD1 The anti-hPD1-attenuated hIL2 fusion protein was evaluated for recovery of hIL2 activity using a target cell line expressing hPD1. Briefly, the TF1+IL2Rβ cell line described in Protocol D of the general method was modified by lentiviral transduction to express the hPD1 receptor (SEQ ID NO: 580). hPD1 expressed by TF1+IL2Rβ cells was detected by flow cytometry using the Brilliant Blue 515-conjugated hPD1 antibody (BD Biosciences Cat#565936). Cells with low hPD1 expression (intensity less than 10 3 in Brilliant Blue 515 fluorophore) were sorted. The pool was sorted two more times to collect cells with an hPD1 expression level close to that of activated primary cells. This cell line (TF1+IL2Rβ+hPD1) was expanded and frozen in aliquots for cell-based proliferation assays. The proliferation assay was performed with a 3-day incubation period as described in Protocol E of the general method. Some of the variants tested had modified human IgG1 or IgG4κ light chain frameworks with L235E or L235A / G237A (LAGA) substitutions that interfere with the effector function of immunoglobulins.

[0252] The results of the proliferation assay in the target TF1 + IL2Rβ + hPD1 cell line are summarized in Table 24. The agonist activity was determined to be complete, partial, or inactive by comparing the maximum luminescence of the antibody-attenuated hIL2 fusion protein and rhIL2. The dose titration curve of the antibody-attenuated hIL2 fusion protein that reached the maximum luminescence as rhIL2 was regarded as a variant with complete activity. The partial activity was calculated as the ratio to the complete activity with the maximum luminescence of rhIL2 as 100%. The antibody-attenuated hIL2 fusion protein with a maximum RLU at the highest concentration of 1200 nM less than 10% of the rhIL2 maximum RLU was regarded as having no or inactive agonist activity. In some variants, since the complete curve was not reached, only the EC 50 value was estimated. In many examples of the anti-hPD1-hIL2 fusion protein with attenuated hIL2, hIL2 activity was restored in the target cell line, but not in the non-target antibody control (designated 1H3). Complete recovery was indicated by a decrease in the fold change from rhIL2 to 0 or 1.

[0253] [Table 24-1]

[0254] [Table 24-2]

[0255] [Table 24-3]

[0256] Evaluation of anti-hPD1-attenuated hIL2 fusion alternative proteins that block or do not block mouse PDL1 in the in vivo mouse colon adenocarcinoma (MC38) model of Example 18 There is no primate model accepted for investigating the in vivo efficacy of cancer therapeutics, so an anti-mPD1-attenuated hIL2 fusion replacement protein was produced and tested in a syngeneic mouse tumor model. This MC38 colon adenocarcinoma model is routinely used for testing the efficacy of immuno-oncology therapeutics. To investigate the in vivo effects of the anti-PD1-attenuated hIL2 fusion protein, anti-mouse PD1 replacement antibodies named RMP1-14 (known to block the binding of mouse PDL1) and RMP1-30 (described as a mouse PDL1 non-blocking agent) were fused to hIL2 attenuated at the C-terminus of the mouse IgG2b-N297A heavy chain and tested in the MC38 colon adenocarcinoma model. The hIL2 portion contained the F42K, Y45R, and V69R substitutions that were tested in the IL2-dependent mouse T lymphoblast cell line (CTLL-2) and shown to attenuate mouse IL2 activity. Human IL2 can stimulate the proliferation of mouse T cells at similar concentrations, but the same substitutions that attenuate activity against human IL2-dependent cell lines do not attenuate activity against the CTLL-2 cell line (data not shown). Therefore, the F42K / Y45R / V69R substitutions were used in hIL2 as an alternative because they showed attenuated IL2 activity in the mouse cell line. Sequences containing the variable region sequences of the heavy and light chains of the anti-mouse PD1 antibodies RMP1-14 and RMP1-30 (described in Matsumoto K et al., J Immunol. 2004 Feb 15;172(4):2530-41) were formatted on a mouse IgG2b-N297A background to produce anti-mPD1 RMP1-14 mIgG2b-N297A (SEQ ID NOs: 564 and 566) and anti-mPD1 RMP1-30 mIgG2b-N297A (SEQ ID NOs: 567 and 568) as well. The mouse IgG2b isotype with the N297A substitution is the mouse equivalent of an Fc isotype that impairs Fc effector function. The antibody-attenuated hIL2 fusion protein and replacement antibodies were produced, expressed, and purified with protein A using standard techniques.

[0257] In this tumor model mouse, 5 × 105 Individual MC38 colorectal cancer cells were injected. When the tumor reached 80 - 120 mm 3 in size, the mice were classified into cohorts (10 mice / group), and treatment was initiated on day 1 of the study. Anti-mPD1 RMP1-14 mIgG2b-N297A, anti-mPD1 RMP1-30 mIgG2b-N297A, anti-mPD1 RMP1-14 mIgG2b-N297A-L6-hIL2(F42K / Y45R / V69R) (SEQ ID NOs: 565 and 566) and anti-mPD1 RMP1-30 mIgG2b-N297A-L6-hIL2(F42K / Y45R / V69R) (SEQ ID NOs: 568 and 569), and solvent control (phosphate buffered saline) were administered intraperitoneally at 5 mg / kg twice a week for 4 weeks. Tumor size was measured twice a week with calipers using the formula (w 2 ×L) / 2 (w = width, L = length). The study endpoint was either when the tumor volume reached 1000 mm 3 or survival of 50 days, whichever came first.

[0258] Figure 8 shows that administration of anti-mPD1 RMP1-14-mIgG2b-N297A or anti-mPD1 RMP1-30-mIgG2b-N297A antibody alone did not result in significant efficacy compared to treatment with solvent control, but administration of the anti-PD1-attenuated hIL2 fusion proteins anti-mPD1 RMP1-14 mIgG2b-N297A-L6-hIL2(F42K / Y45R / V69R) or anti-mPD1 RMP1-30 mIgG2b-N297A-L6-hIL2(F42K / Y45R / V69R) was associated with complete tumor regression of 90% and 100% respectively. These data indicate that the anti-tumor effect of the anti-mPD1-hIL2(F42K / Y45R / V69R) fusion protein does not require PD1 checkpoint inhibition and its effect is dependent on hIL2 activity. Furthermore, this data indicates that targeting with an antibody against PD1-expressing T cells is sufficient to promote a strong anti-tumor effect in the MC38 tumor model.

[0259] Example 19 Anti-hPD1-attenuated hIL2 fusion replacement protein increases effector memory CD8+ T cells in in vivo colon adenocarcinoma model mice To understand the in vivo mechanism of action of the anti-hPD1-attenuated hIL2 fusion replacement protein, an in vivo experiment similar to Example 18 was conducted. Subsequently, after three administrations, immunophenotyping of the T cell population obtained from tumors, blood, spleen, and lymph nodes was performed. Five × 10 5 mouse MC38 colon adenocarcinoma tumor cells were subcutaneously transplanted into the right flanks of 10-week-old female C57BL / 6NCrl mice (Charles River), and tumor growth was monitored. Animals with tumors of 150 - 260 mm 3 were divided into 4 groups of 10 mice per group for the experiment. Twenty-one days after transplantation, on days 1, 4, and 8, 0.2 mL / dose of phosphate-buffered saline (PBS) as a solvent control, 5 mg / kg of anti-KLH-C3-mIgG2b-N297A-L6-hIL2 (F42K / Y45R / V69R), 5 mg / kg of anti-mPD1 RMP1-30 mIgG2b-N297A, or 5 mg / kg of anti-mPD1 RMP1-30 mIgG2b-N297A-L6-hIL2 (F42K / Y45R / V69R) were administered intraperitoneally to the animals. On day 9, tumors, spleens, and inguinal lymph nodes were collected from all mice and processed into single cell suspensions for subsequent flow cytometry analysis.

[0260] Figure 9A is a graph of the growth of tumor volume (mm 3 ) from the first administration on day 1 to day 9, and each point represents the average of 10 mice. By day 8, the tumor volume of anti-mPD1 RMP1-30 mIgG2b-N297A-L6-hIL2 (F42K / Y45R / V69R) decreased compared to other treatment groups. Figure 9B summarizes the contribution of various CD8 + T cell subsets in the tumors of each treatment group, where the phenotypes are CD45 + CD3 + CD4 - CD8 + CD44 + CD127 + CD69 - CD103- is central memory T cells, CD45 + CD3 + CD4 - CD8 + CD44 + CD127 + CD69 - CD103 - CD62L - is effector memory T cells, CD45 + CD3 + CD4 - CD8 + CD44 + CD127 + CD69 + CD103 + is resident memory T cells, CD45 + CD3 + CD4 - CD8 + CD44 + CD62L - is CD44 - CD62L - T cells, and CD45 + CD3 + CD4 - CD8 + CD44 - CD62L + is naive T cells. As shown by the increase in the light gray portion in Figure 9B, in mice treated with anti-mPD1 RMP1-30 mIgG2b-N297A-L6-hIL2(F42K / Y45R / V69R), compared to other treatment groups, the CD8 + effector memory T cell subset increased. This was also shown by the absolute numbers (cells / μL) in the MC38 excised tumors in Figure 9C. Furthermore, within the tumors, the absolute numbers (cells / μL) of regulatory T cells identified by the expression of CD45 + CD3 + CD4 + CD8 - CD25 + FoxP3 + marker decreased.

[0261] CD8 +The increase in effector memory T cells and the decrease in regulatory T cells are associated with effective immunotherapy in both mice and humans.

[0262] Example 20 Anti-hPD1-attenuated hIL2 fusion protein is active in vivo in the NCG-PBMC model NOD-Prkdc lacking functional T cells, B cells and NK cells of human immune cells em26Cd52 IL-2rg em26Cd22 Transplantation into / NjuCrl (NCG) mice has become a valuable tool for evaluating the efficacy of therapeutic agents that are hypothesized to stimulate human T cells. In this model, when a therapeutic agent activates human T cells, T cells increase and graft-versus-host disease (GvHD) is accelerated.

[0263] For human peripheral blood mononuclear cell (hPBMC) transplantation, the infusion rate constants and the expression of human PD1 and human IL2 receptor on T cells of three donors were evaluated for 4 weeks. Among the three donors, the donor that induced the most T cells in the intermediate window of GvHD was selected. 1.5×10 7hPBMC were intravenously administered to NCG mice and divided into 8 groups of 8 - 16 mice each. On days 7, 10, and 14, the mice were intraperitoneally administered 2H7 - hIgG1 - LAGA - df - hIL2(T3A / D20A / R38E / C125A)(SEQ ID NO: 471, 425)(2.5 mg / kg, 5 mg / kg, or 10 mg / kg), 1H3 - hIgG1 - LAGA - df - hIL2(T3A / D20A / R38E / C125A)(SEQ ID NO: 546, 374)(5 mg / kg or 10 mg / kg), 1H3 - hIgG1 - LAGA - df - hIL2(T3A / C125A)(SEQ ID NO: 563, 374)(10 mg / kg), or 2H7 - hIgG1 - LAGA - df - hIL2(T3A / R38E / I92K / C125A)(SEQ ID NO: 474, 425)(5 mg / kg) three times. Anti - DNase fusion proteins as wild - type hIL2 moiety (1H3 - hIgG1 - LAGA - df - hIL2(T3A / C125A)) and as attenuated hIL2 moiety (1H3 - hIgG1 - LAGA - df - hIL2(T3A / D20A / R38E / C125A)) were used as non - target antibody controls. The 1H3 - hIgG1 - LAGA - df - hIL2(T3A / C125A) fusion protein contains T3A and C125A substitutions, respectively, to remove the predicted O - linked glycosylation sites of human IL2 (see, for example, WO 2012 / 107417) and unpaired cysteine residues (see, for example, WO 2018 / 184964), although there is no change in the hIL2 moiety that reduces hIL2 activity. These substitutions do not show a decrease in hIL2 efficacy clinically. On day 21, blood, spleen, and lung were collected. Blood and spleen were processed for immunophenotyping by flow cytometry, and the lung was weighed.

[0264] Twenty - one days later, immunophenotyping by flow cytometry was performed on the blood and spleen of the animals. Markers used to distinguish human T - cell populations in subsequent analyses are summarized in Table 25.

[0265]

Table 25

[0266] As shown in Fig. 10, as an evaluation of graft-versus-host disease (GvHD), the body weight of each animal was measured for 21 days and normalized on day 1. Acceleration of GvHD was observed in the mice treated with 2H7-hIgG1-LAGA-df-hIL2 (T3A / D20A / R38E / C125A) at 10 mg / kg. Slight decreases in body weight were also observed in the mice treated with 2H7-hIgG1-LAGA-df-hIL2 (T3A / D20A / R38E / C125A) at 2.5 mg / kg and 5 mg / kg, and in the mice treated with 2H7-hIgG1-LAGA-df-hIL2 (T3A / R38E / I92K / C125A) at 5 mg / kg. A decrease in body weight was also seen with 1H3-hIgG1-LAGA-df-hIL2 (T3A / C125A), but it was not sustained.

[0267] Flow cytometry analysis was correlated with the observed acceleration of graft-versus-host disease (GvHD). In the flow cytometry analysis of peripheral blood using the phenotypic markers that distinguish human T cell subsets shown in Table 25, only slight increases in the CD3 + , CD4 + and CD8 + T cell subsets were shown (for CD3 + T cells, fold change from the vehicle control was 10-fold to 50-fold). Furthermore, the CD3 + , CD4 + and CD8 + T cell subsets were significantly increased in the peripheral blood of the mice treated with 2H7-hIgG1-LAGA-df-hIL2 (T3A / D20A / R38E / C125A) at 10 mg / kg (for CD3 + T cells, fold change from the vehicle control exceeded 50-fold). The increases in human T cell subsets are summarized in Table 26.

[0268]

Table 26

[0269] CD3 among treatment groups + , CD4 + and CD8 + In addition to the evaluation of CD4 + and CD8 + T cells, the memory and naive subsets of CD4 + T cells and CD8 + T cells were also evaluated. The phenotypic markers used to distinguish naive, effector, effector memory, and central memory of CD4 + T cells and CD8 + T cells are summarized in Table 25. There were no changes in naive, effector, or central memory T cells among treatment groups (data not shown). However, in mice treated with 10 mg / kg of 2H7-hIgG1-LAGA-df-hIL2 (T3A / D20A / R38E / C125A), CD4 + and CD8 + effector memory (EM) T cells were significantly increased, and the average cell number per milliliter was more than 5 million for CD8 + T cells and more than 50 million for CD4 + T cells (Figures 11A and 11B). Box-and-whisker plots were graphed with boxes enclosing the first and third quartiles, a horizontal line as the median, and lines indicating the minimum and maximum values. In animals treated with 2.5 mg / kg and 5 mg / kg of 2H7-hIgG1-LAGA-df-hIL2 (T3A / D20A / R38E / C125A), and in animals treated with 1H3-hIgG1-LAGA-df-hIL2 (T3A / C125A), there was a moderate increase in the average cell number per milliliter of CD8 + effector memory (EM) T cells from 1 million to 5 million. In mice treated with 2.5 mg / kg and 5 mg / kg of 2H7-hIgG1-LAGA-df-hIL2 (T3A / D20A / R38E / C125A), there was a moderate increase in CD4

[0270] In addition to stimulating effector T cells, IL2 has been described to stimulate NK cells and regulatory T cells (Tregs). Since Tregs express CD25 at high levels and NK cells express CD122, these immune cells were also evaluated. Figure 12 shows that animals administered the highest dose of 2H7-hIgG1-LAGA-df-hIL2 (T3A / D20A / R38E / C125A), 10 mg / kg, did not increase human regulatory T cells. Instead, the proportion of regulatory T cells (identified by the phenotype in Table 25) in the peripheral blood of the animals was the lowest. There was a dose-dependent decrease in human regulatory T cells, and human CD3 T cells that are Tregs were, at 10 mg / kg of 2H7-hIgG1-LAGA-df-hIL2 (T3A / D20A / R38E / C125A), 0.16% on average compared to 1.6% on average in the vehicle control. The proportion of human NK cells in the peripheral blood (identified by the phenotype in Table 25) did not change in all treatment groups (data not shown) compared to the vehicle control. + T cells were 0.16% on average at 10 mg / kg of 2H7-hIgG1-LAGA-df-hIL2 (T3A / D20A / R38E / C125A) compared to 1.6% on average in the vehicle control. The proportion of human NK cells in the peripheral blood (identified by the phenotype in Table 25) did not change in all treatment groups (data not shown) compared to the vehicle control.

[0271] Example 21 Non-clinical safety profile of anti-hPD1-attenuated hIL2 fusion protein Previously, cynomolgus monkeys have been used to evaluate the toxicity of unmodified IL2. In cynomolgus monkeys, lethality was observed even at a low dose of 50 μg / kg / day of exogenous recombinant IL2. Since the binding of cynomolgus monkey hPD1 and H7-767 was confirmed by flow cytometry in activated primary PBMCs (Example 12), a single-dose test for preliminary safety evaluation was conducted with both variants of H7-767 (H7-02-hIgG1-LAGA-df-hIL2(T3A / D20A / R38E / C125A) (SEQ ID NOs: 582 and 583) and H7-767). H7-02-hIgG1-LAGA-df-hIL2(T3A / D20A / R38E / C125A) was delivered to monkeys by intravenous injection for 15 minutes at 1 mg / kg (4 animals) or 10 mg / kg (4 animals). Sampling was performed up to 360 hours after injection. No side effects, gross toxicity, weight loss, or lethality were observed (data not shown). A follow-up single-dose test with H7-767 was conducted at high doses of 5 mg / kg and 50 mg / kg in the same manner as the first test, and sampling was performed up to 360 hours after injection. Again, no side effects, gross toxicity, weight loss, or lethality were observed (data not shown).

[0272] Example 22 Attenuation of IL2 Activity of Modified hIL2 Protein The attenuation of IL2 activity of modified hIL2 proteins containing the substitution at position 20 (D20) and the substitution at position 38 (R38) was tested in proliferation assays in NK-92 and TF1+IL2Rβ cell lines as described in Example 5. The modified hIL2 proteins were divided into seven groups (Group 1 to Group 7) based on the level of attenuation of the maximum agonist activity of the modified hIL2 proteins and the potency against both the intermediate-affinity and high-affinity receptors compared to unmodified recombinant hIL2 (Table 27). The criteria used to divide the modified hIL2 proteins were as follows: · Group 1: Variants with the highest attenuation (i.e., more than 10,000-fold) against the intermediate-affinity receptor, showing at least about 80% activity, and also high attenuation against the high-affinity receptor, showing at least about 70% activity, · Group 2: Variants that show at least about 70% activity against the intermediate affinity receptor and more than a 1,000-fold attenuation, and show about 20% to about 30% activity against the high affinity receptor. · Group 3: Variants that show about 50% to about 70% activity against the intermediate affinity receptor and more than a 1,000-fold attenuation, and show about 20% activity against the high affinity receptor. · Group 4: Variants that show at least about 70% activity against the intermediate affinity receptor but show more than a 500-fold attenuation and show about 50% activity against the high affinity receptor. · Group 5: Variants that show at least about 70% activity against both receptors, but show more than a 10-fold to more than a 300-fold attenuation in descending order against the intermediate affinity receptor, and also show a 70-fold to 1,500-fold attenuation in descending order against the high affinity receptor. · Group 6: Variants that show only about 30% activity and more than a 2,500-fold attenuation against the intermediate affinity receptor and have no activity against the high affinity receptor. · Group 7: Variants that have no activity against both the intermediate affinity receptor and the high affinity receptor.

[0273]

Table 27

[0274] Example 23 Activity of the fusion replacement protein in a mouse MC38 colorectal tumor model Isogenic MC38 colorectal cancer cells at 5×10 5 were injected into the right flank of 10-week-old female C57BL / 6NCrl mice. When the tumors reached 80 - 120 mm 3 , the mice were divided into cohorts (10 mice / group), and treatment was initiated on day 1 of the study. All agents except hIL2 were intraperitoneally administered at 5 mg / kg twice a week for 4 weeks starting from day 1. hIL2 at 36,000 international units was intraperitoneally administered once a day from day 1 to day 5. During the study period, the tumor size was measured twice a week with calipers. The study endpoint was either the tumor volume reaching 1000 mm 3 , survival for 50 days, or progression-free survival for 70 days, whichever came first.

[0275] All agents tested that contain antibody molecules and antibody-hIL2 fusion proteins were produced using the Fc region of mouse IgG2b with a single N297A amino acid substitution at position 297 that prevents glycosylation of the Fc region, greatly reduces immune effector function via the Fc region, and thereby prevents cell depletion in vivo. Anti-mPD1 RMP1-14 is a monoclonal antibody antagonist of the mouse PD1 receptor (Matsumoto, J Immunol 172: 2530-2541, 2004). Anti-mPD1 RMP1-14-hIL2 F42K / Y45R / V69R is a bifunctional fusion protein consisting of a monoclonal RMP1-14 antibody antagonist of the mouse PD1 receptor fused at the C-terminus via a flexible 6-amino acid glycine / serine linker to hIL2 F42K / Y45R / V69R (SEQ ID NO: 621), an IL2 variant with reduced potency. This molecule was designed such that the IL2 variant with reduced potency directly targets T cells expressing PD1 in vivo in mice. Anti-KLH-hIL2 F42K, Y45R, V69R is a control fusion protein consisting of an isotype control monoclonal antibody that recognizes a non-mammalian antigen (keyhole limpet hemocyanin, KLH) fused at the C-terminus via a flexible 6-amino acid glycine / serine linker to hIL2 F42K, Y45R, V69R, an IL2 variant with reduced potency.

[0276] The results are shown in Figure 19. The MC38 colorectal tumor model responds particularly to inhibition of the PD1 receptor by antibodies. Tumor growth in vehicle-treated mice rapidly reached the endpoint of the study, and tumors completely regressed in 50% of the mice treated with anti-mPD1 RMP1-14. In contrast, 100% of the mice treated with the anti-mPD1 RMP1-14-hIL2 F42K, Y45R, V69R fusion protein experienced persistent and long-term tumor regression. Mice treated with various combinations of the individual components of the anti-mPD1 RMP1-14-hIL2 F42K, Y45R, V69R fusion protein, including either anti-mPD1 RMP1-14 combined with cytokines other than hIL2 (administered at doses and regimens equivalent to human therapeutic doses), or anti-mPD1 RMP1-14 combined with a non-target anti-KLH-hIL2 F42K, Y45R, V69R fusion protein, did not reproduce the effect seen with anti-mPD1 RMP1-14-hIL2 F42K, Y45R, V69R. These data indicate that targeting hIL2 with reduced potency to PD1-expressing cells significantly improves the anti-tumor effect compared to anti-PD1 receptor antagonists, and that the activity of the fusion protein is not due to the additive effects of the individual components of the molecule.

[0277] Example 24 Evaluation of Anti-Tumor Protective Immunity Induced by Alternative Anti-mPD1 RMP1-14-hIL2 F42K, Y45R, V69R in the MC38 Colorectal Tumor Model Mice that had complete tumor regression and survived until day 50 in the first experiment of Example 23 were given a second tumor challenge without further drug therapy. For the tumor rechallenge, 5 × 10 5 MC38 tumor cells were implanted into the left flank on the opposite side of the location of the mice's first tumor. As a control group, 10 age-matched tumor-naive mice were implanted with MC38 tumor cells.

[0278] Figure 20 shows that all mice that survived until day 50 after treatment with anti-mPD1 RMP1-14-hIL2 F42K, Y45R, V69R, in which the tumors completely regressed in the first tumor experiment, were completely protected from the development of tumors in the second tumor challenge. In contrast, all tumor-naive mice transplanted with MC38 tumor cells developed tumors that rapidly reached the experimental endpoint of a tumor volume of 100 mm 3 . The generation of anti-tumor defensive immunity without continued drug therapy suggests that anti-mPD1 RMP1-14-hIL2 F42K, Y45R, V69R induced a response of anti-tumor memory T cells.

[0279]

Table 28-1

[0280]

Table 28-2

[0281]

Table 28-3

[0282]

Table 28-4

[0283]

Table 28-5

[0284]

Table 28-6

[0285]

Table 28-7

[0286]

Table 28-8

[0287]

Table 28-9

[0288]

Table 28-10

[0289]

Table 29-1

[0290]

Table 29-2

[0291]

Table 29-3

[0292]

Table 29-4

[0293]

Table 29-5

[0294]

Table 29-6

[0295]

Table 29-7

[0296]

Table 29-8

[0297]

Table 29-9

[0298]

Table 29-10

[0299]

Table 29-11

[0300]

Table 29-12

[0301]

Table 29-13

[0302]

Table 29-14

[0303]

Table 29-15

[0304]

Table 29-16

[0305]

Table 29-17

[0306]

Table 29-18

[0307]

Table 29-19

[0308]

Table 29-20

[0309]

Table 29-21

[0310]

Table 29-22

[0311]

Table 29-23

[0312]

Table 29-24

[0313]

Table 29-25

[0314]

Table 29-26

[0315]

Table 29-27

[0316]

Table 29-28

[0317]

Table 29-29

[0318]

Table 29-30

[0319]

Table 29-31

[0320]

Table 29-32

[0321]

Table 29-33

[0322]

Table 29-34

[0323]

Table 29-35

[0324]

Table 29-36

[0325]

Table 29-37

[0326]

Table 29-38

[0327]

Table 29-39

[0328]

Table 29-40

[0329]

Table 29-41

[0330]

Table 29-42

[0331]

Table 29-43

[0332]

Table 29-44

[0333]

Table 29-45

[0334]

Table 29-46

[0335]

Table 29-47

[0336]

Table 29-48

[0337]

Table 29-49

[0338]

Table 29-50

[0339]

Table 29-51

[0340]

Table 29-52

[0341]

Table 29-53

[0342]

Table 29-54

[0343]

Table 29-55

[0344]

Table 29-56

[0345]

Table 29-57

[0346]

Table 29-58

[0347]

Table 29-59

[0348]

Table 29-60

[0349]

Table 29-61

[0350]

Table 29-62

[0351]

Table 29-63

[0352]

Table 29-64

[0353]

Table 29-65

[0354]

Table 29-66

[0355]

Table 29-67

[0356]

Table 29-68

[0357]

Table 29-69

[0358]

Table 29-70

[0359]

Table 29-71

[0360]

Table 29-72

[0361]

Table 29-73

[0362]

Table 29-74

[0363]

Table 29-75

[0364]

Table 29-76

[0365]

Table 29-77

[0366]

Table 29-78

[0367]

Table 29-79

[0368]

Table 29-80

[0369]

Table 29-81

[0370]

Table 29-82

[0371]

Table 29-83

[0372]

Table 29-84

[0373]

Table 29-85

[0374]

Table 29-86

[0375]

Table 29-87

[0376]

Table 29-88

[0377]

Table 29-89

[0378]

Table 29-90

[0379]

Table 29-91

[0380]

Table 29-92

[0381]

Table 29-93

[0382]

Table 29-94

[0383]

Table 29-95

[0384] Those skilled in the art will understand that numerous changes and modifications can be made to the preferred embodiments disclosed in this specification, and such changes and modifications can be made without departing from the spirit of the present invention. Accordingly, the claims are intended to cover all equivalent variants that fall within the true spirit and scope of the present invention.

[0385] The disclosure of each patent, patent application, and publication cited or described in this specification is hereby incorporated by reference in its entirety into this specification.

[0386] Embodiments The following list of aspects is not intended to supersede or replace the previous descriptions, but rather to supplement them. Aspect 1: A modified human interleukin-2 (hIL2) protein comprising substitutions at positions 20 and 38 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345, wherein the modified hIL2 protein has reduced potency against both the high-affinity hIL2 receptor and the intermediate-affinity hIL2 receptor compared to unmodified hIL2. Aspect 2: The modified hIL2 protein according to aspect 1, wherein the substitution at position 20 is selected from D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E. Aspect 3: The modified hIL2 protein according to aspect 1 or 2, wherein the substitution at position 38 is selected from R38E, R38N, R38G, R38H, R38I, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D and R38K. Aspect 4: The modified hIL2 protein according to any one of the preceding aspects, further comprising a deletion or substitution at position 3 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345. Aspect 5: The modified hIL2 protein according to aspect 4, wherein the substitution at position 3 is T3A. Aspect 6: The modified hIL2 protein according to any one of the preceding aspects, further comprising a deletion or substitution at position 125 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345. Aspect 7: The modified hIL2 protein according to aspect 6, wherein the substitution at position 125 is C125A. Aspect 8: The modified hIL2 protein according to any one of the preceding aspects, wherein the potency of the modified hIL2 protein against the high-affinity hIL2 receptor (hIL2Rαβγ) is reduced by about 1,000-fold. Aspect 9: The modified hIL2 protein according to any one of the preceding aspects, wherein the potency of the modified hIL2 protein against the intermediate-affinity hIL2 receptor (hIL2Rβγ) is reduced by about 10,000-fold. Aspect 10: The modified hIL2 protein according to any one of Aspects 1 to 9, which is fused with an anti-PD1 antibody or an antigen-binding fragment thereof. Aspect 11: The modified hIL2 protein according to Aspect 10, wherein the modified hIL2 protein is fused with the antibody or the antigen-binding fragment thereof at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment. Aspect 12: The modified hIL2 protein according to Aspect 10 or 11, wherein the modified hIL2 protein is directly fused with the antibody or the antigen-binding fragment thereof by a peptide bond. Aspect 13: The modified hIL2 protein according to Aspect 12, wherein the modified hIL2 is directly fused with the amino acid residue at the C-terminus of the antibody heavy chain by a peptide bond. Aspect 14: The modified hIL2 protein according to Aspect 10 or 11, wherein the modified hIL2 protein is fused with the antibody or the antigen-binding fragment thereof via a linker. Aspect 15: A modified human interleukin-2 (hIL2) protein, comprising a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitution at position 20 and an R38E substitution at position 38 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345. Aspect 16: The modified hIL2 protein according to Aspect 15, comprising any one of the amino acid sequences shown in SEQ ID NO: 307, 607-611, 614, 617, or 620. Aspect 17: The modified hIL2 protein according to Aspect 15 or 16, comprising a D20A substitution and an R38E substitution. Aspect 18: The modified hIL2 protein according to Aspect 17, comprising the amino acid sequence shown in SEQ ID NO: 149. Aspect 19: The modified hIL2 protein according to any one of Aspects 15 to 18, further comprising a deletion or substitution at position 3 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345. Aspect 20: The modified hIL2 protein according to Aspect 19, wherein the substitution at position 3 is T3A. Aspect 21: The modified hIL2 protein according to Aspect 20, comprising the amino acid sequence shown in SEQ ID NO: 216. Aspect 22: The modified hIL2 protein according to Aspect 19, comprising the amino acid sequence shown in SEQ ID NO: 218. Aspect 23: The modified hIL2 protein according to any one of Aspects 15 to 22, further comprising a deletion or substitution at position 125 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345. Aspect 24: The modified hIL2 protein according to Aspect 23, wherein the substitution at position 125 is C125A. Aspect 25: The modified hIL2 protein according to Aspect 24, comprising the amino acid sequence shown in SEQ ID NO: 215, 217 or 219. Aspect 26: The modified hIL2 protein according to Aspect 25, comprising the amino acid sequence shown in SEQ ID NO: 217. Aspect 27: The modified hIL2 protein according to any one of Aspects 15 to 26, wherein the modified hIL2 protein is fused with an anti-PD1 antibody or an antigen-binding fragment thereof. Aspect 28: The modified hIL2 protein according to Aspect 27, wherein the modified hIL2 protein is fused with the antibody or its antigen-binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment or the C-terminus of the antigen-binding fragment. Aspect 29: The modified hIL2 protein according to Aspect 27 or 28, wherein the modified hIL2 protein is directly fused with the antibody or its antigen-binding fragment by a peptide bond. Aspect 30: The modified hIL2 protein according to Aspect 29, wherein the modified hIL2 is directly fused with the amino acid residue at the C-terminus of the antibody heavy chain by a peptide bond.

[0387] Aspect 31: The modified hIL2 protein according to Aspect 27 or 28, wherein the modified hIL2 protein is fused with the antibody or its antigen-binding fragment via a linker. Aspect 32: A human antibody molecule or an antigen-binding fragment thereof that immunospecifically binds to human programmed cell death protein 1 (hPD1), wherein the human antibody molecule or its antigen-binding fragment a) A heavy chain complementarity determining region 1 (CDR1) comprising the amino acid sequence shown in SEQ ID NO: 418, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 419, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 420, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 421, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 422, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 423; b) A heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 386, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 387, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 388, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 389, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 390, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 391; c) A heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 396, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 397, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 398, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 399, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 400, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 401; or, d) A heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 406, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 407, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 408, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 409, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 410, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 411; A human antibody molecule or an antigen-binding fragment thereof comprising the above. Aspect 33 a) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 416, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 417; b) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 384, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 385; c) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 394, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 395; or, d) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 404 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 405; The human antibody molecule or antigen-binding fragment thereof according to embodiment 32, comprising Embodiment 34 The human antibody molecule or antigen-binding fragment thereof according to embodiment 32 or 33, comprising a human IgG1 heavy chain constant region. Embodiment 35 The human antibody molecule or antigen-binding fragment thereof according to embodiment 34, comprising L235A substitution and G237A substitution according to EU numbering. Embodiment 36 a) A heavy chain comprising the amino acid sequence shown in SEQ ID NO: 414 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 415; b) A heavy chain comprising the amino acid sequence shown in SEQ ID NO: 424 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 425; c) A heavy chain comprising the amino acid sequence shown in SEQ ID NO: 426 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 427; or, d) A heavy chain comprising the amino acid sequence shown in SEQ ID NO: 428 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 429; The human antibody molecule or antigen-binding fragment thereof according to any one of embodiments 32 to 35, comprising Embodiment 37 The human antibody molecule or antigen-binding fragment thereof according to embodiment 36, comprising a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 414 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 415. Embodiment 38 The human antibody molecule or antigen-binding fragment thereof according to any one of embodiments 32 to 37, which is fused with a modified human interleukin-2 (hIL2) protein comprising substitutions at position 20 and position 38 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345. Embodiment 39 The human antibody molecule or antigen-binding fragment thereof according to embodiment 38, wherein the modified hIL2 protein comprises any one of the amino acid sequences shown in SEQ ID NOs: 134 to 150, 307, 344, 607 to 611, 614, 617 or 620. Embodiment 40 The human antibody molecule or antigen-binding fragment thereof according to embodiment 39, wherein the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 149. Aspect 41: The modified hIL2 protein further comprises a deletion or substitution at position 3 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345, and is the human antibody molecule or antigen-binding fragment thereof according to any one of Aspects 38 to 40. Aspect 42: The substitution at position 3 is T3A, and is the human antibody molecule or antigen-binding fragment thereof according to Aspect 41. Aspect 43: The modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 216, and is the human antibody molecule or antigen-binding fragment thereof according to Aspect 42. Aspect 44: The modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 218, and is the human antibody molecule or antigen-binding fragment thereof according to Aspect 41. Aspect 45: The modified hIL2 protein further comprises a deletion or substitution at position 125 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345, and is the human antibody molecule or antigen-binding fragment thereof according to any one of Aspects 38 to 44. Aspect 46: The substitution at position 125 is C125A, and is the human antibody molecule or antigen-binding fragment thereof according to Aspect 45. Aspect 47: The modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 215, 217 or 219, and is the human antibody molecule or antigen-binding fragment thereof according to Aspect 46. Aspect 48: The modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 217, and is the human antibody molecule or antigen-binding fragment thereof according to Aspect 47. Aspect 49: The modified hIL2 protein is fused with the antibody or antigen-binding fragment thereof at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment or the C-terminus of the antigen-binding fragment, and is the human antibody molecule or antigen-binding fragment thereof according to any one of Aspects 38 to 48. Aspect 50: The modified hIL2 protein is directly fused with the antibody or antigen-binding fragment thereof by a peptide bond, and is the human antibody molecule or antigen-binding fragment thereof according to any one of Aspects 38 to 49. Aspect 51: The human antibody molecule or antigen-binding fragment thereof according to Aspect 50, wherein the modified hIL2 protein is directly fused to the amino acid residue at the C-terminus of the antibody heavy chain by a peptide bond. Aspect 52: The human antibody molecule or antigen-binding fragment thereof according to any one of Aspects 38 to 49, wherein the modified hIL2 protein is fused to the antibody or antigen-binding fragment via a linker. Aspect 53: An immune complex comprising: (a) A modified human interleukin 2 (hIL2) protein comprising substitutions at position 20 and position 38 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345; and (b) A human antibody molecule or antigen-binding fragment thereof that immunospecifically binds to human programmed cell death protein 1 (hPD1). The human antibody molecule or antigen-binding fragment thereof comprises: (i) Heavy chain complementarity determining region 1 (CDR1) comprising the amino acid sequence shown in SEQ ID NO: 418, heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 419, heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 420, light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 421, light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 422, and light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 423; (ii) Heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 386, heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 387, heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 388, light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 389, light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 390, and light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 391; (iii) Heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 396, heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 397, heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 398, light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 399, light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 400, and light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 401; or (iv) A heavy-chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 406, a heavy-chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 407, a heavy-chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 408, a light-chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 409, a light-chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 410, and a light-chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 411; An immune complex comprising the same. Aspect 54 The substitution at position 20 of the modified hIL2 protein is selected from D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E, and the immune complex according to aspect 53. Aspect 55 The substitution at position 38 of the modified hIL2 protein is selected from R38E, R38N, R38G, R38H, R38I, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D, and R38K, and the immune complex according to aspect 53 or 54. Aspect 56 The substitution at position 20 of the modified hIL2 protein is selected from D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E, and the substitution at position 38 of the modified hIL2 protein is R38E. The immune complex according to any one of aspects 53 to 55. Aspect 57 The modified hIL2 protein comprises any one of the amino acid sequences shown in SEQ ID NOs: 134 to 150, 307, 344, 607 to 611, 614, 617, or 620, and the immune complex according to any one of aspects 53 to 56. Aspect 58 The modified hIL2 protein comprises D20A and R38E substitutions, and the immune complex according to any one of aspects 53 to 56. Aspect 59 The modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 149, and the immune complex according to aspect 58. Aspect 60 The immune complex according to any one of aspects 53 to 57, which comprises any one of the amino acid sequences shown in SEQ ID NOs: 608, 614, 611, 620, 607, 610, 617, 609, or 307.

[0388] Aspect 61: The immune complex according to any one of Aspects 53 to 60, wherein the modified hIL-2 protein further comprises a deletion or substitution at position 3 of the amino acid sequence of unmodified IL-2 shown in SEQ ID NO: 345. Aspect 62: The immune complex according to Aspect 61, wherein the substitution at position 3 of the modified hIL-2 protein is T3A. Aspect 63: The immune complex according to Aspect 62, wherein the modified hIL-2 protein comprises the amino acid sequence shown in SEQ ID NO: 216. Aspect 64: The immune complex according to Aspect 61, wherein the modified hIL-2 protein comprises the amino acid sequence shown in SEQ ID NO: 218. Aspect 65: The immune complex according to any one of Aspects 53 to 64, wherein the modified hIL-2 protein further comprises a deletion or substitution at position 125 of the amino acid sequence of unmodified IL-2 shown in SEQ ID NO: 345. Aspect 66: The immune complex according to Aspect 65, wherein the substitution at position 125 is C125A. Aspect 67: The immune complex according to Aspect 66, wherein the modified hIL-2 protein comprises the amino acid sequence shown in SEQ ID NO: 215, 217 or 219. Aspect 68: The immune complex according to Aspect 67, wherein the modified hIL-2 protein comprises the amino acid sequence shown in SEQ ID NO: 217. Aspect 69: The immune complex according to any one of Aspects 53 to 68, wherein the modified hIL-2 protein is fused with the antibody or its antigen-binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment or the C-terminus of the antigen-binding fragment. Aspect 70: The immune complex according to any one of Aspects 53 to 69, wherein the modified hIL-2 protein is directly fused with the antibody or its antigen-binding fragment by a peptide bond. Aspect 71: The immune complex according to Aspect 70, wherein the modified hIL-2 protein is directly fused with the amino acid residue at the C-terminus of the antibody heavy chain by a peptide bond. Aspect 72: The immune complex according to any one of Aspects 53 to 69, wherein the modified hIL-2 protein is fused with the antibody or its antigen-binding fragment via a linker. Aspect 73 The human antibody molecule or antigen-binding fragment thereof is a) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 416, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 417; b) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 384, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 385; c) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 394, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 395; or, d) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 404, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 405; and is the immune complex according to any one of Aspects 53 to 72. Aspect 74 The human antibody molecule or antigen-binding fragment thereof is the immune complex according to any one of Aspects 53 to 73, which comprises an IgG1 heavy chain constant region. Aspect 75 The human antibody molecule or antigen-binding fragment thereof is the immune complex according to Aspect 74, which comprises L235A substitution and G237A substitution according to EU numbering. Aspect 76 The human antibody molecule or antigen-binding fragment thereof is a) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 414 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 415; b) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 424 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 425; c) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 426 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 427; or, d) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 428 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 429; and is the immune complex according to any one of Aspects 53 to 75. Aspect 77 The human antibody molecule or antigen-binding fragment thereof is the immune complex according to Aspect 76, which comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 414 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 415. Aspect 78 A light chain comprising the amino acid sequence shown in SEQ ID NO: 415; and, A heavy-chain modified hIL2 protein fusion comprising the amino acid sequence shown in SEQ ID NO: 532 An immune complex according to any one of aspects 53 to 57, comprising . A pharmaceutical composition comprising a modified hIL2 protein according to any one of aspects 1 to 31, a human antibody molecule or an antigen-binding fragment thereof according to any one of aspects 32 to 52, or an immune complex according to any one of aspects 53 to 78. A polynucleotide comprising a nucleic acid sequence encoding a modified hIL2 protein according to any one of aspects 1 to 31, a human antibody molecule or an antigen-binding fragment thereof according to any one of aspects 32 to 52, or an immune complex according to any one of aspects 53 to 78, according to aspect 80. A vector comprising a polynucleotide comprising a nucleic acid sequence encoding a modified hIL2 protein according to any one of aspects 1 to 31, a human antibody molecule or an antigen-binding fragment thereof according to any one of aspects 32 to 52, or an immune complex according to any one of aspects 53 to 78, according to aspect 81. A transformed cell comprising the vector according to aspect 81, according to aspect 82. A method of treating a disease or disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of a modified hIL2 protein according to any one of aspects 10 to 14 and 27 to 31, an immune complex according to any one of aspects 53 to 78, or a pharmaceutical composition according to aspect 79 to treat the disease or disorder, according to aspect 83. The method according to aspect 83, wherein the disease or disorder is cancer, according to aspect 84. The method according to aspect 84, wherein the cancer is melanoma, according to aspect 85. The method according to aspect 84, wherein the cancer is non-small cell lung cancer, according to aspect 86. Use of a modified hIL2 protein according to any one of aspects 10 to 14 and 27 to 31, an immune complex according to any one of aspects 53 to 78, or a pharmaceutical composition according to aspect 79 in the manufacture of a medicament for treating a disease or disorder, according to aspect 87. The use according to aspect 87, wherein the disease or disorder is cancer, according to aspect 88. The use according to aspect 88, wherein the cancer is melanoma, according to aspect 89. Use according to aspect 88, wherein the cancer is non-small cell lung cancer. Use of a modified hIL2 protein according to any one of aspects 10 to 14 and 27 to 31, an immune complex according to any one of aspects 53 to 78, or a pharmaceutical composition according to aspect 79 for the treatment of a disease or disorder. Use according to aspect 91, wherein the disease or disorder is cancer. Use according to aspect 92, wherein the cancer is melanoma. Use according to aspect 82, wherein the cancer is non-small cell lung cancer.

Claims

1. A modified human interleukin-2 (hIL2) protein comprising substitutions at position 20 and position 38 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345, wherein the modified hIL2 protein exhibits reduced efficacy against both high-affinity and medium-affinity hIL2 receptors compared to unmodified hIL2.

2. The modified hIL2 protein according to claim 1, wherein the substitution at position 20 is selected from D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E.

3. The modified hIL2 protein according to claim 1, wherein the substitution at position 38 is selected from R38E, R38N, R38G, R38H, R38I, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D, and R38K.

4. The modified hIL2 protein according to claim 1, further comprising a deletion or substitution at position 3 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO:

345.

5. The modified hIL2 protein according to claim 4, wherein the substitution at the 3rd position is T3A.

6. The modified hIL2 protein according to claim 1, further comprising a deletion or substitution at position 125 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO:

345.

7. The modified hIL2 protein according to claim 6, wherein the substitution at position 125 is C125A.

8. The modified hIL2 protein according to claim 1, wherein the efficacy of the modified hIL2 protein against the high-affinity hIL2 receptor (hIL2Rαβγ) is reduced by approximately 1,000 times.

9. The modified hIL2 protein according to claim 1, wherein the modified hIL2 protein has approximately 10,000 times less potency towards the medium-affinity hIL2 receptor (hIL2Rβγ).

10. The modified hIL2 protein according to claim 1, wherein the modified hIL2 protein is fused with an anti-PD1 antibody or an antigen-binding fragment thereof.

11. The modified hIL2 protein according to claim 10, wherein the modified hIL2 protein is fused with the antibody or its antigen-binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment.

12. The modified hIL2 protein according to claim 10, wherein the modified hIL2 protein is directly fused with the antibody or its antigen-binding fragment by a peptide bond.

13. The modified hIL2 protein according to claim 12, wherein the modified hIL2 is directly fused with the C-terminal amino acid residue of the antibody heavy chain by a peptide bond.

14. The modified hIL2 protein according to claim 10, wherein the modified hIL2 protein is fused with the antibody or its antigen-binding fragment via a linker.

15. A modified human interleukin-2 (hIL2) protein comprising a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitution at position 20 and an R38E substitution at position 38 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO:

345.

16. The modified hIL2 protein according to claim 15, comprising any of the amino acid sequences shown in SEQ ID NOs. 307, 607-611, 614, 617, or 620.

17. The modified hIL2 protein according to claim 15, comprising a D20A substitution and an R38E substitution.

18. The modified hIL2 protein according to claim 17, comprising the amino acid sequence shown in SEQ ID NO:

149.

19. The modified hIL2 protein according to claim 15, further comprising a deletion or substitution at position 3 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO:

345.

20. The modified hIL2 protein according to claim 19, wherein the substitution at position 3 is T3A.

21. The modified hIL2 protein according to claim 20, comprising the amino acid sequence shown in SEQ ID NO:

216.

22. The modified hIL2 protein according to claim 19, comprising the amino acid sequence shown in SEQ ID NO:

218.

23. The modified hIL2 protein according to claim 15, further comprising a deletion or substitution at position 125 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO:

345.

24. The modified hIL2 protein according to claim 23, wherein the substitution at position 125 is C125A.

25. The modified hIL2 protein according to claim 24, comprising the amino acid sequence shown in SEQ ID NO: 215, 217, or 219.

26. The modified hIL2 protein according to claim 25, comprising the amino acid sequence shown in SEQ ID NO:

217.

27. The modified hIL2 protein according to claim 15, wherein the modified hIL2 protein is fused with an anti-PD1 antibody or an antigen-binding fragment thereof.

28. The modified hIL2 protein according to claim 27, wherein the modified hIL2 protein is fused with the antibody or its antigen-binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment.

29. The modified hIL2 protein according to claim 27, wherein the modified hIL2 protein is directly fused with the antibody or its antigen-binding fragment by a peptide bond.

30. The modified hIL2 protein according to claim 29, wherein the modified hIL2 is directly fused with the C-terminal amino acid residue of the antibody heavy chain by a peptide bond.

31. The modified hIL2 protein according to claim 27, wherein the modified hIL2 protein is fused with the antibody or its antigen-binding fragment via a linker.

32. A human antibody molecule or its antigen-binding fragment that immune-specifically binds to human programmed cell death protein 1 (hPD1), wherein the human antibody molecule or its antigen-binding fragment is a) Heavy chain complementarity determination region 1 (CDR1) containing the amino acid sequence shown in SEQ ID NO: 418, heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 419, heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 420, light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 421, light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 422, and light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 423; b) Heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 386, heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 387, heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 388, light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 389, light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 390, and light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 391; c) Heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 396, heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 397, heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 398, light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 399, light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 400, and light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 401; or, d) A human antibody molecule or its antigen-binding fragment comprising: heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 406; heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 407; heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 408; light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 409; light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 410; and light chain CDR3 containing the amino acid sequence shown in SEQ ID NO:

411.

33. a) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 416, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 417; b) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 384, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 385; c) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 394, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 395; or, d) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 404, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 405; A human antibody molecule or antigen-binding fragment thereof according to claim 32, comprising:

34. A human antibody molecule or antigen-binding fragment thereof according to claim 32, comprising a human IgG1 heavy chain constant region.

35. A human antibody molecule or antigen-binding fragment thereof according to claim 34, comprising L235A substitution and G237A substitution by EU numbering.

36. a) A heavy chain containing the amino acid sequence shown in SEQ ID NO: 414 and a light chain containing the amino acid sequence shown in SEQ ID NO: 415; b) A heavy chain containing the amino acid sequence shown in SEQ ID NO: 424 and a light chain containing the amino acid sequence shown in SEQ ID NO: 425; c) A heavy chain containing the amino acid sequence shown in SEQ ID NO: 426 and a light chain containing the amino acid sequence shown in SEQ ID NO: 427; or, d) A heavy chain containing the amino acid sequence shown in SEQ ID NO: 428 and a light chain containing the amino acid sequence shown in SEQ ID NO: 429; A human antibody molecule or antigen-binding fragment thereof according to claim 32, comprising:

37. A human antibody molecule or antigen-binding fragment thereof according to claim 36, comprising a heavy chain containing the amino acid sequence shown in SEQ ID NO: 414 and a light chain containing the amino acid sequence shown in SEQ ID NO:

415.

38. A human antibody molecule or antigen-binding fragment thereof according to claim 32, which is fused with a modified human interleukin 2 (hIL2) protein containing substitutions at position 20 and position 38 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO:

345.

39. The modified hIL2 protein comprises any of the amino acid sequences shown in SEQ ID NOs. 134-150, 307, 344, 607-611, 614, 617, or 620, according to claim 38, a human antibody molecule or antigen-binding fragment thereof.

40. The modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 149, according to claim 39, for the human antibody molecule or antigen-binding fragment thereof.

41. The modified hIL2 protein further comprises a deletion or substitution at position 3 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345, according to claim 38, a human antibody molecule or antigen-binding fragment thereof.

42. The human antibody molecule or antigen-binding fragment thereof according to claim 41, wherein the substitution at the 3rd position is T3A.

43. The modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 216, according to claim 42, for the human antibody molecule or antigen-binding fragment thereof.

44. The modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 218, according to claim 41, a human antibody molecule or antigen-binding fragment thereof.

45. The modified hIL2 protein further comprises a deletion or substitution at position 125 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO: 345, according to claim 38, a human antibody molecule or antigen-binding fragment thereof.

46. The human antibody molecule or antigen-binding fragment thereof according to claim 45, wherein the substitution at position 125 is C125A.

47. The modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 215, 217, or 219, according to claim 46, a human antibody molecule or antigen-binding fragment thereof.

48. The modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 217, according to claim 47, a human antibody molecule or antigen-binding fragment thereof.

49. The modified hIL2 protein is fused with the antibody or its antigen-binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment, according to claim 38, a human antibody molecule or its antigen-binding fragment.

50. The modified hIL2 protein is directly fused with the antibody or its antigen-binding fragment by a peptide bond, according to claim 38, a human antibody molecule or its antigen-binding fragment.

51. The human antibody molecule or antigen-binding fragment thereof according to claim 50, wherein the modified hIL2 protein is directly fused with the C-terminal amino acid residue of the antibody heavy chain by a peptide bond.

52. The modified hIL2 protein is fused with the antibody or antigen-binding fragment via a linker, according to claim 38, for the human antibody molecule or antigen-binding fragment.

53. It is an immune complex, (a) Modified human interleukin 2 (hIL2) protein including substitutions at position 20 and position 38 of the amino acid sequence of unmodified IL2 as shown in SEQ ID NO: 345; and, (b) A human antibody molecule or its antigen-binding fragment that binds immunospecifically to human programmed cell death protein 1 (hPD1), The human antibody molecule or its antigen-binding fragment is (i) Heavy chain complementarity determination region 1 (CDR1) containing the amino acid sequence shown in SEQ ID NO: 418, heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 419, heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 420, light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 421, light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 422, and light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 423; (ii) Heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 386, heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 387, heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 388, light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 389, light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 390, and light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 391; (iii) Heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 396, heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 397, heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 398, light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 399, light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 400, and light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 401; or, (iv) An immune complex comprising: heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 406; heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 407; heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 408; light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 409; light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 410; and light chain CDR3 containing the amino acid sequence shown in SEQ ID NO:

411.

54. The immunocomplex according to claim 53, wherein the substitution at position 20 of the modified hIL2 protein is selected from D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E.

55. The immunocomplex according to claim 53, wherein the substitution at position 38 of the modified hIL2 protein is selected from R38E, R38N, R38G, R38H, R38I, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D, and R38K.

56. The immunocomplex according to claim 53, wherein the substitution at position 20 of the modified hIL2 protein is selected from D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E, and the substitution at position 38 of the modified hIL2 protein is R38E.

57. The immune complex according to claim 53, wherein the modified hIL2 protein comprises any of the amino acid sequences shown in SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620.

58. The immune complex according to claim 53, wherein the modified hIL2 protein includes D20A and R38E substitutions.

59. The immune complex according to claim 58, wherein the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO:

149.

60. The immune complex according to claim 53, comprising any of the amino acid sequences shown in SEQ ID NOs: 608, 614, 611, 620, 607, 610, 617, 609, or 307.

61. The immune complex according to claim 53, wherein the modified hIL2 protein further comprises a deletion or substitution at position 3 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO:

345.

62. The immune complex according to claim 61, wherein the substitution at the 3-position of the modified hIL2 protein is T3A.

63. The immune complex according to claim 62, wherein the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO:

216.

64. The immune complex according to claim 61, wherein the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO:

218.

65. The immune complex according to claim 53, wherein the modified hIL2 protein further comprises a deletion or substitution at position 125 of the amino acid sequence of unmodified IL2 shown in SEQ ID NO:

345.

66. The immunocomplex according to claim 65, wherein the substitution at position 125 is C125A.

67. The immune complex according to claim 66, wherein the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO: 215, 217, or 219.

68. The immune complex according to claim 67, wherein the modified hIL2 protein comprises the amino acid sequence shown in SEQ ID NO:

217.

69. The immune complex according to claim 53, wherein the modified hIL2 protein is fused with the antibody or its antigen-binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment.

70. The immune complex according to claim 53, wherein the modified hIL2 protein is directly fused with the antibody or its antigen-binding fragment by a peptide bond.

71. The immune complex according to claim 70, wherein the modified hIL2 protein is directly fused with the C-terminal amino acid residue of the antibody heavy chain by a peptide bond.

72. The immune complex according to claim 53, wherein the modified hIL2 protein is fused with the antibody or its antigen-binding fragment via a linker.

73. The aforementioned human antibody molecule or its antigen-binding fragment is a) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 416, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 417; b) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 384, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 385; c) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 394, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 395; or, d) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 404, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 405; The immune complex according to claim 53, comprising:

74. The immune complex according to claim 53, wherein the human antibody molecule or its antigen-binding fragment comprises an IgG1 heavy chain constant region.

75. The immune complex according to claim 74, wherein the human antibody molecule or its antigen-binding fragment comprises L235A substitution and G237A substitution according to EU numbering.

76. The aforementioned human antibody molecule or its antigen-binding fragment is a) A heavy chain containing the amino acid sequence shown in SEQ ID NO: 414 and a light chain containing the amino acid sequence shown in SEQ ID NO: 415; b) A heavy chain containing the amino acid sequence shown in SEQ ID NO: 424 and a light chain containing the amino acid sequence shown in SEQ ID NO: 425; c) A heavy chain containing the amino acid sequence shown in SEQ ID NO: 426 and a light chain containing the amino acid sequence shown in SEQ ID NO: 427; or, d) A heavy chain containing the amino acid sequence shown in SEQ ID NO: 428 and a light chain containing the amino acid sequence shown in SEQ ID NO: 429; The immune complex according to claim 53, comprising:

77. The immune complex according to claim 76, wherein the human antibody molecule or its antigen-binding fragment comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 414 and a light chain having the amino acid sequence shown in SEQ ID NO:

415.

78. A light chain containing the amino acid sequence shown in Sequence ID No. 415; and, Heavy chain modified hIL2 protein fusion containing the amino acid sequence shown in SEQ ID NO: 532 The immune complex according to claim 53, comprising:

79. A pharmaceutical composition comprising a modified hIL2 protein according to any one of claims 1 to 31, a human antibody molecule or antigen-binding fragment thereof according to any one of claims 32 to 52, or an immune complex according to any one of claims 53 to 78.

80. The pharmaceutical composition according to claim 79 for treating a target cancer.

81. The pharmaceutical composition according to claim 80, wherein the cancer is melanoma.

82. The pharmaceutical composition according to claim 80, wherein the cancer is non-small cell lung cancer.

83. A polynucleotide comprising a modified hIL2 protein according to any one of claims 1 to 31, a human antibody molecule or antigen-binding fragment thereof according to any one of claims 32 to 52, or a nucleic acid sequence encoding an immune complex according to any one of claims 53 to 78.

84. A vector comprising a modified hIL2 protein according to any one of claims 1 to 31, a human antibody molecule or antigen-binding fragment thereof according to any one of claims 32 to 52, or a polynucleotide comprising a nucleic acid sequence encoding an immune complex according to any one of claims 53 to 78.

85. Transformed cells comprising the vector according to claim 84.