CN116507633A

CN116507633A - Glycosylated IL-2 proteins and uses thereof

Info

Publication number: CN116507633A
Application number: CN202180073386.7A
Authority: CN
Inventors: S·奥科尔斯; D·B·罗森; B·劳费尔; T·克纳佩
Original assignee: Asendis Pharmaceutical Oncology Co ltd
Current assignee: Asendis Pharmaceutical Oncology Co ltd
Priority date: 2020-08-28
Filing date: 2021-08-27
Publication date: 2023-07-28
Also published as: AR123371A1

Abstract

The present invention relates to(Tag) ¹ ) _y ‑(Ala) _x ‑SEQ A‑SEQ B‑SEQ C‑(Tag ² ) _z (I) Wherein SEQ a and SEQ ID NO:1 has at least 89% sequence identity; SEQ B and SEQ ID NO:2 and comprises at least one glycosylation motif and at least 76% sequence identity; SEQ C and SEQ ID NO:4 has at least 91% sequence identity; tag ¹ And Tag ² Independently a label moiety; ala is an alanine residue; x is 0 or 1; y is 0 or 1; and z is 0 or 1; the present invention relates to conjugates thereof and their use in the treatment of cell proliferative disorders.

Description

Glycosylated IL-2 proteins and uses thereof

The invention relates to a composition of the formula (Tag ¹ ) _y -(Ala) _x -SEQ A-SEQ B-SEQ C-(Tag ² ) _z (I) Wherein SEQ a and SEQ ID NO:1 has at least 89% sequence identity; SEQ B and SEQ ID NO:2 and comprises at least one glycosylation motif and at least 76% sequence identity; SEQ C and SEQ ID NO:4 has at least 91% sequence identity; tag ¹ And Tag ² Independently a label moiety; ala is an alanine residue; x is 0 or 1; y is 0 or 1; and z is 0 or 1; the present invention relates to conjugates thereof and their use in the treatment of cell proliferative disorders.

In healthy humans, the immune system can generally distinguish between healthy cells and cancer cells. Once a particular cell is identified as a cancer cell, the immune system will typically eliminate it. However, when the immune system is damaged or overwhelmed by acute or chronic defects, cancers may occur because the damaged immune system is unable to distinguish and eliminate cancer cells. In patients with cancer, administration of an immunomodulatory protein to the patient can help activate the patient's immune system, resulting in an enhanced ability of the immune system to eliminate cancer cells. In patients with viral infections, administration of an immunomodulatory protein to the patient can help activate the patient's immune system, thereby enhancing the ability of the immune system to eliminate viral infections. Likewise, even healthy patients can have an enhanced immune response to the vaccine by the addition of such immunomodulatory proteins.

One such immunomodulatory protein used to treat patients with certain cancers is interleukin-2 (IL-2). Human IL-2 is synthesized as a 153 amino acid precursor polypeptide (full length) and then processed into mature IL-2. Mature human IL-2 is a 15.5kDa 4-alpha-helical bundle glycoprotein consisting of 133 amino acids. IL-2 plays a central role in the production, differentiation, survival and homeostasis of immune effector cells. IL-2 is synthesized by activated CD4+ helper T cells, and through different receptor interactions, IL-2 can modulate immune responses, causing them to go to immunity or tolerance.

IL-2 functions by binding to the IL-2 receptor (IL-2R). Binding of the α - (CD 25), β - (CD 122) and common γ - (yc, CD 132) subunits results in a trimeric high affinity IL-2rαβγ. The intermediate affinity IL-2Rβγ of the dimer consists of β -and γ -subunits, and binds to IL-2 with 50-fold lower affinity. IL-2Rα is not a requirement for IL-2 signaling, but can confer high affinity binding to trimeric receptors, while β -and γ -subunits mediate signal transduction. IL-2Rβγ is expressed on NK cells, monocytes, macrophages, γδ T cells, particularly Vγ9Vδ T cells and resting CD4+ and CD8+ T cells, whereas IL-2Rβγ is transiently induced on activated T cells and NK cells and constitutively expressed on T regulatory cells as well as type 2 congenital lymphocytes (ILC 2 s), eosinophils and endothelial cells. The ability of IL-2 to amplify and activate both innate and adaptive effector cells is the basis for its anti-tumor activity.

IL-2 stimulates antitumor efficacy in patients, and is characterized by an increase in cytotoxic lymphocytes, including effector T and NK cells, when administered in large doses (i.e. in the human body at 600 000-720 000IU/kg body weight, three times per day, up to 14 doses per cycle). It is speculated that during this treatment all T cells will be stimulated with IL-2 after a large dose, however, at the end of the treatment cycle and at the time point after any single dose, the IL-2 level decreases. As a result, IL-2 will become limiting and T regulatory (Treg) cells expressing IL-2rβγ will outcompete effector T cells expressing IL-2rβγ for the remaining wild-type IL-2.

However, the anti-tumor immunity of IL-2 is dose-limited by severe cardiovascular, pulmonary, hepatic, gastrointestinal, nervous system and hematological side effects and therefore can only be used in professional medical centers for patients. Many of these adverse events are characterized by Vascular Leak Syndrome (VLS), also known as capillary leak syndrome. There are several proposals for the mechanism responsible for VLS, many of which involve interactions between wild-type IL-2 and cells expressing IL-2rαβγ, such as ILC2s, eosinophils and endothelial cells.

Effector cd4+ T cells, cd8+ T-cells, γδ T cells, particularly vγ9vδ T cells and NK cells, which significantly enhance anti-tumor immune responses, preferentially express the IL-2rβγ form of IL-2R. Thus, administration of a compound that binds to IL-2rβγ and acts as an agonist thereof is expected to enhance immune responses against tumors (e.g., by increasing the effects of proliferation and activity of cd4+ T cells, cd8+ T-cells, γδ T cells, particularly vγ9vδ T cells and NK cells).

Thus, administration of an IL-2rβγ selective agonist (with or without reduced binding to IL-2rα or enhanced binding to IL-2rβγ) would be beneficial to patients with certain cancers, as doing so would be expected to reduce systemic vascular leakage side effects such as pulmonary edema, hypotension and eosinophilia, thereby providing an improved therapeutic window.

One way to synthesize such biased IL-2 (i.e., IL-2 protein that preferentially binds IL-2Rβγ) is to introduce steric hindrance such that the binding of IL-2 to the IL-2Rα subunit is blocked. This can be achieved by conjugating certain moieties to endogenous or mutated amino acids in the IL-2 sequence to mediate a bias that does not bind IL-2rα, as shown for example in WO2019/185705 A1. However, manufacturing such mutated or biased IL-2 proteins in sufficient yields and quality can be challenging, and conjugating the moiety to IL-2 requires additional synthetic steps, which increases costs and can have a negative impact on quality and overall yield.

It is therefore an object of the present invention to at least partially overcome the above-mentioned drawbacks.

This object is achieved by the IL-2 protein sequence of the formula (I) (Tag ¹ ) _y -(Ala) _x -SEQ A-SEQ B-SEQ C-(Tag ² ) _z (I)，

Wherein the method comprises the steps of

SEQ A and SEQ ID NO:1 has at least 89% sequence identity;

SEQ B and SEQ ID NO:2 and comprises at least one glycosylation motif and at least 76% sequence identity;

SEQ C and SEQ ID NO:4 has at least 91% sequence identity;

Tag ¹ and Tag ² Independently a label moiety;

ala is an alanine residue;

x is 0 or 1;

y is 0 or 1; and is also provided with

z is 0 or 1.

Surprisingly, it was found, without being bound by theory, that the introduction of glycosylation motifs at specific positions of the amino acid sequence of IL-2 variants mediating IL-2rα binding results in efficient site-specific glycosylation and provides efficient IL-2rβγ binding bias, as demonstrated by reduced Treg activation and sustained cd8+ T cell activity, while avoiding any additional conjugation step.

Surprisingly, the insertion of a glycosylation motif into the IL-2rα binding region results in particularly high glycosylation rates at the desired site in certain embodiments, and expression of glycosylated IL-2 variants is surprisingly more efficient than expression of non-glycosylated IL-2 variants. Furthermore, without being bound by theory, this glycosylation effectively blocks IL-2rα ⁺ Activation of the cells without the need for conjugation of any additional moiety, thus making the production of biased IL-2 more efficient.

In the present invention, terms having the following meanings are used.

In general, the term "interleukin-2" or "IL-2" refers to all IL-2 proteins, preferably from mammalian species, more preferably from primate species, most preferably from humans, as well as variants, analogs, orthologs (orthologs), homologs (homolog) and derivatives and fragments and fusion proteins thereof, characterized by having a central role in lymphopoiesis, survival and homeostasis, and also includes naturally occurring variants of IL-2, such as splice variants or allelic variants. In the present invention, the terms "interleukin-2" and "IL-2" particularly refer to proteins having the sequence of formula (I).

Human full-length IL-2 has the following sequence:

the mature form of human IL-2 has the following sequence:

as used herein, the term "biased IL-2" refers to modified IL-2, wherein the bias IL-2 is associated with K of IL-2Rα _D K with said biases IL-2 and IL-2Rβ _D Is greater than the ratio of SEQ ID NO:213 IL-2 and IL-2Rα K _D And SEQ ID NO:2 comparison of IL-2 of 13 with K of IL-2Rbeta _D Is a ratio of (2). This is described by the following formula:

Wherein the method comprises the steps of

“K _D Biasing IL-2 vs IL-2 Ra' is K biasing IL-2 vs IL-2Rα _D ，

“K _D The bias IL-2 vs IL-2Rβ "is K of the bias IL-2 vs IL-2Rβ _D ，

“K _D SEQ ID NO:213 IL-2 vs IL-2rα″ is SEQ ID NO:213 IL-2 vs. K for IL-2Rα _D A kind of electronic device

“K _D SEQ ID NO:213 IL-2 vs IL-2Rβ "is K _D SEQ ID NO:213 IL-2 vs IL-2Rβ.

SEQ ID NO:213 has the following sequence:

k-determination of biased IL-2 versus IL-2Rα can be performed using surface plasmon resonance (surface plasmon resonance, SPR) evaluation _D K of biased IL-2 to IL-2Rbeta _D SEQ ID NO:213 IL-2 vs. K for IL-2Rα _D And SEQ ID NO:213 IL-2 vs. IL-2Rβ K _D The binding affinities/kinetics required were measured on a Biacore instrument (GE Healthcare) as follows: the human Fc capture surface on the CM5 (or alternatively C1 or CM 4) chip, or alternatively the protein a chip, is prepared by covalent coating with an anti-human Fc antibody. Next, IL-2Rβ -Fc or IL2-Rα -Fc was immobilized on the chip. For measuring affinity/kinetic constants, serial dilutions of the analyte are performed starting with, for example, 1 nM-2. Mu.M or 30nM and 500nM (for IL-2 compounds). The analytes are each exposed to the receptor modified chip for a suitable period of time, e.g., 1-30 minutes, which may be, e.g., 2 minutes or may be 3 minutes, and then eluted for a suitable period of time, e.g., 2-60 minutes, which may be, e.g., 10 minutes. The resulting binding curves from the dilution series are fitted to a 1:1 kinetic model to match the observed response units (R) to the association and dissociation rate constants k _a And k _d And (3) associating:

wherein the method comprises the steps of

t is time;

c is the concentration of the analyte; and

R _max is the maximum binding capacity of the surface.

If determined by a dynamic 1:1 model, the ratio of dissociation and association rates provides an equilibrium dissociation constant K _D 。

Alternatively, the binding curves generated by serial dilutions were fitted using a 1:1 steady state interaction model that was developed from steady state binding levels (R _eq ) Calculation of the 1:1 interaction K in a plot against the analyte concentration (C) _D ：

Wherein the method comprises the steps of

R _eq Is the steady state binding level.

C is the concentration of the analyte; and

R _max is the maximum binding capacity of the surface.

It is understood that not every calculation method may be applicable to every biased IL-2 molecule. For example, if the reaction rate is too fast, a 1:1 kinetic model may not be used, and a 1:1 steady state interaction model may be used. For example, if equilibrium is not achieved, a 1:1 interaction model may not be used, and a 1:1 kinetic model may be used.

As used herein, the term "protein amino acid" relates to an amino acid selected from the group consisting of: alanine (Ala, a), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamic acid (Glu, E), glutamine (gin, Q), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, D). Corresponding three-letter codes and one-letter codes are provided in brackets.

As used herein, the term "non-protein amino acid" relates to an amino acid selected from the group consisting of: d-stereoisomers of each proteinogenic amino acid, pyrrolysine (Pyl, O), selenocysteine (Sec, U), 2-aminoadipic Acid (AD), 3-aminoadipic acid (BaaD), beta-alanine (BaLa), 2-aminobutyric acid (Abu), 4-aminobutyric acid (4 Abu), 6-aminocaproic acid (Acp), 2-aminoheptanoic acid (Ahe), 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid (bAib), 2-aminopimelic acid (Apm), 2, 4-diaminobutyric acid (Dbu), desmin (Des), 2' -diaminopimelic acid (Dpm), 2, 3-diaminopropionic acid (Dpr), N-ethylglycine (EtGly), N-ethylasparamide (Easn), hydroxylysine (Hyl), allo-hydroxylysine (aHyl), 3-hydroxyproline (3 Hyp), 4-hydroxyproline (4 Hyp), isodesmin (e), allo-isoleucine (ALL), N-methyl glycine (Me), N-methyl-valine (Me), N-methyl-valine (Me-Val) and N-methylglycine (Me), norleucine (Nle) and ornithine (Om). Corresponding abbreviations are provided in brackets.

It is understood that in the context of amino acid sequences, single letter refers to the single letter code of an amino acid rather than a chemical atom.

It will be appreciated that the deletion or insertion of one or more amino acids in a sequence (e.g. Seq a, seq B, seq C or Seq D) may alter the numbering, i.e. the position, of a particular amino acid within such sequence and in this case includes the corresponding, i.e. homologous, amino acid position. This may be indicated by the phrase "or at a corresponding position of a homologue or variant thereof", but such corresponding position is included even without the phrase.

As used herein, the terms "identical" and "percent identity" in the context of two or more polynucleotide or polypeptide/protein sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of the same nucleotide or amino acid residues, as measured, for example, using a sequence comparison algorithm, when comparing and aligning for maximum correspondence. Examples of such algorithms suitable for determining the percent sequence identity are the BLAST algorithm (Altschul et al, J.mol. Biol.,215:403-410 (1990); henikoff & Henikoff, proc. Natl. Acad. Sci. USA,89:10915 (1989); karlin & Altschul, proc. Natl. Acad. Sci. USA,90:58735787 (1993)). Software for performing BLAST analysis is publicly available through the national center for biotechnology information (National Center for Biotechnology Information).

As used herein, the term "glycosylation" refers to post-translational modification of proteins in which carbohydrates, particularly glycans, are attached to functional groups of amino acids. Glycosylation involves the nitrogen attachment of a carbohydrate to an asparagine or arginine side chain (N-glycosylation), the hydroxy oxygen attachment to a serine, threonine, tyrosine, hydroxylysine or hydroxyproline side chain (O-glycosylation); phosphate linkage to phosphoserine (phosphoglycosylation); or to a carbon on the tryptophan side chain (C-glycosylation). The O-linked glycans can be, for example, N1 (NeuAc (a 2-3) Gal (bl-3) GalNAc-ol) or N2 (NeuAc (a 2-3) Gal (b 1-3) (NeuAc (a 2-6)) GalNAc-ol), where NeuAc is N-acetylneuraminic acid (sialic acid), gal is galactose, and GalNAc-ol is acetylgalactitol. The N-linked glycans can be low mannose glycans (paucimannose glycan) (Man ₃ GlcNAc ₂ ) Wherein the two innermost sugar residues are N-acetylglucosamine (GlcNAc) further surrounded by a trimannosyl core (Man ₃ ) Extending. It may be, for example, a double-antenna G0 glycan (GlcNAc) ₂ Man ₃ GlcNAc ₂ ) Wherein the few mannose is linked to two GlcNAc residues, one GlcNAc being linked at each of the two external mannose residues of the trimannosyl core ₂ Resulting in a double antenna structure. This biantennary glycoform may be further modified by a range of additional sugars, including fucose (Fuc) residues attached at the innermost GlcNAc residues, as well as branches comprising GlcNAc, gal and NeuAc residues. For example, the N-linked glycan may be a G1 glycan carrying one additional galactose and one additional sialic acid residue compared to the G0 structure (NeuAcGalGlcNAc ₂ Man ₃ GlcNAc ₂ ) Or a G2 glycan comprising two additional galactose and two additional sialic acid residues compared to the G0 structure (NeuAc) ₂ Gal ₂ GlcNAc ₂ Man ₃ GlcNAc ₂ ). In addition, G0, G1 and G2 glycans can be fucosylated at the innermost GlcNAc residues, yielding G0F (GlcNAc ₂ Man ₃ GlcNAc ₂ Fuc)、G1F(NeuAcGalGlcNAc ₂ Man ₃ GlcNAc ₂ Fuc) and G2F (NeuAc) ₂ Gal ₂ GlcNAc ₂ Man ₃ GlcNAc ₂ Fuc) glycans. The glycans described above can also be modified with additional GlcNAc residues attached to the trimannosyl core, resulting in G0B (GlcNAc ₃ Man ₃ GlcNAc ₂ )、G1B(NeuAcGalGlcNAc ₃ Man ₃ GlcNAc ₂ ) And G2B (NeuAc) ₂ Gal ₂ GlcNAc ₃ Man ₃ GlcNAc ₂ ) A glycan. Fucosylation and additional GlcNAc linkages at the trimannosyl core can also be combined to produce G0BF (GlcNAc ₃ Man ₃ GlcNAc ₂ Fuc)、G1BF(NeuAcGalGlcNAc ₃ Man ₃ GlcNAc ₂ Fuc) and G2BF (NeuAc) ₂ Gal ₂ GlcNAc ₃ Man ₃ GlcNAc ₂ Fuc) glycans. The N-linked glycans can also be tetradentate glycans formed by transferring additional GlcNAc residues to the biantennary G0 glycans through the action of GlcNAc glycosyltransferases GNTIV and GNTV. The N-linked glycans can also be double-or quad-antennary low mannose glycans modified with GlcNAc-gal extensions (LacNAc). The N-linked glycans can also be bi-or tetra-antennary low mannose glycans modified with GlcNAc-Gal extension (LacNAc) and otherwise coupled to Gal residues and one or more NeuAc residues. Other examples of N-linked glycans can be found in "Antibody glycoengineering strategies in mammalian cells", wang et al Biotechnology and Bioengineering, month 6 of 2018; 115 (6): 1378-1393 and "snappshot: N-Glycosylation Processing Pathways across Kingdoms ", chung et al, cell,2017, 9, 21; 171 (1): 258-258.e1, which is incorporated herein by reference. N-glycosylated proteins typically comprise a mixture of N-glycans, rather than being homogeneously glycosylated with a specific N-glycan. When variations in glycan structure at a particular site are involved, this is often referred to as micro-heterogeneity, and when glycosylation sites and site-occupied variability are involved, this is often referred to as macro-heterogeneity. The degree of heterogeneity of N-glycosylation can vary depending on the protein and the expression host. For example, in human as well as mammalian expression systems, the N-glycosylation site in the Fc domain of IgG1 antibodies is predominantly occupied by G0F, G F and G2F glycans, but although recombinant therapeutic antibodies produced in CHO cells may typically comprise a ratio of G0F>G1F>>G0F, G F and G2F glycans of G2F, the recombinant antibodies produced in mouse myeloma SP2/0 cells may comprise a ratio of G1 F.gtoreq.G0F>>G2F glycans. Removal of the N-linked or O-linked glycans can be accomplished by enzymatic or chemical treatment. Removal of the N-linked glycans by peptide-N-glycosidase F (PNgaseF) causes amino acid changes of arginine (N) to aspartic acid (D), e.g., the glycosylation motif of sequence FGNST (SEQ ID NO: 307) is thereby converted to FGDST (SEQ ID NO: 368).

As used herein, the term "site occupancy" refers to the percentage of glycosylation found at a particular glycosylation motif. It is to be understood that the phrase "at least X% site occupancy" refers to a number of IL-2 molecules or moieties, e.g. present in a formulation or pharmaceutical composition, wherein X% of all IL-2 molecules or moieties are glycosylated at a specific site, but not to a single molecule or moiety. The N-glycan structure and site occupancy of glycoproteins can affect important properties such as receptor binding affinity, stability, solubility, antigenicity, clearance, and half-life. Thus, for recombinant glycoprotein manufacturing processes, it is often important to obtain reproducible and homogeneous glycosylation products. It is therefore advantageous if the product is produced in a fully N-glycosylated form (full site occupancy) by the production host cell. For "complete glycosylation," at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95% of all proteins are glycosylated at a particular site. The N-glycosylated form can be separated from the form lacking N-glycosylation in a downstream purification process, however, if the starting materials are heterogeneous, more complex processes and lower yields of the purified compounds can be expected with a large impact on the overall process costs. Furthermore, if the starting material contains a large fraction of variants lacking N-glycosylation, it may be difficult or even impossible to obtain a highly pure N-glycosylated form. This is particularly relevant where the non-glycosylated form has undesirable properties. For example, for biased N-glycosylated IL-2 proteins, the presence of forms that do not carry N-glycans is highly undesirable because these IL-2 forms may have higher residual potency than the N-glycosylated forms to activate IL-2Rα.

As used herein, the expression amino acid "in the" -X position "or an amino acid in the" +x position "(where X is an integer) refers to the position of an amino acid relative to a specified amino acid in a protein or peptide sequence, wherein the amino acid in the-X position is the amino acid located N-terminal from the specified amino acid by X, and the amino acid in the +x position is the amino acid located C-terminal from the specified amino acid by X. For example, in the peptide sequence "ALMGR" (SEQ ID NO: 253), A is the amino acid at position-2 of M, L is the amino acid at position-1 of M, G is the amino acid at position +1 of M, and R is the amino acid at position +2 of M.

As used herein, the term "glycosylation motif" refers to an amino acid sequence that directs site-specific glycosylation of a protein. The glycosylation motif may be an N-glycosylation motif, an O-glycosylation motif, a phospho-glycosylation motif or a C-glycosylation motif. The N-glycosylation motif can comprise 2, 3, 4, 5, or 6 amino acids. The O-glycosylation motif may comprise one or two amino acids.

As used herein, the term "N-glycosylation site" refers to the position of asparagine (N) of an N-glycosylation motif in an amino acid sequence.

As used herein, the term "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). As used herein, unless otherwise indicated, "affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., between a receptor and a ligand). The affinity of a molecule X for its partner Y can generally be determined by the equilibrium dissociation constant (K _D ) It is shown that it is the dissociation and association rate constant (k respectively _d And k _a ) Is a ratio of (2). Thus, as long as the ratio of the rate constants remains unchanged, equivalent affinity can be achievedTo include different rate constants. Affinity can be measured by established methods known in the art, including the methods described herein.

As used herein, the terms "IL-2 receptor alpha subunit" and "IL-2Ralpha" refer to human CD25.

As used herein, the terms "β subunit of the IL-2 receptor" and "IL-2rβ" refer to human CD122.

As used herein, the terms "gamma subunit of IL-2 receptor" and "IL-2rγ" refer to human CD132.

The term "pattern recognition receptor agonist" ("PRRA") as used herein refers to a molecule that binds to and activates one or more immune cell related receptors that recognize pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMP), resulting in immune cell activation and/or pathogen-or damage-induced inflammatory responses. Pattern recognition receptors are typically expressed by cells of the innate immune system, such as monocytes, macrophages, dendritic Cells (DCs), neutrophils and epithelial cells, as well as by cells of the adaptive immune system.

The terms "cytotoxic agent" and "chemotherapeutic agent" as used herein are synonymous and refer to a compound that is toxic to cells and prevents the replication or growth of the cells, resulting in cell destruction/death. Examples of cytotoxic agents include chemotherapeutic agents and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including synthetic analogs and derivatives thereof.

The terms "immune checkpoint inhibitor" and "immune checkpoint antagonist" as used herein are synonymous and refer to a compound that interferes with the function of a ligand or inhibits ligand-induced signaling by binding to a cell membrane-expressed receptor that inhibits the function of inflammatory immune cells upon activation of the receptor. Such compounds may be, for example, biologicals, such as antibodies, antibody fragments, affibodies (affibodies), affilins, affimers, affitins, alpha monoclonal antibodies (alphamabs), alphabodies, anticalins, avimers, DARPins,Kunitz domain peptides, monobodies, nanoCLAMP, cyclic peptides, heavy chain-only antibodies, VHH antibodies or +.>Single chain variable region fragments (scFvs), natural or modified ligands or binding partners for these receptors or small molecule inhibitors.

The term "immune activation agonist" as used herein refers to a compound that directly or indirectly activates a checkpoint receptor expressed by a cell membrane.

The term "immune activating receptor agonist" as used herein refers to a compound that stimulates immune cell function upon activation or co-stimulatory receptor activation. Examples of such stimulatory receptors include the CD3 subunits CD3 gammase:Sub>A, CD3 deltase:Sub>A, CD3 epsilon and CD3 zetase:Sub>A (CD 247), the T Cell Receptor (TCR) subunits TCRase:Sub>A, TCRbetase:Sub>A, TCRgammase:Sub>A and TCR deltase:Sub>A, the B Cell Receptor (BCR) chains or signal transduction units CD79 ase:Sub>A or CD79B, CD2, CD4, CD8, CD16, CD32 ase:Sub>A, CD64, CD27, CD28, CD134 (OX 40), CD137 (41 BB), CD244 (2B 4), CD278 (ICOS), CD357 (GITR), CRACC (CS 1), LFA-1, NKG2D, NKG C, NKp30, NKp46, NKp44, NKp80, TB-A, activated short forms of KIR (KIR 2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS 1), CD40, ctip-betase:Sub>A, dectin-1, dectin-2, TRKB 1, TRT 2, LIT 6, ILT8, and other immune receptor signaling motifs that induce immune responses viase:Sub>A the protein motif of the protein, or other immune receptor genes. Many multispecific drugs are types of immune activating receptor agonists.

The terms "multispecific" and "multispecific drug" as used herein refer to a compound that binds to two or more different antigens simultaneously and is capable of mediating antagonistic, agonistic or specific antigen binding activity in a target-dependent manner.

The term "antibody-drug conjugate" (ADC) as used herein refers to a compound typically consisting of an antibody linked to a biologically active cytotoxic load, radiation therapy or other drug designed to deliver a cytotoxic agent to the tumor environment. ADCs are particularly effective in reducing tumor burden without significant systemic toxicity and may have the effect of ameliorating checkpoint inhibitor antibody-induced immune responses.

The term "antibody-adjuvant conjugate" (AAC) as used herein refers to a compound consisting of an antibody linked directly or through a linker to a biologically active adjuvant.

As used herein, the term "adjuvant" refers to a substance that enhances the immune response of the body to an antigen.

The term "boltbody" as used herein refers to an antibody-adjuvant conjugate comprising: (a) An antibody moiety comprising (i) an antigen binding domain and (ii) an Fc domain; (b) an adjuvant moiety; and (c) a linker comprising an ethylene glycol or glycine residue, wherein each adjuvant moiety is covalently bound to the antibody moiety through a linker, which may be cleavable or non-cleavable.

The term "radionuclide" as used herein refers to a radioisotope that emits ionizing radiation resulting in cell destruction/death. Radionuclides conjugated to tumor targeting vectors are referred to as "targeted radionuclide therapy".

The term "DNA damage repair inhibitor" as used herein refers to a drug directed against DNA damage repair elements, such as CHK1, CHK2, ATM, ATR and PARP. Due to the presence of mutations or pathway alterations, certain cancers are more susceptible to these pathways, e.g., patients with BRCA1 mutations or patients with defects in homologous recombination pathways, are susceptible to PARP inhibitors due to the concept of synthetic lethality.

The term "tumor metabolic inhibitor" as used herein refers to a compound that interferes with the function of one or more enzymes expressed in the tumor environment that produce metabolic intermediates that may inhibit immune cell function.

The term "protein kinase inhibitor" as used herein refers to a compound that inhibits the activity of one or more protein kinases. Protein kinases are enzymes that phosphorylate proteins, which can thereby regulate the function of proteins. It will be appreciated that a single protein kinase inhibitor may target more than one kinase, and that any classification of protein kinase inhibitor as used herein refers to a primary or most characteristic target.

The term "chemokine receptor and chemoattractant receptor agonist" as used herein refers to compounds that activate chemokine receptors or chemoattractant receptors, which are G-protein coupled receptors or a subset of G-protein coupled receptors, expressed on a wide variety of cells, primarily involved in controlling cell movement (chemotaxis or chemoactivation). These receptors may also be involved in non-cellular migration processes such as angiogenesis, cell maturation or inflammation.

The term "cytokine receptor agonist" as used herein refers to a soluble protein that controls immune cell activation and proliferation. Cytokines include, for example, interferons, interleukins, lymphokines and tumor necrosis factors.

The term "death receptor agonist" as used herein refers to a molecule capable of inducing pro-apoptotic signaling through one or more death receptors, such as DR4 (TRAIL-R1) or DR5 (TRAIL-R2). The death receptor agonist may be selected from antibodies, death ligands, cytokines, vectors expressing death receptor agonists, peptides, small molecule agonists, cells expressing death receptor agonists (e.g., stem cells), and drugs that induce expression of death ligands.

The term "antigen presenting cell" or "APC" as used herein refers to a cell, such as a macrophage, B cell or dendritic cell, that presents a treated antigen peptide to a T cell receptor on a CD4T cell via MHC class II molecules. One skilled in the art can identify APCs by using phenotypic techniques such as flow cytometry. Phenotypic markers used to identify APCs vary by species and tissue, but may include myeloid or dendritic cell surface markers (e.g., CD11B, CD11c, CD14, CD16, CD33, CD34, CD68, CD206, MHC-II, CD163, ly6C, ly6G, GR-1, F4/80, TREM1, TREM 2) or B cell surface markers (e.g., CD19, CD20, B220).

The term "MHC", as used herein, refers to a class of Major Histocompatibility Complex (MHC) molecules that are normally present only in antigen presenting cells, such as myeloid cells, dendritic cells, and B cells. MHCII presents the treated antigenic peptide to T cell receptors on CD4T cells. Expression of MHCII can be measured by one skilled in the art using protein expression analysis techniques (e.g., flow cytometry). Changes in MHC I expression may be determined by analyzing changes in the median fluorescence intensity signal of MHC I or the percentage of MHC II positive cells in a particular cell subset of interest.

The term "T cell" as used herein refers to a class of immune cells that play a central role in the adaptive immune response. T cells differ from other immune cells in the presence of the αβ or γδ T Cell Receptor (TCR) on their cell surface. T cells also express CD3, a protein complex critical for TCR signaling. αβ T cells can be divided into CD4, CD8 or CD4/CD8 double negative subpopulations. Due to CD4 ⁺ And CD8 ⁺ The surface density of CD4 and CD8 on T cells is high and CD4 can be generally recognized using only CD4 and CD8, respectively ⁺ And CD8 ⁺ T cells. γδ T cells are equipped with a TCR consisting of a γ chain and a δ chain, which, like δ3δ4tcr, is the core for antigen recognition and cell activation. Such TCRs are also used to distinguish between different subsets of γδ0t cells, vδ11 and vδ22.Vδ 51T cells are a minority (< 5%) and heterogeneous γδ T cell population with anti-inflammatory and pro-inflammatory functions. vδ2T cells are a single relatively homogenous vγ9vδ2 (vδ2) T cell population, accounting for about 95% of γδt cells in the circulation. Because of their unique properties of TCR and other innate immune receptors, vδ2t cells are endowed with powerful antitumor properties and can be used in immunotherapy. Upon activation by TCR recognition of the corresponding antigen presented by MHC molecules, T cells can mature and divide to produce effector or memory T cells. Memory T cells are a subset of T cells that have previously encountered and responded to a corresponding antigen. Such T cells can recognize pathogenic antigens, such as antigens from bacteria or viruses, as well as cancer-associated antigens. One skilled in the art can identify T cells by using phenotypic techniques such as flow cytometry. Phenotypic markers for T cell recognition are generally conserved in mammals, including CD3, tcra, tcrp, tcrδ, CD4, and CD8. Phenotypic markers for identifying memory T cells may be due to species and tissue And, but may include cell surface markers such as CD45RO, LY6C, CD44, and CD95.

As used herein, the expression "cell therapy" refers to the infusion or transplantation of modified human cells into a patient to treat a disease. The source of cells may be from the patient (autologous) or from a healthy donor (allogeneic).

As used herein, the term "reversible," "degradable," or "degradable" with respect to the attachment of a first moiety to a second moiety refers to the attachment of the first and second moieties being cleavable bonds under physiological conditions, which are aqueous buffers at pH 7.4, 37 ℃, with half-life ranges from 1 hour to 3 months, for example from 1 hour to 2 months, 3 hours to 1 month, 6 hours to 28 days, 12 hours to 21 days, 24 hours to 14 days, or 48 hours to 7 days. Cleavage may be enzymatic or non-enzymatic, and in certain embodiments is non-enzymatic. Thus, the term "stable" or "durable" with respect to the attachment of a first moiety to a second moiety means that the attachment connecting the first and second moiety is a cleavable bond under physiological conditions, wherein the half-life exceeds three months.

As used herein, the term "agent" refers to a compound that includes at least one functional group for reacting with a functional group of another compound or drug. It will be appreciated that a drug containing a functional group (e.g., primary or secondary amine or hydroxyl functionality) is also an agent.

As used herein, the term "moiety" refers to a portion of a molecule that lacks one or more atoms as compared to the corresponding reagent. For example, if a reagent of the formula "HX-H" reacts with another reagent and becomes part of a reaction product, the corresponding part of the reaction product has the structure of "H-X-" or "-X-" and each "-" represents a linkage with another part. Thus, the drug moiety is released from the reversible linkage as a drug.

As used herein, the term "tag moiety" refers to a peptide or protein sequence that is translationally fused to an IL-2 protein. It may perform various functions such as stabilization or half-life extension of the IL-2 protein ("stabilization tag"), facilitating purification of the IL-2 protein ("purification tag") or targeting the IL-2 protein to a specific cell type or tissue ("targeting tag").

It is understood that if a sequence or chemical structure of a set of atoms is provided, the set of atoms is attached to two moieties or to one moiety that is interrupted (intermittence), the sequence or chemical structure may be attached to the two moieties in any orientation unless explicitly stated otherwise. For example, one moiety "-C (O) N (R ¹ ) - "can be used as" -C (O) N (R) ¹ ) - "or as" -N (R ¹ ) C (O) - "is attached to both moieties or interrupts one moiety. Similarly, a part

May be connected to both parts or interrupt one part in the following form,

or as/>or is as follows

The term "substituted" as used herein means that one or more H atoms in a molecule or moiety are replaced with a different atom or group of atoms, which are referred to as "substituents". In certain embodiments, no more than 6-H atoms of the moiety or molecule are substituted. In certain embodiments, no more than 5-H atoms of the moiety or molecule are substituted. In certain embodiments, no more than 4-H atoms of the moiety or molecule are substituted. In certain embodiments, no more than 3-H atoms of the moiety or molecule are substituted. In certain embodiments, no more than 2-H atoms of the moiety or molecule are substituted. In certain embodiments, 1-H atom of a moiety or molecule is substituted.

As used herein, the term "substituent" refers in certain embodiments to a group selected from the group consisting of: halogen, -CNCOOR ^x1 、-OR ^x1 、-C(O)R ^x1 、-C(O)N(R ^x1 R ^x1a )、-S(O) ₂ N(R ^x1 R ^x1a )、-S(O)N(R ^x1 R ^x1a )、-S(O) ₂ R ^x1 、-S(O)R ^x1 、-N(R ^x1 )S(O) ₂ N(R ^xla R ^x1b )、-SR ^x1 、-N(R ^x1 R ^xla )、-NO ₂ 、-OC(O)R ^x1 、-N(R ^x1 )C(O)R ^x1a 、-N(R ^x1 )S(O) ₂ R ^x1a 、-N(R ^x1 )S(O)R ^xla 、-N(R ^x1 )C(O)OR ^xla 、-N(R ^x1 )C(O)N(R ^xla R ^x1b )、-OC(O)N(R ^x1 R ^x1a )、-T ⁰ 、C _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl; wherein-T ⁰ 、C _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl groups optionally being substituted by one or more R, which may be the same or different ^x2 Substituted, and wherein C _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl groups are optionally interrupted by one or more groups selected from: -T ⁰ -、-C(O)O-、-O-、-C(O)-、-C(O)N(R ^x3 )-、-S(O) ₂ N(R ^x3 )-、-S(O)N(R ^x3 )-、-S(O) ₂ -、-S(O)-、-N(R ^x3 )S(O) ₂ N(R ^x3a )-、-S-、-N(R ^x3 )-、-OC(OR ^x3 )(R ^x3a )-、-N(R ^x3 )C(O)N(R ^x3a ) -and-OC (O) N (R) ^x3 )-；

-R ^x1 、-R ^x1a 、-R ^x1b Are independently selected from-H, -T ⁰ 、C _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl, wherein-T ⁰ 、C _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl is optionally substituted with one or more of the same or different-R ^x2 Substitution, wherein C _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl groups are optionally interrupted by one or more groups selected from: -T ⁰ 、-C(O)O-、-O-、-C(O)-、-C(O)N(R ^x3 )-、-S(O) ₂ N(R ^x3 )-、-S(O)N(R ^x3 )-、-S(O) ₂ -、-S(O)-、-N(R ^x3 )S(O) ₂ N(R ^x3a )-、-S-、-N(R ^x3 )-、-OC(OR ^x3 )(R ^x3a )-、-N(R ^x3 )C(O)N(R ^x3a ) -, a part of and-OC (O) N (R) ^x3 )-；

Each T ⁰ Independently selected from the following: phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C _3-10 Cycloalkyl, 3-10 membered heterocyclyl and 8-11 membered heterobicyclic; wherein each T ⁰ Independently by one or more of the same or different-R ^x2 Optionally substituted;

each-R ^x2 Independently selected from the following groups: halogen, -CN, oxo (= O), -COOR ^x4 、-OR ^x4 、-C(O)R ^x4 、-C(O)N(R ^x4 R ^x4a )、-S(O) ₂ N(R ^x4 R ^x4a )、-S(O)N(R ^x4 R ^x4a )、-S(O) ₂ R ^x4 、-S(O)R ^x4 、-N(R ^x4 )S(O) ₂ N(R ^x4a R ^x4b )、-SR ^x4 、-N(R ^x4 R ^x4a )、-NO ₂ 、-OC(O)R ^x4 、-N(R ^x4 )C(O)R ^x4a 、-N(R ^x4 )S(O) ₂ R ^x4a 、-N(R ^x4 )S(O)Rx ^4a 、-N(R ^x4 )C(O)OR ^x4a 、-N(R ^x4 )C(O)N(R ^x4a R ^x4b )、-OC(O)N(R ^x4 R ^x4a ) And C _1-6 An alkyl group; wherein C is _1-6 Alkyl is optionally substituted with one or more halogen, the same or different;

each-R ^x3 、-R ^x3a 、-R ^x4 、-R ^x4a 、-R ^x4b Independently selected from H and C _1-6 An alkyl group; wherein C is _1-6 The alkyl groups are optionally substituted with one or more halogen groups, which may be the same or different.

In certain embodiments, up to 6H atoms in an optionally substituted molecule are independently replaced by substituents, e.g., 5H atoms are independently replaced by substituents, 4H atoms are independently replaced by substituents, 3H atoms are independently replaced by substituents, 2H atoms are independently replaced by substituents, or 1H atom is independently replaced by a substituent.

As used herein, the term "fatty acid" refers to a saturated or unsaturated monocarboxylic acid having an aliphatic tail, which may include from 4 to 28 carbon atoms. The fatty acids may be saturated or unsaturated, linear or branched. The term "fatty acid variant" refers to a modified fatty acid in which certain carbon atoms may be replaced by other atoms or groups of atoms, and may be substituted.

The term "peptide" as used herein refers to a chain of at least 2 and up to and including 50 amino acid monomer moieties linked by peptide (amide) bonds. The term "peptide" also includes peptoids, such as D-peptide, peptide samples or β -peptide, and covers peptoids having a maximum of and including 50 monomeric moieties.

As used herein, the term "protein" refers to a chain of more than 50 amino acid monomer moieties (also referred to as "amino acid residues") linked by peptide chains, wherein in certain embodiments the amino acid monomers linked by peptide bonds are no more than 12000, such as no more than 10000 amino acid monomer moieties, no more than 8000 amino acid monomer moieties, no more than 5000 amino acid monomer moieties, or no more than 2000 amino acid monomer moieties.

The term "about" as used herein in connection with a numerical value is used to mean a range that is plus and minus no more than 25% of the numerical value and includes the endpoints, in some embodiments no more than 20% of the numerical value, and in some embodiments no more than 10% of the numerical value. For example, the phrase "about 200" refers to a range from 200+/-25%, i.e., a range from 150 to 250, inclusive; in certain embodiments, 200+/-20% is meant, i.e., ranging from 160 to 240 and inclusive; and in certain embodiments, ranges from 200+/-10%, i.e., from 180 to 220 and inclusive. It is understood that a percentage of "about 50%" does not mean "50% +/-25%", i.e., does not mean from 25 to 75% and includes the end value, but rather "about 50%" means from 37.5 to 62.5% and includes the end value, i.e., plus or minus 25% of the value of 50.

As used herein, the term "polymer" refers to a molecule comprising repeating structural units, i.e., monomers, linked by chemical bonds in a linear, cyclic, branched, crosslinked, or dendritic fashion, or a combination thereof, which may be synthetic or biological in origin, or a combination of both. It will be appreciated that the polymer may also contain one or more other chemical groups and/or, for example, one or more moieties. Likewise, it is understood that a peptide or protein is also a polymer, even though the side chains of the individual amino acid residues may differ. In certain embodiments, the soluble polymer has a molecular weight of at least 0.5kDa, e.g., a molecular weight of at least 1kDa, a molecular weight of at least 2kDa, a molecular weight of at least 3kDa, or a molecular weight of at least 5kDa. If the polymer is soluble, in certain embodiments, it has a molecular weight of at most 1000kDa, such as at most 750kDa, such as at most 500kDa, such as at most 300kDa, such as at most 200kDa, such as at most 100kDa. It will be appreciated that for insoluble polymers such as hydrogels, a meaningful molecular weight range may not be provided.

As used herein, the term "polymeric" refers to an agent or moiety that comprises one or more polymers or polymeric moieties. The polymeric reagent or moiety may also optionally comprise one or more other moieties, which in certain embodiments are selected from the group consisting of:

·C _1-50 Alkyl, C _2-50 Alkenyl, C _2-50 Alkynyl, C _3-10 Cycloalkyl, 3-to 10-membered heterocyclyl, 8-to 11-membered heterobicycloyl, phenyl, naphthyl, indenyl, indanyl and tetrahydronaphthyl; and

a linker selected from

Wherein the method comprises the steps of

The dotted line indicates the connection to the rest of the moiety or reagent, and

-R and-R ^a Independently selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butylTert-butyl, n-pentyl, 2-methylbutyl, 2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2-dimethylbutyl, 2, 3-dimethylbutyl and 3, 3-dimethylpropyl.

Those skilled in the art will appreciate that the polymerization products obtained from the polymerization are not all of the same molecular weight, but rather exhibit a molecular weight distribution. Thus, as used herein, molecular weight range, molecular weight, range of amounts of monomers in the polymer, and amounts of monomers in the polymer refer to the number average molecular weight and the number average of the monomers, i.e., the arithmetic average of the molecular weight of the polymer or polymeric moiety and the arithmetic average of the amount of monomers of the polymer or polymeric moiety.

Thus, in a polymeric moiety comprising "x" monomer units, any integer of "x" corresponds to the arithmetic average of the monomers. Any integer range given for "x" provides the integer range in which the arithmetic mean of the monomers is located. The integers given for "x" being "about x" means that the arithmetic average of the monomers lies within an integer range of x +/-25%, preferably x +/-20%, more preferably x +/-10%.

As used herein, the term "number average molecular weight" refers to the average, generally arithmetic, of the molecular weights of the individual polymers.

As used herein, the term "PEG-based" in connection with a moiety or agent means that the moiety or agent comprises PEG. In certain embodiments, the PEG-based moiety or agent comprises at least 10% (w/w) PEG, such as at least 20% (w/w) PEG, such as at least 30% (w/w) PEG, such as at least 40% (w/w) PEG, such as at least 50% (w/w) PEG, such as at least 60 (w/w) PEG, such as at least 70% (w/w) PEG, such as at least 80% (w/w) PEG, such as at least 90% (w/w) PEG, such as at least 95%. The remaining weight percent of the PEG-based moiety or reagent is other moieties, which in certain embodiments are selected from the following moieties and linkers:

a linker selected from

Wherein the method comprises the steps of

-R and-Ra are independently selected from the group consisting of H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2-dimethylbutyl, 2, 3-dimethylbutyl and 3, 3-dimethylpropyl.

The term "hyaluronic acid-based" is used accordingly.

As used herein, the term "PEG-based comprises at least X% PEG" for a moiety or agent means that the moiety or agent comprises at least X% (w/w) ethylene glycol units (-CH) ₂ CH ₂ O-), wherein the ethylene glycol units may be arranged in blocks, alternating, or may be randomly distributed within a moiety or reagent, in certain embodiments, all of which are present in one block; the remaining weight percentages of the PEG-based moieties or reagents are other moieties, and in certain embodiments, these moieties are selected from the following moieties and linkers:

a linker selected from

Wherein the method comprises the steps of

-R and-R ^a Independently of one another, from the group H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2-dimethylbutyl, 2, 3-dimethylbutyl and 3, 3-dimethylpropyl.

The term "hyaluronic acid-based comprising at least X% hyaluronic acid" is also used accordingly.

As used herein, the term "hydrogel" refers to a hydrophilic or amphiphilic polymer network comprising homopolymers or copolymers that is insoluble due to the presence of hydrophobic interactions, hydrogen bonds, ionic interactions, and/or covalent chemical crosslinks. In certain embodiments, the hydrogel is insoluble due to the presence of covalent chemical crosslinks. In general, crosslinking provides network structure and physical integrity.

The term "interrupted" refers to the insertion of a moiety between two carbon atoms, or between a carbon atom or heteroatom and a hydrogen atom if the insertion is at one end of the moiety.

As used herein, the term "C _1-4 Alkyl "alone or in combination refers to a straight or branched alkyl moiety having 1 to 4 carbon atoms. If present at the end of the molecule, C, linear or branched ₁ - ₄ Examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. When two parts of a molecule are formed by C _1-4 When alkyl is attached, then such C _1-4 Examples of alkyl groups are-CH ₂ -、-CH ₂ CH ₂ -、-CH(CH ₃ )-、-CH ₂ CH ₂ CH ₂ -、-CH(C ₂ H ₅ )-、-C(CH ₃ ) ₂ -。C _1-4 Each hydrogen of the alkyl carbon is optionally substituted with a substituent as defined above. Optionally C _1-4 The alkyl group may be interrupted by one or more moieties as defined below.

As used herein, the term "C _1-6 Alkyl "alone or in combination refers to a straight or branched alkyl moiety having 1 to 6 carbon atoms.Linear and branched C, if present at the ends of the molecule _1-6 Examples of alkyl are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2-dimethylbutyl, 2, 3-dimethylbutyl and 3, 3-dimethylpropyl. When two parts of a molecule are formed by C _1-6 When alkyl is attached, then such C _1-6 Examples of alkyl groups are-CH ₂ -、-CH ₂ -CH ₂ -、-CH(CH ₃ )-、-CH ₂ -CH ₂ -CH ₂ -、-CH(C ₂ H ₅ ) -and-C (CH) ₃ ) ₂ -。C _1-6 Each hydrogen atom of the carbon is optionally substituted with a substituent as defined above. Optionally C _1-6 The alkyl group may be interrupted by one or more moieties as defined below.

Thus, "C _1-10 Alkyl "," C _1-20 Alkyl "or" C _1-50 Alkyl "means an alkyl chain having 1 to 10, 1 to 20 or 1 to 50 carbon atoms, respectively, wherein C _1-10 、C _1-20 Or C _1-50 Each hydrogen atom of the carbon is optionally substituted with a substituent as defined above. Optionally C _1-10 Or C _1-50 The alkyl group may be interrupted by one or more moieties as defined below.

As used herein, the term "C _2-6 Alkenyl "alone or in combination refers to a straight or branched hydrocarbon group having 2 to 6 carbon atoms containing at least one carbon-carbon double bond. Examples are-ch=ch if present at the end of the molecule ₂ 、-CH＝CH-CH ₃ 、-CH ₂ -CH＝CH ₂ 、-CH＝CHCH ₂ -CH ₃ And-ch=ch ₂ . When two parts of a molecule are formed by C _2-6 When alkenyl is attached, then such C _2-6 An example of an alkenyl group is-ch=ch-. C (C) _2-6 Each hydrogen atom of the alkenyl moiety may be optionally substituted with a substituent as defined above. Optionally C _2-6 Alkenyl groups may be interrupted by one or more moieties as defined below.

Thus, the term "C _2-10 Alkenyl group "" C _2-20 Alkenyl "or" C _2-50 Alkenyl "alone or in combination refers to a straight or branched hydrocarbon moiety containing at least one carbon-carbon double bond, having 2 to 10, 2 to 20, or 2 to 50 carbon atoms. C (C) _2-10 Alkenyl, C _2-20 Alkenyl or C _2-50 Each hydrogen atom of the alkenyl group is optionally substituted with a substituent as defined above. Optionally C _2-10 Alkenyl, C _2-20 Alkenyl or C _2-50 Alkenyl groups may be interrupted by one or more moieties as defined below.

As used herein, the term "C _2-6 Alkynyl "alone or in combination refers to a straight or branched hydrocarbon moiety having 2 to 6 carbon atoms containing at least one carbon-carbon triple bond. Examples of molecules, if present at the ends of the molecule, are: -C.ident.CH, -CH ₂ -C≡CH、-CH ₂ -CH ₂ C.ident.CH and-CH ₂ -C≡C-CH ₃ . When two parts of a molecule are linked by an alkyne group then an example is-C.ident.C-. C (C) _2-6 Each hydrogen atom of an alkynyl group is optionally substituted with a substituent as defined above. Optionally, one or more double bonds may be present. Optionally C _2-6 Alkynyl groups may be interrupted by one or more moieties as defined below.

Thus, for example, as used herein, the term "C _2-10 Alkynyl "," C _2-20 Alkynyl "and" C _2-50 Alkynyl "alone or in combination refers to straight or branched hydrocarbon groups containing at least one carbon-carbon triple bond, having 2 to 10, 2 to 20, or 2 to 50 carbon atoms, respectively. C (C) _2-10 Alkynyl, C _2-20 Alkynyl or C _2-50 Each hydrogen atom of an alkynyl group is optionally substituted with a substituent as defined above. Optionally, one or more double bonds may be present. Optionally C _2-10 Alkynyl, C _2-20 Alkynyl or C _2-50 Alkynyl groups may be interrupted by one or more moieties as defined below.

As described above, C _1-4 Alkyl, C _1-6 Alkyl, C _1-10 Alkyl, C _1-20 Alkyl, C _1-50 Alkyl, C _2-6 Alkenyl, C _2-10 Alkenyl, C _2-20 Alkenyl, C _2-50 Alkenyl groups、C _2-6 Alkynyl, C _2-10 Alkynyl, C _2-20 Alkenyl or C _2-50 Alkynyl groups are optionally interrupted by one or more moieties, preferably selected from the following:

wherein the method comprises the steps of

The dashed line represents the remainder attached to the moiety or reagent; and

-R and-R ^a Independently of one another selected from-H and methyl, ethyl, propyl, butyl, pentyl and hexyl.

As used herein, the term "C _3-10 Cycloalkyl "means a cyclic alkyl chain having 3 to 10 carbon atoms and may be saturated or unsaturated, for example cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl. C (C) _3-10 Each hydrogen atom of the cycloalkyl carbon may be substituted with a substituent as defined above. The term "C _3-10 Cycloalkyl "also includes bridged bicyclic rings, such as norbornane or norbornene.

As used herein, the term "8-30 membered carbon polycyclic group" or "8-30 membered carbon polycyclic" refers to a cyclic moiety having two or more rings of 8-30 ring atoms, wherein two adjacent rings share at least one ring atom and may contain a maximum number of double bonds (fully saturated, partially saturated or unsaturated aromatic or non-aromatic rings). Preferably, an 8-30 membered carbon polycyclic group refers to a cyclic moiety of two, three, four or five rings, more preferably a cyclic moiety of two, three or four rings.

The term "3-10 membered heterocyclyl" or "3-10 membered heterocyclic ring" as used herein refers to a ring having 3, 4, 5, 6, 7, 8, 9 or 10 ring atoms which may contain up to a maximum number of double bonds (fully saturated, partially saturated or unsaturated aromatic or non-aromatic rings), wherein at least one ring atom and up to 4 ring atoms are selected from sulfur (including-S (O) -, -S (O)) ₂ -), oxygen and nitrogen (including =n (O) -), and wherein the ring is substituted by a ringWith each carbon or nitrogen atom being attached to the remainder of the molecule. Examples of 3-10 membered heterocycles include, but are not limited to, aziridine, oxetane, azetidine, oxetane, thietane, furan, thiophene, pyrrole, pyrroline, imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline, isoxazole, isoxazoline, thiazole, thiazoline, isothiazole, isothiazoline, thiadiazole, tetrahydrofuran, tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, timididine, sulfones, pyran, dihydropyran, tetrahydropyran, imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine, piperidine, morpholine, tetrazole, triazole, triazolidine, tetrazolidine, diazepane, azepine, and homopiperazine. Each hydrogen atom of the 3-10 membered heterocyclic group or the 3-10 membered heterocyclic group may be substituted with a substituent defined below.

As used herein, the term "8-11 membered heterobicyclic group" or "8-11 membered heterobicyclic" refers to a heterocyclic moiety consisting of two rings having 8 to 11 ring atoms, at least one of which is common to both rings and may contain up to the number of double bonds (fully saturated, partially saturated or unsaturated aromatic or non-aromatic rings), wherein at least one ring atom and up to 6 ring atoms are selected from sulfur (including-S (O) -, -S (O)) ₂ (-), oxygen and nitrogen (including = N (O) -) and the ring is attached to the rest of the molecule by one carbon or nitrogen atom. Examples of 8-11 membered heterobicyclic rings are indole, indoline, benzofuran, benzothiophene, benzoxazole, benzisoxazole, benzothiazole, benzisothiazole, benzimidazole, benzimidazoline, quinoline, quinazoline. Dihydroquinazolines, quinolines, dihydroquinolines, tetrahydroquinolines, decahydroquinolines, isoquinolines, decahydroisoquinolines, tetrahydroisoquinolines, dihydroisoquinolines, benzazepines, purines, and pteridines. The term 8-11 membered heterobicyclic also includes spiro structures of two rings, for example 1, 4-dioxa-8-azaspiro [4.5 ]]Decane, or bridged heterocycles, e.g. 8-azacyclo [3.2.1]Octane. Each of 8-11 membered heterobicyclic groups or 8-11 membered heterobicyclic carbonsThe hydrogen atoms may be substituted with substituents defined below.

Likewise, the term "8-30 membered heteropolycyclyl" or "8-30 membered heteropolycyclyl" refers to a heterocyclic moiety of two or more rings, preferably 3, 4, or 5 rings, having 8-30 ring atoms, wherein two adjacent rings share at least one ring atom and may contain the greatest number of double bonds (fully saturated, partially saturated, or unsaturated aromatic or non-aromatic rings), wherein at least one ring atom and up to 10 ring atoms are selected from sulfur (including-S (O) -, S (O)) ₂ (-), oxygen and nitrogen (including = N (O) -) and the ring is attached to the rest of the molecule through one carbon or nitrogen atom.

It will be appreciated that the phrase "a pair of Rx/Ry together with the atoms to which they are attached form C in connection with the following structural moieties _3-10 Cycloalkyl or 3-10 membered heterocyclyl

Meaning that Rx and Ry constitute the following structure.

Wherein R is C _3-10 Cycloalkyl or 3 to 10 membered heterocyclyl.

It will also be appreciated that the phrase "a pair of R's in relation to the following structural portions ^x /R ^y Together with the atoms to which they are attached form a ring A

Meaning R ^x And R is ^y The following structure is formed:

as used herein, "halogen" refers to fluorine, chlorine, bromine or iodine. It is generally preferred that the halogen is fluorine or chlorine.

As used herein, the term "functional group" refers to a radical that can react with other radicals. For example, exemplary functional groups are: carboxylic acid (- (c=o) OH), primary or secondary amine (-NH) ₂ -NH-), maleimide, thiol (-SH), sulfonic acid (- (o=s=o) OH), carbonate, carbamate (-O (c=o) N <), hydroxyl (-OH), aldehyde (- (c=o) H), ketone (- (c=o)), hydrazine @>N-N <), isocyanate, isothiocyanate, phosphoric acid (-O (p=o) OHOH), phosphonic acid (-O (p=o) OHH), haloacetyl, haloalkyl, acryl, aryl fluoride, hydroxylamine, disulfide, sulfonamide, sulfuric acid, vinyl sulfone, vinyl ketone, diazoane, ethylene oxide, and azetidine.

If the IL-2 protein or conjugate of the invention comprises one or more acidic or basic groups, the invention also comprises the corresponding pharmaceutically or toxicologically acceptable salts thereof, in particular the pharmaceutically usable salts thereof. Thus, according to the invention, IL-2 proteins or conjugates comprising acidic groups can be used as, for example, alkali metal salts, alkaline earth metal salts or ammonium salts. More precise examples of such salts include sodium, potassium, calcium, magnesium salts or salts with ammonia or organic amines such as ethylamine, ethanolamine, triethanolamine or amino acids. The IL-2 proteins or conjugates of the invention comprise one or more basic groups, i.e. groups which can be protonated, can be present in the form of their addition salts with inorganic or organic acids and are used according to the invention. Examples of suitable acids include hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid. Propionic acid, pivalic acid, diethyl acetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, and other acids known to those skilled in the art. Further methods of converting basic groups into cations are known to those skilled in the art, for example alkylation of amine groups results in positively charged ammonium groups and appropriate counter ions for salts. If the IL-2 proteins or conjugates of the invention contain both acidic and basic groups, the invention includes, in addition to the salt forms mentioned, internal salts or betaines (zwitterionic). The salts may be obtained by conventional methods known to those skilled in the art, for example by contacting the prodrugs with an organic or inorganic acid or base in a solvent or dispersant, or with other salts by anion exchange or cation exchange. The invention also includes all such salts of the IL-2 proteins or conjugates of the invention which, due to their low physiological compatibility, are not directly suitable for use in pharmacy but may for example be intermediates of chemical reactions or for the preparation of pharmaceutically acceptable salts.

As used herein, the term "pharmaceutically acceptable" refers to substances that do not cause injury when administered to a patient, preferably approved by regulatory authorities, such as EMA (europe) and/or FDA (united states) and/or any other national regulatory authority for use with animals, such as for use with humans.

As used herein, the term "excipient" refers to a diluent, adjuvant, or carrier that is administered with a therapeutic agent, such as a drug or prodrug. Such pharmaceutically acceptable excipients may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, mineral oil, sesame oil and the like. When the pharmaceutical composition is to be administered orally, water is a preferred excipient. Saline and dextrose water are preferred excipients when the pharmaceutical composition is to be administered intravenously. Saline and aqueous dextrose are preferred as liquid excipients for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, mannitol, trehalose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The pharmaceutical composition may also contain minor amounts of wetting or emulsifying agents, pH buffering agents, for example, acetates, succinates, tris, carbonates, phosphates, if desired. HEPES (4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid), MES (2- (N-morpholino) ethanesulfonic acid), or may contain detergents such as Tween, polyoxyethylene ether, polyoxyethylene amine, CHAPS, igepal, or amino acids such as glycine, lysine, or histidine. These pharmaceutical compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The pharmaceutical compositions may be formulated as suppositories using conventional binders and excipients such as triglycerides. Oral formulations may include standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Such a composition will comprise a therapeutically effective amount of the drug or biologically active molecule, and an appropriate amount of excipients, in order to provide the appropriate form of administration to the patient. The formulation should be suitable for the mode of administration.

Generally, the terms "comprising" or "including" also include "consisting of …".

SEQ A and SEQ ID NO:1 has at least 89% sequence identity. SEQ ID NO:1 has the following sequence:

SEQ B and SEQ ID NO:2 has at least 76% sequence identity. SEQ ID NO:2 has the following sequence:

SEQ C and SEQ ID NO:4 has at least 91% sequence identity. SEQ ID NO:4 has the following sequence:

unless otherwise indicated, all amino acid positions given herein are based on SEQ ID NO:1, a step of; SEQ ID NO:2 or SEQ ID NO:4.

in certain embodiments, SEQ a and SEQ ID NO:1 has at least 93% sequence identity. In certain embodiments, SEQ a and SEQ ID NO:1 has at least 96% sequence identity.

In certain embodiments, SEQ a and SEQ ID NO:1 comprising three amino acid changes. In certain embodiments, the SEQ a package is identical to SEQ ID NO:1 contains two amino acid changes. In certain embodiments, SEQ a and SEQ ID NO:1 comprising an amino acid change. Such amino acid changes may be amino acid deletions, amino acid additions or amino acid exchanges of one amino acid with another, i.e. mutations. Such mutations may also be the exchange of proteinogenic amino acids for non-proteinogenic amino acids. In certain embodiments, SEQ a and SEQ ID NO:1, i.e. SEQ a has the amino acid sequence of SEQ ID NO: 1. In certain embodiments, SEQ a has the amino acid sequence of SEQ ID NO:36: PASSSTKKTQLQLEHLLLDLQMILNGINN.

In certain embodiments, SEQ a comprises an amino acid mutation that eliminates an endogenous O-glycosylation motif. Preferably, such amino acid mutation is in SEQ ID NO:1, even more preferably, such amino acid mutation is selected from T2A, T2G, T2Q, T2E, T2N, T2D, T2R, T2K and T2P. In certain embodiments, such amino acid is mutated to T2A based on SEQ ID NO:1 or a homologue or variant thereof.

In certain embodiments, SEQ B of formula (I) is identical to SEQ ID NO:2 has at least 78% sequence identity. In certain embodiments, SEQ B of formula (I) is identical to SEQ ID NO:2 has at least 80% sequence identity. In certain embodiments, SEQ B of formula (I) is identical to SEQ ID NO:2 has at least 82% sequence identity. In certain embodiments, SEQ B of formula (I) is identical to SEQ ID NO:2 has at least 84% sequence identity. In certain embodiments, SEQ B of formula (I) is identical to SEQ ID NO:2 has at least 87% sequence identity. In certain embodiments, SEQ B of formula (I) is identical to SEQ ID NO:2 has at least 89% sequence identity. In certain embodiments, SEQ B of formula (I) is identical to SEQ ID NO:2 has at least 91% sequence identity. In certain embodiments, SEQ B of formula (I) is identical to SEQ ID NO:2 has at least 93% sequence identity. In certain embodiments, SEQ B of formula (I) is identical to SEQ ID NO:2 has at least 95% sequence identity.

In certain embodiments, SEQ B and SEQ ID NO:2 comprising eleven amino acid changes. In certain embodiments, SEQ B and SEQ ID NO:2 comprising ten amino acid changes. In certain embodiments, SEQ B and SEQ ID NO:2 comprising nine amino acid changes. In certain embodiments, SEQ B and SEQ ID NO:2 comprising eight amino acid changes. In certain embodiments, SEQ B and SEQ ID NO:2 comprising seven amino acid changes. In certain embodiments, SEQ B comprises a sequence identical to SEQ ID NO:2 compared to 2. Six amino acid changes in certain embodiments, SEQ B and SEQ ID NO:2 comprising five amino acid changes. In certain embodiments, SEQ B comprises a sequence identical to SEQ ID NO:2 compared to four amino acid changes. In certain embodiments, SEQ B and SEQ ID NO:2 comprising three amino acid changes. In certain embodiments, SEQ B and SEQ ID NO:2 comprising two amino acid changes.

SEQ B contains at least one glycosylation motif. In certain embodiments, SEQ B comprises a glycosylation motif. In certain embodiments, SEQ B comprises two identical or different glycosylation motifs. In certain embodiments, SEQ B comprises three identical or different glycosylation motifs.

In certain embodiments, the at least one glycosylation motif is selected from the group consisting of an N-glycosylation motif, an O-glycosylation motif, a phospho-glycosylation motif, and a C-glycosylation motif.

In certain embodiments, at least one glycosylation motif is an N-glycosylation motif.

This N-glycosylation occurs at the amine functionality of the asparagine amino acid. Asparagine having an amine functionality may naturally occur in SEQ ID NO:2 or may be introduced by substitution of an amino acid with an asparagine residue.

In certain embodiments, the N-glycosylation motif comprises the amino acid sequence X ₁ X ₂ NX ₃ X ₄ (SEQ ID NO: 254), wherein

X ₁ Is any proteinogenic or nonproteinogenic amino groupAcid or absence;

X ₂ is any proteinogenic or non-proteinogenic amino acid or is absent;

n is asparagine;

X ₃ is any proteinogenic or non-proteinogenic amino acid other than proline; and

X ₄ selected from threonine, serine, and cysteine.

In certain embodiments, X ₁ Is a proteinogenic amino acid, a non-proteinogenic amino acid, or is absent. In certain embodiments, X ₁ Is a proteinogenic amino acid. In certain embodiments, X ₁ Is a non-proteinogenic amino acid. In certain embodiments, X ₁ Is not present. In certain embodiments, X ₁ Is phenylalanine, glutamic acid or aspartic acid. In certain embodiments, X ₁ Is phenylalanine. In certain embodiments, X ₁ Glutamic acid. In certain embodiments, X ₁ Aspartic acid.

In certain embodiments, X ₂ Is a proteinogenic amino acid, a non-proteinogenic amino acid, or is absent. In certain embodiments, X ₂ Is a proteinogenic amino acid. In certain embodiments, X ₂ Is a non-proteinogenic amino acid. In certain embodiments, X ₂ Is not present. In certain embodiments, X ₂ Glycine or alanine. In certain embodiments, X ₂ Is glycine. In certain embodiments, X ₂ Is alanine.

In certain embodiments, X ₃ Are protein amino acids other than proline. In certain embodiments, X ₃ Is a non-proteinogenic amino acid. In certain embodiments, X ₃ Serine.

In certain embodiments, X ₄ Threonine, serine or cysteine. In certain embodiments, X ₄ Threonine. In certain embodiments, X ₄ Serine. In certain embodiments, X ₄ Is cysteine.

In certain embodimentsThe N-glycosylation motif is the amino acid sequence NX ₃ X ₄ Wherein X is ₃ And X ₄ As described in additional sections herein. In certain embodiments, the N-glycosylation motif is selected from NX ₃ T、NX ₃ S and NX ₃ C. In certain embodiments, the N-glycosylation motif is NX ₃ T. In certain embodiments, the N-glycosylation motif is NX ₃ S, S. In certain embodiments, the N-glycosylation motif is NX ₃ C. In certain embodiments, the N-glycosylation motif is NSX ₄ . In certain embodiments, the N-glycosylation motif is selected from NST, NSS, and NSC. In certain embodiments, the N-glycosylation motif is NST. In certain embodiments, the N-glycosylation motif is NSS. In certain embodiments, the N-glycosylation motif is NSC.

In certain embodiments, the N-glycosylation motif is X ₂ NX ₃ X ₄ (SEQ ID NO: 255), wherein X ₂ 、X ₃ And X ₄ As defined elsewhere herein. In certain embodiments, the N-glycosylation motif is selected from X ₂ NX ₃ T(SEQ ID NO：256)，X ₂ NX ₃ S (SEQ ID NO: 257) and X ₂ NX ₃ C (SEQ ID NO: 258). In certain embodiments, the N-glycosylation motif is X ₂ NX ₃ T (SEQ ID NO: 256). In certain embodiments, the N-glycosylation motif is X ₂ NX ₃ S (SEQ ID NO: 257). In certain embodiments, the N-glycosylation motif is X ₂ NX ₃ C (SEQ ID NO: 258). In certain embodiments, the N-glycosylation motif is X ₂ NSX ₄ (SEQ ID NO: 259). In certain embodiments, the N-glycosylation motif is selected from X ₂ NST(SEQ ID NO：260)，X ₂ NSS (SEQ ID NO: 261) and X ₂ NSC (SEQ ID NO: 262). In certain embodiments, the N-glycosylation motif is X ₂ NST (SEQ ID NO: 260). In certain embodiments, the N-glycosylation motif is X ₂ NSS (SEQ ID NO: 261). In certain embodiments, the N-glycosylation motif is X ₂ NSC (SEQ ID NO: 262). In certain embodiments, the N-glycosylation motif is GNX ₃ X ₄ (SEQ ID NO: 263). In certain embodimentsIn which the N-glycosylation motif is selected from GNX ₃ T(SEQ ID NO：264)、GNX ₃ S (SEQ ID NO: 265) and GNX ₃ C (SEQ ID NO: 266). In certain embodiments, the N-glycosylation motif is GNX ₃ T (SEQ ID NO: 264). In certain embodiments, the N-glycosylation motif is GNX ₃ S (SEQ ID NO: 265). In certain embodiments, the N-glycosylation motif is GNX ₃ C (SEQ ID NO: 266). In certain embodiments, the N-glycosylation motif is GNSX ₄ (SEQ ID NO: 267). In certain embodiments, the N-glycosylation motif is selected from the group consisting of GNST (SEQ ID NO: 268), GNSS (SEQ ID NO: 269), and GNSC (SEQ ID NO: 270). In certain embodiments, the N-glycosylation motif is GNST (SEQ ID NO: 268). In certain embodiments, the N-glycosylation motif is GNSS (SEQ ID NO:269. In certain embodiments, the N-glycosylation motif is GNSC (SEQ ID NO: 270). In certain embodiments, the N-glycosylation motif is ANX ₃ X ₄ (SEQ ID NO: 271). In certain embodiments, the N-glycosylation motif is selected from ANX ₃ T(SEQ ID NO：272)、ANX ₃ S (SEQ ID NO: 273) and ANX ₃ C (SEQ ID NO: 274). In certain embodiments, the N-glycosylation motif is ANX ₃ T (SEQ ID NO: 272). In certain embodiments, the N-glycosylation motif is ANX ₃ S (SEQ ID NO: 273). In certain embodiments, the N-glycosylation motif is ANX ₃ C (SEQ ID NO: 274). In certain embodiments, the N-glycosylation motif is ANSX ₄ (SEQ ID NO: 275). In certain embodiments, the N-glycosylation motif is selected from ANST (SEQ ID NO: 276), ANSS (SEQ ID NO: 277), and ANSC (SEQ ID NO: 278). In certain embodiments, the N-glycosylation motif is ANST (SEQ ID NO: 276). In certain embodiments, the N-glycosylation motif is ANSS (SEQ ID NO: 277). In certain embodiments, the N-glycosylation motif is ANSC (SEQ ID NO: 278).

In certain embodiments, the N-glycosylation motif is X ₁ X ₂ NX ₃ X ₄ (SEQ ID NO: 254), wherein X ₁ 、X ₂ 、X ₃ And X ₄ As defined elsewhere herein. In certain embodiments, the N-glycosylation motif is selected from X ₁ X ₂ NX ₃ T(SEQ ID NO：279)、X ₁ X ₂ NX ₃ S (SEQ ID NO: 280) and X ₁ X ₂ NX ₃ C (SEQ ID NO: 281). In certain embodiments, the N-glycosylation motif has X ₁ X ₂ NX ₃ T (SEQ ID NO: 279). In certain embodiments, the N-glycosylation motif is X ₁ X ₂ NX ₃ S (SEQ ID NO: 280). In certain embodiments, the N-glycosylation motif is X ₁ X ₂ NX ₃ C (SEQ ID NO: 281). In certain embodiments, the N-glycosylation motif is X ₁ X ₂ NSX ₄ (SEQ ID NO: 282). In certain embodiments, the N-glycosylation motif is selected from X ₁ X ₂ NST(SEQ ID NO：283)、X ₁ X ₂ NSS (SEQ ID NO: 284) and X ₁ X ₂ NSC (SEQ ID NO: 285). In certain embodiments, the N-glycosylation motif is X ₁ X ₂ NST (SEQ ID NO: 283). In certain embodiments, the N-glycosylation motif is X ₁ X ₂ NSS (SEQ ID NO: 284). In certain embodiments, the N-glycosylation motif is X ₁ X ₂ NSC (SEQ ID NO: 285). In certain embodiments, the N-glycosylation motif is X ₁ GNX ₃ X ₄ (SEQ ID NO: 286). In certain embodiments, the N-glycosylation motif is selected from X ₁ GNX ₃ T(SEQ ID NO：287)、X ₁ GNX ₃ S (SEQ ID NO: 288) and X ₁ GNX ₃ C (SEQ ID NO: 289). In certain embodiments, the N-glycosylation motif is X ₁ GNX ₃ T (SEQ ID NO: 287). In certain embodiments, the N-glycosylation motif is X ₁ GNX ₃ S (SEQ ID NO: 288). In certain embodiments, the N-glycosylation motif is X ₁ GNX ₃ C (SEQ ID NO: 289). In certain embodiments, the N-glycosylation motif is X ₁ GNSX ₄ (SEQ ID NO: 290). In certain embodiments, the N-glycosylation motif is selected from X ₁ GNST(SEQ ID NO：291)、X ₁ GNSS (SEQ ID NO: 292) and X ₁ GNSC (SEQ ID NO: 293). In certain embodiments, the N-glycosylation motif is X ₁ GNST (SEQ ID NO: 291). In certain embodiments, the N-sugarThe glycosylation motif is X ₁ GNSS (SEQ ID NO: 292). In certain embodiments, the N-glycosylation motif is X ₁ GNSC (SEQ ID NO: 293). In certain embodiments, the N-glycosylation motif is FX ₂ NX ₃ X ₄ (SEQ ID NO: 294). In certain embodiments, the N-glycosylation motif is selected from FX ₂ NX ₃ T(SEQ ID NO：295)、FX ₂ NX ₃ S (SEQ ID NO: 296) and FX ₂ NX ₃ C (SEQ ID NO: 297). In certain embodiments, the N-glycosylation motif is FX ₂ NX ₃ T (SEQ ID NO: 295). In certain embodiments, the N-glycosylation motif is FX ₂ NX ₃ S (SEQ ID NO: 296). In certain embodiments, the N-glycosylation motif is FX ₂ NX ₃ C (SEQ ID NO: 297). In certain embodiments, the N-glycosylation motif is FX ₂ NSX ₄ (SEQ ID NO: 298). In certain embodiments, the N-glycosylation motif is selected from FX ₂ NST(SEQ ID NO：299)、FX ₂ NSS (SEQ ID NO: 300) and FX ₂ NSC (SEQ ID NO: 301). In certain embodiments, the N-glycosylation motif is FX ₂ NST (SEQ ID NO: 299). In certain embodiments, the N-glycosylation motif is FX ₂ NSS (SEQ ID NO: 300). In certain embodiments, the N-glycosylation motif is FX ₂ NSC (SEQ ID NO: 301). In certain embodiments, the N-glycosylation motif is FGNX ₃ X ₄ (SEQ ID NO: 302). In certain embodiments, the N-glycosylation motif is selected from FGNX ₃ T(SEQ ID NO：303)、FGNX ₃ S (SEQ ID NO: 304) and FGNX ₃ C (SEQ ID NO: 305). In certain embodiments, the N-glycosylation motif is FGNX ₃ T (SEQ ID NO: 303). In certain embodiments, the N-glycosylation motif is FGNX ₃ S (SEQ ID NO: 304). In certain embodiments, the N-glycosylation motif is FGNX ₃ C (SEQ ID NO: 305). In certain embodiments, the N-glycosylation motif is FGNSX ₄ (SEQ ID NO: 306). In certain embodiments, the N-glycosylation motif is selected from FGNST (SEQ ID NO: 307), FGNSS (SEQ ID NO: 308), and FGNSC (SEQ ID NO: 309). In certain embodiments, the N-glycosylation motif is FGNST (SEQ ID NO: 307). In certain embodimentsIn (C), the N-glycosylation motif is FGNSS (SEQ ID NO: 308). In certain embodiments, the N-glycosylation motif is FGNSC (SEQ ID NO: 309). In certain embodiments, the N-glycosylation motif is X ₁ ANX ₃ X ₄ (SEQ ID NO: 310). In certain embodiments, the N-glycosylation motif is selected from X ₁ ANX ₃ T(SEQ ID NO：311)、X ₁ ANX ₃ S (SEQ ID NO: 312) and X ₁ ANX ₃ C (SEQ ID NO: 313). In certain embodiments, the N-glycosylation motif is X ₁ ANX ₃ T (SEQ ID NO: 311). In certain embodiments, the N-glycosylation motif is X ₁ ANX ₃ S (SEQ ID NO: 312). In certain embodiments, the N-glycosylation motif is X ₁ ANX ₃ C (SEQ ID NO: 313). In certain embodiments, the N-glycosylation motif is X ₁ ANSX ₄ (SEQ ID NO: 314). In certain embodiments, the N-glycosylation motif is selected from X ₁ ANST(SEQ ID NO：315)、X ₁ ANSS (SEQ ID NO: 316) and X ₁ ANSC (SEQ ID NO: 317). In certain embodiments, the N-glycosylation motif is X ₁ ANST (SEQ ID NO: 315). In certain embodiments, the N-glycosylation motif is X ₁ ANSS (SEQ ID NO: 316). In certain embodiments, the N-glycosylation motif is X ₁ ANSC (SEQ ID NO: 317). In certain embodiments, the N-glycosylation motif is FX ₂ NX ₃ X ₄ (SEQ ID NO: 318). In certain embodiments, the N-glycosylation motif is selected from FX ₂ NX ₃ T(SEQ ID NO：319)、FX ₂ NX ₃ S (SEQ ID NO: 320) and FX ₂ NX ₃ C (SEQ ID NO: 321). In certain embodiments, the N-glycosylation motif is FX ₂ NX ₃ T (SEQ ID NO: 319). In certain embodiments, the N-glycosylation motif is FX ₂ NX ₃ S (SEQ ID NO: 320). In certain embodiments, the N-glycosylation motif is FX ₂ NX ₃ C (SEQ ID NO: 321). In certain embodiments, the N-glycosylation motif is FX ₂ NSX ₄ (SEQ ID NO: 322). In certain embodiments, the N-glycosylation motif is selected from FX ₂ NST(SEQ ID NO：323)、FX ₂ NSS (SEQ ID NO: 324) and FX ₂ NSC (SEQ ID NO: 325). In certain embodiments, the N-glycosylation motif is FX ₂ NST (SEQ ID NO: 323). In certain embodiments, the N-glycosylation motif is FX ₂ NSS (SEQ ID NO: 324). In certain embodiments, the N-glycosylation motif is FX ₂ NSC (SEQ ID NO: 325). In certain embodiments, the N-glycosylation motif is FANX ₃ X ₄ (SEQ ID NO: 326). In certain embodiments, the N-glycosylation motif is selected from FANX ₃ T(SEQ ID NO：327)、FANX ₃ S (SEQ ID NO: 328) and FANX ₃ C (SEQ ID NO: 329). In certain embodiments, the N-glycosylation motif is FANX ₃ T (SEQ ID NO: 327). In certain embodiments, the N-glycosylation motif is FANX ₃ S (SEQ ID NO: 328). In certain embodiments, the N-glycosylation motif is FANX ₃ C (SEQ ID NO: 329). In certain embodiments, the N-glycosylation motif is FANSX ₄ (SEQ ID NO: 330). In certain embodiments, the N-glycosylation motif is selected from FANST (SEQ ID NO: 331), FANSS (SEQ ID NO: 332), and FANSC (SEQ ID NO: 333). In certain embodiments, the N-glycosylation motif is FANST (SEQ ID NO: 331). In certain embodiments, the N-glycosylation motif is FANSS (SEQ ID NO: 332). In certain embodiments, the N-glycosylation motif is FANSC (SEQ ID NO: 333).

In certain embodiments, the N-glycosylation motif is FGNST (SEQ ID NO: 307) or FANST (SEQ ID NO: 331).

In certain embodiments, at least one N-glycosylation motif is an O-glycosylation motif.

This O-glycosylation occurs at amino acids having hydroxyl functionality. Amino acids having hydroxyl functionality may naturally occur in SEQ ID NOs: 2 or may be introduced by substitution of an amino acid comprising a hydroxyl functionality with an endogenous amino acid. The amino acid used for O-glycosylation may be a proteinogenic amino acid or a non-proteinogenic amino acid having a hydroxyl functionality. In certain embodiments, the amino acid used for O-glycosylation is a proteinogenic amino acid. In certain embodiments, the proteinogenic amino acid having a hydroxyl functionality is selected from serine, threonine, tyrosine, hydroxylysine, and hydroxyproline. In certain embodiments, the amino acid in the-6 to-1 or +1 to +4 position is replaced with an amino acid having a hydroxyl functionality for O-glycosylation with proline. In certain embodiments, the amino acid at the-6-position of the amino acid with a hydroxyl functionality for O-glycosylation is substituted with proline. In certain embodiments, the amino acid at the-5 position of the amino acid with a hydroxyl functionality for O-glycosylation is substituted with proline. In certain embodiments, the amino acid at position-4 of the amino acid with a hydroxyl functionality for O-glycosylation is substituted with proline. In certain embodiments, the amino acid at the-3 position of the amino acid with a hydroxyl functionality for O-glycosylation is substituted with proline. In certain embodiments, the amino acid at the-2-position of the amino acid with a hydroxyl functionality for O-glycosylation is substituted with proline. In certain embodiments, the amino acid at the-1-position of the amino acid with a hydroxyl functionality for O-glycosylation is substituted with proline. In certain embodiments, the amino acid at position +1 of the amino acid with a hydroxyl functionality for O-glycosylation is substituted with proline. In certain embodiments, the amino acid at position +2 of the amino acid with a hydroxyl functionality for O-glycosylation is substituted with proline. In certain embodiments, the amino acid at position +3 of the amino acid with a hydroxyl functionality for O-glycosylation is substituted with proline. In certain embodiments, the amino acid at position +4 of the amino acid with a hydroxyl functionality for O-glycosylation is substituted with proline.

In certain embodiments, at least one glycosylation motif is obtained by mutating the amino acid sequence of SEQ ID NO:2, and certain consecutive amino acids. In certain embodiments, the polypeptide is produced by mutating SEQ ID NO:2 into one such glycosylation motif is introduced into the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the polypeptide is produced by mutating SEQ ID NO:2 into the sequence of SEQ ID NO:2, wherein the two glycosylation motifs can be the same or different.

In certain embodiments, SEQ B comprises an N-glycosylation site at an amino acid position selected from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 29, 30, 31, 32, 33, 34, 40, 41, 42, 43, and 44, based on SEQ ID NO:2 or at a homologue or variant thereof. In certain embodiments, SEQ B comprises an N-glycosylation site at position 3, based on SEQ ID NO:2 or at a homologue or variant thereof. In certain embodiments, SEQ B comprises an N-glycosylation site at position 4, based on SEQ ID NO:2 or at a homologue or variant thereof. In certain embodiments, SEQ B comprises an N-glycosylation site at position 5, based on SEQ ID NO:2 or at a homologue or variant thereof. In certain embodiments, SEQ B comprises an N-glycosylation site at position 6, based on SEQ ID NO:2 or at a homologue or variant thereof. In certain embodiments, SEQ B comprises an N-glycosylation site at position 7, based on SEQ ID NO:2 or at a homologue or variant thereof. In certain embodiments, SEQ B comprises an N-glycosylation site at position 8, based on SEQ ID NO:2 or at a homologue or variant thereof. In certain embodiments, SEQ B comprises an N-glycosylation site at position 9, based on SEQ ID NO:2 or at a homologue or variant thereof. In certain embodiments, SEQ B comprises an N-glycosylation site at position 10, based on SEQ ID NO:2 or at a homologue or variant thereof. In certain embodiments, SEQ B comprises an N-glycosylation site at position 11, based on SEQ ID NO:2 or at a homologue or variant thereof. In certain embodiments, SEQ B comprises an N-glycosylation site at position 12, based on SEQ ID NO:2 or at a homologue or variant thereof. In certain embodiments, SEQ B comprises an N-glycosylation site at position 13, based on SEQ ID NO:2 or at a homologue or variant thereof. In certain embodiments, SEQ B comprises an N-glycosylation site at position 14, based on SEQ ID NO:2 or at a homologue or variant thereof. In certain embodiments, SEQ B comprises an N-glycosylation site at position 15, based on SEQ ID NO:2 or at a homologue or variant thereof. In certain embodiments, SEQ B comprises an N-glycosylation site at position 16, based on SEQ ID NO:2 or at a homologue or variant thereof. In certain embodiments, SEQ B comprises an N-glycosylation site at position 17, based on SEQ ID NO:2 or at a homologue or variant thereof. In certain embodiments, SEQ B comprises an N-glycosylation site at position 29, based on SEQ ID NO:2 or at a homologue or variant thereof. In certain embodiments, SEQ B comprises an N-glycosylation site at position 30, based on SEQ ID NO:2 or at a homologue or variant thereof. In certain embodiments, SEQ B comprises an N-glycosylation site at position 31, based on SEQ ID NO:2 or at a homologue or variant thereof. In certain embodiments, SEQ B comprises an N-glycosylation site at position 32, based on SEQ ID NO:2 or at a homologue or variant thereof. In certain embodiments, SEQ B comprises an N-glycosylation site at position 33, based on SEQ ID NO:2 or at a homologue or variant thereof. In certain embodiments, SEQ B comprises an N-glycosylation site at position 34, based on SEQ ID NO:2 or at a homologue or variant thereof. In certain embodiments, SEQ B comprises an N-glycosylation site at position 40, based on SEQ ID NO:2 or at a homologue or variant thereof. In certain embodiments, SEQ B comprises an N-glycosylation site at position 41, based on SEQ ID NO:2 or at a homologue or variant thereof. In certain embodiments, SEQ B comprises an N-glycosylation site at position 42, based on SEQ ID NO:2 or at a homologue or variant thereof. In certain embodiments, SEQ B comprises an N-glycosylation site at position 43, based on SEQ ID NO:2 or at a homologue or variant thereof. In certain embodiments, SEQ B comprises an N-glycosylation site at position 44, based on SEQ ID NO:2 or at a homologue or variant thereof.

In certain embodiments, SEQ B has a sequence selected from the group consisting of:

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wherein X is ₃ And X ₄ As defined elsewhere herein.

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in certain embodiments, SEQ B has a sequence selected from the group consisting of: SEQ ID NO:87, a base; SEQ ID NO:88; SEQ ID NO:89; SEQ ID NO:90; SEQ ID NO:91; SEQ ID NO:92; SEQ ID NO:93; SEQ ID NO:94; SEQ ID NO:95; SEQ ID NO:96; SEQ ID NO:97; SEQ ID NO:98; SEQ ID NO:99, a step of; SEQ ID NO:100; SEQ ID NO: 114. SEQ ID NO:115; SEQ ID NO:116; SEQ ID NO:117; SEQ ID NO:125; SEQ ID NO:126; and SEQ ID NO:127.

in certain embodiments, SEQ B has a sequence selected from the group consisting of: SEQ ID NO:130; SEQ ID NO:131; SEQ ID NO:132, a part of the material; SEQ ID NO:133; SEQ ID NO:134; SEQ ID NO:135; SEQ ID NO:136; SEQ ID NO:137, respectively; SEQ ID NO:138; SEQ ID NO:139, respectively; SEQ ID NO:140; SEQ ID NO:141; SEQ ID NO:142; SEQ ID NO:143, a base; SEQ ID NO:144 (144); SEQ ID NO:156, respectively; SEQ ID NO:157 (157); SEQ ID NO:158; SEQ ID NO:159; SEQ ID NO:160, a step of detecting a position of the base; SEQ ID NO:161; SEQ ID NO:167; SEQ ID NO:168; SEQ ID NO:169 (169); SEQ ID NO:170, a step of; and SEQ ID NO:171.

In certain embodiments, SEQ B has a sequence selected from the group consisting of: SEQ ID NO:89; SEQ ID NO:92; SEQ ID NO:93; SEQ ID NO:95; SEQ ID NO:96; SEQ ID NO:97; SEQ ID NO:98; SEQ ID NO:99, a step of; SEQ ID NO:116; SEQ ID NO:117; and SEQ ID NO:127.

in certain embodiments, SEQ B has a sequence selected from the group consisting of: SEQ ID NO:134; SEQ ID NO:137, respectively; SEQ ID NO:138; SEQ ID NO:140; SEQ ID NO:141; SEQ ID NO:142; SEQ ID NO:143, a base; SEQ ID NO:144 (144); SEQ ID NO:160, a step of detecting a position of the base; SEQ ID NO:161; and SEQ ID NO:171.

in certain embodiments, SEQ B has a sequence selected from the group consisting of SEQ id no

In certain embodiments, the N-glycosylation motif is introduced in such a way that the sequence SEQ ID NO:2, in which case the asparagine need not be substituted, resulting in SEQ B of one of the sequences selected from the group consisting of: SEQ ID NO:87, a base; SEQ ID NO:130; SEQ ID NO:127; SEQ ID NO:168;

In certain embodiments, the O-glycosylation motif is introduced by replacing any amino acid in sequence SEQ B with an amino acid selected from serine, threonine, tyrosine, hydroxylysine, and hydroxyproline.

In certain embodiments, the O-glycosylation motif, i.e., SEQ ID NO:2 into an amino acid selected from serine, threonine, tyrosine, hydroxylysine and hydroxyproline occurs at position K5, R8, M9, T11, F12, K13, F14, Y15, E32 or L42 of the sequence SEQ B.

In certain embodiments, the O-glycosylation motif is introduced by an amino acid substitution in sequence SEQ B selected from: K5S, K5T, K Y, K Hyl, K5Hyp, R8S, R8T, R8Y, R Hy1, R8Hy, M9S, M9T, M9Y, M Hyl, M9Hyp, T11S, T11Y, T Hyl, T11Hyp, F12S, F12T, F12Y, F Hyl, F12Hyp, K13S, K T, K13Y, K Hyl, K13Hyp, Y15S, Y15T, Y Hyl, Y15Hyp, E32S, E T, E Y, E Hyl, E32Hyp, L42S, L42T, L42Y, L Hyl and L42Hyp.

In certain embodiments, SEQ B is selected from SEQ ID NOs: 11. SEQ ID NO: 14. SEQ ID NO: 17. SEQ ID NO: 20. SEQ ID NO: 23. SEQ ID NO: 26. SEQ ID NO: 32. SEQ ID NO: 35. SEQ ID NO:214 and SEQ ID NO:215.

In certain embodiments, SEQ B has the amino acid sequence of SEQ ID NO: 11. In certain embodiments, SEQ B has the amino acid sequence of SEQ ID NO: 14. In certain embodiments, SEQ B has the amino acid sequence of SEQ ID NO: 17. In certain embodiments, SEQ B has the amino acid sequence of SEQ ID NO: 20. In certain embodiments, SEQ B has the amino acid sequence of SEQ ID NO: 23. In certain embodiments, SEQ B has the amino acid sequence of SEQ ID NO: 26. In certain embodiments, SEQ B has the amino acid sequence of SEQ ID NO: 32. In certain embodiments, SEQ B has the amino acid sequence of SEQ ID NO: 35. In certain embodiments, SEQ B has the amino acid sequence of SEQ ID NO: 214. In certain embodiments, SEQ B has the amino acid sequence of SEQ ID NO: 215.

In certain embodiments, insertion of a glycosylation motif into SEQ B results in a site occupancy of at least 50%. In certain embodiments, insertion of a glycosylation motif into SEQ B results in a site occupancy of at least 55%. In certain embodiments, insertion of a glycosylation motif into SEQ B results in a site occupancy of at least 60%. In certain embodiments, inserting a glycosylation motif into SEQ B results in a site occupancy of at least 65%. In certain embodiments, insertion of a glycosylation motif into SEQ B results in a site occupancy of at least 70%. In certain embodiments, inserting a glycosylation motif into SEQ B results in a site occupancy of at least 75%. In certain embodiments, insertion of a glycosylation motif into SEQ B results in a site occupancy of at least 80%. In certain embodiments, insertion of a glycosylation motif into SEQ B results in a site occupancy of at least 85%. In certain embodiments, insertion of a glycosylation motif into SEQ B results in a site occupancy of at least 90%. In certain embodiments, insertion of a glycosylation motif into SEQ B results in a site occupancy of at least 95%.

In certain embodiments, SEQ B further comprises a sequence based on SEQ ID NO:2 at least one amino acid mutation occurring at an amino acid position selected from the group consisting of K5, R8, M9, T11, F12, K13, F14, Y15, E31, E32 and L42 or at a corresponding position of a homolog or variant thereof. Even more preferably, the at least one amino acid mutation comprises a sequence based on SEQ ID NO:2 at an amino acid position selected from F12, Y15, E31, E32 and L42 and or at a corresponding position of a homologue or variant thereof. In certain embodiments, the at least one amino acid mutation comprises a sequence based on SEQ ID NO:2 or a mutation at a corresponding position in a homologue or variant thereof. In certain embodiments, the at least one amino acid mutation comprises a sequence based on SEQ ID NO:2 or a mutation at a corresponding position in a homologue or variant thereof. In certain embodiments, the at least one amino acid mutation comprises a sequence based on SEQ ID NO:2 or a mutation at a corresponding position in a homologue or variant thereof. In certain embodiments, the at least one amino acid mutation comprises a sequence based on SEQ ID NO:2 or a mutation at a corresponding position in a homologue or variant thereof. In certain embodiments, the at least one amino acid mutation comprises a sequence based on SEQ ID NO:2 or a mutation at a corresponding position of a homolog or variant thereof. In certain embodiments, the at least one amino acid mutation comprises a sequence based on SEQ ID NO:2 or a mutation at a corresponding position in a homologue or variant thereof. In certain embodiments, the at least one amino acid mutation comprises a sequence based on SEQ ID NO:2 or a mutation at a corresponding position of a homolog or variant thereof. In certain embodiments, the at least one amino acid mutation comprises a sequence based on SEQ ID NO:2 or a mutation at a corresponding position in a homologue or variant thereof. In certain embodiments, the at least one amino acid mutation comprises a sequence based on SEQ ID NO:2 or a mutation at a corresponding position in a homologue or variant thereof. In certain embodiments, the at least one amino acid mutation comprises a sequence based on SEQ ID NO:2 or a mutation at a corresponding position in a homologue or variant thereof. In certain embodiments, the at least one amino acid mutation comprises a sequence based on SEQ ID NO:2 or a mutation at a corresponding position of a homolog or variant thereof.

In certain embodiments, such mutations are substitutions of proteinogenic naturally occurring amino acids with amino acid residues selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, lysine, serine, threonine, tryptophan, and tyrosine. In certain embodiments, the naturally occurring amino acid is replaced with alanine. In certain embodiments, the naturally occurring amino acid is replaced with arginine. In certain embodiments, the naturally occurring amino acid is replaced with asparagine. In certain embodiments, the naturally occurring amino acid is replaced with aspartic acid. In certain embodiments, the naturally occurring amino acid is replaced with a cysteine. In certain embodiments, the naturally occurring amino acid is replaced with glutamine. In certain embodiments, the naturally occurring amino acid is replaced with glutamic acid. In certain embodiments, the naturally occurring amino acid is replaced with glycine. In certain embodiments, the naturally occurring amino acid is replaced with histidine. In certain embodiments, the naturally occurring amino acid is replaced with lysine. In certain embodiments, the naturally occurring amino acid is substituted for serine. In certain embodiments, the naturally occurring amino acid is replaced with threonine. In certain embodiments, the naturally occurring amino acid is replaced with tryptophan. In certain embodiments, the naturally occurring amino acid is replaced with tyrosine. In certain embodiments, such mutations are substitutions of naturally occurring amino acids with amino acid residues selected from the group consisting of arginine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, lysine, serine, threonine, tryptophan, and tyrosine. In certain embodiments, such mutations are substitutions of naturally occurring amino acids with amino acid residues selected from the group consisting of cysteine, glutamic acid, lysine, serine, threonine, and tyrosine. In certain embodiments, the naturally occurring amino acid is replaced with a non-proteinogenic amino acid. Embodiments of such non-proteinogenic amino acids are described above.

In certain embodiments, SEQ B comprises a sequence based on SEQ ID NO:2 or a homologue or variant thereof, is selected from the group consisting of K5A, K5C, K5G, K5S, K5T, K5Q, K5E, K5N, K5D, K5H, K5W, K5Y and K5R. In certain embodiments, SEQ B comprises a K5A mutation. In certain embodiments, SEQ B comprises a K5C mutation. In certain embodiments, SEQ B comprises a K5G mutation. In certain embodiments, SEQ B comprises a K5S mutation. In certain embodiments, SEQ B comprises a K5T mutation. In certain embodiments, SEQ B comprises a K5Q mutation. In certain embodiments, SEQ B comprises a K5E mutation. In certain embodiments, SEQ B comprises a K5D mutation. In certain embodiments, SEQ B comprises a K5H mutation. In certain embodiments, SEQ B comprises a K5W mutation. In certain embodiments, SEQ B comprises a K5Y mutation. In certain embodiments, SEQ B comprises a K5R mutation.

In certain embodiments, SEQ B has the amino acid sequence of SEQ ID NO: 214. In certain embodiments, SEQ B has the amino acid sequence of SEQ ID NO: 215. In certain embodiments, SEQ B has the amino acid sequence of SEQ ID NO: 235. In certain embodiments, SEQ B has the amino acid sequence of SEQ ID NO: 236.

In certain embodiments, SEQ B comprises a sequence based on SEQ ID NO:2 or a homologue or variant thereof selected from the group consisting of R8A, R8C, R8G, R8S, R8T, R8Q, R8E, R8N, R8D, R8H, R8W, R Y and R8K amino acid mutations at the corresponding positions. In certain embodiments, SEQ B comprises the R8A mutation. In certain embodiments, SEQ B comprises the R8C mutation. In certain embodiments, SEQ B comprises R8G mutations. In certain embodiments, SEQ B comprises R8S mutations. In certain embodiments, SEQ B comprises an R8T mutation. In certain embodiments, SEQ B comprises an R8Q mutation. In certain embodiments, SEQ B comprises R8E mutations. In certain embodiments, SEQ B comprises the R8N mutation. In certain embodiments, SEQ B comprises R8D mutations. In certain embodiments, SEQ B comprises an R8H mutation. In certain embodiments, SEQ B comprises an R8K mutation. In certain embodiments, SEQ B comprises the R8W mutation. In certain embodiments, SEQ B comprises R8Y mutations. In certain embodiments, SEQ B comprises an R8K mutation.

In certain embodiments, SEQ B comprises a sequence based on SEQ ID NO:2 or a homologue or variant thereof selected from the group consisting of F12A, F12C, F12G, F12S, F12T, F12Q, F12E, F12N, F12D, F12R, F12H, F12W, F Y and F12K. In certain embodiments, SEQ B comprises a sequence based on SEQ ID NO:2 or a homologue or variant thereof selected from the group consisting of F12A, F12C, F12G, F3512S, F12T, F12Q, F12E, F12N, F12D, F R and F12K. In certain embodiments, SEQ B comprises the F12A mutation. In certain embodiments, SEQ B comprises the F12C mutation. In certain embodiments, SEQ B comprises the F12G mutation. In certain embodiments, SEQ B comprises an F12S mutation. In certain embodiments, SEQ B comprises an F12T mutation. In certain embodiments, SEQ B comprises the F12Q mutation. In certain embodiments, SEQ B comprises an F12E mutation. In certain embodiments, SEQ B comprises the F12N mutation. In certain embodiments, SEQ B comprises the F12D mutation. In certain embodiments, SEQ B comprises the F12R mutation. In certain embodiments, SEQ B comprises an F12H mutation. In certain embodiments, SEQ B comprises the F12W mutation. In certain embodiments, SEQ B comprises the F12Y mutation.

In certain embodiments, SEQ B comprises the F12K mutation.

In certain embodiments, SEQ B comprises a sequence based on SEQ ID NO:2 or a homologue or variant thereof selected from the group consisting of Y15A, Y15C, Y15G, Y S, Y15T, Y15Q, Y15E, Y15N, Y D, Y15R, Y H, Y W and Y15K. In certain embodiments, SEQ B comprises a sequence based on SEQ ID NO:2 or a homologue or variant thereof, is selected from the group consisting of Y15A, Y15C, Y15G, Y S, Y15T, Y15Q, Y15E, Y15N, Y D, Y R and Y15K. In certain embodiments, SEQ B comprises the Y15A mutation. In certain embodiments, SEQ B comprises the Y15C mutation. In certain embodiments, SEQ B comprises a Y15G mutation. In certain embodiments, SEQ B comprises a Y15S mutation. In certain embodiments, SEQ B comprises a Y15T mutation. In certain embodiments, SEQ B comprises a Y15Q mutation. In certain embodiments, SEQ B comprises a Y15E mutation. In certain embodiments, SEQ B comprises the Y15N mutation. In certain embodiments, SEQ B comprises the Y15D mutation. In certain embodiments, SEQ B comprises a Y15R mutation. In certain embodiments, SEQ B comprises a Y15H mutation. In certain embodiments, SEQ B comprises a Y15W mutation. In certain embodiments, SEQ B comprises a Y15K mutation.

In certain embodiments, SEQ B comprises a sequence based on SEQ ID NO:2 or a homologue or variant thereof selected from the group consisting of L42G, L42C, L A, L42S, L42T, L42Q, L42E, L42N, L D, L42R, L42H, L42W, L Y and L42K. In certain embodiments, SEQ B comprises a sequence based on SEQ ID NO:2 or a homologue or variant thereof selected from the group consisting of L42G, L42C, L42A, L S, L42T, L42Q, L42E, L42N, L42D, L R and L42K. In certain embodiments, SEQ B comprises the L42G mutation. In certain embodiments, SEQ B comprises the L42C mutation. In certain embodiments, SEQ B comprises the L42A mutation. In certain embodiments, SEQ B comprises the L42S mutation. In certain embodiments, SEQ B comprises an L42T mutation. In certain embodiments, SEQ B comprises an L42Q mutation. In certain embodiments, SEQ B comprises an L42E mutation. In certain embodiments, SEQ B comprises the L42N mutation. In certain embodiments, SEQ B comprises the L42D mutation. In certain embodiments, SEQ B comprises the L42R mutation. In certain embodiments, SEQ B comprises the L42H mutation. In certain embodiments, SEQ B comprises the L42W mutation. In certain embodiments, SEQ B comprises the L42Y mutation. In certain embodiments, SEQ B comprises the L42K mutation.

In certain embodiments, SEQ B comprises an amino acid mutation selected from the group consisting of F12A, F12C, F12G, F12S, F12T, F12Q, F12E, F12N, F12D, F R and F12K and an additional amino acid mutation selected from the group consisting of Y15A, Y15C, Y15G, Y15S, Y15T, Y15Q, Y E, Y15N, Y D, Y R and Y15K based on SEQ ID NO:2 or a homologue or variant thereof. In certain embodiments, SEQ B comprises F12A and Y15A mutations. In certain embodiments, SEQ B comprises an F12C mutation and a Y15A mutation. In certain embodiments, SEQ B comprises F12A and Y15C mutations.

In certain embodiments, SEQ B comprises an amino acid mutation selected from the group consisting of F12A, F C, F12G, F12S, F12T, F Q, F12E, F12N, F12D, F R and F12K and an additional amino acid mutation selected from the group consisting of L42G, L42C, L42A, L42S, L42T, L42Q, L42E, L42N, L D, L R and L42K based on SEQ ID NO:2 or a homologue or variant thereof. In certain embodiments, SEQ B comprises F12A and L42G mutations. In certain embodiments, SEQ B comprises F42C and L42G mutations. In certain embodiments, SEQ B comprises F42A and L42C mutations.

In certain embodiments, SEQ B comprises an amino acid mutation selected from the group consisting of Y15A, Y C, Y15G, Y15S, Y15T, Y15Q, Y E, Y15N, Y15D, Y R and Y15K and an additional amino acid mutation selected from the group consisting of L42G, L42C, L42A, L42S, L42T, L42Q, L42E, L42N, L D, L R and L42K based on SEQ ID NO:2 or a homologue or variant thereof. In certain embodiments, SEQ B comprises Y15A and L42G mutations. In certain embodiments, SEQ B comprises Y15C and L42G mutations. In certain embodiments, SEQ B comprises Y15A and L42C mutations.

In certain embodiments, SEQ B comprises an amino acid mutation selected from the group consisting of F12A, F12C, F12G, F12S, F12T, F12Q, F12E, F12N, F12D, F R and F12K; additional amino acid mutations selected from Y15A, Y15C, Y15G, Y15S, Y15T, Y15Q, Y15E, Y15N, Y15D, Y R and Y15K; and a further amino acid mutation selected from L42G, L42C, L42A, L S, L42T, L42Q, L E, L42N, L D, L R and L42K based on SEQ ID NO:2 or a homologue or variant thereof. In certain embodiments, SEQ B comprises F12A, Y a and L42G mutations. In certain embodiments, SEQ B comprises F12C, Y a and L42G mutations. In certain embodiments, SEQ B comprises F12A, Y C and L42G mutations. In certain embodiments, SEQ B comprises F12A, Y C and L42C mutations.

In certain embodiments, SEQ C and SEQ ID NO:4 has at least 93% sequence identity. In certain embodiments, SEQ C and SEQ ID NO:4 has at least 94% sequence identity. In certain embodiments, SEQ C and SEQ ID NO:4 has at least 96% sequence identity. In certain embodiments, SEQ C and SEQ ID NO:4 has at least 98% sequence identity.

In certain embodiments, SEQ C and SEQ ID NO:4 comprising five amino acid changes. In certain embodiments, SEQ C and SEQ ID NO:4 comprising four amino acid changes. In certain embodiments, SEQ C and SEQ ID NO:4 comprising three amino acid changes. In certain embodiments, SEQ C and SEQ ID NO:4 comprising two amino acid changes. In certain embodiments, SEQ C and SEQ ID NO:4 comprising an amino acid change. Such amino acid changes may be amino acid deletions, amino acid additions or exchange of one amino acid for another, i.e. mutations. Such mutations may also be substitutions of proteinogenic amino acids to non-proteinogenic amino acids and to D-stereoisomers of proteinogenic amino acids. In certain embodiments, SEQ C and SEQ ID NO:4 does not comprise an amino acid change, which means that it has the amino acid sequence of SEQ ID NO: 4.

In certain embodiments, SEQ C comprises the amino acid mutations C49A, C49G, C49S, C49T, C49Q, C49E, C49 6749N, C49D, C49H, C W, C49Y, C49R, C49I, C49L, C49K, C49M, C49F, C P and C49V selected from SEQ ID NOs: 4 or a homologue or variant thereof. In certain embodiments, SEQ C comprises the C49A mutation. In certain embodiments, SEQ C comprises a C49G mutation. In certain embodiments, SEQ C comprises a C49S mutation. In certain embodiments, SEQ C comprises a C49T mutation. In certain embodiments, SEQ C comprises a C49Q mutation. In certain embodiments, SEQ C comprises a C49E mutation. In certain embodiments, SEQ C comprises the C49D mutation. In certain embodiments, SEQ C comprises a C49H mutation. In certain embodiments, SEQ C comprises the C49W mutation. In certain embodiments, SEQ C comprises the C49Y mutation. In certain embodiments, SEQ C comprises a C49R mutation. In certain embodiments, SEQ C comprises a C49I mutation. In certain embodiments, SEQ C comprises the C49L mutation. In certain embodiments, SEQ C comprises the C49K mutation. In certain embodiments, SEQ C comprises the C49M mutation. In certain embodiments, SEQ C comprises the C49F mutation. In certain embodiments, SEQ C comprises a C49P mutation. In certain embodiments, SEQ C comprises a C49V mutation.

In certain embodiments, SEQ C is selected from SEQ ID NOs: 3. SEQ ID NO:4 and SEQ ID NO:212.

in certain embodiments, SEQ C has the amino acid sequence of SEQ ID NO:3: NFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 3). In certain embodiments, SEQ C has the amino acid sequence of SEQ ID NO:4: NFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 4). In certain embodiments, SEQ C has the amino acid sequence of SEQ ID NO:212: NFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 212).

In certain embodiments, alanine on the N-terminus of SEQ a of formula (I) is absent, i.e. x is 0. In certain embodiments, alanine at the N-terminus of SEQ a of formula (I) is present, i.e., x is 1.

In certain embodiments, tag1 of formula (I) is a purification Tag selected from the group consisting of albumin binding protein, alkaline phosphatase, AU1 epitope, AU5 epitope, phage T7 epitope (T7-Tag), phage V5 epitope (V5-Tag), biotin-carboxy carrier protein, bluetongue virus Tag (B-Tag), calmodulin binding peptide, chloramphenicol acetyl transferase, cellulose binding domain, chitin binding domain, choline binding domain, dihydrofolate reductase, E2 epitope, FLAG epitope, galactose binding protein, green fluorescent protein, glu-Glu (EE-Tag), glutathione S-transferase, human influenza hemagglutinin, Histidine affinity tag, horseradish peroxidase, HSV epitope, ketosteroid isomerase, KT3 epitope, lacz, luciferase, maltose binding protein, myc epitope, nusA, PDZ domain, PDZ ligand, polyarginine (Arg-tag), polyaspartic acid (Asp-tag), polycystein (Cys-tag), polyhistidine (His-tag), phenylalanine (Phe-tag), proficiency eXact, protein C, S-tag, S-tag, streptavidin binding peptide, staphylococcal protein a, staphylococcal protein G, strep-tag, streptavidin, small ubiquitin-like modifier, tandem affinity purificates (tandem affinity purification), T7 epitope, thioredoxin, trpE, ubiquitin, univorsal and VSV-G.

In certain embodiments, the Tag of formula (I) ¹ Is a purification tag selected from the group consisting of albumin binding protein, alkaline phosphatase, AU1 epitope, AU5 epitope, phage T7 epitope (T7-tag), phage V5 epitope (V5-tag), biotin-carboxy carrier protein, bluetongue virus tag (B-tag), calmodulin binding peptide, chloramphenicol acetyl transferase, cellulose binding domain, chitin binding domain, choline binding domain, dihydrofolate reductase, E2 epitope, FLAG epitope, galactose binding protein, green fluorescent protein, glu- Glu (EE-tag), glutathione S-transferase, human influenza hemagglutinin,Histidine affinity tag, horseradish peroxidase, HSV epitope, ketosteroid isomerase, KT3 epitope, lacz, luciferase, maltose binding protein, myc epitope, nusA, PDZ domain, PDZ ligand, polyarginine (Arg-tag), polyaspartic acid (Asp-tag), polycystein (Cys-tag), polyhistidine (His-tag), phenylalanine (Phe-tag), proficiency eXact, protein C, S-tag, S-tag, streptavidin binding peptide, staphylococcal protein a, staphylococcal protein G, strep-tag, streptavidin, small ubiquitin-like modifier, tandem affinity purificates, T7 epitope, thioredoxin, trpE, ubiquitin, universal and VSV-G.

In certain embodiments, the Tag of formula (I) ¹ Is a poly-histidine (His-tag), for example a His-tag comprising 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 histidines. In certain embodiments, the Tag of formula (I) ¹ Is a His tag of sequence AHHHHHHGSDDDDK (SEQ ID NO: 234).

In certain embodiments, the Tag of formula (I) ² Is a purification tag selected from albumin binding protein, alkaline phosphatase, au1 epitope, au5 epitope, phage T7 epitope (T7-tag), phage V5 epitope (V5-tag), biotin-carboxy carrier protein, bluetongue virus tag (B-tag), calmodulin binding peptide, chloramphenicol acetyl transferase, cellulose binding domain, chitin binding domain, choline binding domain, dihydrofolate reductase, E2 epitope, FLAG epitope, galactose binding protein, green fluorescent protein, glu-Glu (EE-tag), glutathione S-transferase, human influenza hemagglutinin,histidine affinity tag, horseradish peroxidase, HSV epitope. Class fixationAlcohol isomerase, KT3 epitope, lacz, luciferase, maltose binding protein, myc epitope, nusA, PDZ domain, PDZ ligand, poly-arginine (Arg tag), poly-aspartic acid (Asp tag), poly-cysteine (Cys tag), poly-histidine (His tag), poly-phenylalanine (Phe tag), proficiency eXact, protein C, S1-tag, S-tag, streptavidin binding peptide, staphylococcal protein A, staphylococcal protein G, strep-tag, streptavidin, small ubiquitin-like modifier, tandem affinity purification, T7 epitope, thioredoxin, trpE, ubiquitin, unimersal and VSV-G.

In certain embodiments, the Tag of formula (I) ² Is a purification tag selected from albumin binding protein, alkaline phosphatase, au1 epitope, au5 epitope, phage T7 epitope (T7-tag), phage V5 epitope (V5-tag), biotin-carboxy carrier protein, bluetongue virus tag (B-tag), calmodulin binding peptide, chloramphenicol acetyl transferase, cellulose binding domain, chitin binding domain, choline binding domain, dihydrofolate reductase, E2 epitope, FLAG epitope, galactose binding protein, green fluorescent protein, glu-Glu (EE-tag), glutathione S-transferase, human influenza hemagglutinin,histidine affinity tag, horseradish peroxidase, HSV epitope. Steroid isomerase, KT3 epitope, lacz, luciferase, maltose binding protein, myc epitope, nusA, PDZ domain, PDZ ligand, polyarginine (Arg tag), polyaspartic acid (Asp tag), polycysteine (Cys tag), polyhistidine (His tag), polyphenylalanine (Phe tag), proficiency eXact, protein C, S1-tag, S-tag, streptavidin binding peptide, staphylococcal protein a, staphylococcal protein G, strep-tag, streptavidin, small ubiquitin-like modifier, tandem affinity purification, T7 epitope, thioredoxin, trpE, ubiquitin, universal and VSV-G.

In certain embodiments, the Tag of formula (I) ² Is a polyhistidine (His-tag), e.g. comprising 5 histidine, 6 histidine, 7 histidine, 8 groupsAmino acid, 9 histidine, 10 histidine, 11 histidine, 12 histidine, 13 histidine, 14 histidine or 15 histidine. In certain embodiments, the Tag of formula (I) ¹ Is a His tag of sequence AHHHHHHGSDDDDK (SEQ ID NO: 234).

In certain embodiments, the Tag of formula (I) ¹ And Tag ² One of them is a purification Tag, e.g. Tag of formula (I) ¹ Tag for purifying the Tag and of formula (I) ² Either as a different type of tag or not present. In certain embodiments, the Tag of formula (I) ² To purify the Tag, and Tag ¹ Either as a different type of tag or not present. In certain embodiments, the Tag of formula (I) ¹ And Tag ² Are all purification tags, which may be the same or different.

In certain embodiments, the Tag of formula (I) ¹ Is a stable tag selected from Fc, fc fragment, igG fragment, antibody fragment, human serum albumin, albumin binding fragment, transferrin, extended recombinant polypeptide (XTEN), proline-alanine-serine Polymer (PAS), proline-alanine Polymer (PA), elastin-like peptide (ELP), homoamino Acid Polymer (HAP), gelatin-like protein (GLK), or CG B-subunit to antibody fragment in certain embodiments.

In certain embodiments, the Tag of formula (I) ² For stabilizing the tag, which in certain embodiments is selected from Fc, fc fragment, igG fragment, antibody fragment, human serum albumin, albumin binding fragment, transferrin, extended recombinant polypeptide (XTEN), proline-alanine-serine Polymer (PAS), proline-alanine Polymer (PA), elastin-like peptide (ELP), homoamino Acid Polymer (HAP), gelatin-like protein (GLK), or CG B-subunit to antibody fragment.

In certain embodiments, the Tag of formula (I) ¹ And Tag ² One being a stabilizing Tag, e.g. a Tag of formula (I) ¹ Tag which is a stable Tag and of formula (I) ² Either as a different type of tag or not present. In certain embodiments, the Tag of formula (I) ² Is a stable Tag, and Tag ¹ For different types of labels or tagsIs not present. In certain embodiments, the Tag of formula (I) ¹ And Tag ² Are stable labels, which may be the same or different.

In certain embodiments, the Tag of formula (I) ¹ Is a targeting tag, which in certain embodiments is selected from the group consisting of an antibody, an antibody fragment, a Fab, an affibody, affilin, affimer, affitin, an alpha monoclonal antibody, alphabodies, anticalins, avimers, DARPin, Kunitz domain peptides, monobodies, nanoCLAMP, cyclic peptides, heavy chain-only antibodies, VHH antibodies or +.>Single chain variable fragment (scFv) and natural or modified peptide or protein receptor ligands.

In certain embodiments, the Tag of formula (I) ¹ Is a targeting tag, which in certain embodiments is selected from the group consisting of an antibody, an antibody fragment, a Fab, an affibody, affilin, affimer, affitin, an alpha monoclonal antibody, a alphabodies, anticalins, avimers, DARPin, kunitz domain peptide, monobodies, nanoCLAMP, a cyclic peptide, a heavy chain only antibody, a VHH antibody, a single chain variable fragment (scFv), and a native or modified peptide or protein receptor ligand.

In certain embodiments, the Tag of formula (I) ² Is a targeting tag, which in certain embodiments is selected from the group consisting of an antibody, an antibody fragment, an affibody, affilin, affimer, affitin, an alpha monoclonal antibody, alphabodies, anticalins, avimers, DARPin,Kunitz domain peptides, monobodies, nanoCLAMP, cyclic peptides, heavy chain-only antibodies, VHH antibodies or +.>Single chain variable fragment (scFv) and natural or modified peptide or protein receptor ligands.

In some implementationsIn the scheme, tag of formula (I) ² Is a targeting tag, which in certain embodiments is selected from the group consisting of an antibody, an antibody fragment, an affibody, affilin, affimer, affitin, an alpha monoclonal antibody, a alphabodies, anticalins, avimers, DARPin, kunitz domain peptide, monobodies, nanoCLAMP, a cyclic peptide, a heavy chain only antibody, a VHH antibody, a single chain variable fragment (scFv), and a native or modified peptide or protein receptor ligand.

In certain embodiments, the Tag of formula (I) ¹ And Tag ² One of them is a targeting Tag, e.g. a Tag of formula (I) ¹ Tag which is a targeting Tag and of formula (I) ² Either as a different type of tag or not present. In certain embodiments, the Tag of formula (I) ² Is a targeting Tag, and Tag ¹ Either as a different type of tag or not present. In certain embodiments, the Tag of formula (I) ¹ And Tag ² Are all targeting tags, which may be the same or different.

In certain embodiments, "Tag" of formula (I) ¹ "absent, i.e., y is 0. In certain embodiments, "Tag" of formula (I) ¹ "present", i.e. y is 1. In certain embodiments, "Tag" of formula (I) ² "absent, i.e., z is 0. In certain embodiments, "Tag" of formula (I) ² "present", i.e. z is 1.

In certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 11; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0; and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 9:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 11; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 10:

In certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 11; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:37 sequence:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 11; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 38:

in certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 11; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:39 sequence:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 11; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 40:

In certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 11; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:41 sequence:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 11; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:42 sequence:

in certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 214; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0; and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 216:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 214; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:217 sequence:

In certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 214; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 218:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 214; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:219 sequence:

in certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 214; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 220:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 214; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:221 sequence of:

In certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 214; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:222 sequence:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 214; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:223, sequence of seq id no:

in certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 215; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0; and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:224 sequence:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 215; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 225:

In certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 215; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:226 sequence:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 215; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:227 sequence:

in certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 215; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:228 sequence:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 215; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 27:

In certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 215; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 28:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 215; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 29:

in certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 14; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 12:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 14; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 13:

In certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 14; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 43:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 14; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 44:

in certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 14; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:45 sequence:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 14; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 46:

In certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 14; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 47:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 14; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 48:

in certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 17; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 15:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 17; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 16:

In certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 17; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:49 sequence:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 17; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 50:

in certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 17; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:51 sequence:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 17; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:52 sequence:

In certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 17; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:53 sequence:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 17; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:54 sequence:

in certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 20; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 18:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 20; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 19:

In certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 20; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 55:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 20; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 56:

in certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 20; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:57, sequence:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 20; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:58 sequence:

In certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 20; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:59 sequence:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 20; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 60:

in certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 23; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 21:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 23; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 22:

In certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 23; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:61 sequence:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 23; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 62:

in certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 23; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:63 sequence:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 23; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:64 sequence:

In certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 23; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:65 sequence:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 23; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:66 sequence:

in certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 26; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 24:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 26; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 25:

In certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B has the sequence of SEQ ID NO: 26; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:67 sequence:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 26; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 68:

in certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 26; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:69 sequence:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 26; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 70:

In certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 26; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:71 sequence:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 26; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:72 sequence:

in certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 32; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 30:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 32; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 31:

In certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 32; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:73 sequence:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 32; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 74:

in certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 32; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 75:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 32; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:76 sequence:

In certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 32; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 77:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 32; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:78 sequence:

in certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO:35, a sequence of 35; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 33:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO:35, a sequence of 35; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 34:

In certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO:35, a sequence of 35; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:79 sequence:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO:35, a sequence of 35; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:80 sequence:

in certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO:35, a sequence of 35; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:81 sequence:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO:35, a sequence of 35; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:82 sequence:

In certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO:35, a sequence of 35; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:83 sequence:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO:35, a sequence of 35; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0 and z of formula (I) is 0. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:84 sequence:

in certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 11; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0; z of formula (I) is 1, and "Tag" of formula (I) ² "is a His tag comprising 6 histidines. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:204 sequence:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 11; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0; z of formula (I) is 1, and "Tag" of formula (I) ² "is a His tag comprising 6 histidines. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:205, sequence:

in certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 11; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0; z of formula (I) is 1,and "Tag" of formula (I) ² "is a His tag comprising 6 histidines. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:206 sequence:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 11; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0; z of formula (I) is 1, and "Tag" of formula (I) ² "is a His tag comprising 6 histidines. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:207 sequence:

in certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 11; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0; z of formula (I) is 1, and "Tag2" of formula (I) is polyhistidine (His-Tag). Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 208:

In certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 11; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0; z of formula (I) is 1, and "Tag2" of formula (I) is a His Tag comprising 6 histidines. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:209 sequence:

in certain embodiments, x of formula (I) is 0; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 11; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0; z of formula (I) is 1, and "Tag2" of formula (I) is a His Tag comprising 6 histidines. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 210:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO:36, a sequence of seq id no; SEQ B of formula (I) has the sequence SEQ ID NO: 11; SEQ C of formula (I) has the sequence SEQ ID NO: 4; y of formula (I) is 0; z of formula (I) is 1, and "Tag2" of formula (I) is a His Tag comprising 6 histidines. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:211 sequence:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 17; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0; z of formula (I) is 1, and "Tag2" of formula (I) is a His Tag comprising 6 histidines. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:229 sequence:

In certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 23; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 0; z of formula (I) is 1, and "Tag2" of formula (I) is a His Tag comprising 6 histidines. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO: 230:

in certain embodiments, x of formula (I) is 1; SEQ A of formula (I) has the sequence SEQ ID NO: 1; SEQ B of formula (I) has the sequence SEQ ID NO: 11; SEQ C of formula (I) has the sequence SEQ ID NO: 3; y of formula (I) is 1; z of formula (I) is 0, and "Tag1" of formula (I) is a polypeptide having the sequence SEQ ID NO: 234. Thus, the IL-2 protein of formula (I) has the sequence of SEQ ID NO:231 sequence:

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in certain embodiments, the IL-2 protein of formula (I) has a sequence selected from the group consisting of SEQ ID NOs: 9, a step of performing the process; SEQ ID NO:10; SEQ ID NO:12; SEQ ID NO:13; SEQ ID NO:15; SEQ ID NO:16; SEQ ID NO:18; SEQ ID NO:19; SEQ ID NO:21, a step of; SEQ ID NO:22; SEQ ID NO:24, a step of detecting the position of the base; SEQ ID NO:25, a step of selecting a specific type of material; SEQ ID NO:30; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO: 34. SEQ ID NO:216; SEQ ID NO:217; SEQ ID NO:224; and SEQ ID NO: 225.

In certain embodiments, the IL-2 protein of formula (I) has a sequence selected from the group consisting of SEQ ID NOs: 9, a step of performing the process; SEQ ID NO:12; SEQ ID NO:15; SEQ ID NO:18; SEQ ID NO:21, a step of; SEQ ID NO:24, a step of detecting the position of the base; SEQ ID NO:30; SEQ ID NO:33; SEQ ID NO:216; and SEQ ID NO: 224.

In certain embodiments, the IL-2 protein of formula (I) has a sequence selected from the group consisting of SEQ ID NOs: 10; SEQ ID NO:13; SEQ ID NO:16; SEQ ID NO:19; SEQ ID NO:22; SEQ ID NO:25, a step of selecting a specific type of material; SEQ ID NO:31; SEQ ID NO: 34. SEQ ID NO:217; and SEQ ID NO: 225.

In certain embodiments, the IL-2 protein of formula (I) has the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the IL-2 protein of formula (I) has the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the IL-2 protein of formula (I) has the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the IL-2 protein of formula (I) has the amino acid sequence of SEQ ID NO: 13. In certain embodiments, the IL-2 protein of formula (I) has the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the IL-2 protein of formula (I) has the amino acid sequence of SEQ ID NO: 16. In certain embodiments, the IL-2 protein of formula (I) has the amino acid sequence of SEQ ID NO: 18. In certain embodiments, the IL-2 protein of formula (I) has the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the IL-2 protein of formula (I) has the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the IL-2 protein of formula (I) has the amino acid sequence of SEQ ID NO: 22. In certain embodiments, the IL-2 protein of formula (I) has the amino acid sequence of SEQ ID NO:24 sequence. In certain embodiments, the IL-2 protein of formula (I) has the amino acid sequence of SEQ ID NO: 25. In certain embodiments, the IL-2 protein of formula (I) has the amino acid sequence of SEQ ID NO: 30. In certain embodiments, the IL-2 protein of formula (I) has the amino acid sequence of SEQ ID NO: 31. In certain embodiments, the IL-2 protein of formula (I) has the amino acid sequence of SEQ ID NO: 33. In certain embodiments, the IL-2 protein of formula (I) has the amino acid sequence of SEQ ID NO: 34. In certain embodiments, the IL-2 protein of formula (I) has the amino acid sequence of SEQ ID NO: 216. In certain embodiments, the IL-2 protein of formula (I) has the amino acid sequence of SEQ ID NO: 217. In certain embodiments, the IL-2 protein of formula (I) has the amino acid sequence of SEQ ID NO: 224. In certain embodiments, the IL-2 protein of formula (I) has the amino acid sequence of SEQ ID NO: 225.

In certain embodiments, SEQ A of formula (I) may comprise at least one O-linked N1 glycan, at least one O-linked N2 glycan, a combination of at least one O-linked N1 and at least one O-linked N2 glycan, or may be non-glycosylated. In certain embodiments, SEQ A of formula (I) comprises at least one, e.g., one, O-linked N1 glycan. In certain embodiments, SEQ a of formula (I) comprises at least one, e.g., one O-linked N2 glycan. In certain embodiments, SEQ A of formula (I) comprises a combination of at least one, e.g., one, O-linked N1 glycan and at least one, e.g., one, O-linked N2 glycan. In certain embodiments, SEQ A of formula (I) is not O-glycosylated. In certain embodiments, the ratio of O-linked N1 glycans to O-linked N2 glycans is 1:1, 1:2, 1:3, or 1:4. In certain embodiments, the ratio of O-linked N1 glycans to O-linked N2 glycans is 1:1. In certain embodiments, the ratio of O-linked N1 glycans to O-linked N2 glycans is 1:2. In certain embodiments, the ratio of N1 to N2 glycans is 1:3. In certain embodiments, the ratio of N1 to N2 glycans is 1:4.

In certain embodiments, the IL-2 protein of formula (I) may comprise at least one N-linked double-antennary low mannose glycan. In certain embodiments, the IL-2 protein of formula (I) comprises at least one, e.g., one, N-linked G0 glycan. In certain embodiments, the IL-2 protein of formula (I) comprises at least one, e.g., one, N-linked G1 glycan. In certain embodiments, the IL-2 protein of formula (I) comprises at least one, e.g., one, N-linked G2 glycan. In certain embodiments, the IL-2 protein of formula (I) comprises at least one, e.g., one, N-linked G0B glycan. In certain embodiments, the IL-2 protein of formula (I) comprises at least one, e.g., one, N-linked G1B glycan. In certain embodiments, the IL-2 protein of formula (I) comprises at least one, e.g., one, N-linked G2B glycan. In certain embodiments, the IL-2 protein of formula (I) comprises at least one, e.g., one, N-linked G0F glycan. In certain embodiments, the IL-2 protein of formula (I) comprises at least one, e.g., one, N-linked G1F glycan. In certain embodiments, the IL-2 protein of formula (I) comprises at least one, e.g., one, N-linked G2F glycan. In certain embodiments, the IL-2 protein of formula (I) comprises at least one, e.g., one, N-linked G0BF glycan. In certain embodiments, the IL-2 protein of formula (I) comprises at least one, e.g., one, N-linked G1BF glycan. In certain embodiments, the IL-2 protein of formula (I) comprises at least one, e.g., one, N-linked G2BF glycan. In certain embodiments, the protein of formula (I) may comprise a mixture of double-antenna low mannose glycans. In certain embodiments, the protein of formula (I) comprises an N-linked four-antennary low mannose glycan having more than two Gal residues, e.g., three, four, five, or six Gal residues and/or more than two NeuAc residues, e.g., three or four NeuAc residues.

In certain embodiments, one or more N-linked or O-linked glycans extend the half-life of the protein of formula (I).

In certain embodiments, one or more of the N-linked or O-linked glycans mask mutations introduced with the glycosylation motif from immune recognition. In certain embodiments, one or more N-linked or O-linked glycans block recognition of mutations introduced with the glycosylation motif by antibodies or T Cell Receptors (TCRs).

In certain embodiments, one or more N-linked or O-linked glycans can be used for affinity purification. For example, N-linked glycans can be used for affinity purification via lectins.

It will be appreciated that at least one glycan is present in the Seq B portion, but that glycans can also be present in other portions of the IL-2 protein of formula (I), e.g., in Seq A or Seq C.

In certain embodiments, the IL-2 protein of formula (I) is biased IL-2 (biased IL-2).

Another aspect of the invention relates to oligonucleotide sequences encoding IL-2 proteins of formula (I). Such oligonucleotide sequences may be selected from DNA, RNA and cDNA sequences. In certain embodiments, the oligonucleotide sequence is a DNA sequence. In certain embodiments, the oligonucleotide sequence is an RNA sequence. In certain embodiments, the oligonucleotide sequence is a cDNA sequence. In certain embodiments, oligonucleotides encoding IL-2 proteins of formula (I) are used for expression in eukaryotic, prokaryotic, or cell-free systems. In certain embodiments, the oligonucleotide sequences are for expression in eukaryotic systems. In certain embodiments, the oligonucleotide sequences are for expression in a prokaryotic system. In certain embodiments, the oligonucleotide sequences are for expression in a cell-free system. In certain embodiments, the DNA sequence encoding an IL-2 protein of formula (I) has the sequence of SEQ ID NO: 7. In certain embodiments, the DNA sequence encoding an IL-2 protein of formula (I) has the sequence of SEQ ID NO: 8. In certain embodiments, the DNA sequence encoding an IL-2 protein of formula (I) has the sequence of SEQ ID NO: 202. In certain embodiments, the DNA sequence encoding an IL-2 protein of formula (I) has the sequence of SEQ ID NO: 203. In certain embodiments, the DNA sequence encoding an IL-2 protein of formula (I) has the sequence of SEQ ID NO: 233. In certain embodiments, the DNA sequence encoding an IL-2 protein of formula (I) has the sequence of SEQ ID NO:244 sequence. In certain embodiments, the DNA sequence encoding an IL-2 protein of formula (I) has the sequence of SEQ ID NO:245 sequence. In certain embodiments, the DNA sequence encoding an IL-2 protein of formula (I) has the sequence of SEQ ID NO:246 sequence. In certain embodiments, the RNA sequence encoding the IL-2 protein of formula (I) is an mRNA sequence.

In certain embodiments, the oligonucleotide encoding an IL-2 protein of formula (I) is used for expression in a prokaryotic system, e.g. a bacterial system selected from the group consisting of Escherichia coli; bacillus sp, such as Bacillus subtilis Bacillus subtilis, corynebacterium sp, such as Corynebacterium glutamate Corynebacterium glutamicum; and Pseudomonas fluorescens (Pseudomonas fluorescens). In certain embodiments, such oligonucleotides are DNA sequences in the form of plasmids.

In certain embodiments, the oligonucleotide encoding an IL-2 protein of formula (I) is used for expression in a eukaryotic system, e.g., a eukaryotic system selected from the group consisting of: mammalian systems, such as mammalian cells, e.g., chinese hamster ovary Cells (CHO), mouse myeloma lymphoblastic cells (e.g., NS0 cells), sp2/0 cells, mouse fibroblasts (e.g., NIH3T3 cells), and fully human cells, e.g., human embryonic kidney cells HEK 293, heLa cells, human embryonic retina cells (e.g., per.c6 of cricell), or human amniotic cells (e.g., glycotope and CEVEC); yeasts such as Saccharomyces cerevisiae (Saccharomyces cerevisiae), yarrowia lipolytica (Yarrowia lipolytica) or Pichia pastoris; filamentous fungi, such as Aspergillus, trichoderma or myceliophthora thermophila (Myceliophthora thermophila); baculovirus-infected cells, such as baculovirus-infected insect cells, e.g., sf9, sf21, hi-5 strain; non-lytic insect cell expression systems, such as Sf9, sf21, hi-5, schneider2 cells or Schneider 3 cells; and plants or plant cells, such as arabidopsis thaliana (Arabidopsis thaliana), nicotiana benthamiana (Nicotiana benthamiana), nicotiana tabacum (Nicotiana tabacum), medicago sativa (Medicago sativa), lemna minor (Lemna minor) or physcomitrella patens (Physcomitrella patens) and cells thereof. In certain embodiments, the oligonucleotide sequences encoding the IL-2 protein of formula (I) are for expression in CHO cells. In certain embodiments, the oligonucleotide sequences encoding the IL-2 protein of formula (I) are for expression in mammalian systems. In certain embodiments, such oligonucleotides are DNA sequences in the form of plasmids.

In certain embodiments, oligonucleotides encoding IL-2 proteins of formula (I) are useful in the treatment of diseases treatable with IL-2, particularly with preferential IL-2. In certain embodiments, the disease that can be treated with IL-2, particularly with preferential IL-2, is cancer. In certain embodiments, the oligonucleotides encoding the IL-2 proteins of formula (I) for such use are DNA, RNA or cDNA, in particular modified DNA, modified RNA or modified cDNA. In certain embodiments, such oligonucleotides for treating a disease are administered to a patient, which in certain embodiments is a mammal, such as a cat, dog, horse, cow, sheep, non-human primate, or human.

In certain embodiments, the oligonucleotides encoding IL-2 proteins of formula (I) for use in treating diseases treatable with IL-2 are DNA molecules, particularly modified DNA molecules. In certain embodiments, the DNA encoding the IL-2 protein of formula (I) is modified DNA. In certain embodiments, the modification of the DNA is selected from the group consisting of a backbone modification, a conformational constraint modification, and a chemical modification. In certain embodiments, the conformational constraint modification of DNA encoding an IL-2 protein of formula (I) is selected from Locked Nucleic Acid (LNA), constrained 2' -O-ethyl (cEt), and tricyclic-DNA (tcDNA). In certain embodiments, the backbone modification of DNA encoding an IL-2 protein of formula (I) is selected from the group consisting of 2 '-O-methyl (2' -OMe), 2 '-O-methoxy-ethyl (2' -MOE), and 2 '-fluoro (2' -F) substitution. In certain embodiments, the modification of the DNA comprises a combination of at least two modifications selected from the group consisting of backbone modification, conformational constraint modification, and chemical modification.

In certain embodiments, the oligonucleotide encoding an IL-2 protein of formula (I) for use in the treatment of a disease treatable with IL-2 is a cDNA molecule, particularly a modified cDNA molecule.

In certain embodiments, the oligonucleotides encoding IL-2 proteins of formula (I) for use in the treatment of IL-2 treatable diseases are RNA molecules, in particular modified RNA molecules. In certain embodiments, the RNA encoding the IL-2 protein of formula (I) is a modified RNA. In certain embodiments, the modification of the RNA is selected from the group consisting of a backbone modification, a ribose modification, a conformational constraint modification, and a chemical modification. In certain embodiments, the ribose modification of an RNA encoding an IL-2 protein of formula (I) is the replacement of Phosphodiester (PO) with Phosphorothioate (PS) in the RNA backbone. In certain embodiments, the backbone modification of RNA encoding IL-2 protein of formula (I) is selected from the group consisting of 2 '-O-methyl (2' -OMe), 2 '-O-methoxy-ethyl (2' -MOE), and 2 '-fluoro (2' -F) substitution. In certain embodiments, the chemical modification of RNA encoding an IL-2 protein of formula (I) is a nucleobase change. In certain embodiments, the nucleobase change is a phosphorodiamidate morpholino oligomer (phosphorodiamidate morpholino oligomers, PMO) or a Peptide Nucleic Acid (PNA). In certain embodiments, the modification of the RNA comprises a combination of at least two modifications selected from the group consisting of a backbone modification, a ribose modification, a conformational constraint modification, and a chemical modification.

In certain embodiments, the RNA encoding the IL-2 protein of formula (I) is modified to improve delivery. In certain embodiments, the modification for improved delivery is selected from chemical conjugation to certain moieties and incorporation into or attachment to the nanoparticle carrier. In certain embodiments, the chemical conjugate is selected from the group consisting of polymers, cell Penetrating Peptides (CPPs), lipids, antibodies, receptor ligands, and aptamers. In certain embodiments, the polymer is a dendritic polymer. In certain embodiments, the polymer is selected from PEG, poly (lactide-co-glycolic acid) (PLGA), and polyphosphazene. In certain embodiments, the polymer is PEG. In certain embodiments, the nanoparticle carrier is a nanoparticle carrier comprising a lipid, a polymer, and/or a peptide. In certain embodiments, the lipid-containing nanoparticle carrier is selected from the group consisting of Lipid Nanoparticles (LNP) and exosomes (exosomes). In certain embodiments, the nanoparticle carrier comprises a polymeric dendritic polymer. In certain embodiments, the nanoparticle carrier comprises a polymer selected from PEG, poly (lactide-co-glycolic acid) (PLGA), and polyphosphazene.

In certain embodiments, lipid Nanoparticles (LNPs) for delivering RNA encoding IL-2 proteins of formula (I) comprise ionizable or cationic lipid or polymeric materials bearing tertiary or quaternary amines; zwitterionic lipids (e.g., 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine [ DOPE ]); cholesterol; polyethylene glycol (PEG) -lipid or any combination thereof.

In certain embodiments, the oligonucleotide encoding an IL-2 protein of formula (I) is delivered into the cell by a method selected from the group consisting of: cationic lipids, biodegradable ionizable lipids, co-formulated as Lipid Nanoparticles (LNP), nanoliposomes, emulsions, polymeric carriers, electroporation, lipofection, calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection, or transfection based on cationic polymers such as DEAE-dextran or polyethylenimine. In certain embodiments, an oligonucleotide encoding an IL-2 protein of formula (I) is delivered to a cell ex vivo (ex vivo). In certain embodiments, oligonucleotides encoding IL-2 proteins of formula (I) are delivered to cells in vivo.

In certain embodiments, an oligonucleotide encoding an IL-2 protein of formula (I) is used for expression in a host cell of a human subject. In certain embodiments, the oligonucleotide encoding an IL-2 protein of formula (I) is delivered to a host cell in vivo or ex vivo. In certain embodiments, oligonucleotides encoding IL-2 proteins of formula (I) are delivered in vivo into a host cell. In certain embodiments, an oligonucleotide encoding an IL-2 protein of formula (I) is delivered ex vivo into a host cell. In certain embodiments, an oligonucleotide encoding an IL-2 protein of formula (I) is delivered into a host cell of a human subject, which cell subsequently expresses the IL-2 protein of formula (I). In certain embodiments, the host cell is an immune cell. In certain embodiments, the immune cell is selected from a T cell, a Natural Killer (NK) cell, a myeloid cell, a dendritic cell, a red blood cell, a macrophage, a B cell, or any other genetically modified immune cell type.

In certain embodiments, the oligonucleotide encoding the IL-2 protein of formula (I) is delivered into the cell by virus-based transfection using a viral vector. In certain embodiments, the viral vector comprises an oligonucleotide encoding an IL-2 protein of formula (I). In certain embodiments, the viral vector is selected from the group consisting of DNA-based viral vectors and RNA-based viral vectors.

In certain embodiments, the DNA-based viral vector comprising an oligonucleotide encoding an IL-2 protein of formula (I) is selected from the group consisting of poxviruses, adenoviruses, adeno-associated viruses, and herpesviruses. In certain embodiments, the herpes virus is selected from the group consisting of herpes simplex virus type 1 and herpes simplex virus type 2. In certain embodiments, herpes simplex virus type 1 has a deletion of at least one viral gene (e.g., a gene selected from ICP34.5, ICP6/UL39, and ICP 47). In certain embodiments, herpes simplex virus type 1 is selected from the group consisting of G207, HSV1716, NV1020, and Talimogene laherparepvec (Oncovix-GMCSF). In certain embodiments, the DNA-based viral vector is an oncolytic virus. In certain embodiments, the DNA-based oncolytic virus is selected from the group consisting of herpes simplex virus type 1, adenovirus, and poxvirus. In certain embodiments, the oncolytic adenovirus is selected from the group consisting of H101, coloAd1 (Ad 11p/Ad 3) and Onyx-015 (Ad 2/5d 11520).

In certain embodiments, the RNA-based viral vector comprising an oligonucleotide encoding an IL-2 protein of formula (I) is selected from the group consisting of lentivirus, retrovirus, alphavirus, flavivirus, rhabdovirus, measles virus, poliovirus, human foamy virus, and newcastle disease virus. In certain embodiments, the RNA-based viral vector is a retrovirus, such as a retrovirus selected from the group consisting of moloney murine leukemia virus, moloney murine sarcoma virus, and murine stem cell virus. In certain embodiments, the RNA-based viral vector is a lentivirus, e.g., a lentivirus selected from the group consisting of human immunodeficiency virus 1, human immunodeficiency virus 2, and equine infectious anemia virus. In certain embodiments, the RNA-based viral vector is an alphavirus, such as an alphavirus selected from the group consisting of semliki forest virus, sindbis virus, venezuelan equine encephalitis virus, and Mi virus. In certain embodiments, the RNA-based viral vector is a flavivirus, such as a flavivirus selected from the group consisting of kunjin virus, west nile virus, yellow fever virus, and dengue virus. In certain embodiments, the RNA-based viral vector is a rhabdovirus, for example a rhabdovirus selected from the group consisting of rabies virus and vesicular stomatitis virus. In certain embodiments, the RNA-based viral vector is a measles virus, such as MV-Edm. In certain embodiments, the RNA-based viral vector is a picornavirus, e.g., a picornavirus selected from the group consisting of coxsackievirus and saikokumi virus.

In certain embodiments, the RNA-based viral vector is an oncolytic virus. In certain embodiments, the RNA-based oncolytic virus is selected from MV-Edm, newcastle disease virus, vesicular stomatitis virus, saikovirus, and coxsackievirus.

In certain embodiments, the viral vector is a virus-like vesicle (VLV). In certain embodiments, the virus-like vesicle is a hybrid of components from two viruses. In certain embodiments, the virus-like vesicle is a hybrid of components from the Semliki A forest virus (SFV) and the rhabdovirus Vesicular Stomatitis Virus (VSV).

It will be appreciated that upon successful delivery of a viral vector comprising an oligonucleotide encoding an IL-2 protein of formula (I) into a host cell, the host cell will express and secrete the IL-2 protein of formula (I).

In certain embodiments, the oligonucleotide encoding an IL-2 protein of formula (I) is delivered as a vaccine formulation. In certain embodiments, the vaccine formulation is selected from DNA-based, RNA-based, cell-based, or virus-based vaccines.

In certain embodiments, the IL-2 protein of formula (I) is expressed in engineered cells. In certain embodiments, the engineered cell is an engineered immune cell. In certain embodiments, the engineered immune cell is selected from a T-cell, natural Killer (NK) cell, myeloid cell, dendritic cell, erythrocyte, macrophage, B-cell, or any other genetically modified immune cell type.

In certain embodiments, the IL-2 protein of formula (I) is expressed in engineered T-cells. In certain embodiments, the engineered T-cell is selected from the group consisting of a TCR-engineered T (TCR-T) cell, a chimeric antigen receptor-modified T (CAR-T) cell, and a T cell redirected for universal cytokine killing (TRUCK). In certain embodiments, the engineered T-cell is a TCR-T cell. In certain embodiments, the engineered T-cell is a CAR-T cell. In certain embodiments, the engineered T-cell is a TRUCK.

In certain embodiments, the IL-2 protein of formula (I) is expressed in engineered alpha/beta T-cells. In certain embodiments, the IL-2 protein of formula (I) is expressed in engineered gamma/delta T-cells. In certain embodiments, the IL-2 protein of formula (I) is expressed in engineered natural killer T-cells.

In certain embodiments, the IL-2 protein of formula (I) is expressed in engineered NK-cells. In certain embodiments, the engineered NK-cells are chimeric antigen receptor-modified NK (CAR-NK) cells. In certain embodiments, the engineered NK-cells are lymphokine-activated killer (LAK) cells. In certain embodiments, the engineered NK-cells are NK-92 cells.

In certain embodiments, the IL-2 protein of formula (I) is expressed in engineered myeloid cells. In certain embodiments, the engineered macrophage is a CAR myeloid cell.

In certain embodiments, the IL-2 protein of formula (I) is expressed in engineered dendritic cells. In certain embodiments, the engineered dendritic cell is a CAR dendritic cell.

In certain embodiments, the IL-2 protein of formula (I) is expressed in engineered macrophages. In certain embodiments, the engineered macrophage is a CAR macrophage.

In certain embodiments, the IL-2 protein of formula (I) is expressed in engineered erythrocytes. In certain embodiments, the IL-2 protein of formula (I) is expressed in engineered erythrocytes and presented on the cell surface.

In certain embodiments, the IL-2 protein of formula (I) is expressed in engineered B-cells. In certain embodiments, the engineered macrophage is a CAR B-cell.

It will be appreciated that the IL-2 protein of formula (I) expressed in the engineered immune cell may be secreted from the cell.

In certain embodiments, the engineered immune cells of the IL-2 protein of expression (I) are used in cell therapy. In certain embodiments, the cell therapy is adoptive immune cell therapy. In certain embodiments, the adoptive immune cell therapy is autologous or allogenic. In certain embodiments, the adoptive immune cell therapy is autologous. In certain embodiments, the adoptive immune cell therapy is allogeneic.

In certain embodiments, the IL-2 protein of formula (I) may be used to stimulate cells, e.g., immune cells, in vitro or ex vivo. In certain embodiments, the IL-2 protein of formula (I) is used to stimulate an engineered immune cell. In certain embodiments, the IL-2 protein of formula (I) is used to stimulate cells selected from the group consisting of T-cells, NK-cells, macrophages and dendritic cells.

In another aspect, the invention relates to a method of expressing a recombinant IL-2 protein of formula (I), the method comprising: a) Culturing a host cell expressing one or more genes encoding an IL-2 protein of formula (I); and b) isolating the recombinant IL-2 protein of interest from the host cell culture.

In certain embodiments, the host cell is a prokaryotic cell, such as a bacterial cell. In certain embodiments, the host cell is selected from the group consisting of E.coli; bacillus, such as bacillus subtilis; coryneform bacteria such as Corynebacterium glutamicum; and Pseudomonas fluorescens. In certain embodiments, the host cell is E.coli. In certain embodiments, the host cell is a bacillus. In certain embodiments, the host cell is a coryneform bacterium. In certain embodiments, the host cell is pseudomonas fluorescens.

In certain embodiments, the host cell is a eukaryotic cell. In certain embodiments, the host cell is selected from the group consisting of: yeasts such as Saccharomyces cerevisiae (Saccharomyces cerevisiae) or Pichia pastoris (Pichia pastoris); filamentous fungi, such as Aspergillus, trichoderma or myceliophthora thermophila (Myceliophthora thermophila); baculovirus-infected cells, such as baculovirus-infected insect cells, e.g., sf9, sf21, hi-5 strain, or baculovirus-infected mammalian cells, e.g., heLa, human embryonic kidney cells HEK 293, or chinese hamster ovary Cells (CHO). Mammalian systems, such as mouse myeloma lymphocytes (e.g., NS0 cells), mouse fibroblasts (e.g., NIH3T3 cells), CHO cells, and fully human cells, such as HEK 293 cells, human embryonic retinal cells (e.g., crucell's per.c6), and human amniotic cells (e.g., glycotopes and CEVECs); and non-lytic insect cell expression systems, such as Sf9, sf21, hi-5, schneider 2 cells or Schneider 3 cells. In certain embodiments, the host cell is a yeast cell. In certain embodiments, the host cell is a saccharomyces cerevisiae cell. In certain embodiments, the host cell is a pichia pastoris cell. In certain embodiments, the host cell is a cell of a filamentous fungus. In certain embodiments, the host cell is a cell of aspergillus. In certain embodiments, the host cell is a cell of the genus trichoderma. In certain embodiments, the host cell is a myceliophthora thermophila cell. In certain embodiments, the host cell is a baculovirus-infected cell, such as a baculovirus-infected insect cell or a baculovirus-infected mammalian cell. In certain embodiments, the host cell is a baculovirus-infected Sf9 cell. In certain embodiments, the host cell is an Sf21 cell infected with a baculovirus. In certain embodiments, the host cell is a cell of a baculovirus-infected Hi-5 strain. In certain embodiments, the host cell is a baculovirus-infected HeLa cell. In certain embodiments, the host cell is a human kidney cell infected with a baculovirus. In certain embodiments, the host cell is a baculovirus-infected Sf9 cell. In certain embodiments, the host cell is a baculovirus-infected CHO cell. In certain embodiments, the host cell is a mammalian cell. In certain embodiments, the host cell is a mouse myeloma lymphocyte. In certain embodiments, the host cell is a mouse fibroblast. In certain embodiments, the host cell is a CHO cell. In certain embodiments, the host cell is a HEK 293 cell. In certain embodiments, the host cell is a human embryonic retinal cell. In certain embodiments, the host cell is a human amniotic cell. In certain embodiments, the host cell is a mouse fibroblast. In certain embodiments, the host cell is a non-lytic insect cell expression system. In certain embodiments, the host cell is an Sf9 cell. In certain embodiments, the host cell is an Sf21 cell. In certain embodiments, the host cell is a Hi-5 cell. In certain embodiments, the host cell is a Schneider 2 cell. In certain embodiments, the host cell is a Schneider 3 cell. In certain embodiments, the host cell is an arabidopsis thaliana (Arabidopsis thaliana) cell. In certain embodiments, the host cell is a tobacco (Nicotiana benthamiana) cell. In certain embodiments, the host cell is a tobacco (Nicotiana tabacum) cell. In certain embodiments, the host cell is a alfalfa (Medicago sativa) cell. In certain embodiments, the host cell is a duckweed (Lemna minor) cell. In certain embodiments, the host cell is a physcomitrella patens (Physcomitrella patens) cell.

In certain embodiments, the above-described host cells and organisms may be modified (either genetically or otherwise) to alter the glycosylation of the native organism, e.g., to obtain glycosylation that better resembles or is identical to that found in a mammal (e.g., human).

In certain embodiments, the expression host cell is modified to produce a particular glycosylation pattern or more homologous glycosylation pattern, for example, by deleting or overexpressing certain glycosyltransferases, by introducing glycosyltransferases from other organisms or by introducing modified glycosyltransferases with altered substrate and/or receptor specificity. In certain embodiments, the expressed host cells may be modified to synthesize specific carbohydrate residues that are not naturally occurring in the host cells.

One of the methods for increasing the production of secreted proteins of interest is to improve the mechanism of cleavage of the signal sequence or leader sequence that directs secretion of the protein. Proper processing of the signal sequence or leader sequence is a critical step in the secretory pathway, as it releases the N-terminus of the mature secretory protein and is often a requirement for efficient secretion. Incomplete cleavage of the signal or leader sequence typically results in accumulation of the protein within the cell, but in some cases, incompletely processed products may also be secreted.

In most expression systems, secretion is directed by a secretion signal peptide which is fused to the N-terminus of the protein to be secreted and cleaved off by specific processing enzymes of the host cell prior to or together with secretion. Thus, the IL-2 protein of formula (I) is in certain embodiments expressed with a secretion signal peptide that is cleaved off by a specific processing enzyme of the host cell prior to or in conjunction with secretion.

In mammalian expression systems, the signal peptide is in certain embodiments the signal peptide of any naturally secreted protein. In certain embodiments, the signal peptide for use in the mammalian expression system is thus in certain embodiments a signal peptide of a naturally secreted protein. In certain embodiments, the signal peptide for use in the mammalian expression system is a non-natural synthetic signal sequence designed in a computer, or a signal found in an experiment to be effective in directing secretion. In certain embodiments, the signal peptide has a sequence selected from the group consisting of SEQ ID NOs: 238. SEQ ID NO:239 and SEQ ID NO: 240. In certain embodiments, the signal peptide has the amino acid sequence of SEQ ID NO: 238. In certain embodiments, the signal peptide has the amino acid sequence of SEQ ID NO: 239. In certain embodiments, the signal peptide has the amino acid sequence of SEQ ID NO:240 sequence.

In certain embodiments, the IL-2 protein of formula (I) having a signal peptide has a sequence selected from the group consisting of SEQ ID NOs: 248. SEQ ID NO:250 and SEQ ID NO: 252. In certain embodiments, the IL-2 protein of formula (I) having a signal peptide has the amino acid sequence of SEQ ID NO: 248. In certain embodiments, the IL-2 protein of formula (I) having a signal peptide has the amino acid sequence of SEQ ID NO: 250. In certain embodiments, the IL-2 protein of formula (I) having a signal peptide has the amino acid sequence of SEQ ID NO: 252.

In E.coli, the signal sequence directing the secretion of the protein to the periphery may be any signal peptide naturally secreted to the periphery of the bacterium. In certain embodiments, the signal peptide for the IL-2 protein of expression (I) in E.coli is selected from phoA, dsbA, gllI, mal, ompA, ompC, ompT, pelB, torA, torT, EOX, STII, sfmC, lamB, mglB, mmAp and tolB. In certain embodiments, the signal peptide is a non-native sequence designed in silico or a non-native sequence found in experiments to be effective in directing secretion.

In yeast expression systems, such as Saccharomyces cerevisiae and Pichia pastoris, the leader sequence that directs expression may comprise a signal sequence that directs secretion of the protein into the ER and is cleaved off upon transport to the ER, and a propeptide that is cleaved off in the Golgi by Kex2 enzymes prior to secretion into the growth medium. The leader sequence may be a leader sequence of a naturally secreted enzyme or pheromone. In certain embodiments, the leader sequence of the IL-2 protein of formula (I) for expression in a yeast expression system is thus selected from the group consisting of a saccharomyces cerevisiae mating factor alpha leader sequence, a SUC2 leader sequence, and a VOA1 leader sequence. In certain embodiments, the leader sequence is derived from a secreted protein of another yeast or filamentous fungus, or it may be a non-natural leader sequence designed in silico, or it may be a leader sequence found experimentally to be effective in directing folding and secretion. Leader sequences can also be identified experimentally from a large library of leader sequences, for example, a library comprising a combination of many random amino acid substitutions.

Proper cleavage of the signal or leader sequence by the endogenous processing enzyme of the host cell depends on the amino acid sequence immediately following the cleavage site, which constitutes the N-terminus of the mature processed and secreted recombinant protein. In addition to the specific N-terminal amino acid sequence of the protein of interest, the accessibility of the N-terminal in the folded protein of interest may affect the processing efficiency of the signal sequence or leader sequence. For example, the buried N-terminus may not be accessible to the processing protease and thus is problematic for secretion strategies.

Using predictive models built on existing experimental data, the probability of cleavage of a certain amino acid sequence by a signal peptidase complex can be calculated. Such tools can be found on the net, allowing one skilled in the art to predict the likelihood of proper processing of signal peptides in eukaryotes and bacteria. In yeast expression systems, the leader sequence typically includes a signal sequence that is cleaved by a signal peptidase complex in the ER and a propeptide that is cleaved by Kex2 furin in the Golgi apparatus. The Kex2 recognition site, KR, is very conserved across Kex2 substrates of yeast species. It is well known that negatively charged amino acids predominate at the P1', P2' and P4' positions of Kex2 substrates. However, the potential cleavage by Kex2 generally needs to be checked one by one in the experiment.

Those of ordinary skill in the art will recognize that proper processing of the signal sequence or leader sequence is one of several characteristics required to effectively secrete properly folded and soluble proteins. Examples of important features are sufficient transcription and translation rates, co-translation or post-translational translocation into the ER, folding and formation of the correct disulfide bridge in the ER, and extracellular transport of vesicles. Therefore, experimental verification of any computer-aided secretion efficiency prediction is very important.

It is well known that accumulation of incorrectly folded or aggregated proteins within cells may negatively affect the physiology of the host cell, may induce stress responses, and may lead to a decrease in growth rate and cell fitness (cell fitness). Thus, avoiding intracellular accumulation by improving signal or leader processing may result in an increase in growth rate, cell density and cellular material productivity, which may contribute positively to the overall productivity of the protein of interest. Furthermore, a more suitable cell line is more likely to perform robustly at different scales and culture conditions and better cope with in-process disturbances. Furthermore, it is widely recognized by those skilled in the art that cell lines with normal growth rate and cell adaptation have a lower risk of instability than cell lines with reduced growth rate and cell adaptation due to the influence of transgene expression (e.g. product accumulation in the cell). For cell lines in which transgene expression results in a decrease in growth rate, events that reduce transgene expression, such as gene silencing events, mutations, or outturn of the transgene by direct repeated recombination, can result in a competitive growth advantage. Cells with reduced expression will rapidly outweigh other cells in the population that still express the transgene at high levels, resulting in an unstable expression phenotype.

In certain embodiments, a host cell expressing one or more genes encoding an IL-2 protein of formula (I) may comprise in its genome the one or more genes encoding an IL-2 protein of formula (I).

In another aspect, the invention relates to a conjugate comprising one or more IL-2 proteins of formula (I).

In certain embodiments, the conjugate comprises at least one, e.g., one, two, three, or four, moieties M conjugated to an IL-2 protein of formula (I) _mod They may be the same or different. Such part M _mod May be at the N-terminus, C-terminus, amino acid side chain or internal site of the IL-2 protein. In certain embodiments, such a moiety M is attached at the N-terminus of an IL-2 protein of formula (I) _mod . In certain embodiments, such a moiety M is attached at the C-terminus of an IL-2 protein of formula (I) _mod . In certain embodiments, such a moiety M is attached at an internal site of the IL-2 moiety _mod For example on the amino acid side chains of the IL-2 protein of formula (I). If more than one part M _mod The ligation to the IL-2 protein of formula (I) occurs at any combination of ligation sites selected from the group consisting of the N-terminus, the C-terminus and the internal site.

In certain embodiments, M _mod Is a substituent. Preferably, such substituents have a molecular weight of 15Da to 1kDa.

In certain embodiments, M _mod Is a polymeric moiety. Such polymer moieties may include linear, branched or multi-arm polymers. In one embodiment, the polymer is a linear polymer. In another embodiment, the polymer is a branched polymer. In certain embodiments, such branched polymers have one, two, three, four, or five branching points. Two, three or four polymer arms may extend from each branch point. In another embodiment, the polymer is a multi-arm polymer. Such multi-arm polymers may have 3, 4, 5, 6, 7 or 8 polymer arms.

If M _mod Is a polymer partIn certain embodiments, such polymer moieties have a molecular weight of from 0.5kDa to 1000kDa, e.g., from 1kDa to 1000kDa, e.g., from 2kDa to 500kDa, from 3kDa to 200kDa, from 5kDa to 120kDa or from 7 to 40kDa. In one embodiment, the molecular weight of such a polymer is about 0.5kDa. In one embodiment, the molecular weight of such a polymer is about 1kDa. In one embodiment, the molecular weight of such a polymer is about 2kDa. In one embodiment, the molecular weight of such a polymer is about 3kDa. In one embodiment, the molecular weight of such a polymer is about 4kDa. In one embodiment, the molecular weight of such a polymer is about 5kDa. In one embodiment, the molecular weight of such a polymer is about 7.5kDa. In another embodiment, the molecular weight of such polymer fraction is about 10kDa. In another embodiment, the molecular weight of such polymer fraction is about 15kDa. In another embodiment, the molecular weight of the polymer portion is about 20kDa. In another embodiment, the molecular weight of the polymer portion is about 30kDa. In another embodiment, the molecular weight of the polymer portion is about 40kDa. In another embodiment, the molecular weight of the polymer portion is about 50kDa. In another embodiment, the molecular weight of the polymer portion is about 70kDa. In another embodiment, the molecular weight of the polymer portion is about 80kDa. In another embodiment, the molecular weight of the polymer portion is about 90kDa. In another embodiment, the molecular weight of such polymer fraction is about 100kDa. In one embodiment, the molecular weight of this polymer is 0.5kDa. In one embodiment, the molecular weight of this polymer is 1kDa. In one embodiment, the molecular weight of this polymer is 2kDa. In one embodiment, the molecular weight of this polymer is 3kDa. In one embodiment, the molecular weight of this polymer is 4kDa. In one embodiment, the molecular weight of this polymer is 5kDa. In one embodiment, the molecular weight of this polymer is 7.5kDa. In another embodiment, the molecular weight of this polymer is 10kDa. In another embodiment, the molecular weight of such polymer fraction is 15kDa. In another embodiment In one embodiment, the polymer fraction has a molecular weight of 20kDa. In another embodiment, the molecular weight of the polymer fraction is 30kDa. In another embodiment, the molecular weight of the polymer fraction is 40kDa. In another embodiment, the molecular weight of the polymer portion is 50kDa. In another embodiment, the molecular weight of the polymer portion is 70kDa. In another embodiment, the molecular weight of the polymer portion is 80kDa. In another embodiment, the molecular weight of the polymer portion is 90kDa. In another embodiment, the molecular weight of such polymer fraction is 100kDa.

If M _mod Is a polymer moiety, such polymer moiety preferably comprises a polymer selected from the group consisting of: 2-methacryloyl-oxyethyl phosphorylcholine, poly (acrylic acid), poly (acrylate), poly (acrylamide), poly (alkoxy) polymer, poly (amide), poly (amide-amine), poly (amino acid), poly (anhydride), poly (asparagine), poly (butyric acid), poly (glycolic acid), poly (butylene terephthalate, poly (caprolactone), poly (carbonate), poly (cyanoacrylate), poly (dimethylacrylamide), polyester, polyethylene glycol, polyethylene oxide, poly (ethyl phosphate), poly (ethyl oxazoline), poly (glycolic acid), poly (hydroxyethyl acrylate), poly (hydroxyethyl oxazoline), poly (hydroxymethyl acrylate), poly (hydroxypropyl methacrylamide), poly (hydroxypropyl methacrylate), poly (iminocarbonate), poly (lactic acid-glycolic acid) copolymer, poly (methacrylamide), poly (methacrylate), poly (methyl oxazoline), poly (organophosphazene), poly (orthoester), poly (oxazoline), poly (propylene glycol), poly (siloxane), polyurethane, poly (vinyl alcohol), poly (vinyl amine), poly (vinyl methyl ether), poly (vinyl pyrrolidone), (poly) siloxane, cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid and derivatives thereof, functionalized hyaluronic acid, alginate, mannans, pectins, rhamnogalacturons, starches, hydroxyalkyl starches, hydroxyethyl starches and other carbohydrate-based polymers, wood Polysaccharides and copolymers thereof.

In certain embodiments, M _mod Is a PEG-based polymer.

In certain embodiments, M _mod Is a hyaluronic acid-based polymer.

In certain embodiments, M _mod Comprising a peptide or protein moiety which can be chemically conjugated to an IL-2 protein of formula (I). Preferably, such peptide or protein moiety M _mod Not a fragment of IL-2 or an IL-2 moiety.

M in the form of peptide or protein moieties _mod May be a synthetic or natural protein molecule or a portion or variant thereof. Exemplary peptides and proteins include albumin; an antibody binding domain, such as an Fc domain of an immunoglobulin or an antigen binding domain; CTP and CD25; each may be in its naturally occurring form or a variant or fragment thereof.

M _mod The IL-2 protein of formula (I) may be linked by a stable linker.

In certain embodiments, M _mod Comprises a targeting moiety selected from the group consisting of an antibody, an antibody fragment, an affibody, affilin, affimer, affitin, an alpha monoclonal antibody (alphamab), alphabodies, anticalins, avimers, DARPin,Kunitz domain peptides, monocams, nano-CLAMP, cyclic peptides, heavy chain-only antibodies, VHH antibodies or Single chain variable region fragments (scFvs), natural or modified peptide or protein receptor ligands and small molecule inhibitors.

In certain embodiments, the conjugate is an IL-2 conjugate of formula (Ia) or (Ib) or a pharmaceutically acceptable salt thereof

Wherein the method comprises the steps of

-D comprises an IL-2 protein of formula (I);

-L ¹ -a linker moiety covalently and reversibly linked to-D;

-L ² -a chemical bond or a spacer moiety;

-Z is a polymer moiety or a substituted fatty acid moiety;

x is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16; and is also provided with

y is an integer selected from 2, 3, 4 and 5.

In certain embodiments, the conjugates of formulas (Ia) and (Ib) release a preferential IL-2 moiety or preferential IL-2 protein of formula (I), wherein the ratio _{Biasing IL-2} Ratio to (V/V) _{SEQ ID NO:213 IL-2} The ratio of (2) is greater than 1, preferably greater than 2, preferably greater than 3, preferably greater than 4, even more preferably greater than 5. In certain embodiments, the ratio _{Biasing IL-2} Ratio to (V/V) _{SEQ ID NO:213 IL-2} Greater than 10, greater than 20, greater than 50, greater than 70, greater than 100, greater than 150, or greater than 200.

It is to be understood that-D of formulae (Ia) and (Ib) may comprise at least one moiety M as described elsewhere herein _mod 。

The IL-2 conjugate of formula (Ia) or (Ib) comprises at least one covalently and reversibly linked polymer moiety and/or substituted fatty acid moiety-Z.

The addition of such at least one covalently and reversibly linked polymer moiety and/or substituted fatty acid moiety is capable of extending the circulating half-life of the IL-2 moiety of formula (I) while its reversible linkage ensures sufficient pharmaceutical activity.

In certain embodiments, the IL-2 conjugate belongs to formula (Ia) and comprises one moiety-Z, which is a substituted fatty acid or polymer moiety. In certain embodiments, -Z is a substituted fatty acid. In certain embodiments, -Z is a polymeric moiety.

In certain embodiments, the IL-2 conjugate belongs to formula (Ib) and comprises two moieties-Z, which may be the same or different. In certain embodiments, both moieties-Z are substituted fatty acids, which may be the same or different. In certain embodiments, both moieties-Z are polymeric moieties, which may be the same or different. In certain embodiments, one moiety-Z is a substituted fatty acid and the other moiety-Z is a polymeric moiety.

In certain embodiments, the IL-2 conjugate belongs to formula (Ib) and comprises three moieties-Z, which may be the same or different. In certain embodiments, all three moieties-Z are substituted fatty acids, which may be the same or different. In certain embodiments, all three moieties-Z are polymeric moieties, which may be the same or different. In certain embodiments, one or two moieties-Z are substituted fatty acids, while the remaining one or more moieties-Z are polymeric moieties.

In certain embodiments, the IL-2 conjugate belongs to formula (Ib) and comprises four moieties-Z, which may be the same or different. In certain embodiments, all four moieties-Z are substituted fatty acids, which may be the same or different. In certain embodiments, all four moieties-Z are polymeric moieties, which may be the same or different. In certain embodiments, one, two, or three moieties-Z are substituted fatty acids, while the remaining one or more moieties-Z are polymeric moieties.

if-Z is a substituted fatty acid moiety, it is preferably a substituted fatty acid moiety as disclosed in WO 2005/027978A2 and WO 2014/060512A1, which are incorporated herein by reference.

if-Z is a polymer moiety, in certain embodiments, such polymer moiety has a molecular weight in the range of 1 kDa to 1000kDa, such as 2kDa to 500kDa, 3kDa to 200kDa, 5kDa to 120kDa, 10kDa to 100kDa, or 15kDa to 80kDa. In certain embodiments, -Z is a polymer moiety having a molecular weight of about 2 kDa. In certain embodiments, -Z is a polymer moiety having a molecular weight of about 5 kDa. In certain embodiments, -Z is a polymer moiety having a molecular weight of about 10 kDa. In certain embodiments, -Z is a polymer moiety having a molecular weight of about 15 kDa. In certain embodiments, -Z is a polymer moiety having a molecular weight of about 20 kDa. In certain embodiments, -Z is a polymer moiety having a molecular weight of about 30 kDa. In certain embodiments, -Z is a polymer moiety having a molecular weight of about 40 kDa. In certain embodiments, -Z is a polymer moiety having a molecular weight of about 50 kDa. In certain embodiments, -Z is a polymer moiety having a molecular weight of about 60 kDa. In certain embodiments, -Z is a polymer moiety having a molecular weight of about 70 kDa. In certain embodiments, -Z is a polymer moiety having a molecular weight of about 80kDa. In certain embodiments, -Z is a polymer moiety having a molecular weight of about 90 kDa. In certain embodiments, -Z is a polymer moiety having a molecular weight of about 100 kDa. In certain embodiments, -Z is a polymer moiety having a molecular weight of 2 kDa. In certain embodiments, -Z is a polymer moiety having a molecular weight of 5 kDa. In certain embodiments, -Z is a polymer moiety having a molecular weight of 10 kDa. In certain embodiments, -Z is a polymer moiety having a molecular weight of 15 kDa. In certain embodiments, -Z is a polymer moiety having a molecular weight of 20 kDa. In certain embodiments, -Z is a polymer moiety having a molecular weight of 30 kDa. In certain embodiments, -Z is a polymer moiety having a molecular weight of 40 kDa. In certain embodiments, -Z is a polymer moiety having a molecular weight of 50 kDa. In certain embodiments, -Z is a polymer moiety having a molecular weight of 60 kDa. In certain embodiments, -Z is a polymer moiety having a molecular weight of 70 kDa. In certain embodiments, -Z is a polymer moiety having a molecular weight of 80kDa. In certain embodiments, -Z is a polymer moiety having a molecular weight of 90 kDa. In certain embodiments, -Z is a polymer moiety having a molecular weight of 100 kDa.

In certain embodiments, -Z is a polymer moiety comprising a polymer selected from the group consisting of: 2-methacryloyl-oxyethyl phosphorylcholine, poly (acrylic acid), poly (acrylate), poly (acrylamide), poly (alkoxy) polymer, poly (amide), poly (amide-amine), poly (amino acid), poly (anhydride), poly (asparagine), poly (butyric acid), poly (glycolic acid), polybutylene terephthalate, polycaprolactone, polycarbonate, polycyanoacrylate, polydimethyl acrylamide, polyester, polyethylene glycol, polyethylene oxide, poly (ethyl phosphate), poly (ethyl oxazoline), poly (glycolic acid), poly (hydroxyethyl acrylate), poly (hydroxyethyl oxazoline), poly (hydroxymethyl acrylate), poly (hydroxypropyl methacrylamide), poly (hydroxypropyl methacrylate), poly (hydroxypropyl oxazoline), poly (iminocarbonate), poly (lactic acid-glycolic acid) copolymer, poly (methacrylamide), poly (methacrylate), poly (methyl oxazoline), poly (organophosphazene), poly (orthoester), poly (oxazoline), poly (propylene glycol), poly (siloxane), polyurethane, poly (vinyl alcohol), poly (vinyl ether), poly (vinyl pyrrolidone), (poly) siloxanes, celluloses, carboxymethyl cellulose, hydroxypropyl methyl cellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid and its derivatives, functionalized hyaluronic acid, alginates, mannans, pectins, rhamnogalacturons, starches, hydroxyalkyl starches, hydroxyethyl starch and other carbohydrate based polymers, xylans and their copolymers.

In certain embodiments, -Z is a peptide or protein moiety. Preferably, such peptide or protein moiety is not an IL-2 moiety or fragment thereof. Such peptide or protein moiety-Z may pass through-L ¹ -L ² Chemical conjugation to-D, or through a reversible linker moiety-L ¹ Fusion with-D translation, in which case-L ¹ -is a peptide or protein moiety, and-L ² Preferably a chemical bond. In certain embodiments, such peptide or protein moieties-Z pass-L ¹ -L ² -chemical conjugation with-D. In certain embodiments, such peptide or protein moiety-Z is linked to the moiety-L by a reversible linker ¹ Fusion with-D translation, in which case-L ¹ -is a peptide or protein moiety, and-L ² Preferably a chemical bond. It will be appreciated that this peptide or protein reversible linker moiety-L ¹ Can be enzymatically or non-enzymatically degradable. In order to promote enzymatic degradation, -L ¹ May comprise a protease recognition site.

if-Z is a peptide or protein moiety, it is in certain embodiments selected from the group consisting of the carboxy-terminal peptide portion of chorionic gonadotrophin as described in US 2012/0035101 Al, which is incorporated herein by reference; an albumin moiety; a random coil protein moiety and an Fc fusion protein moiety.

In certain embodiments, -Z comprises a random coil peptide or protein moiety.

In certain embodiments, such a random coil peptide or protein moiety comprises at least 25 amino acid residues and up to 2000 amino acids, for example 30 amino acids to 1500 amino acid residues or 50 to 500 amino acid residues.

In certain embodiments, -Z comprises a random coil protein moiety, wherein at least 80%, such as at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the total number of amino acids forming the random coil protein moiety is selected from the group consisting of alanine and proline. In certain embodiments, at least 10%, but less than 75%, e.g., less than 65%, of the total number of amino acid residues of such a random coil protein moiety are proline residues. In certain embodiments, the random coil protein moiety is as described in WO2011/144756 A1, which is incorporated herein by reference in its entirety. In certain embodiments, -Z comprises at least one moiety selected from the group consisting of: SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO:51 and SEQ ID NO:61, for example as disclosed in WO 2011/144756. The portion comprising such alanine and proline containing random coil proteins is referred to herein as the "PA" or "PA portion".

Thus, in certain embodiments, -Z comprises a PA moiety.

In certain embodiments, -Z comprises a random coil protein moiety, wherein at least 80%, such as at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the total number of amino acids forming the random coil protein moiety is selected from the group consisting of alanine, serine, and proline. In certain embodiments, at least 4%, but less than 40% of the total number of amino acid residues of such a random coil protein moiety are proline residues. In certain embodiments, the random coil protein moiety is as described in WO 2008/155134 A1, which is incorporated herein by reference. In certain embodiments, -Z comprises at least one moiety selected from the group consisting of: SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO: 14. SEQ ID NO: 16. SEQ ID NO: 18. SEQ ID NO: 20. SEQ ID NO: 22. SEQ ID NO: 24. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30. SEQ ID NO: 32. SEQ ID NO: 34. SEQ ID NO: 36. SEQ ID NO: 40. SEQ ID NO: 42. SEQ ID NO: 44. SEQ ID NO: 46. SEQ ID NO: 50. SEQ ID NO: 52. SEQ ID NO:54 and SEQ ID NO:56, for example as disclosed in WO 2008/155134 A1. The portion comprising such a random coil protein moiety comprising alanine, serine and proline is referred to herein as "PAS" or "PAS portion".

Thus, in certain embodiments, -Z comprises a PAS moiety.

In certain embodiments, -Z comprises a random coil protein moiety, wherein at least 80%, such as at least 85%, at least 90%, at least 95%, at least 98% or at least 99% of the total number of amino acids forming the random coil protein moiety is selected from the group consisting of alanine, glycine, serine, threonine, glutamic acid, and proline. In certain embodiments, the random coil protein moiety is as described in WO2010/091122A1, which is incorporated herein by reference. In certain embodiments, -Z comprises at least one moiety selected from the group consisting of: SEQ ID NO: 182. SEQ ID NO: 183. SEQ ID NO:184; SEQ ID NO: 185. SEQ ID NO: 186. SEQ ID NO: 187. SEQ ID NO: 188. SEQ ID NO: 189. SEQ ID NO: 190. SEQ ID NO: 191. SEQ ID NO: 192. SEQ ID NO: 193. SEQ ID NO: 194. SEQ ID NO: 195. SEQ ID NO: 196. SEQ ID NO: 197. SEQ ID NO: 198. SEQ ID NO: 199. SEQ ID NO: 200. SEQ ID NO: 201. SEQ ID NO: 202. SEQ ID NO: 203. SEQ ID NO: 204. SEQ ID NO: 205. SEQ ID NO: 206. SEQ ID NO: 207. SEQ ID NO: 208. SEQ ID NO: 209. SEQ ID NO: 210. SEQ ID NO: 211. SEQ ID NO: 212. SEQ ID NO: 213. SEQ ID NO: 214. SEQ ID NO: 215. SEQ ID NO: 216. SEQ ID NO: 217. SEQ ID NO: 218. SEQ ID NO: 219. SEQ ID NO: 220. SEQ ID NO: 221. SEQ ID NO: 759. SEQ ID NO: 760. SEQ ID NO: 761. SEQ ID NO: 762. SEQ ID NO: 763. SEQ ID NO: 764. SEQ ID NO: 765. SEQ ID NO: 766. SEQ ID NO: 767. SEQ ID NO: 768. SEQ ID NO: 769. SEQ ID NO: 770. SEQ ID NO: 771. SEQ ID NO: 772. SEQ ID NO: 773. SEQ ID NO: 774. SEQ ID NO: 775. SEQ ID NO: 776. SEQ ID NO: 777. SEQ ID NO: 778. SEQ ID NO: 779. SEQ ID NO: 1715. SEQ ID NO: 1716. SEQ ID NO: 1718. SEQ ID NO: 1719. SEQ ID NO: 1720. SEQ ID NO:1721 and SEQ ID NO:1722, for example as disclosed in WO2010/091122 A1. The portion comprising such random coil protein portions containing alanine, glycine, serine, threonine, glutamic acid, and proline is referred to herein as "XTEN" or "XTEN portion".

Thus, in certain embodiments, -Z comprises an XTEN moiety.

In certain embodiments, -Z is a hyaluronic acid-based polymer.

In certain embodiments, -Z is a PEG-based moiety, such as a linear, branched, or multi-arm PEG-based moiety. In certain embodiments, -Z is a branched PEG-based moiety, e.g., a branched PEG-based moiety having one, two, three, four, five, or six branch points. In certain embodiments, -Z is a branched PEG-based moiety having one, two, or three branch points. In certain embodiments, -Z is a branched PEG-based moiety having one branching point. In certain embodiments, -Z is a branched PEG-based moiety having two branch points. In certain embodiments, -z is a branched PEG-based moiety having three branch points.

Each branching point may be independently selected from-N <, -CH < and > C <.

In certain embodiments, -Z comprises a moiety of formula (A)

Wherein the method comprises the steps of

-BP ¹ ＜、-BP ² ＜、-BP ³ < independently of one another selected from the group consisting of-N < and-C (R ⁸ )＜；

-R ⁸ Selected from-H, C _1-6 Alkyl, C _2-6 Alkenyl and C _2-6 Alkynyl;

-P ¹ 、-P ² 、-P ³ 、-P ⁴ are, independently of each other, PEG-based chains comprising at least 40% PEG and having a molecular weight of 3 to 40kDa;

-C ¹ 、-C ² Independently of each other selected from: c (C) _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl groups. Wherein C is _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl is optionally substituted with one or more-R ⁹ Substituted, these radicals being identical or different and wherein C _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl groups are optionally interrupted by one or more groups selected from the group consisting of: -T-, -C (O) O-, -C (O) N (R) ¹⁰ )-、-S(O) ₂ N(R ¹⁰ )-、-S(O)N(R ¹⁰ )-、-S(O) ₂ -、-S(O)-、-N(R ¹⁰ )S(O) ₂ N(R ^10a )-、-S-、-N(R ¹⁰ )-、-OC(OR ¹⁰ )(R ^10a )-、-N(R ¹⁰ )C(O)N(R ^10a ) -and-OC (O) N (R) ¹⁰ )；

T is each independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C _3-10 Cycloalkyl, 3-to 10-membered heterocyclyl, 8-to 11-membered heterobicyclo, 8-to 30-membered carbopolycyclic and 8-to 10-membered heteropolycyclic; wherein T are each independently optionally substituted with one or more R's, which may be the same or different ⁹ Substitution;

each-R ⁹ Independently selected from: halogen, -CN, oxo (=o), COOR ¹¹¹¹ 、-OR ¹¹ 、-C(O)R ¹¹ 、-C(O)N(R ¹¹ R ^11a )、-S(O) ₂ N(R ¹¹ R ^11a )、-S(O)N(R ¹¹ R ^11a )、-S(O) ₂ R ¹¹ 、-S(O)R ¹¹ 、-N(R ¹¹ )S(O) ₂ N(R ^11a R ^11b )、-SR ¹¹¹¹ 、-N(R ¹¹ R ^11a )、-NO ₂ 、-OC(O)R ¹¹ 、-N(R ¹¹ )C(O)R ^11a 、-N(R ¹¹ )S(O) ₂ R ^11a 、-N(R ¹¹ )S(O)R ^11a 、-N(R ¹¹ )C(O)OR ^11a 、-N(R ¹¹ )C(O)N(R ^11a R ^11b )、-OC(O)N(R ¹¹ R ^11a ) And C _1-6 An alkyl group; wherein C is _1-6 Alkyl is optionally substituted with one or more halogen, the same or different; and

each-R ¹⁰ 、-R ^10a 、R ¹¹ 、R ^11a And R is ^11b Independently selected from H and C _1-6 Alkyl, wherein C _1-6 The alkyl groups are optionally substituted with one or more halogen groups, which may be the same or different.

In certain embodiments, -P ¹ 、-P ² 、-P ³ 、-P ⁴ Independently of each other are PEG-based chains comprising at least 50% PEG, with a molecular weight in the range of 3 to 40kDa. In certain embodiments, -P ¹ 、-P ² 、-P ³ 、-P ⁴ Independently of each other are PEG-based chains comprising at least 60% PEG, with a molecular weight in the range of 3 to 40kDa. In certain embodiments, -P ¹ 、-P ² 、-P ³ 、-P ⁴ Independently of each other are PEG-based chains comprising at least 70% PEG, with a molecular weight in the range of 3 to 40kDa. In certain embodiments, -P ¹ 、-P ² 、-P ³ 、-P ⁴ Independently of each other are PEG-based chains comprising at least 80% PEG, with a molecular weight in the range of 3 to 40kDa.

In certain embodiments, the compound of formula (a) is-P ¹ 、-P ² 、-P ³ and-P ⁴ The molecular weight ranges of the fractions are independently of each other in the range 5-30kDa, for example 5-25kDa or 8-20kDa. In certain embodiments, part-P ¹ 、-P ² 、-P ³ or-P ⁴ May have a molecular weight of about 5kDa. In certain embodiments, part-P ¹ 、-P ² 、-P ³ or-P ⁴ May have a molecular weight of about 7kDa. In certain embodiments, part-P ¹ 、-P ² 、-P ³ or-P ⁴ May have a molecular weight of about 10kDa. In certain embodiments, part-P ¹ 、-P ² 、-P ³ or-P ⁴ May have a molecular weight of about 12kDa. In certain embodiments, part-P ¹ 、-P ² 、-P ³ or-P ⁴ The molecular weight of (2) may be about 15kDa. In certain embodiments, the moiety has a molecular weight of P ¹ 、-P ² 、-P ³ or-P ⁴ May be about 20kDa. In certain embodiments, part-P ¹ 、-P ² 、-P ³ or-P ⁴ May have a molecular weight of about 25kDa. In certain embodiments, part- β ¹ 、-P ² 、-P ³ or-P ⁴ May have a molecular weight of about 30kDa. In certain embodiments, part-P ¹ 、-P ² 、-P ³ or-P ⁴ The molecular weight of (2) may be 7kDa. In certain embodiments, part-P ¹ 、-P ² 、-P ³ or-P ⁴ The molecular weight of (2) may be 10kDa. In certain embodiments, part-P ¹ 、-P ² 、-P ³ Or-p ⁴ May have a molecular weight of 12kDa. In certain embodiments, part-p ¹ 、-P ² 、-P ³ Or-p ⁴ The molecular weight of (2) may be 15kDa. In certain embodiments, part-P ¹ 、-P ² 、-P ³ or-P ⁴ May have a molecular weight of 20kDa. In certain embodiments, part-P ¹ 、-P ² 、-P ³ or-P ⁴ The molecular weight of (2) may be 25kDa. In certain embodiments, part-P ¹ 、-P ² 、-P ³ or-P ⁴ May have a molecular weight of 30kDa.

In certain embodiments, the compound of formula (a) is-P ¹ 、-P ² 、-P ³ And-p ⁴ Has the same structure.

In some casesIn an embodiment, the-Bp of formula (A) ¹ <is-N <.

In certain embodiments, the-Bp of formula (A) ² < sum-Bp ³ < have the same structure. In certain embodiments, the-BP of formula (A) ² <and-BP ³ <Are all-CH <.

In certain embodiments, -C of formula (A) ¹ -and-C ² -having the same structure. In certain embodiments, -C of formula (A) ¹ -and-C ² -C interrupted by one or more groups _1-50 Alkyl, said radical being selected from O-, -C (O) N (R) ¹⁰ ) -and 3-to 10-membered heterocyclyl; wherein the 3-to 10-membered heterocyclyl is substituted with at least one oxo (=o).

In certain embodiments, -C of formula (A) ¹ -and-C ² -having the formula (A-a)

Wherein the method comprises the steps of

The dotted line marked with an asterisk indicates the linkage to-BP ¹ ＜；

The unlabeled dashed lines indicate the connection to-Bp, respectively ² < or-BP ³ ＜；

q1 is selected from 1, 2, 3, 4, 5, 6, 7 and 8;

q2 is selected from 1, 2, 3, 4 and 5;

q3 is selected from 1, 2, 3, 4, 5, 6, 7 and 8; and is also provided with

q4 is selected from 1, 2 and 3.

In certain embodiments, q1 of formula (a-a) is selected from 4, 5, 6, 7, and 8. In certain embodiments, q1 of formula (a-a) is selected from 5, 6, and 7. In certain embodiments, q1 of formula (a-a) is 1. In certain embodiments, q1 of formula (a-a) is 2. In certain embodiments, q1 of formula (a-a) is 3. In certain embodiments, q1 of formula (a-a) is 4. In certain embodiments, q1 of formula (a-a) is 5. In certain embodiments, q1 of formula (a-a) is 6. In certain embodiments, q1 of formula (a-a) is 7. In certain embodiments, q1 of formula (a-a) is 8.

In certain embodiments, q2 of formula (a-a) is selected from 1, 2, and 3. In certain embodiments, q2 of formula (a-a) is 1. In certain embodiments, q2 of formula (a-a) is 2. In certain embodiments, q2 of formula (a-a) is 3. In certain embodiments, q2 of formula (a-a) is 4. In certain embodiments, q2 of formula (a-a) is 5.

In certain embodiments, q3 of formula (a-a) is selected from 2, 3, 4, and 5. In certain embodiments, q3 of formula (a-a) is selected from 2, 3, and 4. In certain embodiments, q3 of formula (a-a) is 1. In certain embodiments, q3 of formula (a-a) is 2. In certain embodiments, q3 of formula (a-a) is 3. In certain embodiments, q3 of formula (a-a) is 4. In certain embodiments, q3 of formula (a-a) is 5. In certain embodiments, q3 of formula (a-a) is 6. In certain embodiments, q3 of formula (a-a) is 7. In certain embodiments, q3 of formula (a-a) is 8.

In certain embodiments, q4 of formula (a-a) is 1. In certain embodiments, q4 of formula (a-a) is 2. In certain embodiments, q4 of formula (a-a) is 3.

In certain embodiments, the compound of formula (a) is-P ¹ 、P ² 、-P ³ and-P ⁴ Is of the independent formula (A-b)

Wherein the method comprises the steps of

The dashed line represents the connection to the rest of-Z;

m is 0 or 1;

p is an integer between 70 and 900; and

q is selected from 1, 2, 3, 4, 5 and 6.

In certain embodiments, m of formula (a-b) is 0. In certain embodiments, m of formula (a-b) is 1.

In certain embodiments, p of formula (a-b) is an integer from 115 to 680. In certain embodiments, p of formula (A-b) is an integer from 115 to 560. In certain embodiments, p of formula (a-b) is an integer from 185 to 450. In certain embodiments, p of formula (A-b) is about 115. In certain embodiments, p of formula (A-b) is about 160. In certain embodiments, p of formula (A-b) is about 225. In certain embodiments, p of formula (A-b) is about 270. In certain embodiments, p of formula (A-b) is about 340. In certain embodiments, p of formula (A-b) is about 450. In certain embodiments, p of formula (A-b) is about 560.

In certain embodiments, q of formula (a-b) is 1. In certain embodiments, q of formula (a-b) is 2. In certain embodiments, q of formula (a-b) is 3. In certain embodiments, q of formula (a-b) is 4. In certain embodiments, q of formula (a-b) is 5. In certain embodiments, q of formula (a-b) is 6.

In certain embodiments, -Z comprises a moiety of formula (a-c):

wherein the method comprises the steps of

p1, p2, p3, p4 are independently integers from 70 to 900.

In certain embodiments, p1 of formula (a-c) is an integer from 115 to 680. In certain embodiments, p1 of formula (A-c) is an integer from 115 to 560. In certain embodiments, p1 of formula (A-c) is an integer from 185 to 450. In certain embodiments, p1 of formula (A-c) is an integer from 220 to 240. In certain embodiments, p1 of formula (A-c) is about 115. In certain embodiments, p1 of formula (A-c) is about 160. In certain embodiments, p1 of formula (a-c) is about 225. In certain embodiments, p1 of formula (A-c) is about 270. In certain embodiments, p1 of formula (A-c) is about 340. In certain embodiments, p1 of formula (A-c) is about 450. In certain embodiments, p1 of formula (A-c) is about 560.

In certain embodiments, p2 of formula (a-c) is an integer from 115 to 680. In certain embodiments, p2 of formula (A-c) is an integer from 115 to 560. In certain embodiments, p2 of formula (A-c) is an integer from 185 to 450. In certain embodiments, p2 of formula (A-c) is an integer from 220 to 240. In certain embodiments, p2 of formula (a-c) is about 115. In certain embodiments, p2 of formula (a-c) is about 160. In certain embodiments, p2 of formula (a-c) is about 225. In certain embodiments, p2 of formula (a-c) is about 270. In certain embodiments, p2 of formula (a-c) is about 340. In certain embodiments, p2 of formula (a-c) is about 450. In certain embodiments, p2 of formula (A-c) is about 560.

In certain embodiments, p3 of formula (a-c) is an integer from 115 to 680. In certain embodiments, p3 of formula (a-c) is an integer from 115 to 560. In certain embodiments, p3 of formula (a-c) is an integer from 185 to 450. In certain embodiments, p3 of formula (a-c) is an integer from 220 to 240. In certain embodiments, p3 of formula (A-c) is about 115. In certain embodiments, p3 of formula (a-c) is about 160. In certain embodiments, P3 of formula (A-c) is about 225. In certain embodiments, p3 of formula (a-c) is about 270. In certain embodiments, P3 of formula (A-c) is about 340. In certain embodiments, P3 of formula (A-c) is about 450. In certain embodiments, p3 of formula (A-c) is about 560.

In certain embodiments, p4 of formula (a-c) is an integer from 115 to 680. In certain embodiments, p4 of formula (A-c) is an integer from 115 to 560. In certain embodiments, p4 of formula (A-c) is an integer from 185 to 450. In certain embodiments, p4 of formula (A-c) is an integer from 220 to 240. In certain embodiments, p4 of formula (a-c) is about 115. In certain embodiments, p4 of formula (a-c) is about 160. In certain embodiments, p4 of formula (a-c) is about 225. In certain embodiments, p4 of formula (a-c) is about 270. In certain embodiments, p4 of formula (a-c) is about 340. In certain embodiments, p4 of formula (a-c) is about 450. In certain embodiments, p4 of formula (A-c) is about 560.

In certain embodiments, p1, p2, p3 and p4 of formula (a-c) are the same. In certain embodiments, p1, p2, p3, and p4 are between 220 and 240.

In certain embodiments, -Z is a part of the disclosure in WO 2012/02047 A1, which is incorporated herein by reference.

In certain embodiments, -Z is a part of the disclosure in WO 2013/024948 A1, which is incorporated herein by reference.

In certain embodiments, a conjugate comprising one or more IL-2 proteins of formula (I) or a pharmaceutically acceptable salt thereof comprises a plurality of moieties-D, which is the passage of the IL-2 protein of formula (I) through at least one moiety-L ¹ -L ² Conjugation to at least one moiety Z', wherein one moiety-L ¹ Conjugation to-D by reversible linkage, and wherein one moiety-L ² Conjugation to Z', wherein-L ¹ -and-L ² -as defined for formulae (Ia) and (Ib), and wherein Z' is a water insoluble hydrogel.

In certain embodiments, such hydrogels Z' comprise a polymer selected from the group consisting of 2-methacryloyl-oxyethyl phosphorylcholine, poly (acrylic acid), poly (acrylic acid ester), poly (acrylamide), poly (alkoxy) polymer, poly (amide), poly (amide-amine), poly (amino acid), poly (anhydride), poly (asparagine), poly (butyric acid), poly (glycolic acid), poly (butylene terephthalate), polycaprolactone, polycarbonate, polycyanoacrylate, polydimethyl acrylamide, polyester, polyethylene, polyalkylene glycol (such as polyethylene glycol and polypropylene glycol), polyethylene oxide, poly (ethyl phosphate), poly (ethyl oxazoline), poly (glycolic acid), poly (hydroxyethyl acrylate), poly (hydroxyethyl oxazoline), poly (hydroxymethyl acrylate), poly (hydroxypropyl methacrylamide), poly (hydroxypropyl methacrylate), poly (hydroxypropyl oxazoline), poly (iminocarbonate), poly (lactic acid-glycolic acid copolymer, poly (methacrylamide), poly (methyl oxazoline), poly (organophosphazene), poly (orthoester), poly (oxazoline), poly (vinyl alcohol), poly (propylene glycol), poly (vinyl alcohol), poly (siloxane, poly (vinylamine), poly (vinyl methyl ether), poly (vinyl pyrrolidone)), (poly) siloxanes, cellulose, carboxymethyl cellulose, hydroxypropyl methylcellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid and derivatives thereof, functionalized hyaluronic acid, mannans, pectins, rhamnogalactan, starch, hydroxyalkyl starch, hydroxyethyl starch and other carbohydrate-based polymers, xylans and copolymers thereof.

In certain embodiments, Z' is a poly (alkylene glycol) -based or hyaluronic acid-based hydrogel.

In certain embodiments, z' is a polypropylene glycol-based hydrogel.

In certain embodiments, Z' is a PEG-based hydrogel.

In certain embodiments, Z' is a PEG-based hydrogel as disclosed in WO 2011/012615 A1 or WO2014/056926A1, both of which are incorporated herein by reference.

In certain embodiments, Z' is a hyaluronic acid-based hydrogel.

In certain embodiments, Z' is a hyaluronic acid-based hydrogel as disclosed in WO2018/175788A1, which is incorporated herein by reference.

In certain embodiments, Z' is a hydrogel as disclosed in WO2013/036847 A1. In particular, in certain embodiments, Z' is a hydrogel produced by a process comprising the step of reacting at least a first reactive polymer with a cleavable crosslinker compound, wherein the cleavable crosslinker compound comprises a first functional group-Y that reacts with the first reactive polymer ¹ And further comprising a moiety that is cleaved by elimination under physiological conditions, wherein the moiety comprises a second functional group-Y that reacts with a second reactive polymer ² . In certain embodiments, the cleavable crosslinker compound is of formula (PL-1)

Wherein the method comprises the steps of

m is 0 or 1;

-X comprises a functional group capable of linking to the reactive polymer, which functional group can be eliminated under physiological conditions, and said second functional group-Y ² ；

-R ¹ 、-R ² and-R ⁵ At least one of which contains the first functional group-Y ¹ Which is capable of being attached to a polymer;

-R ¹ and-R ² One and only one of them is selected from the group consisting of-H, alkyl, arylalkyl and heteroarylalkyl;

optionally, -R ¹ and-R ² Can be joined to form a 3-8 membered ring;

-R ¹ and-R ² At least one or both of which are independently selected from-CN, -NO ₂ Aryl, heteroaryl, alkenyl, alkynyl, -COR ³ 、-SOR ³ 、-SO ₂ R ³ and-SR ⁴ ；

-R ³ Selected from the group consisting of-H, alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -OR ⁹ and-NR ⁹ ₂ ；

-R ⁴ Selected from the group consisting of alkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl;

each-R ⁵ Independently selected from the group consisting of-H, alkyl, alkenyl alkyl, alkynyl alkyl, (OCH) ₂ CH ₂ ) _p O-alkyl (p is an integer from 1 to 1000), aryl, arylalkyl, heteroaryl, and heteroarylalkyl;

each-R ⁹ Independently selected from-H and alkyl, or two-R ⁹ Together with the nitrogen to which they are attached, form a heterocyclic ring;

and wherein the moiety of formula (PL-1) is optionally further substituted.

The following paragraphs describe this hydrogel in more detail.

In certain embodiments, -X of formula (PL-1) is selected from the group consisting of succinimide carbonate, thiosuccinimide carbonate halides, thioethers, esters, nitrophenyl carbonate, chloroformates, optionally substituted phenols, and formula (PL-2).

Wherein the method comprises the steps of

The dotted line indicates the connection to the rest of formula (PL-1);

-T is selected from the group consisting of-O-, -S-and-NR ⁶ -；

z is an integer selected from 1, 2, 3, 4, 5 and 6.

-X' -is absent OR selected from-OR ⁷ and-SR ⁷ ；

-Y ² Is a functional group capable of linking with a reactive polymer;

-R ⁶ selected from-H, alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl; and

-R ⁷ selected from the group consisting of alkylene, phenylene and (OCH) ₂ CH ₂ ) _p Wherein p is an integer of 1 to 1000.

In certain embodiments, -X of formula (PL-1) comprises an activated carbonate, such as succinimidyl carbonate, sulfosuccinimidyl carbonate, or nitrophenyl carbonate. In certain embodiments, -X of formula (PL-1) comprises a carbonyl halide, such as O (c=o) Cl or O (c=o) F. In certain embodiments, -X of formula (PL-1) has formula (PL-2). In certain embodiments, X of formula (PL-1) is-OR ⁷ or-SR ⁷ wherein-R is ⁷ Is optionally substituted alkylene, optionally substituted phenylene Or (OCH) ₂ CH ₂ ) _p Wherein p is 1 to 1000.

In certain embodiments, p of formula (PL-2) is an integer from 1 to 100. In certain embodiments, p of formula (PL-2) is an integer from 1 to 10.

In certain embodiments, -Y of formula (PL-1) ¹ and-Y of the formula (PL-2) ² Independently include-N ₃ 、-NH ₂ 、-NH-CO ₂ ^t Bu、-SH、-S ^t Bu, maleimide, -CO ₂ H、-CO ₂ ^t Bu, 1, 3-diene, cyclopentadiene, furan, alkyne, cyclooctyne, acrylate or acrylamide, where ^t Bu is tert-butyl, and wherein when-Y ¹ or-Y ² One of them contains N ₃ When the other does not contain alkyne or cyclooctyne. when-Y ¹ or-Y ² When one of them contains-SH, the other oneDoes not contain maleimide, acrylate or acrylamide; when-Y ¹ or-Y ² One of them comprises-NH ₂ When another does not contain-CO ₂ H is formed; when-Y ¹ or-Y ² When one of them comprises a 1, 3-diene or cyclopentadiene, the other does not comprise furan.

In certain embodiments, the cleavable crosslinker compound is of formula (PL-3).

Wherein the method comprises the steps of

m is 0 or 1;

n is an integer selected from 1 to 1000;

s is 0, 1 or 2;

t is selected from 2, 4, 8, 16 and 32;

-W-is selected from-O (c=o) O-, -O (c=o) NH-, -O (c=o) S-, -O (c=o) NR ⁶ CH ₂ O-and-O (c=o) NR ⁶ S-；

-Q is a core group having a valence = t; which links the arms of the cleavable crosslinking compound;

wherein t is an integer selected from 2, 4, 8, 16 and 32, and

wherein-R ¹ 、-R ² and-R ⁵ As defined by formula (PL-1).

In certain embodiments, t of formula (PL-3) is 2. In certain embodiments, t of formula (PL-3) is 4. In certain embodiments, t of formula (PL-3) is 8. In certain embodiments, t of formula (PL-3) is 16. In certain embodiments, t of formula (PL-3) is 32.

In certain embodiments, the-Q of formula (PL-3) has a structure selected from the group consisting of

/>Wherein the dashed line represents the linkage to the remainder of the cleavable crosslinker compound.

In certain embodiments, -Q of formula (PL-3) has the structure of (PL-3-i). In certain embodiments, -Q of formula (PL-3) has the structure of (PL-3-ii). In certain embodiments, -Q of formula (PL-3) has the structure of (PL-3-iii).

In certain embodiments, the cleavable crosslinker compound is of formula (PL-3) wherein m is 0, n is about 100, s is 0, t is 4, -W-is-O (c=o) NH-, Q has the structure (PL-3 i), -R ² is-H, a-R ⁵ Is H and the other is-R ⁵ Is- (CH) ₂ ) ₅ N ₃ and-R ¹ Is (4-chlorophenyl) SO ₂ Is covered by-SO ₂ morpholinyl-SO ₂ or-CN substituted phenyl.

In certain embodiments, -Y of formula (PL-3) ¹ comprising-N ₃ 、-NH ₂ 、-NH-CO ₂ ^t Bu、-SH、-S ^t Bu, maleimide, -CO ₂ H、-CO ₂ ^t Bu, 1, 3-diene, cyclopentadiene, furan, alkyne, cyclooctyne, acrylate or acrylamide, where ^t Bu is tert-butyl.

In certain embodiments, each-Y of formula (PL-1) or (PL-3) ¹ and-Y of the formula (PL-2) ² Independently include-N ₃ 、-NH ₂ 、-NH-CO ₂ ^t Bu、-SH、-S ^t Bu, maleimide, -CO ₂ H、-CO ₂ ^t Bu, 1, 3-diene, cyclopentadiene, furan, alkyne, cyclooctyne, acrylate or acrylamide.

In certain embodiments, -Y ¹ and-Y ² One of which is an azide and the other is a reactive functional group selected from acetylene, cyclooctyne and maleimide. In certain embodiments, -Y ¹ and-Y ² One of which is a thiol and the other is a reactive functional group selected from maleimide, acrylate, acrylamide, vinyl sulfone, vinyl sulfonamide and halocarbonyl. In certain embodiments, -Y ¹ and-Y ² One of which is an amine and the other isA selectively reactive functional group selected from carboxylic acids and reactive carboxylic acids. In certain embodiments, -Y ¹ and-Y ² One is maleimide and the other is a selectively reactive functional group selected from 1, 3-diene, cyclopentadiene and furan.

In certain embodiments, the first and any second polymer are selected from the group consisting of homo-or co-poly polyethylene glycol, polypropylene glycol, poly (N-vinylpyrrolidone), polymethacrylate, polyphosphazene, polylactide, polyacrylamide, polyethylene glycol ester, polyethyleneimine, agarose, dextrin, gelatin, collagen, polylysine, chitosan, alginate, hyaluronic acid, pectin, and carrageenan, which contain suitable reactive functional groups, or are of formula [ Y ] ³ -(CH ₂ ) _s (CH ₂ CH ₂ O) _n ] _t Q, wherein-Y ³ Is a reactive functional group, s is 0, 1 or 2, n is an integer selected from 10 to 1000, -Q is a core group having a valence t, and t is an integer selected from 2, 4, 8, 16 and 32.

In certain embodiments, the first polymer comprises a multi-arm polymer. In certain embodiments, the first polymer comprises at least three arms. In certain embodiments, the first polymer comprises at least four arms. In certain embodiments, the first polymer comprises at least five arms. In certain embodiments, the first polymer comprises at least six arms. In certain embodiments, the first polymer comprises at least seven arms. In certain embodiments, the first polymer comprises at least eight arms.

In certain embodiments, the second polymer comprises a multi-arm polymer. In certain embodiments, the second polymer comprises at least three arms. In certain embodiments, the second polymer comprises at least four arms. In certain embodiments, the second polymer comprises at least five arms. In certain embodiments, the second polymer comprises at least six arms. In certain embodiments, the second polymer comprises at least seven arms. In certain embodiments, the second polymer comprises at least eight arms.

In certain embodiments, the first polymer comprises a 2-arm polyethylene glycol polymer. In certain embodiments, the first polymer comprises a 4-arm polyethylene glycol polymer. In certain embodiments, the first polymer comprises an 8-arm polyethylene glycol polymer. In certain embodiments, the first polymer comprises a 16-arm polyethylene glycol polymer. In certain embodiments, the first polymer comprises a 32-arm polyethylene glycol polymer.

In certain embodiments, the second polymer comprises a 2-arm polyethylene glycol polymer. In certain embodiments, the second polymer comprises a 4-arm polyethylene glycol polymer. In certain embodiments, the second polymer comprises an 8-arm polyethylene glycol polymer. In certain embodiments, the second polymer comprises a 16-arm polyethylene glycol polymer. In certain embodiments, the second polymer comprises a 32-arm polyethylene glycol polymer.

In certain embodiments, the first and second reactive polymers are reacted with the cleavable crosslinker compound sequentially or simultaneously.

In certain embodiments, the first and second functional groups are the same.

Only the terms used in the ranges of formulas (PL-1), (PL-2) and (PL-3) have the following meanings.

The term "moiety capable of cleavage by elimination under physiological conditions" means a moiety comprising the group H-C- (ch=ch) _m -C-X ' wherein m is 0 or 1 and X ' is a leaving group, wherein the elimination reaction removes HX ' elements as described above at such a rate that the half-life of the reaction is between 1 and 10,000 hours under physiological conditions of pH and temperature. Preferably, the half-life of the reaction is between 1 and 5,000 hours, more preferably between 1 and 1,000 hours, under physiological conditions of pH and temperature. By physiological conditions of pH and temperature is meant a pH between 7 and 8 and a temperature between 30 and 40 degrees Celsius.

The terms "reactive polymer and reactive oligomer" refer to polymers or oligomers comprising functional groups reactive with other functional groups, most preferably under mild conditions compatible with the stability requirements of peptides, proteins and other biological macromolecules. Suitable functional groups present in the reactive polymer include maleimides, thiols or protected thiols, alcohols, acrylates, acrylamides, amines or protected amines, carboxylic acids or protected carboxylic acids, azides, alkynes including cyclic olefins, 1, 3-dienes including cyclopentadiene and furan, alpha-halohydrocarbons, and N-hydroxysuccinimides, N-hydroxysulfosuccinimides, or nitrophenyl esters or carbonates.

The term "functional group capable of linking with a reactive polymer" refers to a functional group capable of reacting with a corresponding functional group of a reactive polymer, thereby forming a covalent bond with the polymer. Suitable functional groups capable of attachment to the reactive polymer include maleimides, thiols or protected thiols, acrylates, acrylamides, amines or protected amines, carboxylic acids or protected carboxylic acids, azides, alkynes including cycloalkynes, 1, 3-dienes including cyclopentadiene and furan, alpha-halohydrocarbons, and N-hydroxysuccinimides, N-hydroxysulfosuccinimides, or nitrophenyl esters or carbonates.

The term "substituted" refers to an alkyl, alkenyl, alkynyl, aryl, or heteroaryl group containing one or more substituents in place of one or more hydrogen atoms. The substituents may generally be selected from halogen, including-F, -CI, -Br and-I; lower alkyl including straight chain, branched, and cyclic; lower haloalkyl including fluoroalkyl, chloroalkyl, bromoalkyl, and iodoalkyl; -OH; lower alkoxy groups include straight chain, branched, and cyclic. -SH; lower alkylthio, including straight, branched, and cyclic; amino, alkylamino, dialkylamino, silyl, including alkylsilane, alkoxysilane, and arylsilane; a nitro group; cyano group; a carbonyl group; carboxylic acids, carboxylic acid esters, carboxylic acid amides; an aminocarbonyl group; an aminoacetyl group; a carbamate; urea; a thiocarbamate; thiourea; a ketone; sulfone; sulfonamide; aryl groups include phenyl, naphthyl and anthracenyl; heteroaryl includes 5-membered heteroaryl groups including pyrrole, imidazole, furan, thiophene, oxazole, thiazole, isoxazole, isothiazole, thiadiazole, triazole, oxadiazole and tetrazole, 6-membered heteroaryl groups including pyridine, pyrimidine, pyrazine, and fused heteroaryl groups including benzofuran, benzothiophene, benzoxazole, benzimidazole, indole, benzothiazole, benzisoxazole and benzisothiazole.

-R ¹ and-R ² The properties of (c) can be adjusted by optionally adding electron donating or electron withdrawing substituents. The term "electron donating group" refers to the group that causes R ¹ R ² Substituents with reduced CH acidity; electron donating groups are generally associated with negative Hammett σ or Taft σ constants and are well known in the physical organic chemistry arts. (Hammett constant refers to aryl/heteroaryl substituents, taft constant refers to substituents on non-aromatic moieties). Examples of suitable electron donating substituents include lower alkyl, lower alkoxy, lower alkylthio, amino, alkylamino, dialkylamino, and silyl.

The term "electron withdrawing group" refers to a group that causes R ¹ R ² A substituent having an increased acidity of the CH group; electron withdrawing groups are generally associated with positive Hammett σ or Taft σ constants and are well known in the art of physical organic chemistry. Examples of suitable electron withdrawing substituents include halogen, difluoromethyl, trifluoromethyl, nitro, cyano, C (=o) -R ^x wherein-R is ^x Is H, lower alkyl, lower alkoxy or amino, or S (O) _m R ^Y Wherein m is 1 or 2, R ^Y Is lower alkyl, aromatic or heteroaryl. As is well known in the art, the electronic influence of substituents may depend on the position of the substituent. For example, an alkoxy substituent at the ortho or para position of an aromatic ring is electron donating and is characterized by a negative Hammett sigma constant, while an alkoxy substituent at the meta position of the aromatic ring is electron withdrawing and is characterized by a positive Hammett sigma constant.

The terms "alkyl", "alkenyl" and "alkynyl" include straight, branched or cyclic hydrocarbon groups of 1 to 8 carbons or 1 to 6 carbons or 1 to 4 carbons, wherein alkyl is a saturated hydrocarbon, alkenyl includes one or more carbon-carbon double bonds, and alkynyl includes one or more carbon-carbon triple bonds. Unless otherwise specified, these contain 1 to 6 carbon atoms.

The term "aryl" includes aromatic hydrocarbon groups of 6 to 18 carbons, preferably 6 to 10 carbons, including groups such as phenyl, naphthyl and anthracenyl. "heteroaryl" includes aromatic rings consisting of 3 to 15 carbons containing at least one N, O or S atom, preferably 3 to 7 carbons containing at least one N, O or S atom, including, for example, pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolinyl, indolyl, indenyl, and the like.

The term "halogen" includes fluorine, chlorine, bromine and iodine.

The term "maleimide" refers to a group of the formula

In certain embodiments, Z' is a hydrogel as disclosed in WO2020/206358 A1. In particular, in certain embodiments, Z' is a hydrogel, the method of manufacture comprising the steps of:

(a) Providing a polymer comprising multiple arms-P ² Wherein the first prepolymer is of the formula (PL-4)

Wherein the method comprises the steps of

n is an integer selected from 0, 1, 2, 3, 4, 5 and 6;

r is an integer greater than 2;

-Y is a reactive functional group for linking the first prepolymer and the second prepolymer;

-R ¹ and-R ² Independently an electron withdrawing group, an alkyl group, or-H, and wherein-R ¹ and-R ² At least one of which is an electron withdrawing group;

each-R ⁴ Are all independently C ₁ -C ₃ Alkyl, or two-R4 together with the carbon atom to which they are attached form a 3 to 6 membered ring;

w-is absent or is

Wherein the dotted line marked with an asterisk represents the connection to-NH-, and the non-marked dotted line represents the connection to-P ² Is connected with the connecting part of the connecting part;

x, y and z are each independently integers selected from 0, 1, 2, 3, 4, 5 and 6;

-B' is-NH ₂ 、-ONH ₂ Ketones, aldehydes, -SH, -OH, -CO ₂ H. Carboxamide groups, or groups comprising cyclooctyne or bicyclononene; and

-C is carboxamide, thioether, thiosuccinimide, triazole or oxime;

(b) Providing a polymer comprising multiple arms-P ¹ Wherein each arm is terminated by a reactive functional group-Y "that reacts with-Y of step (a);

(c) Mixing the two prepolymers of steps (a) and (b) under conditions wherein-Y and-Y "react to form a linkage-Y; optionally, a plurality of

(d) The resulting hydrogel was isolated.

Thus, -Z' is a hydrogel obtainable from the above process. In certain embodiments, the hydrogels produced by the foregoing methods are degradable.

In certain embodiments, -Y and-Y "are reacted under step (c) to form an insoluble hydrogel matrix comprising crosslinks of formula (PL-4'):

wherein n, r, -P ¹ 、-Y*-、-R ⁴ 、-R ¹ 、-R ² (W) and (P) ² As defined above.

In certain embodiments, n of formula (PL-4) or (PL-4') is an integer selected from 1, 2, 3, 4, 5 and 6. In certain embodiments, n of formula (PL-4) or (PL-4') is an integer selected from 1, 2 and 3. In certain embodiments, n of formula (PL-4) or (PL-4') is an integer selected from 0, 1, 2, and 3. In certain embodiments, n of formula (PL-4) or (PL-4') is 1. In certain embodiments, n of formula (PL-4) is 2. In certain embodiments, n of formula (PL-4) or (PL-4') is 3.

In certain embodiments, a multi-arm-p of formula (PL-4) or (PL-4') ² Is an r-arm polymer, wherein r is an integer selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In certain embodiments, r of formula (PL-4) or (PL-4') is an integer selected from 2, 3, 4, 5, 6, 7 and 8. In certain embodiments, r of formula (PL-4) or (PL-4') is an integer selected from 2, 4, 6 and 8. In certain embodiments, r of formula (PL-4) or (PL-4') is 2. In certain embodiments, r of formula (PL-4) or (PL-4') is 4. In certain embodiments, r of formula (PL-4) or (PL-4') is 6. In certain embodiments, r of formula (PL-4) or (PL-4') is 8.

In certain embodiments, the moiety-P of formula (PL-4) or (PL-4') ² Has a molecular weight of at least 1kDa. In certain embodiments, the moiety-P of formula (PL-4) or (PL-4') ² Has a molecular weight of 1 to 100kDa. In certain embodiments, the moiety-P of formula (PL-4) or (PL-4') ² Has a molecular weight of 1 to 80kDa. In certain embodiments, the moiety-P of formula (PL-4) or (PL-4') ² Has a molecular weight of 1 to 60kDa. In certain embodiments, the moiety-P of formula (PL-4) or (PL-4') ² Has a molecular weight of 1 to 40kDa. In certain embodiments, the moiety-P of formula (PL-4) or (PL-4') ² Has a molecular weight of 1 to 20kDa. In certain embodiments, the moiety-P of formula (PL-4) or (PL-4') ² Has a molecular weight of 1 to 10kDa. In certain embodiments, the moiety-P of formula (PL-4) or (PL-4') ² Has a molecular weight of 1 to 5kDa. In certain embodiments, the moiety-P of formula (PL-4) or (PL-4') ² Has a molecular weight of about 20kDa. In certain embodiments, the moiety-P of formula (PL-4) or (PL-4') ² Has a molecular weight of about 40kDa. In certain embodiments, the moiety-P of formula (PL-4) or (PL-4') ² Has a molecular weight of about 60kDa. In certain embodiments, the moiety-P of formula (PL-4) or (PL-4') ² Has a molecular weight of about 80kDa.

In certain embodiments, the multi-arm polymer of step (b) -P ¹ Is an r-arm polymer, wherein r is an integer selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In certain embodiments, the multi-arm-P of step (b) ¹ Is an r-arm polymer, wherein r is an integer selected from 2, 3, 4, 5, 6, 7 and 8. In certain embodiments, the multi-arm-P of step (b) ¹ Is an r-arm polymer, wherein r is an integer selected from 2, 4, 6 and 8. In certain embodiments, the multi-arm-P of step (b) ¹ Is an r-arm polymer, where r is 2. In certain embodiments, the multi-arm-P of step (b) ¹ Is an r-arm polymer, where r is 4. In certain embodiments, the multi-arm P of step (b) ¹ Is an r-arm polymer, where r is 6. In certain embodiments, the multi-arm P of step (b) ¹ -an r-arm polymer, wherein r is 8.

In certain embodiments, -P of step (b) ¹ Has a molecular weight of at least 1kDa. In certain embodiments, the multi-arm polymer-P of step (b) ¹ Has a molecular weight of 1 to 100kDa. In certain embodiments, the multi-arm polymer-P of step (b) ¹ Has a molecular weight of 1 to 80kDa. In certain embodiments, the multi-arm polymer-P of step (b) ¹ Has a molecular weight of 1 to 60kDa. In certain embodiments, the multi-arm polymer-P of step (b) ¹ Has a molecular weight of 1 to 40kDa. In certain embodiments, the multi-arm polymer-P of step (b) ¹ Has a molecular weight of 1 to 20kDa. In certain embodiments, the multi-arm polymer-P of step (b) ¹ Has a molecular weight of 1 to 10kDa. In certain embodiments, the multi-arm polymer-P of step (b) ¹ Has a molecular weight of 1 to 5kDa. In certain embodiments, the multi-arm polymer-P of step (b) ¹ Has a molecular weight of about 20kDa. In certain embodiments, the multi-arm polymer-P of step (b) ¹ Has a molecular weight of about 40kDa. In certain embodiments, the multi-arm polymer-P of step (b) ¹ Has a molecular weight of about 60kDa. In certain embodiments, the multi-arm polymer-P of step (b) ¹ Has a molecular weight of about 80kDa.

In certain embodiments, -P of step (b) ¹ and-P of the formula (PL-4) or (PL-4') ² Comprises poly (ethylene glycol) (PEG), poly (ethylene oxide) (PEO), poly (ethyleneimine) (PEI), dextran, hyaluronic acid or copolymers thereof. In certain embodiments, -P of step (b) ¹ and-P of the formula (PL-4) or (PL-4') ² Is a PEG-based polymer. In certain embodiments, -P of step (b) ¹ and-P of the formula (PL-4) or (PL-4') ² Is a hyaluronic acid-based polymer.

In certain embodiments, the group-R of formula (PL-4) or (PL-4') ¹ and-R ² Independently an electron withdrawing group, an alkyl group, or-H, wherein-R ¹ and-R ² At least one of which is an electron withdrawing group.

In certain embodiments, the group-R of formula (PL-4) or (PL-4') ¹ and-R ² is-CN, -NO ₂ Optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, -COR ³ -SOR3 or-SO ₂ R ³ wherein-R3 is-H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR ⁸ or-NR ⁸ ₂ Wherein each-R ⁸ Independently are-H or optionally substituted alkyl, or two-R ⁸ The groups together with the nitrogen to which they are attached form a heterocyclic ring; or SR (S.J) ⁹ wherein-R is ⁹ Is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl or optionally substituted heteroarylalkyl.

In certain embodiments, the group-R of formula (PL-4) or (PL-4') ¹ and-R ² is-CN. In certain embodiments, the group-R of formula (PL-4) or (PL-4') ¹ and-R ² is-NO ₂ . In certain embodiments, the group-R of formula (PL-4) or (PL-4') ¹ and-R ² Is an optionally substituted aryl group containing 6 to 10 carbons. In certain embodiments, the group-R in formula (PL-4) or (PL-4') ¹ and-R ² Is an optionally substituted phenyl, naphthyl or anthracenyl group. In certain embodiments-R of formula (PL-4) or (PL-4') ¹ and-R ² Is an optionally substituted heteroaryl group comprising 3 to 7 carbon atoms and containing at least one N, O or S atom. In certain embodiments, the group-R of formula (PL-4) or (PL-4') ¹ and-R ² Is an optionally substituted pyrrolyl, pyridinyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolinyl, indolyl or indenyl group. In certain embodiments, the group-R of formula (PL-4) or (PL-4') ¹ and-R ² Is an optionally substituted alkenyl group containing 2 to 20 carbon atoms. In certain embodiments, the group-R of formula (PL-4) or (PL-4') ¹ and-R ² Is an optionally substituted alkyne containing from 2 to 20 carbon atoms. In certain embodiments, the group-R of formula (PL-4) or (PL-4') ¹ and-R ² is-COR ³ 、-SOR ³ or-S0 ₂ R ³ wherein-R is ³ is-H, optionally substituted alkyl having 1 to 20 carbon atoms, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR ⁸ or-NR ⁸ ₂ Wherein each-R ⁸ Independently are-H or optionally substituted alkyl containing 1 to 20 carbon atoms, or two-R ⁸ The groups together with the nitrogen to which they are attached form a heterocyclic ring. In certain embodiments, the group-R of formula (PL-4) or (PL-4') ¹ and-R ² is-SR ⁹ wherein-R9 is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl or optionally substituted heteroarylalkyl containing from 1 to 20 carbon atoms. In certain embodiments, -R ¹ and-R ² At least one of them is-CN or-SO ₂ R ³ 。

In certain embodiments, the group-R of formula (PL-4) or (PL-4') ¹ and-R ² At least one of them is-CN, -SOR ³ or-SO ₂ R ³ . In certain embodiments, the group-R of formula (PL-4) or (PL-4') ¹ and-R ² At least one of them is-CN or-SO ₂ R ³ . In certain embodiments, the group-R of formula (PL-4) or (PL-4') ¹ and-R ² At least one of them is-CN or-SQ ₂ R ³ wherein-R is ³ Is optionally substituted alkyl, optionally substituted aryl or-NR ⁸ ₂ . In certain embodiments, the group-R of formula (PL-4) or (PL-4') ¹ and-R ² At least one of them is-CN, -SO ₂ N(CH ₃ ) ₂ 、-SO ₂ CH ₃ is-S0 ₂ Substituted phenyl, substituted-SO ₂ and-Cl-substituted phenyl, -SO ₂ N(CH ₂ CH ₂ ) ₂ O、-SO ₂ CH(CH ₃ ) ₂ 、-SO ₂ N(CH ₃ )(CH ₃ ) or-SO ₂ N(CH ₂ CH ₂ OCH ₃ ) ₂ 。

In certain embodiments, each-R in formula (PL-4) or (PL-4') ⁴ Independently C ₁ -C ₃ Alkyl groups, or taken together may form a 3 to 6 membered ring. In certain embodiments, each R4 of formula (PL-4) or (PL-4') is independently C ₁ -C ₃ An alkyl group. In certain embodiments, two-R of formula (PL-4) or (PL-4') ⁴ Are all methyl groups.

In certain embodiments, -Y and-Y "are independently selected from the group consisting of amines, aminooxy, ketones, aldehydes, maleimides, thiols, alcohols, azides, 1,2,4, 6-tetrazinyl, trans-cyclooctenyl, bicyclononyl, cyclooctynyl, and protected variants thereof.

In certain embodiments, Y and Y "may react with each other, e.g., in a selective manner. For example, when-Y is an amine, -Y "is a carboxylic acid, an active ester or an active carbonate to produce a residual linking functionality-Y-, which is an amide or a carbamate. For another example, when one Y is azide, -Y "is alkynyl, bicyclononyl or cyclooctynyl, the remaining linking functionality produced-Y-is 1,2, 3-triazole. As another example, when-Y is-NH ₂ At O, -Y "is a ketone or aldehyde and the resulting residual linking functionality-Y-is an oxime. As another example, when-Y is-SH, -Y' is maleimide or halocarbonyl, yielding The remaining linking functional groups-Y-are thiosuccinimide groups or thioethers. Likewise, the effects of-Y and-Y "may be reversed, yielding-Y in the opposite direction.

In certain embodiments, -Y-comprises an amide, an oxime, a 1,2, 3-triazole, a thioether, a thiosuccinimide, or an ether. In certain embodiments, -Y is-L ² -。

These conjugation reactions can be carried out under conditions known in the art, for example, when-Y is azide and-Y "is ring Xin Guishi, the conjugation reaction occurs in any solvent in which both components exhibit sufficient solubility, although aqueous solutions are known to exhibit more favorable reaction rates. When mixed in a suitable solvent, the-Y and-Y 'groups react to form an insoluble hydrogel matrix comprising crosslinks of formula (PL-4') when-Y and-Y 'are azide/cyclooctyne, typically in an aqueous buffer having a pH of 2 to 7, or in a buffer having a pH of 6 to 9 when-Y and-Y' are active esters and amines. This process can be carried out in bulk phase or under emulsifying conditions in an organic/aqueous mixed system to form a suspension of microparticles, such as microspheres, suitable for injection.

In certain embodiments, the conjugate comprising hydrogel Z' is produced by a method comprising the steps of

(a) Providing a first prepolymer of formula (PL-4)

(b) Reacting the prepolymer of formula (PL-4) with the linker-drug of formula (PL-5)

Wherein the method comprises the steps of

n、R ¹ 、R ² 、R ⁴ and-Y is as defined in formula (PL-4);

-D is a drug moiety;

when-D is a drug moiety linked through an amine, -X-is absent, or when-D-is a drug moiety linked through a phenol, alcohol, thiol, thiophene, imidazole or non-basic amine, -X-is-N (R ⁶ )CH ₂ The method comprises the steps of carrying out a first treatment on the surface of the wherein-R ⁶ Is optionally substituted C ₁ -C ₆ An alkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group;

thereby reacting-Y of formula (PL-5) with-B' of formula (PL-4);

(c) Providing a polymer comprising multiple arms-P ¹ Wherein each arm is terminated by a reactive functional group-Y' which reacts with-Y of step (a), wherein-P ¹ The embodiments of (a) are as described above;

(d) Mixing the two prepolymers of steps (a) and (b) under conditions wherein-Y and-Y "react to form the remaining linking functionality-Y; optionally, a plurality of

(e) The resulting hydrogel was isolated.

In certain embodiments, the conjugate is obtained by a method comprising the step of reacting a hydrogel Z ' with a linker-drug of formula (PL-5), wherein-B ' on the hydrogel Z ' is reacted with-Y of formula (PL-5).

Only within the ranges of formulas (PL-4), (PL-4') and (PL-5), the terms used have the following meanings.

The term "alkyl" refers to a straight, branched or cyclic saturated hydrocarbon group of 1 to 20, 1 to 12, 1 to 8, 1 to 6 or 1 to 4 carbon atoms. In some cases, the alkyl group is linear or branched. Examples of straight or branched alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl and n-decyl. In certain embodiments, the alkyl group is cyclic. Examples of cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, and cyclohexyl.

The term "alkoxy" refers to an alkyl group bonded to oxygen and includes methoxy, ethoxy, isopropoxy, cyclopropyloxy and cyclobutoxy groups.

The term "alkenyl" refers to non-aromatic unsaturated hydrocarbons having a carbon-carbon double bond and 2 to 20, 2 to 12, 2 to 8, 2 to 6, or 2 to 4 carbon atoms.

The term "alkyne" refers to a non-aromatic unsaturated hydrocarbon having a carbon-carbon triple bond and from 2 to 20, 2 to 12, 2 to 8, 2 to 6, or 2 to 4 carbon atoms.

The term "aryl" refers to aromatic hydrocarbon groups of 6 to 18 carbons, preferably 6 to 10 carbons, including groups such as phenyl, naphthyl and anthracenyl. The term "heteroaryl" refers to an aromatic ring containing 3 to 15 carbons and containing at least one N, O or S atom, preferably 3 to 7 carbons and containing at least one N, O or S atom, including, for example, pyrrolyl, pyridinyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolinyl, indolyl, and indenyl.

In certain embodiments, the alkenyl, alkynyl, aryl, or heteroaryl moiety may be coupled to the remainder of the molecule through an alkyl linkage. In these cases, the substituent will be referred to as an alkenyl alkyl, alkynyl alkyl, arylalkyl or heteroarylalkyl, indicating that there is an alkylene moiety between the alkenyl, alkynyl, aryl or heteroaryl moiety and the molecule to which the alkenyl, alkynyl, aryl or heteroaryl is coupled.

The term "halogen" or "halo" refers to bromine, fluorine, chlorine and iodine.

The term "heterocycle" or "heterocyclyl" refers to a 3 to 15 membered aromatic or non-aromatic ring containing at least one N, O or S atom. Examples include piperidinyl, piperazinyl, tetrahydropyranyl, pyrrolidinyl, and tetrahydrofuranyl, as well as exemplary groups provided above for the term "heteroaryl". In certain embodiments, the heterocycle or heterocyclyl is non-aromatic. In certain embodiments, the heterocycle or heterocyclyl is aromatic.

The term "optionally substituted" means that one group may be unsubstituted or may be substituted with one or more (e.g., 1, 2, 3, 4, or 5) substituents, which may be the same or different. Examples of substituents include alkyl, alkenyl, alkynyl, halogen, -CN, -OR ^aa 、-SR ^aa 、-NR ^aa R ^bb 、-NO ₂ 、-C＝NH(OR ^aa )、-C(O)R ^aa 、-OC(O)R ^aa 、-C(O)OR ^aa 、-C(O)NR ^aa R ^bb 、-OC(O)NR ^aa R ^bb 、-NR ^aa C(O)R ^bb 、-NR ^aa C(O)OR ^bb 、-S(O)R ^aa 、-S(O) ₂ R ^aa 、-NR ^aa S(O)R ^bb 、-C(O)NR ^aa S(O)R ^bb 、-NR ^aa S(O) ₂ R ^bb 、-C(O)NR ^aa S(O) ₂ R ^bb 、-S(O)NR ^aa R ^bb 、-S(O) ₂ NR ^aa R ^bb 、-P(O)(OR ^aa )(OR ^bb ) A heterocyclic group, a heteroaryl group or an aryl group, wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, heteroaryl and aryl groups are each independently substituted with-R ^cc Substitution, wherein-R ^aa and-R ^bb Each independently is-H, alkyl, alkenyl, alkynyl, heterocyclyl, heteroaryl, or aryl, or-R ^aa and-R ^bb Together with the nitrogen atom to which they are attached, form a heterocyclic group optionally substituted with alkyl, alkenyl, alkynyl, halogen, hydroxy, alkoxy or —cn, and wherein: -R ^cc Each independently is alkyl, alkenyl, alkynyl, halo, heterocyclyl, heteroaryl, aryl, -CN, or NO ₂ 。

part-L ¹ -can be linked to-D by an IL-2 moiety of formula (I), in particular by one amino acid residue of said IL-2 moiety present in-D. In certain embodiments, -L ¹ -through the IL-2 moiety, in particular through one amino acid residue of the IL-2 moiety.

In certain embodiments, all of the moieties-L present in the IL-2 conjugate ¹ All linked to the amino acid residue of-D.

if-L ¹ -an amino acid residue attached to the IL-2 molecule, which amino acid residue may be a proteinogenic (proteinogenic) or nonproteinaceous amino acid residue of-D. In certain embodiments, -L ¹ -linking to a non-proteinogenic amino acid residue. In certain embodiments, -L ¹ To proteinogenic amino acid residues. If the linkage occurs at a proteinogenic amino acid residue selected from the group consisting of cysteine, methionine, histidine, lysine, tryptophan, serine, threonine, tyrosine, aspartic acid, glutamic acid, glutamine, and arginine in certain embodimentsAcid groups. In certain embodiments, such proteinaceous amino acid residues are selected from the group consisting of cysteine, histidine, lysine, tryptophan, serine, threonine, tyrosine, aspartic acid, glutamic acid, and arginine.

In certain embodiments, -L ¹ -to a cysteine residue of-D. In certain embodiments, -L ¹ -to a histidine residue of-D. In certain embodiments, -L ¹ -linked to a lysine residue. In certain embodiments, -L ¹ -a tryptophan residue. In certain embodiments, -L ¹ -to serine residues. In certain embodiments, -L ¹ -linkage to threonine residues. In certain embodiments, -L ¹ -linked to a tyrosine residue. In certain embodiments, -L ¹ -linked to an aspartic acid residue. In certain embodiments, -L ¹ -linking to a glutamic acid residue. In certain embodiments, -L ¹ -linked to an arginine residue.

In certain embodiments, at least one molecule-L ¹ -one or more additional molecules-L linked to one amino acid residue of-D ¹ -linking to a modification present in-D.

The molecule-L ¹ The connection to-D may be through any type of connection, provided that it is reversible. In certain embodiments, -L ¹ -linked to-D by a linkage selected from amide, ester, carbamate, acetal, aminal, imine, oxime, hydrazone, disulfide and acylguanidine. In certain embodiments, -L ¹ -to-D by a linkage selected from the group consisting of amide, ester, carbamate and acylguanidine. It will be appreciated that these connections may themselves be irreversible, but the reversibility may be-L ¹ -the effect of certain groups or moieties present in the molecule.

In certain embodiments, -L ¹ -to-D by an ester linkage. In certain embodiments, -L ¹ -to-D by a carbamate linkage. In certain embodiments, -L ¹ -linked to-D by means of an acylguanidine. In certain embodiments, -L ¹ -to-D by an amide linkage.

In certain embodiments, -L ¹ -attached to-D through the nitrogen of the amine function of the lysine residue side chain of-D. In certain embodiments, -L ¹ -attached to-D through the nitrogen of the amine function of the lysine residue side chain of-D. and-D and-L ¹ The bond formed between them is a carbamate.

In certain embodiments, -L ¹ Has the structure disclosed in WO 2009/095479 A2. Thus, in certain embodiments, the moiety-L ¹ -is of formula (II):

wherein the dashed line represents the linkage to the nitrogen of-D through the formation of an amide bond;

-X-is-C (R) ⁴ R ^4a )-；-N(R ⁴ )-；-O-；-C(R ⁴ R ^4a )-C(R ⁵ R ^5a )-；-C(R ⁵ R ^5a )-C(R ⁴ R ^4a )-；-C(R ⁴ R ^4a )-N(R ⁶ )-；-N(R ⁶ )-C(R ⁴ R ^4a )；-C(R ⁴ R ^4a )-O-；-O-C(R ⁴ R ^4a ) -; or-C (R) ⁷ R ^7a )-；

X ¹ Is C; or S (O);

-X ² -is-C (R) ⁸ R ^8a ) -; or-C (R) ⁸ R ^8a )-C(R ⁹ R ^9a )-；

＝X ³ Is=o; =s; or = N-CN;

-R ¹ 、-R ^1a 、-R ² 、-R ^2a 、-R ⁴ 、-R ^4a 、-R ⁵ 、-R ^5a 、-R ⁶ 、-R ⁸ 、-R ^8a 、-R ⁹ 、-R ^9a independently selected from-H; and C _1-6 An alkyl group;

-R ³ 、-R ^3a independently selected from-H; and C _1-6 Alkyl, provided that at-R ³ 、R ^3a In the case where one or both are not-H, they pass through an SP ³ The hybridized carbon atoms are linked to the N to which they are attached.

-R ⁷ is-N (R) ¹⁰ R ^10a ) The method comprises the steps of carrying out a first treatment on the surface of the or-NR ¹⁰ -(C＝O)-R ¹¹¹¹ ；

-R ^7a 、-R ¹⁰ 、-R ^10a 、-R ¹¹ Independently of each other, -H; or C _1-6 An alkyl group;

optionally, one or more pairs of-R ^1a /-R ^4a 、-R ^1a /-R ^5a 、-R ^1a /-R ^7a 、-R ^4a /-R ^5a 、-R ^8a /-R ^9a Forming a chemical bond;

optionally, one or more pairs of-R ¹ /-R ^1a 、-R ² /-R ^2a 、-R ⁴ /-R ^4a 、-R ⁵ /-R ^5a 、-R ⁸ /-R ^8a 、-R ⁹ /-R ^9a Together with the atoms to which they are attached form C _3-10 Cycloalkyl; or a 3 to 10 membered heterocyclyl;

optionally, one or more pairs of-R ¹ /-R ⁴ 、-R ¹ /-R ⁵ 、-R ¹ /-R ⁶ 、-R ¹ /-R ^7a 、-R ⁴ /-R ⁵ 、-R ⁴ /-R ⁶ 、-R ⁸ /-R ⁹ 、-R ² /-R ³ Together with the atoms to which they are attached form a ring a;

optionally, -R ³ /-R ^3a Together with the nitrogen atom to which they are attached, form a 3-to 10-membered heterocyclic ring;

a is selected from: a phenyl group; a naphthyl group; an indenyl group; indanyl; tetrahydronaphthyl; c (C) _3-10 Cycloalkyl; 3-10 membered heterocyclyl; and 8-11 membered heterobicyclic groups; and

wherein-L ¹ -is at least one-L ² -Z or-L ² -Z' substitution, and wherein-L ¹ -optionally further substituted, provided that the hydrogen marked with an asterisk in formula (II) is not replaced by-L ² -Z、-L ² -Z' or a substituent.

In some embodimentsIn case, -L ¹ -having formula (II), wherein the dashed line represents the nitrogen of the amine attached to the lysine side chain of-D.

In certain embodiments, -L ¹ -having formula (II), wherein the dashed line represents the nitrogen of the amine attached to the N-terminal end of-D.

Preferably, the radical of formula (II) -L ¹ -is covered by a part-L ² -Z or-L ² -Z' substitution.

In certain embodiments, -L of formula (II) ¹ -not further substituted.

It will be appreciated that if-R of formula (II) ³ /-R ^3a Together with the nitrogen atom to which it is attached, form a 3-to 10-membered heterocyclic ring, only 3-to 10-membered heterocyclic rings can be formed in which the atom directly attached to the nitrogen is sp ³ -a hybridised carbon atom. In other words, -R ³ /-R ^3a Such 3-to 10-membered heterocyclic ring formed together with the attached nitrogen atom has the following structure:

Wherein the method comprises the steps of

The dotted line indicates the connection to-L ¹ -the remainder of;

the ring contains 3-10 atoms containing at least one nitrogen; and is also provided with

R ^# And R is ^## Representing sp ³ -a hybridised carbon atom.

It is also understood that 3-to 10-membered heterocycles may be further substituted.

-R of formula (II) ³ /-R ^3a Exemplary embodiments of suitable 3-to 10-membered heterocycles formed with the attached nitrogen atom are as follows:

wherein the method comprises the steps of

The dashed line represents the remainder of the linkage to the molecule; and is also provided with

-R is selected from-H and C _1-6 An alkyl group.

-L of formula (II) ¹ May optionally be further substituted. In general, any substituent may be used as long as the cleavage principle is not affected, i.e. the hydrogen marked with an asterisk in formula (II) is not replaced and the nitrogen of the moiety of formula (II)

Retaining primary, secondary or tertiary amine moieties, i.e. -R ³ and-R ^3a Independently of one another-H or by sp ³ -the hybridised carbon atom is attached to-N <.

In certain embodiments, the group-R of formula (II) ¹ or-R ^1a quilt-L ² -Z or-L ² -Z' substitution. In certain embodiments, the group-R of formula (II) ² or-R ^2a quilt-L ² -Z or-L ² -Z' substitution. In certain embodiments, the group-R of formula (II) ³ or-R ^3a quilt-L ² -Z or-L ² -Z' substitution. In certain embodiments, the group-R of formula (II) ⁴ quilt-L ² -Z or-L ² -Z' substitution. In certain embodiments, the group-R of formula (II) ⁵ or-R ^5a quilt-L ² -Z or-L ² -Z' substitution. In certain embodiments, the group-R of formula (II) ⁶ quilt-L ² -Z or-L ² -Z' substitution. In certain embodiments, the group-R of formula (II) ⁷ or-R ^7a quilt-L ² -Z or-L ² -Z' substitution. In certain embodiments, the group-R of formula (II) ⁸ or-R ^8a quilt-L ² -Z or-L ² -Z' substitution. In certain embodiments, the group-R of formula (II) ⁹ or-R ^9a quilt-L ² -Z or-L ² -Z' substitution.

In certain embodiments, -L ¹ Has the structure as disclosed in WO2016/020373A 1. Thus, in certain embodiments, part-L ¹ -having formula (III):

wherein the method comprises the steps of

The dashed line represents a primary or secondary amine or hydroxyl group attached to-D by formation of an amide or ester linkage, respectively;

-R ¹ 、-R ^1a 、-R ² 、-R ^2a 、-R ³ and-R ^3a Are independently selected from-H, -C (R) ⁸ R ^8a R ^8b )、-C(＝O)R ⁸ 、-C＝N、-C(＝NR ⁸ )R ^8a 、-CR ⁸ (＝CR ^8a R ^8b )、-C≡CR ⁸ and-T;

-R ⁴ 、-R ⁵ and-R ^5a Are independently selected from-H, -C (R) ⁹ R ^9a R ^9b ) and-T;

a1 and a2 are independently 0 or 1;

-R ⁶ 、-R ^6a 、-R ⁷ 、-R ^7a 、-R ⁸ 、-R ^8a 、-R ^8b 、-R ⁹ 、-R ^9a 、-R ^9b each independently of the others is selected from the group consisting of-H, halogen, -CN, -COOR ¹⁰ 、-OR ¹⁰ ，-C(O)R ¹⁰ 、-C(O)N(R ¹⁰ R ^10a )、-S(O) ₂ N(R ¹⁰ R ^10a )、-S(O)N(R ¹⁰ R ^10a )、-S(O) ₂ R ¹⁰ ，-S(O)R ¹⁰ 、-N(R ¹⁰ )S(O) ₂ N(R ^10a R ^10b )、-SR ¹⁰ 、-N(R ¹⁰ R ^10a )、-NO ₂ 、-OC(O)R ¹⁰ ，-N(R ¹⁰ )C(O)R ¹⁰ a、-N(R ¹⁰ )S(O) ₂ R ^10a 、-N(R ¹⁰ )S(O)R ^10a 、-N(R ¹⁰ )C(O)OR ^10a 、-N(R ¹⁰ )C(O)N(R ^10a R ^10b )、-OC(O)N(R ¹⁰ R ^10a )、-T、C _1-20 Alkyl, C _2-20 Alkenyl and C _2-20 Alkynyl; wherein-T, C _1-20 Alkyl, C _2-20 Alkenyl and C _2-20 Alkynyl is optionally substituted with one or more of the same or different-R ¹¹ Substituted, and wherein C _1-20 Alkyl, C _2-20 Alkenyl and C _2-20 Alkynyl groups are optionally interrupted by one or more groups selected from the group consisting of-T-, -C (O) O-, -C (O) N (R) ¹² )-、-S(O) ₂ N(R ¹² )-、-S(O)N(R ¹² )-，-S(O) ₂ -、-S(O)-、-N(R ¹² )S(O) ₂ N(R ¹² a)-、-S-、-N(R ¹² )-、-OC(OR ¹² )(R ^12a )-，-N(R ¹² )C(O)N(R ^12a ) -and-OC (O) N (R) ¹² )-；

-R ¹⁰ 、-R ^10a 、-R ^10b Each independently selected from-H, -T, C _1-20 Alkyl, C _2-20 Alkenyl and C _2-20 Alkynyl; wherein-T, C _1-20 Alkyl, C _2-20 Alkenyl and C _2-20 Alkynyl is optionally substituted with one or more of the same or different-R ¹¹ Substituted, and wherein C _1-20 Alkyl, C _2-20 Alkenyl and C _2-20 Alkynyl groups are optionally interrupted by one or more groups selected from the group consisting of-T-, -C (O) O-, -C (O) N (R) ¹² )-、-S(O) ₂ N(R ¹² )-、-S(O)N(R ¹² )-、-S(O) ₂ -、-S(O)-、-N(R ¹² )S(O) ₂ N(R ^12a )-、-S-、-N(R ¹² )-、-OC(OR ¹² )(R ¹² a)-、-N(R ¹² )C(O)N(R ^12a ) -and-OC (O) N (R) ¹² )-；

T are each independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C _3-10 Cycloalkyl, 3-to 10-membered heterocyclyl and 8-to 11-membered heterobicyclic; wherein T is each independently optionally substituted with one or more of the same or different-R ¹¹ Substitution;

-R ¹¹ each independently of the others is selected from halogen, -CN, oxo (= O), -COOR ¹³ 、-OR ¹³ 、-C(O)R ¹³ 、-C(O)N(R ¹³ R ^13a )、-S(O) ₂ N(R ¹³ R ¹³⁴ )、-S(O)N(R ¹³ R ^13a )、-S(O) ₂ R ¹³ 、-S(O)R ¹³ 、-N(R ¹³ )S(O) ₂ N(R ^13a R ^13b )、-SR ¹³ 、-N(R ¹³ R ^13a )、-NO ₂ 、-OC(O)R ¹³ 、-N(R13)C(O)R ^13a 、-N(R ¹³ )S(O) ₂ R ^13a 、-N(R ¹³ )S(O)R ^13a 、-N(R ¹³ )C(O)OR ^13a 、-N(R ¹³ )C(O)N(R ^13a R1 ^3b )、-OC(O)N(R ¹³ R ^13a ) And C _1-6 An alkyl group; wherein C is _1-6 Alkyl is optionally substituted with one or more halogen, the same or different;

-R ¹² 、-R ^12a 、-R ¹³ 、-R ^13a、 -R ^13b each independently selected from-H and C ₁ - ₆ An alkyl group; wherein C is ₁ - ₆ Alkyl is optionally substituted with one or more halogen, the same or different;

optionally, one or more pairs of-R ¹ /-R ^la 、-R ² /-R ^2a 、-R ³ /-R ^3a 、-R ⁶ /-R ^6a 、-R ⁷ /-R ^7a Together with the atoms to which they are attached form C _3-10 Cycloalkyl or 3-to 10-membered heterocyclyl;

optionally, one or more pairs of-R ¹ /-R ² 、-R ¹ /-R ³ 、-R ¹ /-R ⁴ 、-R ¹ /-R ⁵ ，-R ¹ /-R ⁶ 、-R ¹ /-R ⁷ 、-R ² /-R ³ 、-R ² /-R ⁴ 、-R ² /-R ⁵ 、-R ² /-R ⁶ 、-R ² /-R ⁷ 、-R ³ /-R ⁴ 、-R ³ /-R ⁵ 、-R ³ /-R ⁶ 、-R ³ /-R ⁷ 、-R ⁴ /-R ⁵ 、-R ⁴ /-R ⁶ 、-R ⁴ /-R ⁷ 、-R ⁵ /-R ⁶ 、-R ⁵ /-R ⁷ 、-R ⁶ /-R ⁷ Together with the atoms to which they are attached form a ring a;

a is selected from phenyl; a naphthyl group; an indenyl group; indanyl; tetrahydronaphthyl; c (C) _3-10 Cycloalkyl; 3-to 10-membered heterocyclyl; and 8-to 11-membered heterobicycloyl;

wherein-L ¹ -is at least one-L ² -Z or-L ² -Z' substitution, and wherein-L ¹ Optionally further substituted.

-L of formula (III) ¹ The optional further substituents are preferably as described above.

Preferably, -L of formula (III) ¹ -is covered by a part-L ² -Z or-L ² -Z' substitution.

In certain embodiments, -L of formula (III) ¹ -not further substituted.

In certain embodiments, -L ¹ Having the structure as disclosed in EP1536334B1, WO2009/009712A1, WO2008/034122A1, WO2009/143412A2, WO2011/082368A2 and US8618124B2, which are incorporated herein by reference.

In certain embodiments, -L ¹ Having the structure as disclosed in US8946405B2 and US8754190B 2. Thus, in certain embodiments, -L ¹ -having formula (IV):

wherein the method comprises the steps of

The dotted line is represented by a chain selected from the group consisting of-OH, -SH, and-NH ₂ The functional group of-D of (c) is attached to-D;

m is 0 or 1;

-R ¹ and-R ² Independently of one another, from-CN, -NO ₂ Optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, -C (O) R ³ 、-S(O)R ³ 、-S(O) ₂ R ³ and-SR ⁴ ；

-R ¹ and-R ² One and only one selected from-H, optionally substituted alkyl, optionally substituted arylalkyl and optionally substituted heteroarylalkyl;

-R ³ Selected from-H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR ⁹ and-N (R) ⁹ ) ₂ ；

-R ⁴ Selected from optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl;

-R ⁵ each independently selected from-H, optionally substituted alkyl, optionally substituted alkenyl alkyl, optionally substituted alkynyl alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl;

-R ⁹ selected from-H and optionally substituted alkyl;

-Y-is absent and-X-is-O-or-S-; or (b)

Y-is-N (Q) CH ₂ -and-X-is-O-;

q is selected from optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl;

optionally, -R ¹ and-R ² Can be linked into a 3 to 8-membered ring; and is also provided with

Optionally, two-R ⁹ Together with the nitrogen to which they are attached, form a heterocyclic ring;

wherein-L ¹ -quilt-L ² -Z or-L ² -Z' substitution, and wherein-L ¹ Optionally further substituted.

Only in the context of formula (IV), the terms used have the following meanings:

The term "alkyl" as used herein includes straight, branched or cyclic saturated hydrocarbon groups of 1 to 8 carbon atoms, or in some embodiments 1 to 6 or 1 to 4 carbon atoms.

The term "alkoxy" includes oxygen-bonded alkyl groups including methoxy, ethoxy, isopropoxy, cyclopropyloxy, cyclobutoxy, and the like.

The term "alkenyl" includes non-aromatic unsaturated hydrocarbons having a carbon-carbon double bond.

The term "alkyne" includes non-aromatic unsaturated hydrocarbons having a carbon-carbon triple bond.

The term "aryl" includes aromatic hydrocarbon groups of 6 to 18 carbons, preferably 6 to 10 carbons, including, for example, phenyl, naphthyl and anthracenyl. The term "heteroaryl" includes aromatic rings consisting of 3 to 15 carbons containing at least one N, O or S atom, preferably 3 to 7 carbons containing at least one N, O or S atom, including, for example, pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolinyl, indolyl, indenyl, and the like.

In some cases, the alkenyl, alkynyl, aryl, or heteroaryl moiety may be coupled to the remainder of the molecule through an alkylene linkage. In these cases, the substituent will be referred to as an alkenyl alkyl, alkynyl alkyl, arylalkyl or heteroarylalkyl, indicating that there is an alkylene moiety between the alkenyl, alkynyl, aryl or heteroaryl moiety and the molecule to which the alkenyl, alkynyl, aryl or heteroaryl is coupled.

The term "halogen" includes bromine, fluorine, chlorine and iodine.

The term "heterocycle" refers to a 4 to 8 membered aromatic or non-aromatic ring containing 3 to 7 carbon atoms and at least one N, O or S atom. For example, piperidinyl, piperazinyl, tetrahydropyranyl, pyrrolidinyl, and tetrahydrofuranyl, as well as exemplary groups provided above for the term "heteroaryl".

When one ring system is optionally substituted, suitable substituents are selected from alkyl, alkenyl, alkynyl or additional rings, each optionally further substituted. Optional substituents on any group (including those described above) include halogen, nitro, cyano, -OR, -SR, -NR ₂ 、-OCOR、-NRCOR、-COOR、-CONR ₂ 、-SOR、-SO ₂ R、-SONR ₂ 、-SO ₂ NR ₂ Wherein each R is independently alkyl, alkenyl, alkynyl, aryl, or heteroaryl, or two R groups together with the atoms to which they are attached form a ring.

Preferably, -L of formula (IV) ¹ -is covered by a part-L ² -Z or-L ² -Z' substitution.

In certain embodiments, -L ¹ Having a structure as disclosed in WO2013/036857 A1. Thus, in certain embodiments, -L ¹ -having formula (V):

wherein the method comprises the steps of

The dashed line indicates the attachment to-D through the amine functionality of-D;

-R ¹ selected from optionally substituted C ₁ -C ₆ Linear, branched or cyclic alkyl; optionally substituted aryl; optionally substituted heteroaryl; an alkoxy group; and-NR ⁵ ₂ ；

-R ² Selected from-H; optionally substituted C ₁ -C ₆ An alkyl group; optionally substituted aryl; and optionally substituted heteroaryl;

-R ³ selected from-H; optionally substituted C ₁ -C ₆ An alkyl group; optionally substituted aryl; and optionally substituted heteroaryl;

-R ⁴ selected from-H; optionally substituted C ₁ -C ₆ An alkyl group; optionally substituted aryl; and optionally substituted heteroaryl;

-R ⁵ each independently of the others is selected from-H; optionally substituted C ₁ -C ₆ An alkyl group; optionally substituted aryl; and optionally substituted heteroaryl; or two-R when taken together ⁵ May be cycloalkyl or cycloheteroalkyl;

Only in the context of formula (V), the terms used have the following meanings:

"alkyl", "alkenyl" and "alkynyl" include straight, branched or cyclic hydrocarbon groups of 1 to 8 carbons or 1 to 6 carbons or 1 to 4 carbons, where alkyl is a saturated hydrocarbon, alkenyl includes one or more carbon-carbon double bonds, and alkynyl includes one or more carbon-carbon triple bonds. Unless otherwise specified, these all contain 1-6C.

"aryl" includes aromatic hydrocarbon groups of 6 to 18 carbons, preferably 6 to 10 carbons, including groups such as phenyl, naphthyl and anthracenyl. "heteroaryl" includes aromatic rings containing 3 to 15 carbons, containing at least one N, O or S atom, preferably 3 to 7 carbons, including, for example, pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolinyl, indolyl, indenyl, and the like.

The term "substituted" refers to an alkyl, alkenyl, alkynyl, aryl, or heteroaryl group containing one or more substituents to replace one or more hydrogen atoms. The substituents may generally be selected from halogen, including-F, -CI, -Br and-I; lower alkyl including straight chain, branched, and cyclic; lower haloalkyl including fluoroalkyl, chloroalkyl, bromoalkyl, and iodoalkyl; -OH; lower alkoxy groups include straight chain, branched, and cyclic. -SH; lower alkylthio, including straight, branched, and cyclic; amino, alkylamino, dialkylamino, silyl, including alkylsilane, alkoxysilane, and arylsilane; a nitro group; cyano group; a carbonyl group; carboxylic acids, carboxylic acid esters, carboxylic acid amides; an aminocarbonyl group; an aminoacetyl group; a carbamate; urea; a thiocarbamate; thiourea; a ketone; sulfone; sulfonamide; aryl groups include phenyl, naphthyl and anthracenyl; heteroaryl includes 5-membered heteroaryl groups including pyrrole, imidazole, furan, thiophene, oxazole, thiazole, isoxazole, isothiazole, thiadiazole, triazole, oxadiazole and tetrazole, 6-membered heteroaryl groups including pyridine, pyrimidine, pyrazine, and fused heteroaryl groups including benzofuran, benzothiophene, benzoxazole, benzimidazole, indole, benzothiazole, benzisoxazole and benzisothiazole.

In certain embodiments, -L of formula (V) ¹ -is covered by a part-L ² -Z or-L ² -Z' substitution.

In certain embodiments, -L ¹ Having a structure as disclosed in US7585837B 2. Thus, in certain embodiments, -L ¹ -having formula (VI):

wherein the method comprises the steps of

The dashed line indicates that one amine function through-D is attached to-D;

R ¹ and R is ² Independently selected from hydrogen, alkyl, alkoxy, alkoxyalkyl, aryl, alkylaryl, arylalkyl, halogen, nitro, -SO ₃ H、-SO ₂ NHR ⁵ Amino, ammonium, carboxyl, PO ₃ H ₂ And OPO ₃ H ₂ ；

R ³ 、R ⁴ And R is ⁵ Independently selected from hydrogen, alkyl, and aryl;

Suitable substituents of formula (VI) are alkyl groups (e.g. C _1-6 Alkyl), alkenyl (e.g. C _2-6 Alkenyl), alkynyl (e.g. C _2-6 Alkynyl), aryl (e.g., phenyl), heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl (e.g., an aromatic 4-to 7-membered heterocycle), or a halogen moiety.

Only in the context of formula (VI), the terms used have the following meanings.

The terms "alkyl", "alkoxy", "alkoxyalkyl", "aryl", "alkylaryl" and "aralkyl" refer to alkyl groups of 1 to 8, preferably 1 to 4, carbon atoms, such as methyl, ethyl, propyl, isopropyl and butyl, and aryl groups of 6 to 10 carbon atoms, such as phenyl and naphthyl. The term "halogen" includes bromine, fluorine, chlorine and iodine.

In certain embodiments, -L of formula (VI) ¹ -is covered by a part-L ² -Z or L ² -Z' substitution.

In certain embodiments, -L ¹ Having a structure as disclosed in WO2002/089789A 1. Thus, in certain embodiments, -L ¹ -having formula (VII):

wherein the method comprises the steps of

The dashed line indicates that one amine function through-D is attached to-D;

Y ₁ and Y ₂ Is independently O, S or NR ⁷ ；

R ² 、R ³ 、R ⁴ 、R ⁵ 、R ⁶ And R is ⁷ Independently selected from hydrogen, C _1-6 Alkyl, C _3-12 Branched alkyl, C _3-8 Cycloalkyl, C _1-6 Substituted alkyl, C _3-8 Substituted cycloalkyl, aryl, substituted aryl, aralkyl, C _1-6 Heteroalkyl, substituted C _1-6 Heteroalkyl, C _1-6 Alkoxy, phenoxy and C _1-6 A heteroalkoxy group;

ar is a moiety which, when included in formula (VII), forms a polysubstituted aromatic hydrocarbon or a polysubstituted heterocyclic group;

x is a chemical bond or a moiety actively transported to the target cell, a hydrophobic moiety, or a combination thereof,

y is 0 or 1;

wherein-L ¹ -quilt-L ² -Z or L ² -Z' substitution, and wherein-L ¹ Optionally further substituted.

Only in the context of formula (VII), the terms used have the following meanings:

the term "alkyl" is understood to include, for example, straight, branched, substituted C _1-12 Alkyl groups, including alkoxy groups, C _3-8 Cycloalkyl or substituted cycloalkyl, and the like.

The term "substitution" is understood to include the addition or substitution of one or more atoms contained in a functional group or compound with one or more different atoms.

Substituted alkyl groups include carboxyalkyl, aminoalkyl, dialkylamino, hydroxyalkyl, and mercaptoalkyl groups; substituted cycloalkyl groups include moieties such as 4-chlorocyclohexyl and the like; aryl includes moieties such as naphthyl; substituted aryl groups include moieties such as 3-bromophenyl. Aralkyl includes moieties such as tolyl; heteroalkyl groups include moieties such as ethyl thiophene; substituted heteroalkyl groups include, for example, moieties such as 3-methoxythiophene; alkoxy includes moieties such as methoxy; and phenoxy groups include moieties such as 3-nitrophenoxy. Halogen is understood to include fluorine, chlorine, iodine and bromine.

In certain embodiments, -L of formula (VII) ¹ -is covered by a part-L ² -Z or-L ² -Z' substitution.

In certain embodiments, -L ¹ -a substructure comprising formula (VIII)

Wherein the method comprises the steps of

The dotted line marked with an asterisk indicates the nitrogen attached to-D by the formation of an amide bond;

the unlabeled dashed line indicates the attachment to-L ¹ -the remainder of; and is also provided with

In certain embodiments, -L ¹ -having formula (VIII), wherein the dotted line marked with an asterisk represents the nitrogen of the amine attached to the lysine side chain of-D.

In certain embodiments, -L ¹ -having formula (VIII), wherein the dotted line marked with an asterisk represents the nitrogen of the amine attached to the N-terminus of-D.

In certain embodiments, -L of formula (VIII) ¹ -is covered by a part-L ² -Z or-L ² -Z' substitution.

In certain embodiments, -L of formula (VIII) ¹ -not further substituted.

In certain embodiments, -L ¹ -a substructure comprising formula (IX)

Wherein the method comprises the steps of

The dotted line marked with an asterisk indicates the nitrogen linked to-D by the formation of a urethane linkage;

In certain embodiments, -L ¹ -having (IX), wherein the dotted line marked with an asterisk represents the nitrogen of the amine attached to the lysine side chain of-D.

In certain embodiments, -L ¹ -having (IX), wherein the dotted line marked with an asterisk represents the nitrogen of the amine attached to the N-terminus of-D.

In certain embodiments, -L of formula (IX) ¹ -is covered by a part-L ² -Z or-L ² -Z' substitution.

In certain embodiments, -L of formula (IX) ¹ -not further substituted.

In certain embodiments, -L ¹ -having formula (IX-a):

wherein the method comprises the steps of

The dotted line marked with an asterisk indicates the nitrogen attached to-D, and the unlabeled dotted line indicates the nitrogen attached to-L ² -Z or-L ² -Z’；

n is 0, 1, 2,3 or 4;

＝Y ₁ selected from = O and = S;

-Y ₂ -selected from-O-and-S-;

-Y ₃ -selected from-O-and-S-;

-Y ₄ -is selected from-O-, -NR ⁵ -and-C (R) ⁶ R ^6a )-；

＝Y ₅ Selected from = O and = S;

-R ³ 、-R ⁵ 、-R ⁶ 、-R ^6a independently of one another, selected from the group consisting of-H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2-dimethylbutyl, 2, 3-dimethylbutyl and3, 3-dimethylpropyl;

-R4 is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2-dimethylbutyl, 2, 3-dimethylbutyl and 3, 3-dimethylpropyl;

-W-is selected from C _1-20 Alkyl optionally interrupted by one or more groups selected from: c (C) _3-10 Cycloalkyl, 8-to 30-membered carbocyclyl, 3-to 10-membered heterocyclyl, -C (O) N (R7) -, -O-, -S-, and-N (R7) -;

-Nu is selected from-N (R ⁷ R ^7a )、-N(R ⁷ OH)、-N(R ⁷ )-N(R ^7a R ^7b )、-S(R ⁷ )、-COOH、

/>

Ar-is selected from

Wherein the method comprises the steps of

The dotted line represents the sum-L ¹ Connection of the remaining part of the module,

-Z ¹ -is selected from-O-, -S-and-N (R) ⁷ ) -, and

-Z ² -is-N (R) ⁷ ) -; and is also provided with

-R ⁷ 、-R ^7a 、-R ^7b Independently of one another, from-H, C _1-6 Alkyl, C _2-6 Alkenyl and C _2-6 Alkynyl;

wherein, -L ¹ Optionally further substituted.

In certain embodiments, -L ¹ -having the formula (IX-a) wherein the dotted line marked with an asterisk represents the nitrogen of the amine attached to the lysine side chain of-D.

In certain embodiments, -L ¹ -having formula (IX-a) wherein stars are usedThe dashed line marked with a number represents the nitrogen of the amine attached to the N-terminus of-D.

In certain embodiments, -L of formula (IX-a) ¹ -not further substituted.

In certain embodiments, -L ¹ -having formula (IX-b):

wherein the method comprises the steps of

n is 0, 1, 2,3 or 4;

＝Y ₁ selected from = O and = S;

-Y ₂ -selected from-O-and-S-;

-Y ₃ -selected from-O-and-S-;

-Y ₄ -is selected from-O-, -NR ⁵ -and-C (R) ⁶ R ^6a )-；

＝Y ₅ Selected from = O and = S;

-R ² 、-R ³ 、-R ⁵ 、-R ⁶ 、-R ^6a independently of each other, selected from the group consisting of-H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2-dimethylbutyl, 2, 3-dimethylbutyl and 3, 3-dimethylpropyl;

-R ⁴ selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2-dimethylbutyl, 2, 3-dimethylbutyl and 3, 3-dimethylpropyl;

-W-is selected from C _1-20 Alkyl optionally interrupted by one or more groups selected from: c (C) _3-10 Cycloalkyl, 8-to 30-membered carbon polycyclic group, 3-to 10-membered heterocyclic group、-C(O)-、-C(O)N(R ⁷ ) -, -O-, -S-and-N (R) ⁷ )-；

-Nu is a nucleophile selected from the group consisting of-N (R ⁷ R ^7a )、-N(R ⁷ OH)、-N(R ⁷ )-N(R ^7a R ^7b )、-S(R ⁷ )、-COOH、

Ar-is selected from

Wherein the method comprises the steps of

The dotted line indicates the connection to-L ¹ The remaining part of the process-is,

-Z ¹ -selected from-O-, -S-and-N (R7) -, and

-Z ² -is-N (R) ⁷ ) -; and is also provided with

-R ⁷ 、-R ^7a 、-R ^7b Independently of one another selected from-H, C _1-6 Alkyl, C _2-6 Alkenyl and C _2-6 Alkynyl;

wherein-L ¹ Optionally further substituted.

In certain embodiments, -L ¹ -having formula (IX-b), wherein the dotted line marked with an asterisk represents the nitrogen of the amine attached to the lysine side chain of-D.

In certain embodiments, -L ¹ -having the formula (IX-b), wherein the dotted line marked with an asterisk represents the nitrogen of the amine attached to the N-terminus of-D.

In certain embodiments, -L of formula (IX-b) ¹ -not further substituted.

In certain embodiments, formula (IX-a) and (IX-b) = Y ¹ Is=o.

In certain embodiments, -Y of formulas (IX-a) and (IX-b) ² -is-O-.

In certain embodiments, -Y of formulas (IX-a) and (IX-b) ³ -is-O-.

In certain embodiments, -Y of formulas (IX-a) and (IX-b) ⁴ -is-NR ⁵ -。

In certain embodiments, formula (IX-a) and (IX-b) = Y ⁵ Is=o.

In certain embodiments, n of formulas (IX-a) and (IX-b) is 0 or 1. In certain embodiments, n of formulas (IX-a) and (IX-b) is 0. In certain embodiments, n of formulas (IX-a) and (IX-b) is 1.

In certain embodiments, the-R of formula (IX-b) ² Selected from the group consisting of-H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. In certain embodiments, the-R of formula (IX-b) ² Selected from-H, methyl, ethyl, n-propyl and isopropyl. In certain embodiments, the-R of formula (IX-b) ² Selected from-H, methyl and ethyl. In certain embodiments, the-R of formula (IX-b) ² is-H.

In certain embodiments, the-R3 of formulas (IX-a) and (IX-b) is selected from the group consisting of-H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. In certain embodiments, the-R3 of formulas (IX-a) and (IX-b) is selected from the group consisting of-H, methyl, ethyl, n-propyl, and isopropyl. In certain embodiments, the-R3 of formulas (IX-a) and (IX-b) is selected from-H, methyl, and ethyl. In certain embodiments, the groups of formula (IX-a) and (IX-b) are-R ³ is-H.

In certain embodiments, -R4 of formulas (IX-a) and (IX-b) are each independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. In certain embodiments, -R4 of formulas (IX-a) and (IX-b) is selected from methyl, ethyl, n-propyl, and isopropyl. In certain embodiments, the groups of formula (IX-a) and (IX-b) are-R ⁴ Selected from methyl and ethyl.

In certain embodiments, the groups of formula (IX-a) and (IX-b) are-R ⁵ Selected from the group consisting of-H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. In certain embodiments, the groups of formula (IX-a) and (IX-b) are-R ⁵ Selected from-H, methyl, ethyl, n-propyl and isopropyl. In certain embodiments, the formulaIX-a) and (IXb) ⁵ Selected from methyl and ethyl. In certain embodiments, the groups of formula (IX-a) and (IX-b) are-R ⁵ Is methyl.

In certain embodiments, the groups of formula (IX-a) and (IX-b) are-R ⁶ and-R ^6a Independently selected from the group consisting of-H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. In certain embodiments, the groups of formula (IX-a) and (IX-b) are-R ⁶ and-R ^6a Independently selected from-H, methyl, ethyl, n-propyl and isopropyl. In certain embodiments, the groups of formula (IX-a) and (IX-b) are-R ⁶ and-R ^6a Independently selected from-H, methyl and ethyl. In certain embodiments, the groups of formula (IX-a) and (IX-b) are-R ⁶ and-R ^6a Are all-H.

In certain embodiments, ar of formulas (IX-a) and (IX-b) is phenyl. In certain embodiments, ar of formulas (IX-a) and (IX-b) is

Wherein the dotted line represents the remainder of the moiety attached to formulas (IX-a) and (IX-b).

In certain embodiments, W of formulas (IX-a) and (IX-b) is C _1-20 Alkyl, optionally C _3-10 Cycloalkyl, -C (O) -, -C (O) N (R) ⁷ ) -, -O-, -S-and-N (R) ⁷ ) -breaking. In certain embodiments, W of formulas (IX-a) and (IX-b) is C _1-10 Alkyl, optionally C _3-10 Cycloalkyl, -C (O) -, -C (O) N (R) ⁷ ) -, -O-, -S-and-N (R) ⁷ ) -breaking. In certain embodiments, W of formulas (IX-a) and (IX-b) is C _1-6 Alkyl, optionally C _3-10 Cycloalkyl, -C (O) -, -C (O) N (R) ⁷ ) -, -O-, -S-and-N (R) ⁷ ) -breaking. In certain embodiments, W of formulas (IX-a) and (IX-b) is

Wherein the method comprises the steps of

The dotted line represents the remainder of the moiety attached to formula (IX-a) or (IX-b), respectively.

In certain embodiments, the-Nu of formulas (IX-a) and (IX-b) is-N (R) ⁷ R ^7a )。

In certain embodiments, the groups of formula (IX-a) and (IX-b) are-R7, -R ^7a and-R ^7b Independently of one another, selected from the group consisting of-H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. In certain embodiments, the groups of formula (IX-a) and (IX-b) are-R ⁷ 、-R ^7a and-R ^7b Independently of each other selected from-H, methyl, ethyl, n-propyl and isopropyl. In certain embodiments, the groups of formula (IX-a) and (IX-b) are-R ⁷ 、-R ^7a and-R ^7b Independently of each other selected from methyl or ethyl. In certain embodiments, the groups of formula (IX-a) and (IX-b) are-R ⁷ 、-R ^7a and-R ^7b Are all methyl groups.

In certain embodiments, -L ¹ -having the formula (IX-c)

Wherein the method comprises the steps of

The nitrogen attached to-D is indicated by the dotted line marked with an asterisk;

the unlabeled dashed line indicates the attachment to-L ² -Z or-L ² -Z'; and is also provided with

s1 is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

In certain embodiments, -L ¹ -having the formula (IX-c) wherein the dotted line marked with an asterisk represents the nitrogen of the amine attached to the lysine side chain of-D.

In certain embodiments, -L ¹ -having the formula (IX-c), wherein the dotted line marked with an asterisk represents the nitrogen of the amine attached to the N-terminus of-D.

In certain embodiments, s1 of formula (IX-c) is an integer selected from 1, 2, 3, 4 and 5. In certain embodiments, s1 of formula (IX-c) is 1. In certain embodiments, s1 of formula (IX-c) is 2. In certain embodiments, s1 of formula (IX-c) is 3. In certain embodiments, s1 of formula (IX-c) is 4. In certain embodiments, s1 of formula (IX-c) is 5.

In certain embodiments, -L ¹ -having the formula (IX-d)

Wherein the method comprises the steps of

The nitrogen attached to-D is indicated by the dotted line marked with an asterisk; and is also provided with

The unlabeled dashed line indicates the attachment to-L ² -Z or-L ² -Z’.

In certain embodiments, -L ¹ -having (IX-D) wherein the dotted line marked with an asterisk represents the nitrogen of the amine attached to the lysine side chain of-D.

In certain embodiments, -L ¹ -have (IX-D) wherein the dotted line marked with an asterisk represents the nitrogen of the amine attached to the N-terminus of-D.

In certain embodiments, -L ¹ Having a structure as disclosed in WO2020/206358A 1. Thus, in certain embodiments, part-L ¹ -having formula (X):

wherein the method comprises the steps of

The unlabeled dashed line represents a connection to-D;

the dotted line marked with an asterisk indicates the attachment to-L ² -Z or-L ² -Z’；

n is an integer selected from 0, 1, 2, 3, 4, 5 and 6;

-R ¹ and-R ² Independently an electron withdrawing group, an alkyl group, or-H, and wherein-R ¹ or-R ² At least one of which is an electron withdrawing group;

-R ⁴ each independently is C ₁ -C ₃ Alkyl, or two-R ⁴ Together with the carbon atoms to which they are attached, form a 3 to 6 membered ring; and

when-D is a drug moiety linked through an amine, -Y-is absent; or when-D is a drug moiety linked through a phenol, alcohol, thiol, thiophene, imidazole or non-basic amine, -Y-is-N (R ⁶ )CH ₂ The method comprises the steps of carrying out a first treatment on the surface of the wherein-R ⁶ Is optionally substituted C ₁ -C ₆ An alkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group.

In certain embodiments, n of formula (X) is an integer selected from 1, 2, 3, 4, 5, and 6. In certain embodiments, n of formula (X) is an integer selected from 1, 2, and 3. In certain embodiments, n of formula (X) is an integer selected from 0, 1, 2, and 3. In certain embodiments, n of formula (X) is 1. In certain embodiments, n of formula (X) is 2. In certain embodiments, n of formula (X) is 3.

In certain embodiments, the group-R of formula (X) ¹ and-R ² Is selected from the group consisting of: -CN; -NO ₂ The method comprises the steps of carrying out a first treatment on the surface of the Optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; -COR ³ 、-SOR ³ or-SO ₂ R ³ wherein-R is ³ is-H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR ⁸ Or NR (NR) ⁸ ₂ wherein-R is ⁸ Each independently is-H or optionally substituted alkyl, or two-R ⁸ The groups together with the nitrogen to which they are attached form a heterocyclic ring; or-SR ⁹ wherein-R is ⁹ Is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl or optionally substituted heteroarylalkyl.

In certain embodiments, the group-R of formula (X) ¹ and-R ² is-CN. In certain embodiments, the group-R in formula (X) ¹ and-R ² is-NO ₂ . In certain embodiments, the group-R in formula (X) ¹ and-R ² Is an optionally substituted aromatic group comprising 6 to 10 carbonsA base. In certain embodiments, the group-R in formula (X) ¹ and-R ² Is an optionally substituted phenyl, naphthyl or anthracenyl group. In certain embodiments, the group-R of formula (X) ¹ and-R ² Is an optionally substituted heteroaryl group comprising 3 to 7 carbons and comprising at least one N, O or S atom. In certain embodiments, the group-R of formula (X) ¹ and-R ² Is an optionally substituted pyrrolyl, pyridinyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolinyl, indolyl or indenyl group. In certain embodiments, the group-R of formula (X) ¹ and-R ² Is an optionally substituted alkenyl group comprising 2 to 20 carbon atoms. In certain embodiments, the group-R of formula (X) ¹ and-R ² Is an optionally substituted alkyne containing from 2 to 20 carbon atoms. In certain embodiments, the group-R of formula (X) ¹ and-R ² is-COR ³ 、-SOR ³ or-SO ₂ R ³ wherein-R is ³ is-H, optionally substituted alkyl containing 1 to 20 carbon atoms, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR ⁸ or-NR ⁸ ₂ wherein-R is ⁸ Each independently is-H or an optionally substituted alkyl group containing 1 to 20 carbon atoms, or two-R ⁸ The groups together with the nitrogen to which they are attached form a heterocyclic ring. In certain embodiments, the group-R of formula (X) ¹ and-R ² is-SR ⁹ wherein-R is ⁹ Is an optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl or optionally substituted heteroarylalkyl group comprising from 1 to 20 carbon atoms.

In certain embodiments, the group-R of formula (X) ¹ or-R ² At least one of them is-CN, -SOR ³ or-SO ₂ R ³ . In certain embodiments, the group-R of formula (X) ¹ and-R ² At least one of them is-CN or-SO ₂ R ³ . In certain embodiments, the group-R of formula (X) ¹ and-R ² At least one of them is-CN or-SO ₂ R ³ wherein-R is ³ Is optionally substituted alkyl, optionally substituted aryl, or-NR ⁸ ₂ . In certain embodiments, the group-R of formula (X) ¹ and-R ² At least one of them is-CN, -SO ₂ N(CH ₃ ) ₂ 、-SO ₂ CH ₃ by-SO ₂ Substituted phenyl groups, with-SO ₂ And Cl-substituted phenyl, -SO ₂ N(CH ₂ CH ₂ ) ₂ O、-SO ₂ CH(CH ₃ ) ₂ 、-SO ₂ N(CH ₃ )(CH ₂ CH ₃ ) or-SO ₂ N(CH ₂ CH ₂ OCH ₃ ) ₂ 。

In certain embodiments, the group-R in formula (X) ⁴ Each independently is C ₁ -C ₃ An alkyl group. In certain embodiments, both-R4 are methyl.

In certain embodiments, -Y-of formula (X) is absent. In certain embodiments, the-Y-of formula (X) is-N (R) ⁶ )CH ₂ 。

In certain embodiments, -L ¹ -is of formula (X), wherein n is 1, -R ¹ is-CN, -R ² is-H, and-R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 1, -R ¹ Is SO ₂ CH ₃ ，-R ² is-H, and-R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 1, -R ¹ is-SO ₂ N(CH ₂ CH ₂ ) ₂ CHCH ₃ ，-R ² is-H, and-R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 1, -R ¹ Is a quilt-SO ₂ Substituted phenyl, -R ² is-H, and-R4 is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 1, -R ¹ Is covered by-SO ₂ and-Cl-substituted phenyl, -R ² is-H, and-R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is ¹ ，-R ¹ is-SO ₂ N(CH ₂ CH ₂ ) ₂ O，-R ² is-H, and-R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 1, -R ¹ is-SO ₂ CH(CH ₃ ) ₂ ，-R ² is-H, and-R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 1, -R ¹ is-SO ₂ N(CH ₃ )(CH ₂ CH ₃ )，-R ² is-H, and-R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 1, -R ¹ is-SO ₂ N(CH ₂ CH ₂ OCH ₃ ) ₂ ，-R ² is-H, and-R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 1, -R ¹ Is covered by-SO ₂ and-CH ₃ Substituted phenyl, -R ² is-H, and-R ⁴ is-CH ₃ 。

In certain embodiments, -L ¹ -is of formula (X), wherein n is 2, -R ¹ is-CN, -R ² is-H, and-R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 2, -R ¹ is-SO ₂ N(CH ₃ ) ₂ ，-R ² is-H, and-R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 2, -R ¹ is-SO ₂ CH ₃ ，-R ² is-H, and-R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 2, -R ¹ is-SO ₂ N(CH ₂ CH ₂ ) ₂ CHCH ₃ ，-R ² is-H, and-R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 2, -R ¹ Is covered by-SO ₂ Substituted phenyl, -R ² is-H, and-R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 2, -R ¹ To be SO ₂ And Cl-substituted benzeneRadical, -R ² is-H, and-R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 2, -R ¹ is-SO ₂ N(CH ₂ CH ₂ ) ₂ O，-R ² is-H, and-R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 2, -R ¹ is-SO ₂ CH(CH ₃ ) ₂ ，-R ² is-H, and-R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 2, -R ¹ is-SO ₂ N(CH ₃ )(CH ₂ CH ₃ )，-R ² is-H, -R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 2, -R ¹ is-SO ₂ N(CH ₂ CH ₂ OCH ₃ ) ₂ ，-R ² is-H, -R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 2, -R ¹ Is covered by-SO ₂ and-CH ₃ Substituted phenyl, -R ² is-H, and-R ⁴ is-CH ₃ 。

In certain embodiments, -L ¹ -is of formula (X), wherein n is 3, -R ¹ is-CN, -R ² is-H, and-R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 3, -R ¹ is-SO ₂ N(CH ₃ ) ₂ ，-R ² is-H, and-R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 3, -R ¹ Is sO ₂ CH ₃ ，-R ² is-H, and-R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 3, -R ¹ is-SO ₂ N(CH ₂ CH ₂ ) ₂ CHCH ₃ ，-R ² is-H, and-R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 3, -R ¹ Is covered by-SO ₂ Substituted phenyl, -R ² is-H, and-R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 3, -R ¹ Is covered by-SO ₂ and-Cl-substituted phenyl, -R ² is-H, -R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 3, -R ¹ is-SO ₂ N(CH ₂ CH ₂ ) ₂ O，-R ² is-H, and-R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 3, -R ¹ is-SO ₂ CH(CH ₃ ) ₂ ，-R ² is-H, and-R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 3, -R ¹ is-SO ₂ N(CH ₃ )(CH ₂ CH ₃ )，-R ² is-H, and-R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 3, -R ¹ is-SO ₂ N(CH ₂ CH ₂ OCH ₃ ) ₂ ，-R ² is-H, and-R ⁴ is-CH ₃ . In certain embodiments, -L ¹ -is of formula (X), wherein n is 3, -R ¹ Is a quilt-SO ₂ and-CH ₃ Substituted phenyl, -R ² is-H, and-R ⁴ is-CH ₃ 。

Only in the context of formula (X) the terms used have the following meanings.

The term "alkyl" refers to a straight, branched or cyclic saturated hydrocarbon group of 1 to 20, 1 to 12, 1 to 8, 1 to 6 or 1 to 4 carbon atoms. In certain embodiments, the alkyl group is linear or branched. Examples of straight or branched alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl and n-decyl. In certain embodiments, the alkyl group is cyclic. Examples of cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, and cyclohexyl.

The term "alkoxy" refers to an oxygen-bonded alkyl group including methoxy, ethoxy, isopropoxy, cyclopropyloxy and cyclobutoxy.

The term "aryl" refers to aromatic hydrocarbon groups of 6 to 18 carbons, preferably 6 to 10 carbons, including, for example, phenyl, naphthyl and anthracenyl. The term "heteroaryl" refers to an aromatic ring containing 3 to 15 carbons and containing at least one N, O or S atom, preferably 3 to 7 carbons and containing at least one N, O or S atom, including for example pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolinyl, indolyl and indenyl.

The term "halogen" or "halo" refers to bromine, fluorine, chlorine and iodine.

The term "heterocycle" or "heterocyclyl" refers to a 3 to 15 membered aromatic or non-aromatic ring containing at least one N, O or S atom. Examples include piperidinyl, piperazinyl, tetrahydropyranyl, pyrrolidine and tetrahydrofuranyl, as well as exemplary groups provided above for the term "heteroaryl". In certain embodiments, the heterocycle or heterocyclyl is non-aromatic. In certain embodiments, the heterocycle or heterocyclyl is aromatic.

The term "optionally substituted" refers to a substituent wherein one group may be unsubstituted or substituted one or more times (e.g., 1, 2, 3, 4, or 5 times), and the substituents may be the same or different. Examples of substituents include alkyl, alkenyl, alkynyl, halogen, -CN, -OR ^aa 、-SR ^aa 、-NR ^aa R ^bb 、-NO ₂ 、-C＝NH(OR ^aa )、-C(O)R ^aa 、-OC(O)R ^aa 、-C(O)OR ^aa 、-C(O)NR ^aa R ^bb 、-OC(O)NR ^aa R ^bb 、-NR ^aa C(O)R ^bb 、-NR ^aa C(O)OR ^bb 、-S(O)R ^aa 、-S(O) ₂ R ^aa 、-NR ^aa S(O)R ^bb 、-C(O)NR ^aa S(O)R ^bb 、-NR ^aa S(O) ₂ R ^bb 、-C(O)NR ^aa S(O) ₂ R ^bb 、-S(O)NR ^aa R ^bb 、-S(O) ₂ NR ^aa R ^bb 、-P(O)(OR ^aa )(OR ^bb ) A heterocyclic group, a heteroaryl group or an aryl group, wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, heteroaryl and aryl groups are each independently substituted with-R ^cc Substitution, wherein-R ^aa and-R ^bb Each independently is-H, alkyl, alkenyl, alkynyl, heterocyclyl, heteroaryl, or aryl, or-R ^aa and-R ^bb Together with the nitrogen atom to which they are attached, form a heterocyclic group which is optionally substituted with alkyl, alkenyl, alkynyl, halogen, hydroxy, alkoxy or-CN, and wherein-R ^cc Each independently is alkyl, alkenyl, alkynyl, halo, heterocyclyl, heteroaryl, aryl, -CN, or-NO ₂ 。

In certain embodiments, -L ² -is a chemical bond. In certain embodiments, -L ² Is a spacer portion.

In certain embodiments, -L ² -selected from-T-, -C (O) O-, -C (O) N (R) ^y1 )-、-S(O) ₂ N(R ^y1 )-、-S(O)N(R ^y1 )-、-S(O) ₂ -、-S(O)-、-N(R ^y1 )S(O) ₂ N(R ^y1a )-、-S-、-N(R ^y1 )-、-OC(OR ^y1 )(R ^yla )-、-N(R ^y1 )C(O)N(R ^y1a )-、-OC(O)N(R ^y1 )-、C _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl; wherein-T-, C _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl is optionally substituted with one or more of the same or different-R ^y2 Substituted andand wherein C _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl groups are optionally interrupted by one or more groups selected from: -T-, -C (O) O-, -C (O) N (R) ^y3 )-、-S(O) ₂ N(R ^y3 )-、-S(O)N(R ^y3 )-、-S(O) ₂ -、-S(O)-、-N(R ^y3 )S(O) ₂ N(R ^y3a )-、-S-、-N(R ^y3 )-、-OC(OR ^y3 )(R ^y3a )-、-N(R ^y3 )C(O)N(R ^y3a ) -and-OC (O) N (R) ^y3 )-；

-R ^y1 and-R ^yla Are independently selected from-H, -T, C _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl groups. wherein-T, C _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl is optionally substituted with one or more of the same or different-R ^y2 Substituted, and wherein C _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl groups are optionally interrupted by one or more groups selected from: -T-, -C (O) O-, -C (O) N (R) ^y4 )-、-S(O) ₂ N(R ^y4 )-、-S(O)N(R ^y4 )-、-S(O) ₂ -、-S(O)-、-N(R ^y4 )S(O) ₂ N(R ^y4a )-、-S-、-N(R ^y4 )-、-OC(OR ^y4 )(R ^y4a )-、-N(R ^y4 )C(O)N(R ^y4a ) -and-OC (O) N (R) ^y4 )-；

T are each independently selected from the group consisting of: phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C _3-10 Cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclic, 8-30 membered carbopolycyclic and 8-30 membered heteropolycyclic; wherein T is each independently substituted with one or more of the same or different-R ^y2 And (3) substitution.

-R ^y2 Each independently selected from the group consisting of: halogen, -CN, oxo (= O), -COOR ^y5 、-OR ^y5 、-C(O)R ^y5 、-C(O)N(R ^y5 R ^y5a )、-S(O) ₂ N(R ^y5 R ^y5a )、-S(O)N(R ^y5 R ^y5a )、-S(O) ₂ R ^y5 、-S(O)R ^y5 、-N(R ^y5 )S(O) ₂ N(R ^y5a R ^y5b )、-SR ^y5 、-N(R ^y5 R ^y5a )、-NO ₂ 、-OC(O)R ^y5 、-N(R ^y5 )C(O)R ^y5a 、-N(R ^y5 )S(O) ₂ R ^y5a 、-N(R ^y5 )S(O)R ^y5a 、-N(R ^y5 )C(O)OR ^y5a 、-N(R ^y5 )C(O)N(R ^y5a R ^y5b )、-OC(O)N(R ^y5 R ^y5a ) And C _1-6 An alkyl group; wherein C is _1-6 Alkyl is optionally substituted with one or more halogen, the same or different; and

-R ^y3 、-R ^y3a 、-R ^y4 、-R ^y4a 、-R ^y5 、-Ry ^5a and-R ^y5b Each independently selected from-H and C _1-6 Alkyl, wherein C _1-6 The alkyl groups are optionally substituted with one or more halogen groups, which may be the same or different.

In certain embodiments, -L ² -selected from-T-, -C (O) O-, -C (O) N (R) ^y1 )-、-S(O) ₂ N(R ^y1 )-、-S(O)N(R ^y1 )-、-S(O) ₂ -、-S(O)-、-N(R ^y1 )S(O) ₂ N(R ^y1a )-、-S-、-N(R ^y1 )-、-OC(OR ^y1 )(R ^yla )-、-N(R ^y1 )C(O)N(R ^y1a )-、-OC(O)N(R ^y1 )-、C _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl groups. wherein-T-, C _1-20 Alkyl, C _2-20 Alkenyl and C _2-20 Alkynyl is optionally substituted by one or more of the same or different-R ^y2 Substituted, and wherein C _1-20 Alkyl, C _2-20 Alkenyl and C _2-20 Alkynyl groups are optionally interrupted by one or more groups selected from: -T-, -C (O) O-, -C (O) N (R) ^y3 )-、-S(O) ₂ N(R ^y3 )-、-S(O)N(R ^y3 )-、-S(O) ₂ -、-S(O)-、-N(R ^y3 )S(O) ₂ N(R ^y3a )-、-S-、-N(R ^y3 )-、-OC(OR ^y3 )(R ^y3a )-、-N(R ^y3 )C(O)N(R ^y3a ) -and-OC (O) N (R) ^y3 )-；

-R ^y1 and-R ^y1a Are independently selected from-H, -T, C _1-10 Alkyl, C _2-10 Alkenyl and C _2-10 Alkynyl groups. wherein-T, C _1-10 Alkyl, C _2-10 Alkenyl and C _2-10 Alkynyl is optionally substituted by one or more of the same or different-R ^y2 Substituted, and wherein C _1-10 Alkyl, C _2-10 Alkenyl and C _2-10 Alkynyl groups are optionally interrupted by one or more groups selected from: -T-, -C (O) O-, -C (O) N (R) ^y4 )-、-S(O) ₂ N(R ^y4 )-、-S(O)N(R ^y4 )-、-S(O) ₂ -、-S(O)-、-N(R ^y4 )S(O) ₂ N(R ^y4a )-、-S-、-N(R ^y4 )-、-OC(OR ^y4 )(R ^y4a )-、-N(R ^y4 )C(O)N(R ^y4a ) -and-OC (O) N (R) ^y4 )-；

T are each independently selected from the group consisting of: phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C _3-10 Cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclic, 8-30 membered carbopolycyclic and 8-30 membered heteropolycyclic; wherein T is each independently substituted with one or more of the same or different-R ^y2 Substitution;

-R ^y2 selected from halogen, -CN, oxo (= O), -COOR ^y5 、-OR ^y5 、-C(O)R ^y5 、-C(O)N(R ^y5 R ^y5a )、-S(O) ₂ N(R ^y5 R ^y5a )、-S(O)N(R ^y5 R ^y5a )、-S(O) ₂ R ^y5 、-S(O)R ^y5 、-N(R ^y5 )S(O) ₂ N(R ^y5a R ^y5b )、-SR ^y5 、-N(R ^y5 R ^y5a )、-N0 ₂ 、-OC(O)R ^y5 、-N(R ^y5 )C(O)R ^y5a 、-N(R ^y5 )S(O) ₂ R ^y5a 、-N(R ^y5 )S(O)R ^y5a 、-N(R ^y5 )C(O)OR ^y5a 、-N(R ^y5 )C(O)N(R ^y5a R ^y5b )、-OC(O)N(R ^y5 R ^y5a ) And C _1-6 An alkyl group; wherein C is _1-6 Alkyl is optionally substituted with one or more halogen, the same or different; and

-R ^y3 、-R ^y3a 、-R ^y4 、-R ^y4a 、-R ^y5 、-R ^y5a and-R ^y5b Each independently of the others is selected from-H and C _1-6 An alkyl group; wherein C is _1-6 The alkyl groups are optionally substituted with one or more of the same or different halogens.

In certain embodiments, -L ² -selected from-T-, -C (O) O-, -C (O) N (R) ^y1 )-、-S(O) ₂ N(R ^y1 )-、-S(O)N(R ^y1 )-、-S(O) ₂ -、-S(O)-、-N(R ^y1 )S(O) ₂ N(R ^y1a )-、-S-、-N(R ^y1 )-、-OC(OR ^y1 )(R ^yla )-、-N(R ^y1 )C(O)N(R ^y1a )-、-OC(O)N(R ^y 1)-、C _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl groups. wherein-T-, C _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl groups optionally being substituted by one or more R's, identical or different ^y2 Substituted, and wherein C _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl groups are optionally interrupted by one or more groups selected from: -T-, -C (O) O-, -C (O) N (R) ^y3 )-、-S(O) ₂ N(R ^y3 )-、-S(O)N(R ^y3 )-、-S(O) ₂ -、-S(O)-、-N(R ^y3 )S(O) ₂ N(R ^y3a )-、-S-、-N(R ^y3 )-、-OC(OR ^y3 )(R ^y3a )-、-N(R ^y3 )C(O)N(R ^y3a ) -and-OC (O) N (R) ^y3 )-；

-R ^y1 and-R ^yla Independently selected from-H, -T, C _1-10 Alkyl, C _2-10 Alkenyl and C _2-10 Alkynyl;

t are each independently selected from the group consisting of: phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C _3-10 Cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclic, 8-30 membered carbopolycyclic and 8-30 membered heteropolycyclic;

-R ^y2 Each independently selected from halogen and C _1-6 An alkyl group; and

-R ^y3 、-R ^y3a 、-R ^y4 、-R ^y4a 、-R ^y5 、-R ^y5a and-R ^y5b Each independently of the others is selected from H and C _1-6 An alkyl group; wherein C is _1-6 Alkyl is optionally covered withOne or more identical or different halogen substitutions.

In certain embodiments, -L ² -is C _1-20 Alkyl chains optionally substituted with one or more groups independently selected from the group consisting of-O-, -T-and-C (O) N (R) ^y1 ) -a group interruption; and the C is _1-20 The alkyl chain is optionally interrupted by one or more groups independently selected from-OH, -T and-C (O) N (R) ^y6 R ^y6a ) Is substituted with a group of (a). wherein-R ^y1 、-R ^y6 、-R ^y6a Independently selected from H and C ₁ - ₄ Alkyl, and wherein T is selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C _3-10 Cycloalkyl, 3 to 10 membered heterocyclyl, 8 to 11 membered heterobicyclic, 8 to 30 membered carbopolycyclic and 8 to 30 membered heteropolycyclic.

In certain embodiments, -L ² The molecular weight is between 14g/mol and 750 g/mol.

In certain embodiments, -L ² -comprising a moiety selected from the group consisting of

Wherein the method comprises the steps of

Dashed lines represent the respective and-L ¹ -、-L ² -the remainder being either attached to-Z; and

-R and-R ^a Independently of one another selected from-H, methyl, ethyl, propyl, butyl, pentyl and hexyl.

In certain embodiments, -L ² Having the formula (IX-e)

/>

Wherein the method comprises the steps of

The dotted line marked with an asterisk indicates the attachment to-L ¹ -；

The unlabeled dashed line represents a link to-Z; and is also provided with

s2 is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20.

In certain embodiments, s2 of formula (IX-e) is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. In certain embodiments, s2 of formula (IX-e) is an integer selected from 1, 2, 3, 4, 5, 6, 7 and 8. In certain embodiments, s2 of formula (IX-e) is 1. In certain embodiments, s2 of formula (IX-e) is 2. In certain embodiments, s2 of formula (IX-e) is 3. In certain embodiments, s2 of formula (IX-e) is 4. In certain embodiments, s2 of formula (IX-e) is 5. In certain embodiments, s2 of formula (IX-e) is 6. In certain embodiments, s2 of formula (IX-e) is 7. In certain embodiments, s2 of formula (IX-e) is 8.

In certain embodiments, part-L ¹ -L ² -having the formula (IX-f)

Wherein the method comprises the steps of

the unlabeled dashed line represents a link to-Z;

s1 is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; and is also provided with

In certain embodiments, -L ¹ -having the formula (IX-f) wherein the dotted line marked with an asterisk represents the nitrogen of the amine attached to the lysine side chain of-D.

In certain embodiments, -L ¹ -having the formula (IX-f), wherein the dotted line marked with an asterisk represents the nitrogen of the amine attached to the N-terminus of-D.

Thus, in the compounds of formula (IX-f), -L ¹ The bond between the moiety formed with-D is a carbamateAcid esters.

In certain embodiments, s1 of formula (IX-f) is an integer selected from 1, 2, 3, 4 and 5. In certain embodiments, s1 of formula (IX-f) is 1. In certain embodiments, s1 of formula (IX-f) is 2. In certain embodiments, s1 of formula (IX-f) is 3. In certain embodiments, s1 of formula (IX-f) is 4. In certain embodiments, s1 of formula (IX-f) is 5.

In certain embodiments, s2 of formula (IX-f) is selected from integers of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. In certain embodiments, s2 of formula (IX-f) is an integer selected from 1, 2, 3, 4, 5, 6, 7 and 8. In certain embodiments, s2 of formula (IX-f) is 1. In certain embodiments, s2 of formula (IX-f) is 2. In certain embodiments, s2 of formula (IX-f) is 3. In certain embodiments, s2 of formula (IX-e) is 4. In certain embodiments, s2 of formula (IX-f) is 5. In certain embodiments, s2 of formula (IX-e) is 6. In certain embodiments, s2 of formula (IX-f) is 7. In certain embodiments, s2 of formula (IX-f) is 8.

In certain embodiments, s1 of formula (IX-f) is 3 and s2 of formula (IX-f) is 3.

In certain embodiments, the IL-2 conjugate is of formula (Ia). In certain embodiments, x is 1. In certain embodiments, x is 2. In certain embodiments, x is 3. In certain embodiments, x is 4.

In certain embodiments, the IL-2 conjugate is of formula (Ib). In certain embodiments, y is 2. In certain embodiments, y is 3. In certain embodiments, y is 4.

In certain embodiments, part-L ¹ -L ² Z is of formula (XI)

Wherein the dashed line represents the-D nitrogen attached;

s1 is an integer selected from 1, 2,3, 4, 5, 6, 7, 8, 9 and 10;

s2 is an integer selected from 0, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 5, 16, 17, 18, 19 and 20; and is also provided with

p1, p2, p3, p4 are independently integers from 70 to 900.

In certain embodiments, -L ¹ -L ² -Z has formula (XI) wherein the dotted line represents the nitrogen of the amine attached to the lysine side chain of-D.

In certain embodiments, -L ¹ -L ² -Z has formula (XI) wherein the dashed line represents the nitrogen of the amine attached to the N-terminal end of-D.

Thus, the moiety-L formed by the compound of formula (XI) ¹ The linkage between-and-D is a carbamate.

In certain embodiments, s1 of formula (XI) is an integer selected from 1, 2, 3, 4, and 5. In certain embodiments, s1 of formula (XI) is 1. In certain embodiments, s1 of formula (XI) is 2. In certain embodiments, s1 of formula (XI) is 3. In certain embodiments, s1 of formula (XI) is 4. In certain embodiments, s1 of formula (XI) is 5.

In certain embodiments, s2 of formula (XI) is selected from integers of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In certain embodiments, s2 of formula (XI) is an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8. In certain embodiments, s2 of formula (XI) is 1. In certain embodiments, s2 of formula (XI) is 2. In certain embodiments, s2 of formula (XI) is 3. In certain embodiments, s2 of formula (XI) is 4. In certain embodiments, s2 of formula (XI) is 5. In certain embodiments, s2 of formula (XI) is 6. In certain embodiments, s2 of formula (XI) is 7. In certain embodiments, s2 of formula (XI) is 8.

In certain embodiments, s1 of formula (XI) is 3 and s2 of formula (XI) is 3.

In certain embodiments, p1 of formula (XI) is an integer from 115 to 680. In certain embodiments, p1 of formula (XI) is an integer from 115 to 560. In certain embodiments, p1 of formula (XI) is an integer from 185 to 450. In certain embodiments, p1 of formula (XI) is an integer from 220 to 240. In certain embodiments, p1 of formula (XI) is about 115. In certain embodiments, p1 of formula (XI) is about 160. In certain embodiments, p1 of formula (XI) is about 225. In certain embodiments, p1 of formula (XI) is about 270. In certain embodiments, p1 of formula (XI) is about 340. In certain embodiments, p1 of formula (XI) is about 450. In certain embodiments, p1 of formula (XI) is about 560.

In certain embodiments, p2 of formula (XI) is an integer 115-680. In certain embodiments, p2 of formula (XI) is an integer from 115 to 560. In certain embodiments, p2 of formula (XI) is an integer from 185 to 450. In certain embodiments, p2 of formula (XI) is an integer from 220 to 240. In certain embodiments, p2 of formula (XI) is about 115. In certain embodiments, p2 of formula (XI) is about 160. In certain embodiments, p2 of formula (XI) is about 225. In certain embodiments, p2 of formula (XI) is about 270. In certain embodiments, p2 of formula (XI) is about 340. In certain embodiments, p2 of formula (XI) is about 450. In certain embodiments, p2 of formula (XI) is about 560.

In certain embodiments, p3 of formula (XI) is an integer 115-680. In certain embodiments, p3 of formula (XI) is an integer from 115 to 560. In certain embodiments, p3 of formula (XI) is an integer from 185 to 450. In certain embodiments, p3 of formula (XI) is an integer from 220 to 240. In certain embodiments, p3 of formula (XI) is about 115. In certain embodiments, p3 of formula (XI) is about 160. In certain embodiments, p3 of formula (XI) is about 225. In certain embodiments, p3 of formula (XI) is about 270. In certain embodiments, p3 of formula (XI) is about 340. In certain embodiments, p3 of formula (XI) is about 450. In certain embodiments, p3 of formula (XI) is about 560.

In certain embodiments, p4 of formula (XI) is an integer 115-680. In certain embodiments, p4 of formula (XI) is an integer from 115 to 560. In certain embodiments, p4 of formula (XI) is an integer from 185 to 450. In certain embodiments, p4 of formula (XI) is an integer from 220 to 240. In certain embodiments, p4 of formula (XI) is about 115. In certain embodiments, p4 of formula (XI) is about 160. In certain embodiments, p4 of formula (XI) is about 225. In certain embodiments, p4 of formula (XI) is about 270. In certain embodiments, p4 of formula (XI) is about 340. In certain embodiments, p4 of formula (XI) is about 450. In certain embodiments, p4 of formula (XI) is about 560.

In certain embodiments, p1, p2, p3 of formula (XI) are the same as p 4. In certain embodiments, p1, p2, p3, and p4 are 220-240.

In certain embodiments, part-L ¹ -L ² Z has the formula (XI-a)

Wherein the dashed line represents nitrogen attached to-D; and is also provided with

p1, p2, p3, p4 are independently of each other integers from 70 to 900.

In certain embodiments, -L ¹ -L ² Z has the formula (XI-a) where the dotted line represents the nitrogen of the amine attached to the lysine side chain of-D.

In certain embodiments, -L ¹ -L ² Z has the formula (XI-a) where the dotted line represents the nitrogen of the amine attached to the N-terminal end of-D.

Thus, part-L formed in the compound of formula (XI-a) ¹ The bond between-and-D is a carbamate.

In certain embodiments, p1 of formula (XI-a) is an integer 115-680. In certain embodiments, p1 of formula (XI-a) is an integer from 115 to 560. In certain embodiments, p1 of formula (XI-a) is an integer from 185 to 450. In certain embodiments, p1 of formula (XI-a) is an integer from 220 to 240. In certain embodiments, p1 of formula (XI-a) is about 115. In certain embodiments, p1 of formula (XI-a) is about 160. In certain embodiments, p1 of formula (XI-a) is about 225. In certain embodiments, p1 of formula (XI-a) is about 270. In certain embodiments, p1 of formula (XI-a) is about 340. In certain embodiments, p1 of formula (XI-a) is about 450. In certain embodiments, p1 of formula (XI-a) is about 560.

In certain embodiments, p2 of formula (XI-a) is an integer 115-680. In certain embodiments, p2 of formula (XI-a) is an integer from 115 to 560. In certain embodiments, p2 of formula (XI-a) is an integer from 185 to 450. In certain embodiments, p2 of formula (XI-a) is an integer from 220 to 240. In certain embodiments, p2 of formula (XI-a) is about 115. In certain embodiments, p2 of formula (XI-a) is about 160. In certain embodiments, p2 of formula (XI-a) is about 225. In certain embodiments, p2 of formula (XI-a) is about 270. In certain embodiments, p2 of formula (XI-a) is about 340. In certain embodiments, p2 of formula (XI-a) is about 450. In certain embodiments, p2 of formula (XI-a) is about 560.

In certain embodiments, p3 of formula (XI-a) is an integer 115-680. In certain embodiments, p3 of formula (XI-a) is an integer 115-560. In certain embodiments, p3 of formula (XI-a) is an integer 185-450. In certain embodiments, p3 of formula (XI-a) is an integer from 220 to 240. In certain embodiments, p3 of formula (XI-a) is about 115. In certain embodiments, p3 of formula (XI-a) is about 160. In certain embodiments, p3 of formula (XI-a) is about 225. In certain embodiments, p3 of formula (XI-a) is about 270. In certain embodiments, p3 of formula (XI-a) is about 340. In certain embodiments, p3 of formula (XI-a) is about 450. In certain embodiments, p3 of formula (XI-a) is about 560.

In certain embodiments, p4 of formula (XI-a) is an integer 115-680. In certain embodiments, p4 of formula (XI-a) is an integer 115-560. In certain embodiments, p4 of formula (XI-a) is an integer 185-450. In certain embodiments, p4 of formula (XI-a) is an integer from 220 to 240. In certain embodiments, p4 of formula (XI-a) is about 115. In certain embodiments, p4 of formula (XI-a) is about 160. In certain embodiments, p4 of formula (XI-a) is about 225. In certain embodiments, p4 of formula (XI-a) is about 270. In certain embodiments, p4 of formula (XI-a) is about 340. In certain embodiments, p4 of formula (XI-a) is about 450. In certain embodiments, p4 of formula (XI-a) is about 560.

In certain embodiments, p1, p2, p3 of formula (XI-a) are the same as p 4. In certain embodiments, p1, p2, p3, and p4 are 220-240.

In certain embodiments, the conjugates of the invention comprise SEQ ID NO:10, for the IL-2 moiety, the moiety of formula (XIa) is conjugated to the nitrogen of the primary amine of the N-terminal or lysine side chain residue of the IL-2 moiety, and p1, p2, p3 and p4 are 220-240. In certain embodiments, b3 is about 112. In certain embodiments, the moiety of formula (XI-a) is conjugated to the nitrogen of a primary amine of a lysine side chain residue of the IL-2 moiety.

In certain embodiments, the conjugates of the invention comprise SEQ ID NO:13, for the IL-2 moiety, the moiety of formula (XIa) is conjugated to the nitrogen of the primary amine of the N-terminal or lysine side chain residue of the IL-2 moiety, and p1, p2, p3 and p4 are 220-240. In certain embodiments, b3 is about 112. In certain embodiments, the moiety of formula (XI-a) is conjugated to the nitrogen of a primary amine of a lysine side chain residue of the IL-2 moiety.

In certain embodiments, the conjugates of the invention comprise SEQ ID NO:16, for the IL-2 moiety, the moiety of formula (XIa) is conjugated to the nitrogen of the primary amine of the N-terminal or lysine side chain residue of the IL-2 moiety, and p1, p2, p3 and p4 are 220-240. In certain embodiments, b3 is about 112. In certain embodiments, the moiety of formula (XI-a) is conjugated to the nitrogen of a primary amine of a lysine side chain residue of the IL-2 moiety.

In certain embodiments, the conjugates of the invention comprise SEQ ID NO:19, for the IL-2 moiety, the moiety of formula (XIa) is conjugated to the nitrogen of the primary amine of the N-terminal or lysine side chain residue of the IL-2 moiety, and p1, p2, p3 and p4 are 220-240. In certain embodiments, b3 is about 112. In certain embodiments, the moiety of formula (XI-a) is conjugated to the nitrogen of a primary amine of a lysine side chain residue of the IL-2 moiety.

In certain embodiments, the conjugates of the invention comprise SEQ ID NO:22, for the IL-2 moiety, the moiety of formula (XIa) is conjugated to the nitrogen of the primary amine of the N-terminal or lysine side chain residue of the IL-2 moiety, and p1, p2, p3 and p4 are 220-240. In certain embodiments, b3 is about 112. In certain embodiments, the moiety of formula (XI-a) is conjugated to the nitrogen of a primary amine of a lysine side chain residue of the IL-2 moiety.

In certain embodiments, the conjugates of the invention comprise SEQ ID NO:25, for the IL-2 moiety, the moiety of formula (XIa) is conjugated to the nitrogen of the primary amine of the N-terminal or lysine side chain residue of the IL-2 moiety, and pl, p2, p3 and p4 are 220-240. In certain embodiments, b3 is about 112. In certain embodiments, the moiety of formula (XI-a) is conjugated to the nitrogen of a primary amine of a lysine side chain residue of the IL-2 moiety.

In certain embodiments, the conjugates of the invention comprise SEQ ID NO: the IL-2 moiety of 31, for the IL-2 moiety, the moiety of formula (XIa) is conjugated to the nitrogen of the primary amine of the N-terminal or lysine side chain residue of the IL-2 moiety, and p1, p2, p3 and p4 are 220-240. In certain embodiments, b3 is about 112. In certain embodiments, the moiety of formula (XI-a) is conjugated to the nitrogen of a primary amine of a lysine side chain residue of the IL-2 moiety.

In certain embodiments, the conjugates of the invention comprise SEQ ID NO:34, for the IL-2 moiety, the moiety of formula (XIa) is conjugated to the nitrogen of the primary amine of the N-terminal or lysine side chain residue of the IL-2 moiety, and p1, p2, p3 and p4 are 220-240. In certain embodiments, b3 is about 112. In certain embodiments, the moiety of formula (XI-a) is conjugated to the nitrogen of a primary amine of a lysine side chain residue of the IL-2 moiety.

In certain embodiments, the conjugates of the invention comprise SEQ ID NO: the IL-2 moiety of 217, for the IL-2 moiety, the moiety of formula (XIa) is conjugated to the nitrogen of the primary amine of the N-terminal or lysine side chain residue of the IL-2 moiety, and p1, p2, p3 and p4 are 220-240. In certain embodiments, b3 is about 112. In certain embodiments, the moiety of formula (XI-a) is conjugated to the nitrogen of a primary amine of a lysine side chain residue of the IL-2 moiety.

In certain embodiments, the conjugates of the invention comprise SEQ ID NO:225, for the IL-2 moiety, the moiety of formula (XIa) is conjugated to the nitrogen of the primary amine of the N-terminal or lysine side chain residue of the IL-2 moiety, and p1, p2, p3 and p4 are 220-240. In certain embodiments, b3 is about 112. In certain embodiments, the moiety of formula (XI-a) is conjugated to the nitrogen of a primary amine of a lysine side chain residue of the IL-2 moiety.

Another aspect of the invention is a pharmaceutical composition comprising at least one IL-2 protein of formula (I) or at least one IL-2 conjugate as described herein and at least one excipient. In certain embodiments, such pharmaceutical compositions have and include a pH in the range of pH 3-pH 8.

In certain embodiments, such pharmaceutical compositions are liquid formulations. In certain embodiments, the pharmaceutical composition is a suspension formulation. In certain embodiments, the pharmaceutical composition is a dry formulation.

Such liquid, suspension or dry formulations comprise at least one excipient. Excipients used in parenteral formulations may be classified as, for example, buffers, isotonicity adjusting agents, preservatives, stabilizers, anti-adsorbents, oxidation protectants, viscosity enhancing agents or other adjuvants. However, in some cases, one excipient may have dual or triple functions. In certain embodiments, at least one excipient included in the pharmaceutical compositions of the present invention is selected from the group consisting of:

(i) Buffering agent: physiologically tolerated buffers to maintain pH in a desired range, such as sodium phosphate, bicarbonate, succinate, histidine, citrate and acetate, sulfate, nitrate, chloride, pyruvate; antacids such as Mg (OH) may also be used ₂ Or ZnCO ₃ ；

(ii) Isotonic regulator: minimizing pain that may result from cell damage due to osmotic pressure differences at the injection reservoir; glycerol and sodium chloride are examples; the effective concentration can be determined by osmometry using a putative osmolality of 285-315mOsmol/kg of serum;

(iii) Preservatives and/or antimicrobial agents: multi-dose parenteral formulations require the addition of sufficient concentrations of preservative to minimize the risk of infection to the patient upon injection, and corresponding regulatory requirements have been established; typical preservatives include m-cresol, phenol, methylparaben, ethylparaben, propylparaben, butylparaben, chlorobutanol, benzyl alcohol, phenylmercuric nitrate, thimerosal, sorbic acid, potassium sorbate, benzoic acid, chlorocresol, and benzalkonium chloride;

(iv) Stabilizing agent: stabilization is achieved by enhancing the protein stabilizing force, by destabilization of the denatured state or by direct binding of excipients to the protein; stabilizers may be amino acids such as alanine, arginine, aspartic acid, glycine, histidine, lysine, proline, sugars such as glucose, sucrose, trehalose, polyols such as glycerol, mannitol, sorbitol, salts such as potassium phosphate, sodium sulfate, chelating agents such as EDTA, hexaphosphate, ligands such as divalent metal ions (zinc, calcium, etc.), other salts or organic molecules such as phenol derivatives; furthermore, oligomers or polymers may be used, such as cyclodextrin, dextran, dendrimers, PEG or PVP or protamine or HSA;

(v) Anti-adsorption agent: the use of ionic or nonionic surfactants or other proteins or soluble polymers to competitively coat or adsorb to the interior surfaces of the formulation vessel; such as poloxamers (Pluronic F-68), PEG dodecyl ethers (Brij 35), polysorbates 20 and 80, dextran, polyethylene glycol, PEG-polyhistidine, BSA and HSA, and gelatin; the selected concentration and type of excipient depends on the effect to be avoided, but typically a monolayer of surfactant is formed at the interface just above the CMC value;

(vi) Oxidation protectant: antioxidants such as ascorbic acid, ectoin, methionine, glutathione, thioglycerol, morin, polyethylenimine (PEI), propyl gallate and vitamin E; chelating agents such as citric acid, EDTA, hexaphosphate and thioglycollic acid may also be used;

(vii) Tackifier or viscosity enhancer: delaying sedimentation of the particles in the vial and syringe, and serving to promote mixing and re-suspension of the particles and make the suspension easier to inject (i.e., small forces on the syringe plunger); suitable tackifiers or viscosity enhancers are, for example, carbomer tackifiers such as Carbopol 940, carbopol Ultrez 10, cellulose derivatives such as hydroxypropyl methylcellulose (hydroxypropyl methylcellulose, HPMC) or diethylaminoethyl cellulose (DEAE or DEAE-C), colloidal magnesium silicate (magnesium aluminum silicate) or sodium silicate, hydroxyapatite gels, tricalcium phosphate gels, xanthan gum, carrageenans such as Satia gum UTC 30, aliphatic poly (hydroxy acids) such as poly (D, L-or L-milk) Acid) (PLA) and poly (glycolic acid) (PGA) and their copolymers (PLGA), terpolymers of D, L-lactide, glycolide and caprolactone, poloxamers, hydrophilic poly (oxyethylene) blocks and hydrophobic poly (oxypropylene) blocks to form triblock (e.g.)) Polyether ester copolymers, such as polyethylene terephthalate/polybutylene terephthalate copolymers, sucrose Acetate Isobutyrate (SAIB), dextran or derivatives thereof, combinations of dextran and PEG, polydimethylsiloxane, collagen, polyacetyl chitin, polyvinyl alcohol (PVA) and derivatives, polyalkyl imides, poly (acrylamide-co-diallyldimethylammonium (DADMA)), polyvinylpyrrolidone (PVP), glycosaminoglycans (GAGs) such as dermatan sulfate, chondroitin sulfate, keratan sulfate, heparin, heparan sulfate, hyaluronan, ABA triblock or AB block copolymers composed of hydrophobic a blocks such as Polylactide (PLA) or poly (lactide-co-glycolide) (PLGA) and hydrophilic B blocks such as polyethylene glycol (PEG) or polyvinylpyrrolidone; such block copolymers, as well as the poloxamers described above, may exhibit reversible thermogelation behavior (fluid state at room temperature to facilitate administration, and gel state above sol-gel transition temperature at body temperature after injection);

(viii) Spreading or diffusing agents: altering the permeability of connective tissue by hydrolyzing components of the extracellular matrix in the interstices, such as, but not limited to, hyaluronic acid, a polysaccharide found in the intercellular spaces of connective tissue; spreading agents, such as, but not limited to, hyaluronidase, temporarily reduce the viscosity of the extracellular matrix and facilitate diffusion of the injected drug; and

(ix) Other auxiliaries: such as wetting agents, viscosity modifiers, antibiotics, hyaluronidase; acids and bases such as hydrochloric acid and sodium hydroxide are essential adjuvants for pH adjustment in manufacturing processes.

Another aspect relates to an IL-2 protein, IL-2 conjugate or pharmaceutical composition of formula (I) as described herein for use as a medicament.

Another aspect relates to an IL-2 protein, IL-2 conjugate or pharmaceutical composition of formula (I) as described herein for use in the treatment of a disease treatable with IL-2.

Another aspect relates to an IL-2 protein, IL-2 conjugate or pharmaceutical composition of formula (I) as described herein for use in the manufacture of a medicament for the treatment of a disease treatable with IL-2.

Another aspect relates to a method of treating, controlling, delaying or preventing one or more diseases in a mammalian subject, preferably a human patient, in need of treatment, which diseases may be treated with IL-2, the method comprising the steps of: administering to said patient in need thereof a therapeutically effective amount of an IL-2 protein, IL-2 conjugate or pharmaceutical composition of formula (I) as described herein.

In certain embodiments, the disease that can be treated with IL-2 is cancer. Such cancers may be selected from liquid tumors, solid tumors, and lymphomas.

The liquid lymphoma may be leukemia or bone marrow neoplasm, such as Chronic Lymphocytic Leukemia (CLL), chronic Myelogenous Leukemia (CML), hairy cell leukemia, lymphoblastic leukemia, myelogenous leukemia, plasma cell leukemia, acute Lymphoblastic Leukemia (ALL), acute Myelogenous Leukemia (AML), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), post-MPN AML, post-MDS AML, del (5 q) -associated high risk MDS or AML, acute stage chronic myelogenous leukemia (blast-phase chronic myelogenous leukemia), multiple myeloma, myelodysplastic syndrome, chronic myeloproliferative disease, plasma cell neoplasm, and waldenstrom macroglobulinemia.

The solid tumor or lymphoma may be selected from: lip and oral cancer (oral cavity cancer), oral cancer (oral cancer), liver cancer/hepatocellular carcinoma, primary liver cancer, lung cancer, lymphoma, malignant mesothelioma, malignant thymoma, skin cancer, intraocular melanoma, metastatic squamous neck cancer with occult primary, childhood multiple endocrine neoplasia syndrome, mycosis fungoides, nasal and sinus cancer, nasopharyngeal carcinoma, neuroblastoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, pheochromocytoma, pituitary tumor, adrenocortical carcinoma, AIDS-related malignancy, anal cancer, cholangiocarcinoma, bladder cancer, brain and nervous system cancer, breast cancer bronchial adenoma/carcinoid, gastrointestinal carcinoid, carcinoma, colorectal cancer, endometrial carcinoma, esophageal carcinoma, extracranial blastoma, extragonadal germ cell tumor, extrahepatic bile duct carcinoma, gallbladder carcinoma, gastric (gastric) carcinoma, gestational trophoblastoma, head and neck carcinoma, hypopharynx carcinoma, islet cell carcinoma (endocrine pancreas), renal carcinoma/renal cell carcinoma, laryngeal carcinoma, pleural pneumoblastoma, prostate carcinoma, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, salivary gland carcinoma, sarcoma, sezary syndrome, small intestine carcinoma, genitourinary tumor, malignant thymoma, thyroid carcinoma, wilms tumor, cholangiocarcinoma, and their associated early abnormal cell growth such as dysplasia, adenoma and carcinoma in situ.

In certain embodiments, the cancer is liver cancer/hepatocellular carcinoma. In certain embodiments, the cancer is lung cancer. In certain embodiments, the cancer is lymphoma. In certain embodiments, the cancer is a malignant thymoma. In certain embodiments, the cancer is skin cancer. In certain embodiments, the cancer is metastatic squamous neck cancer with suppressed primary. In certain embodiments, the cancer is a neuroblastoma. In certain embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is pancreatic cancer. In certain embodiments, the cancer is cholangiocarcinoma. In certain embodiments, the cancer is bladder cancer. In certain embodiments, the cancer is brain and nervous system cancer. In certain embodiments, the cancer is breast cancer. In certain embodiments, the cancer is a gastrointestinal carcinoid. In certain embodiments, the cancer is a cancer. In certain embodiments, the cancer is colorectal cancer. In certain embodiments, the cancer is extrahepatic cholangiocarcinoma. In certain embodiments, the cancer is gallbladder cancer. In certain embodiments, the cancer is gastric (stomach) cancer. In certain embodiments, the cancer is a head and neck cancer. In certain embodiments, the cancer is renal cell carcinoma/renal cell carcinoma. In certain embodiments, the cancer is prostate cancer. In certain embodiments, the cancer is a sarcoma. In certain embodiments, the cancer is small intestine cancer. In certain embodiments, the cancer is a genitourinary tumor.

Examples of lung cancer are non-small cell lung cancer and small cell lung cancer. In certain embodiments, the cancer is non-small cell lung cancer. In certain embodiments, the cancer is small cell lung cancer.

Examples of lymphomas are AIDS-related lymphomas, primary central nervous system lymphomas, T cell lymphomas, cutaneous T cell lymphomas, hodgkin's lymphomas during pregnancy, non-hodgkin's lymphomas during pregnancy and angioimmunoblastic lymphomas.

Examples of skin cancers are melanoma and merck cell carcinoma. In certain embodiments, the cancer is skin cancer. In certain embodiments, the cancer is merck cell cancer.

Ovarian cancer may be, for example, an epithelial cancer, a germ cell tumor, or a low grade malignant potential tumor. In certain embodiments, the cancer is an epithelial cancer. In certain embodiments, the cancer is a germ cell tumor. In certain embodiments, the cancer is a low grade malignant potential tumor.

Pancreatic cancer can be, for example, an exocrine tumor/adenocarcinoma, a Pancreatic Endocrine Tumor (PET), or a neuroendocrine tumor (NET). In certain embodiments, the cancer is an exocrine tumor/adenocarcinoma. In certain embodiments, the tumor is a pancreatic endocrine tumor. In certain embodiments, the cancer is a neuroendocrine tumor.

Examples of brain and nervous system cancers are medulloblastoma (medullobastoma), such as childhood medulloblastoma, astrocytoma, ependymoma, neuroectodermal tumor, schwannoma, meningioma, pituitary adenoma, and glioma. In certain embodiments, the cancer is a medulloblastoma. In certain embodiments, the cancer is childhood medulloblastoma. In certain embodiments, the cancer is astrocytoma. In certain embodiments, the cancer is ependymoma. In certain embodiments, the cancer is neuroectodermal tumor. In certain embodiments, the tumor is a schwannoma. In certain embodiments, the cancer is a meningioma. In certain embodiments, the cancer is pituitary adenoma. In certain embodiments, the cancer is glioma.

The astrocytoma may be selected from giant cell glioblastoma, secondary glioblastoma, primary adult glioblastoma, primary pediatric glioblastoma, oligodendroglioma, anaplastic oligodendroglioma, oligoastrocytoma, anaplastic oligodendroglioma, oligoastrocytoma, anaplastic oligoastrocytoma, glioblastoma multiforme, fibrous astrocytoma subependymal, diffuse astrocytoma, polymorphic yellow astrocytoma, and cerebellar astrocytoma.

Examples of neuroectodermal tumors (neuroectodermal tumor) are pineal primitive neuroectodermal tumors and supratentorial primary neuroectodermal tumors.

The ependymoma may be selected from the group consisting of ependymoma, mucous papillary ependymoma, and anaplastic ependymoma.

Meningiomas may be atypical meningiomas or degenerative meningiomas.

Gliomas may be selected from the group consisting of glioblastoma multiforme, paragangliomas, supratentorial primitive neuroectodermal tumors (sPNET), brain stem gliomas, childhood brain stem gliomas, hypothalamic and visual pathway gliomas, childhood hypothalamic and visual pathway gliomas, and malignant gliomas.

Examples of breast cancer are breast cancer during pregnancy, triple negative breast cancer, ductal Carcinoma In Situ (DCIS), invasive Ductal Carcinoma (IDC), ductal carcinoma of the breast, medullary carcinoma of the breast, mucous carcinoma of the breast. Papillary carcinoma of the breast, wedge-shaped carcinoma of the breast, invasive Lobular Carcinoma (ILC), inflammatory breast carcinoma, lobular Carcinoma In Situ (LCIS), male breast carcinoma, paget's disease of the nipple, dermatophytes tumors of the breast, and metastatic breast cancer. In certain embodiments, the cancer is breast cancer during pregnancy. In certain embodiments, the cancer is a triple negative breast cancer. In certain embodiments, the cancer is an in situ catheter cancer. In certain embodiments, the cancer is an invasive ductal cancer. In certain embodiments, the cancer is a breast tubular cancer. In certain embodiments, the cancer is breast medullary cancer. In certain embodiments, the cancer is breast mucilage cancer. In certain embodiments, the cancer is breast papillary cancer. In certain embodiments, the cancer is breast wedge cancer. In certain embodiments, the cancer is invasive lobular cancer. In certain embodiments, the cancer is inflammatory breast cancer. In certain embodiments, the cancer is a lobular carcinoma. In certain embodiments, the cancer is male breast cancer. In certain embodiments, the cancer is paget's disease of the nipple. In certain embodiments, the cancer is a plant tumor of the breast. In certain embodiments, the cancer is metastatic breast cancer.

Examples of cancers are neuroendocrine, adrenocortical and islet cell cancers. In some cases, the cancer is a neuroendocrine cancer. In certain embodiments, the cancer is adrenocortical cancer. In certain embodiments, the cancer is islet cell cancer.

Examples of colorectal cancer are colon cancer and rectal cancer. In some cases, the cancer is colon cancer. In some cases, the cancer is rectal cancer.

The sarcoma is selected from Kaposi's sarcoma, osteosarcoma/osteomalignant fibrous histiocytoma, soft tissue sarcoma, ewing's tumor/sarcoma family, rhabdomyosarcoma, tenosynovial clear cell sarcoma, central chondrosarcoma, central and periosteal chondria, fibrosarcoma and uterine sarcoma. In certain embodiments, the cancer may be kaposi's sarcoma. In certain embodiments, the cancer may be osteosarcoma/bone malignant fibrous histiocytoma. In certain embodiments, the cancer may be a soft tissue sarcoma. In certain embodiments, the cancer may be a tumor/sarcoma of the ewing family. In certain embodiments, the cancer may be rhabdomyosarcoma. In certain embodiments, the cancer may be tenosynovial clear cell sarcoma. In certain embodiments, the cancer may be central chondrosarcoma. In certain embodiments, the cancer may be central and periosteal osteoma. In certain embodiments, the cancer may be fibrosarcoma. In certain embodiments, the cancer may be uterine sarcoma.

Examples of genitourinary cancers are testicular, urethral, vaginal, cervical, penile and vulvar cancers. In some cases, the cancer may be testicular cancer. In some cases, the cancer may be a urinary tract cancer. In certain embodiments, the cancer may be vaginal cancer. In certain embodiments, the cancer may be cervical cancer. In certain embodiments, the cancer may be penile cancer. In certain embodiments, the cancer may be vaginal cancer.

In certain embodiments, treatment with the IL-2 proteins, IL-2 conjugates or pharmaceutical compositions of formula (I) of the invention may be initiated prior to, concurrent with or subsequent to surgical removal of a tumor or radiation therapy. Furthermore, such treatment is optionally combined with at least one other cancer treatment method, such as systemic immunotherapy or local intratumoral immunotherapy or intralymphatic immunotherapy. At least one cancer treatment method, such as an example of systemic immunotherapy, such as one or more other drugs provided elsewhere herein, may be present in the pharmaceutical compositions of the invention in certain embodiments. In certain embodiments, the IL-2 protein of formula (I), IL-2 conjugate or pharmaceutical composition of the invention is administered systemically before, simultaneously with or after combination with at least one systemic immunotherapy or local intratumoral immunotherapy or intralymphatic immunotherapy, prior to radiation therapy or surgical excision of the injected tumor. In certain embodiments, the IL-2 protein, IL-2 conjugate or pharmaceutical composition of formula (I) of the invention is administered intratumorally prior to radiation therapy or surgical excision of the injected tumor, either before, simultaneously with or after combination with at least one systemic immunotherapy or local intratumoral immunotherapy or intralymphatic immunotherapy. In certain embodiments, the conjugates of the invention, pharmaceutically acceptable salts or pharmaceutical compositions thereof, are administered intratumorally after radiation or surgical removal of the tumor, either prior to, concurrent with or subsequent to combination with at least one systemic immunotherapy or local intratumoral immunotherapy or intralymphatic immunotherapy. In certain embodiments, the IL-2 protein, IL-2 conjugate or pharmaceutical composition of formula (I) of the invention is administered into the tumor draining lymph nodes prior to, concurrent with, or after surgical removal of the tumor or radiation therapy. In certain embodiments, the IL-2 protein, IL-2 conjugate, or pharmaceutical composition of the invention of formula (I) enters the tumor draining lymph nodes before, simultaneously with, or after combination with at least one systemic immunotherapy or local intratumoral immunotherapy or intralymph node immunotherapy, and before, simultaneously with, or after surgical excision of the tumor or radiotherapy. In certain embodiments, the IL-2 protein, IL-2 conjugate or pharmaceutical composition of formula (I) of the invention is administered to a metastatic tumor within the tumor either before or after surgical excision or radiotherapy of the primary tumor. In certain embodiments, the IL-2 protein of formula (I), IL-2 conjugate or pharmaceutical composition of the invention is administered intratumorally, which tumors may occur before, simultaneously with or after combination with at least one systemic or local intratumoral or intralymphatic immunotherapy, and before, simultaneously with or after surgical excision or radiation therapy of the primary tumor. In certain embodiments, at least one systemic treatment is performed prior to surgical removal of the tumor or radiation therapy, followed by systemic or intratumoral or intralymphatic administration of the IL-2 protein of formula (I), IL-2 conjugate or pharmaceutical composition of the invention. In certain embodiments, the IL-2 protein of formula (I), IL-2 conjugate, or pharmaceutical composition of the invention is first intratumorally administered, followed by subsequent treatment in combination with at least one systemic treatment or local intratumoral immunotherapy or intralymphatic immunotherapy. In certain embodiments, at least one systemic treatment is administered prior to surgical removal of the tumor, followed by systemic administration of the IL-2 protein of formula (I), IL-2 conjugate or pharmaceutical composition of the invention, or to draining lymph nodes or tumor beds after surgery, or by intratumoral administration in tumors that have not been surgically removed.

In certain embodiments, the IL-2 protein, IL-2 conjugate, or pharmaceutical composition of formula (I) of the invention is administered to a patient prior to, concurrently with, or after administration of one or more other agents, which in certain embodiments are selected from Pattern Recognition Receptor Agonists (PRRA). Cytotoxic/chemotherapeutic drugs, immune checkpoint inhibitors or antagonists, immune checkpoint agonists, immune activating receptor agonists, multispecific drugs, antibody-drug conjugates (ADCs), antibody-adjuvant conjugates (AACs), radionuclide or targeted radionuclide therapeutics, DNA damage repair inhibitors, tumor metabolism inhibitors, protein kinase inhibitors. Chemokines and chemoattractant receptor agonists, chemokine or chemokine receptor antagonists, cytokine receptor agonists, death receptor agonists, CD47 or sirpa antagonists, oncolytic drugs, signal transduction proteins, epigenetic modifiers, tumor peptides or tumor vaccines, heat Shock Protein (HSP) inhibitors, proteolytic enzymes, ubiquitin and proteasome inhibitors, adhesion molecule antagonists, hormones, including hormone peptides and synthetic hormones, and cell therapies such as Tumor Infiltrating Lymphocyte (TIL) therapy, chimeric Antigen Receptor (CAR) therapy, T cell therapy, natural Killer (NK) cell therapy, CAR-T therapy, CAR-NK therapy, CAR- γδ therapy, CAR-macrophage therapy, or any other cell therapy employing genetically modified or non-genetically modified immune cell types.

PRRA may be selected from Toll-like receptor (TLR) agonists, NOD-like receptor agonists (NLR), RIG-I-like receptor agonists, cytoplasmic DNA sensor agonists, STING agonists and aromatic hydrocarbon receptor agonists (AhR).

In certain embodiments, PRRA is a Toll-like receptor agonist, e.g., a Toll-like receptor agonist selected from the group consisting of: agonists of TLR1/2, such as peptidoglycan, lipoprotein, pam3CSK4, amplivant, SLP-AMPLIVANT, HESPECTA, ISA101 and ISA201; TLR2 agonists such as LAM-MS, LPS-PG, LTA-BS, LTA-SA, PGN-BS, PGN-EB, PGN-EK, PGN-SA, CIA29, FSL-1, pam2CSK4, pam3CSK4, zymosan, CBLB612, SV-283, ISA204, SMP105, heat-inactivated listeria monocytogenes (Listeria monocytogenes); agonists of TLR3, e.g., poly (A: U), poly (I: C) (poly-ICLC), rintatolimod, apoxxim, IPH3102, poly-ICR, PRV300, RGCL2, RGIC.1, Riboxxim (RGC 100, RGIC 100), riboxxol (RGIC 50)), synthetic natural or modified double stranded RNAs, synthetic natural or modified nucleic acid oligomers and agonists of Riboxxon, TLR4, such as Lipopolysaccharide (LPS), neoxetine-3, glucopyranosyl Lipid Adjuvant (GLA), GLA-SE, G100, GLA-aF, clinical Center Reference Endotoxin (CCRE), monophosphoryl lipid, grass MATA MPL, PEPA10, ONT-10 (PET-lipid a, oncothyrion), G-305, ALD046, CRX527, CRX675 (RC 527, RC 590), GSK1795091, OM197MPAC, OM294DP, tumor targeting agonists, and agonists of SAR439794, TLR2/4, such as lipid A, OM174 and PGN007; agonists of TLR5, such as flagellin, entolimod, mobilan, proteon CBLB501; agonists of TLR6/2, such as diacylated lipoproteins, diacylated lipopeptides, FSL-1, MALP-2 and CBLB613; agonists of TLR7, such as CL264, CL307, imiquimod (R837), TMX-101, TMX-201, TMX-202, TMX302, calirimmod, S-27609, 851, UC-IV150, 852A (3M-001, PF-04878691), loxoribine, polyuridinic acid, GSK2245035, GS-9620, RO6864018 (ANA 773, RG 7795), RO7020531, isotoibine, AN0331, ANA245, ANA971, ANA975, DSP0509, DSP3025 (AZD 8848), GS986, MBS2, MBS5, RG7863 (RO 6870868), sotimod, SZU101, synthetic natural or modified single stranded RNA, synthetic nucleic acids, synthetic natural or modified nucleic acid oligomers, tumor targeted TLR7 agonists, and TQA3334; agonists of TLR8, e.g. TransCon ^TM Agonists of TLR7/8, CL075, CL097, poly (dT), requimod (R-848, VML600, S28463), MEDI9197 (3M-052), NKTR262, DV1001, IMO4200, IPH3201, synthetic natural or modified single-stranded RNA, synthetic nucleic acids, synthetic natural or modified nucleic acid oligomers, synthetic nucleic acid oligomers, BDC-1001, other tumor-targeted TLR7/8 agonists, and VTX1463; agonists of TLR9, such as CpG DNA, cpG ODN, lefitolimod (MGN 1703), SD-101, qbG, CYT003-QbG, DUK-CpG-001, cpG-7909 (PF-3512676), GNKG168, EMD 1201081, IMO-2125, IMO-2055, cpG10104, AZD1419, AST008, IMO2134, MGN1706, IRS 954, 1018ISS, actilon (CPG 10101), ATP00001, AVE0675, AVE7279, CMP001, DIMS0001, DIMS9022, DIMS9054, DIMS9059, DV230, DV281, enanDIM, he)plissav (V270), kappa tact (DIMS 0150), jp834, NPI503, SAR21609, synthetic natural or modified nucleic acid oligomers and tolamba, and agonists of TLR7/9, e.g., DV1179.

In certain embodiments, the one or more additional drugs are agonists of TLR 1/2. In certain embodiments, the one or more additional drugs are agonists of TLR 2. In certain embodiments, the one or more additional drugs is the agonist TLR3. In certain embodiments, the one or more additional drugs are agonists of TLR 4. In certain embodiments, the one or more additional drugs are agonists of TLR 2/4. In certain embodiments, the one or more additional drugs are agonists of TLR 5. In one embodiment, the one or more additional agents are agonists of TLR 6/2. In certain embodiments, the one or more additional drugs are agonists of TLR 7. In certain embodiments, the one or more additional drugs are agonists of TLR 8. In certain embodiments, the one or more additional drugs are agonists of TLR 7/8. In certain embodiments, the one or more additional drugs are agonists of TLR 9.

Examples of CpG ODNs are ODN 1585, ODN 2216, ODN 2336, ODN 1668, ODN 1826, ODN 2006, ODN 2007, ODN BW006, ODN D-SL01, ODN 2395, ODN M362 and ODN D-SL03.

In certain embodiments, the one or more additional drugs is resiquimod. In certain embodiments, the one or more additional drugs is imiquimod.

In certain embodiments, the one or more additional drugs is resiquimod in free form. In certain embodiments, the one or more additional drugs are conjugates comprising a polymer to which one or more moieties of formula (a-i) are conjugated

Wherein the method comprises the steps of

The dashed line indicates attachment to the polymer; and is also provided with

n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

In certain embodiments, the polymer to which one or more moieties of formula (a-i) are conjugated is a water soluble polymer. In certain embodiments, such water-soluble polymers are PEG-based or hyaluronic acid-based polymers. In certain embodiments, the polymer is a PEG-based polymer. In certain embodiments, the polymer is a hydrogel, such as a PEG-based or hyaluronic acid-based hydrogel. In certain embodiments, the hydrogel is a PEG-based hydrogel. In certain embodiments, n of formula (A-i) is 1. In certain embodiments, n of formula (A-i) is 2. In certain embodiments, n of formula (A-i) is 3. In certain embodiments, n of formula (A-i) is 4.

In certain embodiments, the one or more additional drugs are conjugates comprising a plurality of PEG-based hydrogels conjugated with moieties of formula (a-i), wherein n of formula (a-i) is 2. In certain embodiments, the one or more additional agents are compounds 12 or 14 from WO2020/141221A1 as shown on pages 217 and 219, respectively, which are incorporated herein by reference. In certain embodiments, the one or more additional agents is compound 12 from WO2020/141221A1 as shown on page 217. In certain embodiments, the one or more additional agents is compound 14 from WO2020/141221A1 as shown on page 219. Compounds 12 and 14 may be synthesized as disclosed in WO2020/141221A 1.

In certain embodiments, PRRA is a NOD-like receptor agonist. If one or more additional drugs are NOD-like receptor agonists, such NOD-like receptor agonists may be selected from the group consisting of agonists of NOD1, such as C12-iE-DAP, C14-Tri-LAN-Gly, iE-DAP, iE-Lys and Tri-DAP; and agonists of NOD2, such as L18-MDP, MDP, M-TriLYS, moraxel butyl and N-glycolyl-MDP. In certain embodiments, the one or more additional agents are agonists of NOD 1. In certain embodiments, the one or more additional agents are agonists of NOD 2.

In certain embodiments, PRRA is a RIG-I-like receptor agonist. If the one or more additional agents are RIG-I-like receptor agonists, such RIG-I-like receptor agonists may be selected from the group consisting of 3p-hpRNA, 5'ppp-dsRNA, 5' ppp RNA (M8), 5'OH RNA with kinks (CBS-13-BPS), 5' PPP SLR, KIN100, KIN 101, KIN1000, KIN1400, KIN1408, KIN1409, KIN1148, KIN131A, poly (dA: dT), SB9200, RGT100 and Hiertanol.

In certain embodiments, PRRA is a cytoplasmic DNA sensor agonist. If the one or more additional drugs are cytoplasmic DNA sensor agonists, such cytoplasmic DNA sensor agonists may be selected from the group consisting of cGAS agonists, dsDNA-EC, G3-YSD, HSV-60, ISD, ODN TTAGGG (A151), poly (dG: dC), and VACV-70.

In certain embodiments, PRRA is a STING agonist. If one or more additional drugs are STING agonists, such STING agonists may be selected from MK-1454, ADU-S100 (MIW 815), 2'3' -cGAMP, 3' -cGAMP, c-di-AMP, c-di-GMP, cAIMP (CL 592), cAIMP difluoro (CL 614), cAIM (PS) 2 difluoro (Rp/Sp) (CL 656), 2' -cGAMP, 2'3' -cGAM (PS) 2 (Rp/Sp), 3' -cGAM fluoride, c-di-AMP fluoride, 2'3' -c-di-AMP, 2'3' -c-di-AM (PS) 2 (Rp, rp), c-di-GMP fluoride, 2'3' -c-di-GMP, c-di-UMP and DMXAA (vard Messa, ASA 404). In certain embodiments, the one or more additional agents is MK-1454. In certain embodiments, the one or more additional agents is ADU-S100 (MIW 815). In certain embodiments, the one or more additional agents is 2'3' -cGAMP.

In certain embodiments, PRRA is an arene receptor agonist. If the one or more additional agents are aromatic hydrocarbon receptor (AhR) agonists, such AhR agonists may be selected from FICZ, ITE and L-kynurenine.

In certain embodiments, the one or more additional agents are cytotoxic/chemotherapeutic agents. In certain embodiments, the one or more other agents are immune checkpoint inhibitors or antagonists. In certain embodiments, the one or more additional agents are immune activation receptor agonists. In certain embodiments, the one or more other drugs are multispecific drugs. In certain embodiments, the one or more additional drugs is an antibody-drug conjugate (ADC). In certain embodiments, the one or more other drugs is an antibody-adjuvant conjugate (AAC). In certain embodiments, the one or more other drugs are radionuclides or targeted radionuclide therapeutics. In certain embodiments, the one or more additional agents are DNA damage repair inhibitors. In certain embodiments, the one or more other agents are tumor metabolism inhibitors. In certain embodiments, the one or more other agents are pattern recognition receptor agonists. In certain embodiments, the one or more additional agents are protein kinase inhibitors. In certain embodiments, the one or more other agents are chemokines and chemoattractant receptor agonists. In certain embodiments, the one or more additional agents are chemokines or chemokine receptor antagonists. In certain embodiments, the one or more additional agents are cytokine receptor agonists. In certain embodiments, the one or more additional agents are death receptor agonists. In certain embodiments, the one or more additional agents are CD47 antagonists. In certain embodiments, the one or more additional agents are sirpa antagonists. In certain embodiments, the one or more other agents are oncolytic agents. In certain embodiments, the one or more additional agents is a signal transduction protein. In certain embodiments, the one or more additional agents are epigenetic modifiers. In certain embodiments, the one or more additional drugs is a tumor peptide or tumor vaccine. In certain embodiments, the one or more additional agents are Heat Shock Protein (HSP) inhibitors. In certain embodiments, the one or more additional agents are proteolytic enzymes. In certain embodiments, the one or more additional agents are ubiquitin and proteasome inhibitors. In certain embodiments, the one or more additional agents are adhesion molecule antagonists. In certain embodiments, the one or more other drugs are hormones, including hormone peptides and synthetic hormones.

The cytotoxic or chemotherapeutic agent may be selected from the following: alkylating agents, anthracyclines, pyrrole diazacyclic agents, nitrogen mustards, platinum agents, antimetabolites, antimicrotubule agents, topoisomerase inhibitors, cytotoxic antibiotics, auristatins, enediynes, leptins (lexitropsins), docarpimides, cyclopropyl pyrroloindoles, puromycin, dolastatins, maytansine derivatives, alkylsulfonates, triazenes and piperazines.

The alkylating agent may be selected from the following: nitrogen mustards such as trimethoprim, cyclophosphamide, melphalan, chloramphenicol, ifosfamide, and cloth Su Fen; nitrosoureas such as N-nitroso-N-methyl urea, carmustine, lomustine, semustine, fortemustine and streptomycin. Tetrazine drugs, such as dacarbazine, mi Tuoluo midate and temozolomide; vinylamine drugs, such as atratan; a Ji Lide classes of drugs, such as thiotepa, mitomycin and dieziquinone; cisplatin and its derivatives, such as cisplatin, carboplatin, oxaliplatin; and non-classical alkylating agents such as procarbazine and hexamethylmethylamine.

The antimetabolite may be selected from the group consisting of: anticholinergic esters, such as methotrexate and pemetrexed; fluoropyrimidines such as fluorouracil and capecitabine. Deoxynucleoside analogues such as cytarabine, gemcitabine, decitabine, azacytidine, fludarabine, nelarabine, clarithromycin, clofarabine and pennisetum; and thiopurines such as thioguanine and mercaptopurine.

The anti-microtubule agent may be selected from vinca alkaloids such as vincristine, vinblastine, vinorelbine, vindesine and vinflunine; taxanes, such as paclitaxel and docetaxel; podophyllotoxins and derivatives, such as podophyllotoxin, etoposide, and teniposide; stilbene phenols and derivatives, such as zybrestat (CA 4P); and BNCs 105.

The topoisomerase inhibitor may be selected from topoisomerase I inhibitors, for example irinotecan, topotecan and camptothecin; and topoisomerase II inhibitors such as etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, moebaron and aclabicin.

The cytotoxic antibiotic may be selected from the group comprising anthracyclines, such as doxorubicin, danofloxacin, epirubicin and idarubicin; pirubicin, aclarubicin, bleomycin, mitomycin C, mitomycin, actinomycin, daptomycin, doxorubicin, mitomycin, and tirapamide.

The auristatin may be selected from monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).

The enediyne may be selected from the group consisting of neocasinos, lidamycin (C-1027), carbo Li Kamei, epothilone, danamicin and Golgi A.

The maytansine derivative may be selected from the group consisting of ansamitocin, maytansine (DM 1) and raffmin (Sha Lafu tamoxin, DM 4).

The immune checkpoint inhibitor or antagonist may be selected from inhibitors of CTLA-4 (cytotoxic T lymphocyte-associated protein 4), such as ipilimumab, tremelimumab, MK-1308, FPT155, PRS010, BMS-986249, BPI-002, CBT509, JS007, ONC392, TE1254, IBI310, BR02001, CG0161, KN044, PBI5D3H5, BCD145, ADU1604, AGEN1884, AGEN1181, CS1002 and CP675206; inhibitors of PD-1 (programmed death 1), such as palbociclizumab, nivolumab, rituximab, AMP-224, BMS-936559, telithromycin and PDR001; inhibitors of PD-L1 (programmed cell death protein 1), such as MDX-1105, MEDI4736, alemtuzumab, avermectin, BMS-936559, and duvalizumab; inhibitors of PD-L2 (programmed death ligand 2); inhibitors of KIR (killer cell immunoglobulin-like receptor), such as Li Lushan anti (IPH 2102) and IPH2101; inhibitors of B7-H3, such as MGA271; inhibitors of B7-H4, such as FPA150; inhibitors of BTLA (B and T lymphocyte attenuators); inhibitors of LAG3 (lymphocyte activation gene 3), such as IMP21 (etilamode α), rilamelizumab, MK-4280, AVA017, BI754111, ENUM006, GSK2831781, INCAGN2385, LAG3Ig, LAG525, reg 3767, sym016, sym022, TSR033, TSR075 and XmAb22841; inhibitors of TIM-3 (T cell immunoglobulins and mucin-containing domain-3), such as LY3321367, MBG453 and TSR-022; inhibitors of VISTA (V-domain Ig inhibitors of T cell activation), such as JNJ-61610588; inhibitors of ILT2/LILRB1 (Ig-like transcript 2/leukocyte Ig-like receptor 1); inhibitors of ILT3/LILRB4 (Ig-like transcript 3/leukocyte Ig-like receptor 4); inhibitors of ILT4/LILRB2 (Ig-like transcript 4/leukocyte Ig-like receptor 2), such as MK-4830; inhibitors of TIGIT (T cell immune receptors with Ig and ITIM domains), such as MK-7684, PTZ-201, RG6058 and COM902; inhibitors of NKG2A, such as IPH-2201; PVRIG inhibitors, such as COM701; TREM1 inhibitors, such as PY314; and inhibitors of TREM2, such as PY159.

An example of a CTLA-4 inhibitor is an anti-CTLA 4 conjugate or a pharmaceutically acceptable salt thereof, wherein the conjugate comprises a plurality of anti-CTLA 4 moieties-D _CTLA4 By at least one part-L ¹ -L ² Covalently conjugated to a polymer moiety Z, wherein-L ¹ -and-D _CTLA4 Covalent and reversible conjugation, -L ² Covalent conjugation to Z, wherein-L ¹ -is a linker moiety, -L ² -is a chemical bond or a spacer moiety, wherein moiety-L ¹ -、-L ² -and Z are as described elsewhere herein for the conjugates of the invention. In certain embodiments-D _CTLA4 Selected from the group consisting of wild type F _c anti-CTLA 4 antibodies, fc-enhanced effector function/fcγr binding anti-CTLA 4 antibodies, anti-CTLA 4 antibodies that are conditionally active in the tumor microenvironment, anti-CTLA 4 small molecules, CTLA4 antagonist fusion proteins, anti-CTLA 4 anti-tilins, anti-CTLA 4 nanobodies, and antibody-based, scFVs or other forms of anti-CTLA 4 multispecific biologics. In certain embodiments, -D _CTLA4 Is ipilimumab. In certain embodiments, D _CTLA4 Is tremelimumab. In certain embodiments, the anti-CTLA 4 conjugate has the structure:

wherein the method comprises the steps of

Dotted line marked with asterisks indicates the symbol-D _CTLA4 Nitrogen linkage of amine functions, in particular with ipineNitrogen linkage of the amine functional group of the mumab; and

The unlabeled dashed line represents the connection to Z, such as a hydrogel, in particular a crosslinked hyaluronic acid hydrogel.

It will be appreciated that if Z is a hydrogel, such as a crosslinked hyaluronic acid hydrogel, then numerous moieties-D _CTLA4 -L ¹ -L ² Is connected with Z.

In certain embodiments, -D _CTLA4 The nitrogen of the amine function of (particularly ipilimumab) is the amine of the lysine residue. In certain embodiments, -D _CTLA4 The nitrogen of the amine function of (particularly ipilimumab) is the N-terminal amine.

In certain embodiments, the one or more additional agents are inhibitors of CTLA4 described above.

The immune activating receptor agonist may be selected from: agonists of CD27, such as recombinant CD70, e.g., HERA-CD27L and varluzumab (CDX-1127); CD28 agonists such as recombinant CD80, recombinant CD86, TGN1412 and FPT155; CD40 agonists, such as recombinant CD40L, CP-870, 893, dactyluzumab (SGN-40), chi Lob 7/4, ADC-1013 and CDX1140;4-1BB (C D137) agonists, such as recombinant 4-1BBL, wu Ruilu mab (urelumab), wu Tuolu mab (utomilumab) and ATOR-1017; OX40 agonists such as recombinant OX40L, MEDI0562, GSK3174998, MOXR0916 and PF-04548600; GITR agonists such as recombinant GITRL, TRX518, MEDI1873, INCAGN01876, MK-1248, MK-4166, GWN323, and BMS-986156; and ICOS agonists such as recombinant ICOSL, JTX-2011, and GSK3359609.

The multispecific drug may be selected from the group consisting of biological agents and small molecule immune checkpoint inhibitors. Examples of biologies are multispecific immune checkpoint inhibitors, such as CD137/HER2 multispecific drugs, PD- (L) 1/LAG3 antagonists (e.g. FS118, MGD 013), CTLA4/LAG3 antagonists (e.g. XmAb 22841) and CTLA4/PD- (L) 1 antagonists (e.g. XmAb20717, MGD 019); multispecific immune activating receptor agonists, immune cytokines and multispecific immune checkpoint agonists. Such multispecific immune checkpoint agonists may be selected from the group comprising Ig superfamily agonists such as ALPN-202, FPT155, TGN1412, GSK3359609, JTX-2011; TNF superfamily agonists such as FAP-4-1BBL (RG 7826), OX40-41BB (FS 120) ATOR-1015, ATOR-1144, ALG.APV-527, lipocalin/PRS-343, PRS344/ONC0055, FAP-CD40 DARDIN, MP0310 DARDIN, FAP-0X40 DARDIN, EGFR-CD40 DARDIN, EGFR41BB/CD137 DARDIN, EGFR-0X 40/DARDIN, HER2-CD40 DARDIN, HER2-41BB/CD137 DARDIN, HER2-0X40 DARDIN, FIBRONNECTIN ED-B-CD40 DARDIN, FIBNECTIN ED-B-41BB/CD137 and FIBRONNECTIN-B-0X 40 DARDIN; CD3 multispecific agonists such as Bonauzumab (blinatumomab), sorituximab (solitomab), MEDI-565, erttuuzumab (ertumaxomab), anti-HER 2/CD3, 1Fab-immunoblobulin G TDB, GBR 1302, MGD009, MGD007, EGFR Bi, EGFR-CD Probody, RG7802, PF-06863135, PF-06671008, AMG212/BAY2010112, CD3-5T4, xmAb14045, xmAb13676, xmAb18087, S80880, REGN1979, REGN5458, REGN4018, RG6026, mo Tuozhu monoclonal antibody (Mosuneuumab), EM801, ERY974, RG6194, AMG420, AMG330, AMG212, AMG 396, AMG 160, AMG 427, AMG 362, AMG 673, AMG 757, AFM13, AMF24, AFF 26, MB 24, XYN-35, JN-J, JN-7435, JN-J-3, JN-J-35, JN-J-3, JN-J-35, JN-J-3, or other cell-specific agonists (JN-J-3) such as TVs) may be included, comprising a γδ TCR agonist that targets T cell activity on a tumor cell antigen or viral antigen or an expression cell; natural Killer (NK) cell receptor multispecific agonists, targeted activated NK receptors and target tumor cell antigens, such as NKG2D multispecific agonists, NKp30 multispecific agonists, NKp44 multispecific agonists, NKp46 multispecific agonists, NKp80 multispecific agonists, NKG2C multispecific agonists, 2B4 (CD 244) multispecific agonists, CD32a multispecific agonists, CD64 multispecific agonists, multispecific agonists that bind to tumor antigens and activated receptors such as NKG2D or NKp30 or other NK receptors described above and bind to Fc receptors such as TriNKeTs, and CD16 multispecific agonists such as 1633BiKE, 161533 ke, OXS-3550, OXS-C3550, AFM13 and 24; and other therapeutic antibodies capable of binding to the antigen of interest as well as Fc receptors (e.g., CD16, CD32a, CD 64).

Other examples of immune-activating receptor agonists include Dectin agonists (Imprime PGG), recombinant NKG2D ligands, ligands or modifiers of the γδ TCR signal, such as anti-BTN 3A1 mAbs or anti-BTN 2A1 mAbs or vγ9/vδ2 TCR activating ligands, such as phosphoantigen and pyrophosphatase antigen, or agents that increase endogenous vγ9/vδ2 ligands, such as bisphosphates, such as pamidronate sodium and zolidronate sodium.

An example of a small molecule immune checkpoint inhibitor is CA-327 (TIM 3/PD-L1 antagonist).

The antibody-drug conjugate may be selected from: ADCs targeting hematopoietic cancers such as gemtuzumab ozuzumab, veltuximab, oxtuzumab, SAR3419, BT062, SGN-CD19A, IMGN529, MDX-1203, polotouzumab (RG 7596), vildazomet-pinacolumab (RG 7593), RG7598, milatuzumab-doxorubin, and OXS-1550; and ADCs targeting solid tumor antigens, such as Enmetrastuzumab, glembatumomab vedotin, SAR56658, AMG-172, AMG-595, BAY-94-9343, BIIB015, vorsetuzumab mafodotin (SGN-75), ABT-414, ASG-5ME, enfortumab vedotin (ASG-22 ME), ASG-16M8F, IMGN853, statin-infliximab (MLN-0264), vadortuzumab vedotin (RG 7450), statin-Sofostuzumab (RG 7458), statin-rituximab (RG 7599), RG7600, DEDN6526A (RG 7636), PSMA TTC, 1095 from Progeneics pharmaceutical company, morcin-Wo Tuozhu mab, lorvotuzumab emtansine, IMMU-130, gong Sha Tuozhu mab (IMMU-132), PF-06263507, and MEDI0641.

The antibody-adjuvant conjugate may be a boltbody, such as the boltbody described in WO2018112108A1 and WO2018009916 A1. In certain embodiments, the boltbody is selected from BDC-1001 and BDC-2034. In certain embodiments, the boltbody is BDC-1001. In certain embodiments, the boltbody is BDC-2034.

In certain embodiments, the boltbody has the structure of formula (BT-I)

Wherein Ab is an antibody moiety;

a is an unmodified amino acid side chain in the antibody moiety or a modified amino acid side chain in the antibody moiety;

z is a linking moiety;

adj is an adjuvant moiety; and

r is an integer selected from 1 to 10.

It will be appreciated that the r amino acid side chains of the moiety Ab of formula (BT-I) are attached to the moiety Adj-Z.

In certain embodiments, a of formula (BT-I) comprises an amino acid side chain in the antibody moiety, which side chain comprises an amine functional group.

In certain embodiments, the boltbody has the structure of formula (BT-II)

Wherein the method comprises the steps of

Ab is an antibody moiety;

a side chain representing a lysine residue of Ab, wherein the unlabeled dashed line represents a linkage to Z, and the dashed line labeled asterisk represents an alpha carbon linked to the lysine residue;

adj is an adjuvant moiety;

r is an integer selected from 1-10;

and Z is a divalent linking moiety having an ethylene glycol group or a glycine residue.

It will be appreciated that the moiety of formula (BT-11)Corresponds to formula (BT-I) A. Likewise, it will be appreciated that the r lysine side chain moieties of moiety Ab of formula (BT-II) are linked to moiety AdjZ.

In certain embodiments, Z in formulas (BT-I) and (BT-II) is bound to Adj via an amide bond, a C-N single bond, a C-O single bond, or a C-C single bond, and is bound to Ab via an amide bond or a C-N single bond.

In certain embodiments, Z of formulas (BT-I) and (BT-II) is bound to the nitrogen group of Adj and the nitrogen group of Ab. In such embodiments, Z of formulas (BT-I) and (BT-II) is bonded to an adjacent nitrogen group via an amide bond, a C-N single bond, or a combination thereof.

In some embodiments, Z of formulas (BT-I) and (BT-II) comprises a PEG moiety.

In certain embodiments, Z of formulas (BT-I) and (BT-II) comprises at least 2 ethylene glycol groups, such as at least 3 ethylene glycol groups, at least 4 ethylene glycol groups, at least 5 ethylene glycol groups, at least 6 ethylene glycol groups, at least 7 ethylene glycol groups, at least 8 ethylene glycol groups, at least 9 ethylene glycol groups, at least 10 ethylene glycol groups, at least 11 ethylene glycol groups, at least 12 ethylene glycol groups, at least 13 ethylene glycol groups, at least 14 ethylene glycol groups, at least 15 ethylene glycol groups, at least 16 ethylene glycol groups, at least 17 ethylene glycol groups, at least 18 ethylene glycol groups, at least 19 ethylene glycol groups, at least 20 ethylene glycol groups, at least 21 ethylene glycol groups, at least 22 ethylene glycol groups, at least 23 ethylene glycol groups, at least 24 ethylene glycol groups, or at least 25 ethylene glycol groups.

In certain embodiments, Z of formulas (BT-I) and (BT-II) comprises 2 ethylene glycol groups, 3 ethylene glycol groups, 4 ethylene glycol groups, 5 ethylene glycol groups, 6 ethylene glycol groups, 8 ethylene glycol groups, 12 ethylene glycol groups, 24 ethylene glycol groups, or 25 ethylene glycol groups.

In certain embodiments, Z of formulas (BT-I) and (BT-II) comprises a glycine residue.

In certain embodiments, Z of formulas (BT-I) and (BT-II) comprises at least 2 glycine residues, e.g., at least 3 glycine residues, at least 4 glycine residues, at least 5 glycine residues, at least 6 glycine residues, at least 7 glycine residues, at least 8 glycine residues, at least 9 glycine residues, at least 10 glycine complexes, at least 11 glycine complexes, at least 12 glycine complexes, at least 13 glycine residues, at least 14 glycine residues, at least 15 glycine residues, at least 16 glycine residues, at least 17 glycine residues, at least 18 glycine residues, at least 19 glycine residues, at least 20 glycine residues, at least 21 glycine residues, at least 22 glycine residues, at least 23 glycine residues, at least 24 glycine residues, or at least 25 glycine residues.

In certain embodiments, Z of formulas (BT-I) and (BT-II) comprises 2 glycine residues, 3 glycine residues, 4 glycine residues, 5 glycine residues, 6 glycine residues, 8 glycine residues, 12 glycine residues, 24 glycine residues, or 25 glycine residues.

In certain embodiments, Z of formulas (BT-I) and (BT-II) further comprises a divalent cyclohexylene group.

In certain embodiments, abs in formulas (BT-I) and (BT-II) comprise an antibody binding domain that binds to an antigen selected from the group consisting of: CDH1, CD19, CD20, CD29, CD30, CD38, CD40, CD47, CEA, epCAM, MUC1, MUC16, EGFR, VEGF, HER2, SLAMF7, PDGFRa, gp75, CTLA4, PD-1, PD-L2, LAG-3, B7-H4, KIR, TNFRSF4, OX40L, IDO-1, IDO-2, CEACAM1, BTLA, TIM3, A2Ar, VISTA, CLEC4C (BDCA-2, DLEC, CD303, CLECSF 7), CLEC4D (MCL, CLECSF 8), CLEC4E (Mincle), CLEC6A (Dectin-2), CLEC5A (MDL-1, CLECSF 5), CLEC1B (CLEC-2), CLEC9A (DNGR-1) and CLEC7A (Dectin-1).

In certain embodiments, abs of formulas (BT-I) and (BT-II) comprise an antibody binding domain that binds to HER 2. In certain embodiments, abs in formulas (BT-I) and (BT-II) comprise an antibody binding domain that binds EGFR. In certain embodiments, abs of formulas (BT-I) and (BT-II) comprise an antibody binding domain that binds to CCR 8. In certain embodiments, abs of formulas (BT-I) and (BT-II) comprise an antibody binding domain that binds to PD-L1. In certain embodiments, abs of formulas (BT-I) and (BT-II) comprise an antibody binding domain that binds CEA.

In certain embodiments, abs of formulae (BT-I) and (BT-II) comprise antibodies selected from the group consisting of: palbociclib, nivolumab, alemtuzumab, avilamab, ipilimumab, atocyzumab, trastuzumab, cetuximab, rituximab, pertuzumab, bevacizumab, darimumab, etanercept, noduzumab, enoxamab, ma Jituo ximab and bioimitated pharmaceuticals thereof.

In certain embodiments, abs of formulas (BT-I) and (BT-II) comprise trastuzumab. In certain embodiments, abs of formulas (BT-I) and (BT-II) comprise a pamoic Li Zhushan antibody. In certain embodiments, ab in formulas (BT-I) and (BT-II) comprises nivolumab.

In certain embodiments, the Adj in formulas (BT-I) and (BT-II) comprises PRRA.

In certain embodiments, the Adj in formulas (BT-I) and (BT-II) is PRRA selected from the group consisting of toll-like receptor (TLR) agonists, c-type lectin receptor (CLR) agonists, NOD-like receptor (NLR) agonists, rig-I-like receptor (RLR) agonists, interferon stimulating agent (STING) agonists, and combinations thereof.

In certain embodiments, the Adj in formulas (BT-I) and (BT-II) is a TLR agonist, e.g., a TLR agonist selected from the group consisting of: TLR1 agonists, TLR2 agonists, TLR3 agonists, TLR4 agonists, TLR5 agonists, TLR6 agonists, TLR7 agonists, TLR8 agonists, TLR9 agonists, TLR10 agonists, TLR11 agonists, and combinations thereof.

In certain embodiments, the Adj in formulas (BT-I) and (BT-II) is selected from the group consisting of CL264, CL401, CL413, CL419, CL553, CL572, pam ₃ CSK ₄ And Pam ₂ CSK ₄ Is a TLR agonist of (b).

In certain embodiments, the Adj in formulas (BT-I) and (BT-II) is selected from

Wherein the method comprises the steps of

-J are each independently selected from the group consisting of-H, -OR ⁴ and-R ⁴ ；

-R ⁴ Each independently selected from the group consisting of-H, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkylComprising 1, 2, 3, 4, 5, 6, 7 or 8 carbon units;

-Q-is absent or selected from alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl and heteroarylalkyl containing 1, 2, 3, 4, 5, 6, 7 or 8 carbon units; and

the dashed line indicates the connection to Z.

In certain embodiments, the Adj in formulas (BT-I) and (BT-II) have the formula

Wherein the method comprises the steps of

-J are each independently selected from the group consisting of-H, -OR ⁴ or-R ⁴ ；

-R4 are each independently selected from-H, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl containing 1, 2, 3, 4, 5, 6, 7, or 8 carbon units;

-U-are each independently-CH-or-N-, wherein at least one-U-is-N-;

t is each independently an integer selected from 1, 2 and 3;

-Q-is absent or selected from alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl and heteroarylalkyl containing 1, 2, 3, 4, 5, 6, 7 or 8 carbon units, and

the dashed line indicates the connection to Z.

In certain embodiments, the Adj in formulas (BT-I) and (BT-II) is selected from the following formulas

/>

Wherein the dashed line represents the connection to Z.

In certain embodiments, the Adj of formulas (BT-I) and (BT-II) are selected from the adjuvant moieties disclosed in paragraphs [118] - [136] of WO2018112108A 1.

In certain embodiments, the boltbody comprises more than one distinct adjuvant moiety.

In certain embodiments, the boltbody has the structure of formula (BT-VI)

Wherein the method comprises the steps of

Ab is an antibody moiety;

r is an integer selected from 1 to 10; and

z is a divalent linking moiety comprising at least one ethylene glycol group or at least one glycine residue.

In certain embodiments, Z of formula (BT-VI) is as defined for formulas (BT-I) and (BT-II).

In certain embodiments, the boltbody has the structure of formula (BT-VII)

Wherein the method comprises the steps of

Ab is an antibody moiety comprising (i) an antigen binding domain and (ii) an Fc domain;

Adj is an adjuvant moiety of formula (BT-IVb)

Wherein the method comprises the steps of

-R ⁴ Selected from alkyl groups containing 1-8 carbons, heteroalkyl groups, and,Cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl;

-J are each-H;

-U-are each-N-;

each t is 2;

-Q-absent;

the dotted line indicates the connection to G ₁ ；

-G ₁ -a valence bond;

a is an integer selected from 1-40; and is also provided with

r is an integer selected from 1-10.

In certain embodiments, the boltbody has the structure of formula (BT-VII)

Wherein the method comprises the steps of

Ab is trastuzumab;

adj is an adjuvant moiety of formula (BT-IVb)

Wherein the method comprises the steps of

-R4 is butyl;

-J are each-H;

-U-are each-N-;

each t is 2;

-Q-absent;

the dotted line indicates the attachment to-G ₁ -；

-G ₁ -a valence bond;

a is an integer selected from 1-40; and is also provided with

r is an integer of 1 to 4.

In certain embodiments, the boltbody has the structure of formula (BT-VIII)

Wherein the method comprises the steps of

r is an integer selected from 1 to 10;

n is an integer selected from about 2 to about 25; and

ab is an antibody moiety.

In certain embodiments, r of formula (BT-VIII) is 1. In certain embodiments, r of formula (BT-VIII) is 2. In certain embodiments, r of formula (BT-VIII) is 3. In certain embodiments, r of formula (BT-VIII) is 4.

In certain embodiments, n of formula (BT-VIII) is an integer selected from 6, 7, 8, 9, 10, 11, and 12. In certain embodiments, n of formula (BT-VIII) is an integer selected from 8, 9, 10, 11, and 12. In certain embodiments, n of formula (BT-VIII) is 10.

In certain embodiments, the Ab of formula (BT-VIII) comprises an antigen binding domain that binds to HER2, EGFR, PD-L1 or CEA. In certain embodiments, the antibody moiety of formula (BT-VIII) comprises an antigen binding domain that binds HER 2. In certain embodiments, the Ab of formula (BT-VIII) comprises an antigen binding domain that binds EGFR. In certain embodiments, an Ab of formula (BT-VIII) comprises an antigen binding domain that binds PD-L1 self. In certain embodiments, an Ab of formula (BT-VIII) comprises an antigen binding domain that binds CEA.

Only in the context of the formulae (BT-IIIa), (BT-IIIb), (BT-IIIc), (BT-IIId), (BT-IVa) and (BT-IVb) are the following meanings used:

the term "alkyl" refers to a straight or branched, saturated aliphatic group having the indicated number of carbon atoms. The alkyl group may contain any number of carbons, e.g. C _1-2 、C _1-3 、C _1-4 、C _1-5 、C _1-6 、C _1-7 、C _1-8 、C _1-9 、C _1-10 、C _2-3 、C _2-4 、C _2-5 、C _2-6 、C _3-4 、C _3-5 、C _3-6 、C _4-5 、C _4-6 And C _5-6 . For example, C _1-6 Alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl.The alkyl group may also comprise alkyl groups having up to 30 carbon atoms, such as heptyl, octyl, nonyl, decyl. Alkyl groups may be substituted or unsubstituted. "substituted alkyl" may be substituted with one or more groups selected from halogen, hydroxy, amino, oxo (=o), alkylamino, amino, acyl, nitro, cyano and alkoxy.

The term "aryl" refers to an aromatic ring system having any suitable number of ring atoms and any suitable number of rings. Aryl groups may contain any suitable number of ring atoms, for example 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring atoms, and 6 to 10, 6 to 12 or 6 to 14 ring members. Aryl groups may be monocyclic, may be fused to bicyclic or tricyclic groups, or may be linked to biaryl groups by a bond. Representative aryl groups include phenyl, naphthyl, and biphenyl. Other aryl groups include benzyl groups having a methylene linkage. Some aryl groups have 6 to 12 ring members, such as phenyl, naphthyl, or biphenyl. Other aryl groups have 6 to 10 ring members, such as phenyl or naphthyl.

The term "carbocycle" refers to a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring assembly containing 3 to 12 ring atoms or the indicated number of atoms. Carbocycles may contain any number of carbons, e.g. C _3-6 、C _4-6 、C _5-6 、C _3-8 、C _4-8 、C _5-8 、C _6-8 、C _3-9 、C _3-10 、C _3-11 And C _3-12 . Saturated monocyclic carbocycles include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic carbocycles include, for example, norbornane, [2.2.2 ]]Bicyclooctane, decalin and adamantane. The carbocyclic groups may also be partially unsaturated, having one or more double or triple bonds in the ring. Representative carbocyclic groups that are partially unsaturated include, but are not limited to, cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1, 3-and 1, 4-isomers), cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1, 3-, 1, 4-and 1, 5-isomers), norbornene, and norbornadiene.

The term "heteroalkyl" refers to an alkyl group in which one or more carbon atoms are optionally independently replaced by a heteroatom selected from N, O and S.

The term "heterocycle" refers to heterocycloalkyl and heteroaryl. "heteroaryl" by itself or as part of another substituent means a monocyclic or fused bicyclic or tricyclic aromatic ring combination containing 5 to 16 ring atoms, wherein 1 to 5 ring atoms are heteroatoms, such as N, O or S. Additional heteroatoms may also be useful, such as B, al, si, and P. Heteroatoms can be oxidized to form, for example, -S (O) -and-S (O) ₂ -a portion. Heteroaryl groups may contain any number of ring atoms, for example 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 3 to 9, 3 to 10, 3 to 11, or 3 to 12 ring members. Any suitable number of heteroatoms may be included in the heteroaryl group, for example 1,2,3, 4, or 5, or 1 to 2, 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, 2 to 5, 3 to 4, or 3 to 5. Heteroaryl groups may include, for example, pyrrole, pyridine, imidazole, pyrazole, triazole, tetrazole, pyrazine, pyrimidine, pyridazine, triazine (1, 2,3-, 1,2, 4-and 1,3, 5-isomers), thiophene, furan, thiazole, isothiazole, oxazole and isoxazole. Heteroaryl groups may also be fused to an aromatic ring system (e.g., benzene ring) to form members, such as benzopyrroles, e.g., indoles and isoindoles; benzopyridines such as quinoline and isoquinoline; benzopyrazine (quinoxaline); benzopyrimidines (quinazolines); benzopyridazines, such as phthalazine and cinnoline; benzothiophenes and benzofurans. Other heteroaryl groups include heteroaryl rings linked by a bond, such as bipyridine. Heteroaryl groups may be substituted or unsubstituted. The "substituted heteroaryl" group may be substituted with one or more groups selected from halogen, hydroxy, amino, oxo (=o), alkylamino, amino, acyl, nitro, cyano and alkoxy. Heteroaryl groups may be attached at any position on the ring. For example, pyrrole includes 1-, 2-, and 3-pyrrole, pyridine includes 2-, 3-, and 4-pyridine, imidazole includes 1-, 2-, 4-, and 5-imidazole, pyrazole includes 1-, 3-, 4-, and 5-pyrazole, triazole includes 1-, 4-, and 5-triazole, tetrazole includes 1-and 5-tetrazole, pyrimidine includes 2-, 4-, 5-, and 6-pyrimidine, pyridazine includes 3-and 4-pyridazine, 1,2, 3-triazine includes 4-and 5-triazine, 1,2, 4-triazine includes 3-, 5-, and 5-triazine 6-triazine, 1,3, 5-triazine includes 2-triazine, thiophene includes 2-and 3-thiophene, furan includes 2-and 3-furan, thiazole includes 2-, 4-and 5-thiazole, isothiazole includes 3-, 4-and 5-isothiazole, oxazole includes 2-, 4-and 5-oxazole, isoxazole includes 3-, 4-and 5-isoxazole, indole such as 1-, 2-and 3-indole, isoindole such as 1-and 2-isoindole, quinoline such as 2-, 3-and 4-quinoline, isoquinoline such as 1-, 3-and 4-isoquinoline, quinazoline such as 2-and 4-quinazoline, cinnoline such as 3-and 4-cinnoline, benzothiophene such as 2-and 3-benzothiophene, and benzofuran such as 2-and 3-benzofuran.

The term "heterocycloalkyl" by itself or as part of another substituent refers to a saturated ring system having 3 to 12 ring members and 1 to 4 heteroatoms N, O and S. Other heteroatoms may also be useful, such as B, al, si, and P. Heteroatoms may be oxidized to form moieties such as-S (O) -and-S (O) ₂ -. The heterocycloalkyl group can contain any number of ring atoms, for example, 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 3 to 9, 3 to 10, 3 to 11, or 3 to 12 ring members. Any suitable number of heteroatoms may be included in the heterocycloalkyl group, for example 1,2, 3 or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4 or 3 to 4. Heterocycloalkyl groups can include, for example, aziridine, azetidine, pyrrolidine, piperidine, cyclohexylimine, cycloheptimine, quinuclidine, pyrazolidine, imidazolidine, piperazine (1, 2-, 1, 3-and 1, 4-isomers), oxirane, oxetane, tetrahydrofuran, oxirane (tetrahydropyran), oxetane, thiirane, thietane (tetrahydrothiophene), thiacyclohexane (tetrahydrothiopyran), oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, dioxolane, dithiolane, morpholine, thiomorpholine, dioxane or dithiane. Heterocycloalkyl groups can also be fused with aromatic or non-aromatic ring systems to form members, such as indolines. Heterocycloalkyl groups may be unsubstituted or substituted. "substituted heterocycloalkyl" may be substituted with one or more groups selected from halogen, hydroxy, amino, oxo (=o), alkylamino, amino, acyl, nitro, cyano and alkoxy. Heterocyclyl can be attached by any of the rings And (5) connecting positions. For example, the aziridine may be a 1-or 2-aziridine, the azetidine may be a 1-or 2-azetidine, the pyrrolidine may be a 1-, 2-or 3-pyrrolidine, the piperidine may be a 1-, 2-, 3-or 4-piperidine, the pyrazolidine may be a 1-, 2-, 3-or 4-pyrazolidine, the imidazolidine may be a 1-, 2-, 3-or 4-imidazolidine, and the piperazine may be a 1, 2-. 3-or 4-piperazine, tetrahydrofuran may be 1-or 2-tetrahydrofuran, oxazolidines may be 2-, 3-, 4-or 5-oxazolidines, isoxazolidines may be 2-, 3-, 4-or 5-isoxazolidines, thiazolidines may be 2-, 3-, 4-or 5-thiazolidines, isothiazolidines may be 2-, 3-, 4-or 5-isothiazolidines, and morpholine may be 2-, 3-, or 4-morpholine.

The term "arylalkyl" refers to any aryl derivative of an alkyl group. In certain embodiments, one or more aryl moieties may be coupled to the remainder of the molecule through an alkyl linkage. In these cases, the substituent will be referred to as arylalkyl, indicating that there is an alkylene moiety between the aryl moiety and the molecule to which the aryl is coupled. Representative arylalkyl groups include phenylmethyl, phenylethyl, phenylpropyl, phenylisopropyl, phenylbutyl, phenylsec-butyl, phenyltert-butyl, phenylpentyl, phenylisopentyl, phenylhexyl, naphthylmethyl, naphthylethyl, naphthylpropyl, naphthylisopropyl, naphthylbutyl, naphthylisobutyl, naphthylsec-butyl, naphthyltert-butyl, naphthylpentyl, naphthylisopentyl, naphthylhexyl, biphenylmethyl, biphenylethyl, biphenylpropyl, biphenylisopropyl, biphenylbutyl, biphenylisobutyl, biphenyl sec-butyl, biphenyl tert-butyl, biphenylpentyl, biphenylisopentyl, and biphenylhexyl.

The term "heteroarylalkyl" refers to an arylalkyl group wherein one or more carbon atoms are optionally independently replaced by heteroatoms selected from N, O and S.

In certain embodiments, the one or more other drugs are radionuclides, which may be selected from the group consisting of: beta-emitters, e.g. ¹⁷⁷ Lutetium (lutetium), ¹⁶⁶ Holmium (holmium), ¹⁸⁶ Rhenium (Re), ¹⁸⁸ Rhenium (Re), ⁶⁷ Copper (Cu), ¹⁴⁹ Promethium (promethium), ¹⁹⁹ Gold (gold), ⁷⁷ Bromine (bromine), ¹⁵³ Samarium, ¹⁰⁵ Rhodium, ⁸⁹ Strontium (strontium), ⁹⁰ Yttrium, ¹³¹ Iodine. Alpha-emitters, e.g. ²¹³ Bismuth (Bi), ²²³ Radium (radium), ²²⁵ Actinium(s), ²¹¹ Astatine; and Oger electron emitters, e.g. ⁷⁷ Bromine (bromine), ¹¹¹ Indium (indium), ¹²³ Iodine and ¹²⁵ iodine.

The targeted radionuclide therapeutic agent may be selected from the following: zewa ling (Chinese character) ⁹⁰ Y-Tilmizumab, bexxar ] ¹³¹ I-Toximomab), oncolym @ and ¹³¹ I-Lym 1)、lymphocide( ⁹⁰ Y-Epalzhuzumab and cotara @ ¹³¹ I-chTNT-1/B) and pull Bei Zhushan ⁹⁰ Y or ¹³¹ I-CEA)、theragyn( ⁹⁰ Y-day Qu Moshan antibody, li Kating% ¹³¹ I-rituximab), lei Tuoshan anti ¹³¹ I-L19)PAM4( ⁹⁰ Y-clivatuzumab tetraxetan), duofigo @ and ²²³ ra dichloride), lutetium oxyoctreotide ] ¹⁷⁷ Lu-DOTA-Tyr ³ -Octreotate) and ¹³¹ I-MIBG。

the DNA damage repair inhibitor may be selected from poly (ADP-ribose) polymerase (PARP) inhibitors, such as olapari, lu Kapa, nilapari, vitamin Li Pa, CEP 9722, and E7016; dual CHK1/CHK2 inhibitors such as AZD7762, V158411, CBP501 and XL844; CHK1 selective inhibitors such as PF477736, MK8776/SCH900776, CCT244747, CCT245737, LY2603618, LY 2606368/primeverin, AB-IsoG, ARRY575, AZD7762, CBP93872, ESP01, GDC0425, SAR020106, SRA737, V158411, and VER250840; CHK2 inhibitors such as CCT241533 and PV1019; ATM inhibitors such as AZD0156, AZD1390, KU55933, M3541 and SX-RDS1; ATR inhibitors, such as AZD6738, BAY1895344, M4344 and M6620 (VX-970); and DNA-PK inhibitors such as M3814.

The tumor metabolism inhibitor may be selected from an inhibitor of the adenosine pathway, an inhibitor of tryptophan metabolism, and an inhibitor of the arginine pathway.

Examples of inhibitors of the adenosine pathway are inhibitors of the A2AR (adenosine A2A receptor), such as ATL-444, itrafylline (KW-6002), MSX-3, praziquantel Ding Nai (SCH-420, 814), SCH-58261, SCH412, 348, SCH-442, 416, ST-1535, caffeine, VER-6623, VER-6947, VER-7835, verapamil (BIIB-014), ZM-241, 385, PBF-509 and V81444; inhibitors of CD73, such as IPH53 and SRF373; and inhibitors of CD39, such as IPH52.

Examples of tryptophan metabolic inhibitors are inhibitors of IDO such as indomethacin (NLG 8189), epacadotril, natamod, BMS-986205 and MK-7162; inhibitors of TDO, such as 680C91; dual IDO/TDO inhibitors.

Examples of inhibitors of the arginine pathway are inhibitors of arginase, such as INCB001158.

The protein kinase inhibitor may be selected from the group consisting of receptor tyrosine kinase inhibitors, intracellular kinase inhibitors, cyclin-dependent kinase inhibitors, phosphocarnosine-3 kinase inhibitors, mitogen activated protein kinase inhibitors, nuclear factor kappa-beta kinase (IKK) inhibitors, and Wee-1 inhibitors.

Examples of receptor tyrosine kinase inhibitors are EGF receptor inhibitors such as afatinib, cetuximab, erlotinib, gefitinib, pertuzumab and rituximab; VEGF receptor inhibitors such as acitinib, lenvatinib, etanercept and Li Nifa (ABT-869); C-KIT receptor inhibitors, such as CDX0158 (KTN 0158); ERBB2 (HER 2) inhibitors, such as herceptin (trastuzumab); ERBB3 receptor inhibitors such as CDX3379 (MEDI 3379, KTN 3379) and AZD8931 (sapitinib); FGF receptor inhibitors such as adafiltinib; AXL receptor inhibitors such as BGB324 (BGB 324, R428, benzene Xin Nibu) and SLC391; and MET receptor inhibitors, such as CGEN241.

Examples of intracellular kinase inhibitors are Bruton's Tyrosine Kinase (BTK) inhibitors such as ibutenib, acarb Ding Ni, GS-4059, selutinib, BGB-3111, HM71224, zabrutinib, ARQ531, BI-BTK1 and Vikab Ding Ni; spleen tyrosine kinase inhibitors, such as fositinib; bcr-Ab1 tyrosine kinase inhibitors such as imatinib and nilotinib; janus kinase inhibitors such as rucotinib, tofacitinib and fet Lei Tini; and multispecific tyrosine kinase inhibitors such as bosutinib, crizotinib, cabotinib, dasatinib, emtrictinib, lapatinib, mo Li tinib, pazopanib, sorafenib, sutinib 6656, and vandetanib.

An example of a tyrosine kinase inhibitor is a tyrosine kinase inhibitor ("TKI") conjugate or a pharmaceutically acceptable salt thereof, wherein the conjugate comprises a plurality of TKI moieties-D _TKI Through at least one part-L ¹ -L ² Covalent conjugation to the polymer moiety Z, wherein-L ¹ -and D _TKI Covalent and reversible conjugation, -L ² Covalent conjugation to Z, and wherein-L ¹ -is a linker moiety, -L ² -is a chemical bond or a spacer moiety, wherein moiety-L ¹ -、-L ² -and Z are as described elsewhere herein for the conjugates of the invention. In certain embodiments, -D _TKI Selected from the group consisting of receptor tyrosine kinase inhibitors, intracellular kinase inhibitors, cyclin dependent kinase inhibitors, phosphocarnosine-3 kinase (PI 3K) inhibitors, mitogen activated protein kinase inhibitors, nuclear factor kappa-beta kinase (IKK) inhibitors, and Wee-1 inhibitors. In certain embodiments, -D _TKI Is acitinib. In certain embodiments, -D _TKI Is lenvatinib. In certain embodiments, -D _TKI Is peganib. In certain embodiments, -D _TKI Is Li Nifa Ni.

Examples of cyclin-dependent kinase inhibitors are rebaudinib, pamoxb, abbe-cili, qu Laxi b, pravastatin a, ol Lu Meisu II and MK-7965.

Examples of phosphatidylinositol-3-kinase inhibitors are IPI549, GDc-0326, P Li Xibu, sila Bei Lixi, IC-87114, AMG319, silita Li Xibu, aldrich, and CUDC907.

Examples of mitogen-activated protein kinase inhibitors are Ras/farnesyl transferase inhibitors, such as tipiranib and LB42708; raf inhibitors such as regorafenib, enkola fenib, vemurafenib, dabrafenib, sorafenib, PLX-4720, GDC-0879, AZ628, li Feila fenib, PLX7904 and RO5126766; MEK inhibitors such as, for example, comparable Mi Ni, trametetinib, dimetinib, semetinib, pimetinib, lei Feia tinib and PD0325901; ERK inhibitors such as MK-8353, GDC-0994, wu Lisha tinib and SCH772984.

Examples of inhibitors of nuclear factor kappa-beta kinase (IKK) are BPI-003 and AS602868.

An example of a Wee-1 inhibitor is adapalene.

Chemokine receptors and chemoattractant receptor agonists can be selected from CXC chemokine receptors, CC chemokine receptors, C chemokine receptors, CX3C chemokine receptors and chemoattractant receptors.

CXC chemokine receptors may be selected from: CXCR1 agonists, such as recombinant CXCL8 and recombinant CXCL6; CXCR2 agonists, such as recombinant CXCL8, recombinant CXCL1, recombinant CXCL2, recombinant CXCL3, recombinant CXCL5, recombinant CXCL6, MGTA 145, and SB251353; CXCR3 agonists, such as recombinant CXCL9, recombinant CXCL10, recombinant CXCL11, and recombinant CXCL4; CXCR4 agonists, such as recombinant CXCL12, ATI2341, CTCE0214, CTCE0324 and NNZ4921; CXCR5 agonists, such as recombinant CXCL13; CXCR6 agonists, such as recombinant CXCL16; and CXCL7 agonists, such as recombinant CXCL11.

The CC chemokine receptor may be selected from: CCR1 agonists such as recombinant CCL3, ECI301, recombinant CCL4, recombinant CCL5, recombinant CCL6, recombinant CCL8, recombinant CCL9/10, recombinant CCL14, recombinant CCL15, recombinant CCL16, recombinant CCL23, PB103, PB105, and MPIF1; CCR2 agonists, such as recombinant CCL2, recombinant CCL8, recombinant CCL16, PB103, and PB105; CCR3 agonists, such as recombinant CCL11, recombinant CCL26, recombinant CCL7, recombinant CCL13, recombinant CCL15, recombinant CCL24, recombinant CCL5, recombinant CCL28, and recombinant CCL18; CCR4 agonists such as recombinant CCL3, ECI301, recombinant CCL5, recombinant CCL17, and recombinant CCL22; CCR5 agonists, such as recombinant CCL3, ECI301, recombinant CCL5, recombinant CCL8, recombinant CCL11, recombinant CCL13, recombinant CCL14, recombinant CCL16, PB103, and PB105; CCR6 agonists, such as recombinant CCL20; CCR7 agonists, such as recombinant CCL19 and recombinant CCL21; CCR8 agonists, such as recombinant CCL1, recombinant CCL16, PB103, and PB105; CCR9 agonists, such as recombinant CCL25; CCR10 agonists, such as recombinant CCL27 and recombinant CCL28; and CCR11 agonists such as recombinant CCL19, recombinant CCL21, and recombinant CCL25.

The C chemokine receptor can be an XCR1 agonist, e.g., recombinant XCL1 or recombinant XCL2.

The CX3C chemokine receptor can be a CX3CR1 agonist such as recombinant CX3C L1.

Chemoattractant receptors can be selected from: formyl peptide receptor agonists such as N-formyl peptide, N-formyl methionine-leucyl-phenylalanine, enfuvirtide, T21/DP107, annexin A1, ac2-26 and Ac9-25; c5a receptor agonists; and chemokine-like receptor 1 agonists such as, for example, merrelin.

Chemokine antagonists may be selected from: inhibitors of CXCL chemokines, such as ubs 5162; inhibitors of CXCL8, such as BMS986253 and PA620; inhibitors of CXCL10, such as TM110, edilumab (elderlumab), and NI0801; inhibitors of CXCL12, such as NOX-a12 and JVS100; inhibitors of CXCL13, such as VX5; inhibitors of CCL2, such as PA508, ABN912, AF2838, BN83250, BN83470, C243, CGEN54, CNTO888, NOXE36, VT224, and SSR150106. Inhibitors of CCL5, such as HGS1025 and NI0701; inhibitors of CCL2/CCL5, such as BKTP46; inhibitors of the CCL5/FMLP receptor, such as RAP160; inhibitors of CCL11, such as Bai Ti mab and RAP701; inhibitors of CCL5/CXCL4, such as CT2008 and CT2009; inhibitors of CCL20, such as GSK3050002; and inhibitors of CX3CL1, such as quinuclidine Mo Lishan.

Chemokine receptor antagonists may be selected from: inhibitors of CXCR1, such as rapalixin, CCX832, FX68 and KB03; inhibitors of CXCR2, for example AZD5069, AZD5122, AZD8309, GSK1325756, GSK1325756H, PS291822, SB332235 and SB656933. Inhibitors of CXCR1/CXCR2 such as DF1970, DF2156A, DF2162, DF2755A, rapamycin, SX576, SX682, PACG31P, AZD4721 and PA401; inhibitors of CXCR 3; inhibitors of CXCR4, such as BL8040; inhibitors of CXCR 4/E-selectin, such as GMI1359; inhibitors of CXCR6, such as CCX5224; inhibitors of CCR1, such as AZD4818, BAY865047, BMS817399, CCX354, CCX634, CCX9588, CP481715, MLN3701, MLN3897, PS031291, PS375179, and PS386113; inhibitors of CCR2, such as AZD2423, BL2030, BMS741672, CCX140, CCX598, CCX872, CCX915, CNTX6970, INCB3284, INCB3344, INCB8696, JNJ17166864, JNJ27141491, MK0812, oplcl 2LPM, PF4136309, serocion, STIB0201, STIB0211, STIB0221, STIB0232, STIB0234, TAK202, TPI526; inhibitors of CCR2/CCR5, such as PF04634817, RAP103 and TBR652; inhibitors of CCR2/CCR5/CCR8, such as RAP310; inhibitors of CCR3, such as ASM8, AXP1275, BMS639623, CM101, DPC168, GW766994, GW824575, MT0814, oplcl 11LPM and QAP642; inhibitors of CCR4, such as AT008, AZD2098, CCX6239, FLX193, FLX475, GBV3019, GSK2239633, IC487892, and Mo Geli bead mab (poteligeo); inhibitors of CCR5, such as 5P12-RANTES, AZD5672, AZD8566, CMPD167, ESN196, GSK706769, GW873140, HGS004, INCB15050, INCB9471, L872, microbiocides, PF232798, PRO140, RAP101, SAR113244, SCH350634, SCH351125, SCH417690, maraviroc, TAK779, TBR220, TD0232 and VX286. Inhibitors of CCR5/CXCR4, such as AMD887, ND401 and SP01A. Inhibitors of CCR6, such as CCX507, CCX9664 and STIB100X; inhibitors of CCR6, e.g., CCX025, CCX507, CCX807, eut, MLN3126, POL7085, trahcet-EN; inhibitors of CXCR3, such as AMG487, AT010, STIA120X; inhibitors of CXCR4, such as AD114, AD214, ALX0651, ALX40-4C, AMD070, AT007, AT009, BKT170, BMS936564, plug Li Shafu, CTCE9908, GBV4086, GSK812397, KRH2731, KRH3140, LY2510924, LY2624587, mozobil, OPLCXCL12LPM, PF06747143, POL6326, Q122, revixil, TG0054, USL311, X4P001 and X4P002; and inhibitors of CXCR7, such as CCX650 and CCX662.

Cytokine receptor agonists may be selected from: mRNA, DNA or plasmid encoding IL-2, IL-15, IL-7, IL-10, IL-12, IL-21, IFNα IL-17, IFNβ, IFNγ, IL-18, IL-27, TNF α, GM-CSF, FLT3L, LT α, LT β and TRAIL, and recombinant protein genes, such as agonists of the IL-2/IL-15 β/γ receptor, agonists of the IL-10 receptor, agonists of the IL-12 receptor, agonists of the IL-18 receptor, agonists of the IL-21 receptor, agonists of the IL-7 receptor, agonists of the IFNα/β receptor, agonists of the IFNγ receptor, agonists of the FLT3 receptor, agonists of the GM-CSF receptor, agonists of the LT α receptor, agonists of the LT β receptor and agonists of the TNF α receptor.

Examples of IL-10 receptor agonists are AG011, dekavil, EG10, IL10Nanocap, ilointerleukin, AM0010, tainovir and VT310 VIRON.

Examples of IL-12 receptor agonists are recombinant IL-12p70, recombinant IL-12p35, AM0012, AS1409, dodekin, hemaMax, lipoVIL, MSB0010360N, ad-RTS-hIL-12, tavokinogene telseplasmid, exoIL-12 and NHS-IL12.

An example of an IL-18 receptor agonist is SB485232.

An example of an IL-21 receptor agonist is BMS982470 (Denican).

Examples of IL-7 receptor agonists are CYT107, CYT99007 and GX-I7.

An example of an FLT3R agonist is CDX-301.

Examples of TNFα receptor agonists are L19-TNFα, aurimune, beromun, breMel/TNFα, fibromun, refnot and TNFPEG20.

Death receptor agonists may be selected from trail 1/DR4 agonists, such as AMG951 (Du Lale min), APG350, APG880, HGSETR1 (Ma Pamu mab) and SL231; and TRAILR2/DR5 agonists such as AMG655, DS8273, HGSETR2 (Leishmaniab), HGSTR2J, IDD004/GEN1029, INBRX109, LBY135, MEDI3039, PRQ95780, RG7386 and TAS266.

The CD47 antagonist may be selected from the group consisting of ALX148, CC-90002, hu5F9G4, SRF231, TI061, TTI-621, TTI-622, AO 1 76, IBI 1 88, IMC002, recombinant SIRP lambda and LYN0030 1.

Examples of sirpa antagonists are FSI89 or recombinant CD47.

Examples of oncolytic agents are CAVATAK, BCG, mobilan, TG4010, pexa-Vec (JX-594), JX-900, JX-929 and JX-970.

Examples of signal-converting proteins are Fn14-TRAIL (KAHR 101), CD80-Fc (FTP 155), CTLA4-FasL (KAHR 102), PD1-41BBL (DSP 105), PD-L1-41BB (PRS-344, NM21-1480, FS 222), PD1-CD70 (DSP 106) and SIRPalpha-41 BBL (DSP 107).

The epigenetic modifications may be selected from the group consisting of DNA methyltransferase inhibitors, lysine-specific demethylase 1 inhibitors, zeste homolog 2 inhibitors, bromodomain and terminal external motif (BET) protein inhibitors such as GSK525762, and Histone Deacetylase (HDAC) inhibitors such as resveratrol, SNDX275 and CKD-M808.

Examples of tumor peptides/vaccines are NY-ESO, WT1, MART-1, IO102 and PF-06753512, personalized cancer vaccines using patient derived tumor sequences or neoantigens.

An example of a Heat Shock Protein (HSP) inhibitor is an HSP90 inhibitor, such as PF-04929113 (SNX-5422).

Examples of proteolytic enzymes are recombinant hyaluronidases, such as rHuPH20 and PEGPH20.

Ubiquitin and proteasome inhibitors can be selected from Ubiquitin Specific Protease (USP) inhibitors, such as P005091;20S proteasome inhibitors such as bortezomib, carfilzomib, iferum Shami, olprine Luo Mibu, delazomib and celastrol; and immunoprotease inhibitors such as ONX-0914.

The adhesion molecule antagonist may be selected from the group consisting of a β2-integrin antagonist and a selectin antagonist.

The hormone may be selected from hormone receptor agonists and hormone receptor antagonists.

Examples of hormone receptor agonists are somatostatin receptor agonists such as somatostatin, lanreotide, octreotide, FX125L, FX141L and FX87L.

Examples of hormone receptor antagonists are antiandrogens, antiestrogens and antiprogestins. Examples of antiandrogens are steroidal antiandrogens, such as cyproterone acetate, megestrol acetate, spironolactone, osendolone and ao Sha Telong acetate. Non-steroidal antiandrogens, such as flutamide, bicalutamide, nilutamide, topiramate, enzalutamide, and aclinamide; androgen synthesis inhibitors such as ketoconazole, abiraterone acetate, lingual vitamin, aminoglutethimide, finasteride, dutasteride, eplerenone and alfaestrol. Examples of antiestrogens are Selective Estrogen Receptor Modulators (SERMs), such as tamoxifen, clomiphene, farrerest and raloxifene; ER silencing antagonists and selective estrogen receptor degrading agents (SERDs), such as fulvestrant. Aromatase inhibitors such as anastrozole, letrozole, exemestane, fu Luo, formestane and fazodone; and anti-gonadotropins such as testosterone, progestin and GnRH analogues. Examples of antiprogestins are mifepristone, li Luopu pristone and onapristone.

Examples of cell therapies include CAR therapies such as tisagenlectleicel, axicabtagene ciloleucel, bb21217, LCAR-B38M, JCARH, MCARH171, JNJ-4528, idecabtagene vicleucel (bb 2121), SCRI-CAR19x22; a CAR therapy targeting a tumor antigen, e.g., a CAR therapy targeting a CD19 expressing cell, a CAR therapy targeting a CD22 expressing cell, a CAR therapy targeting a BCMA expressing cell, a CAR therapy targeting a HER2 expressing cell, a CAR therapy targeting a CD138 expressing cell, a CAR therapy targeting a CD133 expressing cell, a CAR therapy targeting a BCMA expressing cell, a CAR therapy targeting a CEA expressing cell, a CAR therapy targeting a claudin18.2 expressing cell, a CAR therapy targeting an EGFR expressing cell, a CAR therapy targeting an EGFRvIII expressing cell, a CAR therapy targeting an Eph2A expressing cell, a CAR therapy targeting an EpCAM expressing cell, a CAR therapy targeting a GD2 expressing cell, a CAR therapy targeting a GPC3 expressing cell, a CAR therapy targeting a MSLN expressing cell, a CAR therapy targeting a 5T4 expressing cell, a CAR therapy targeting a LMP1 expressing cell, a CAR therapy targeting a PD-L1 expressing cell, a PSMA expressing cell, a CAR therapy targeting an FRa expressing cell, and a MUC1 expressing cell. Examples of cell therapies include TIL therapy, NK therapy, cytokine-induced memory NK cell therapy, NK cell therapy with in vitro expansion of cells. Examples of cell therapies include treatment with αβ or γδ T cells that may be engineered to express a tumor antigen or tumor neoantigen specific T cell receptor or expanded in the context of a tumor antigen or tumor neoantigen.

In certain embodiments, the patient is a mammalian patient, such as a human patient.

Administration of the IL-2 protein of formula (I), IL-2 conjugate or pharmaceutical composition described herein may be by external application, injection or infusion, including intra-articular, periarticular, intradermal, subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal, intrathecal, intracapsular, intraorbital, intratumoral, intravitreal, intraventricular, intravesical, intracapsular, subcapsular, subarachnoid, intraspinal, intracerebroventricular, intrasternal injection and infusion, directly to the brain via an implant device that allows for delivery of the invention or analog to brain tissue or brain fluid (e.g., ommaya reservoir), directly into the brain, into brain or brain-related areas, into the subcuticular space, post-orbital injection and ocular instillation, preferably by subcutaneous injection. In certain embodiments, administration is by subcutaneous injection.

In another aspect, the invention relates to IL-2 protein sequences of formula (I-I)

(Tag ¹ ) _y -(X ₁ )x-SEQ A-SEQ B-SEQ C-(Tag ² ) _z (I-i)，

Wherein the method comprises the steps of

SEQ A and SEQ ID NO:1 has at least 89% sequence identity;

SEQ C and SEQ ID NO:4 has at least 91% sequence identity;

Tag ¹ and Tag ² Independently a label moiety;

X ₁ amino acid residues selected from the group consisting of arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine; and is also provided with

x is 0 or 1;

y is 0 or 1; and is also provided with

z is 0 or 1.

SEQ A、SEQ B、SEQ C、Tag ¹ 、Tag ² Specific embodiments of x, y and z are as described for IL-2 proteins of formula (I).

In certain embodiments, X of formula (I-I) ₁ Is an arginine residue. In certain embodiments, X of formula (I-I) ₁ Is an asparagine residue. At the position ofIn certain embodiments, X of formula (I-I) ₁ Is an aspartic acid residue. In certain embodiments, X of formula (I-I) ₁ Are cysteine residues. In certain embodiments, X of formula (I-I) ₁ Is a glutamine residue. In certain embodiments, X of formula (I-I) ₁ Is a glutamic acid residue. In certain embodiments, X of formula (I-I) ₁ Is a glycine residue. In certain embodiments, X of formula (I-I) ₁ Is a histidine residue. In certain embodiments, X of formula (I-I) ₁ Is an isoleucine residue. In certain embodiments, X of formula (I-I) ₁ Is a leucine residue. In certain embodiments, X of formula (I-I) ₁ Is a lysine residue. In certain embodiments, X of formula (I-I) ₁ Is a methionine residue. In certain embodiments, X of formula (I-I) ₁ Is a phenylalanine residue. In certain embodiments, X of formula (I-I) ₁ Serine residues. In certain embodiments, X of formula (I-I) ₁ Is a threonine residue. In certain embodiments, X of formula (I-I) ₁ Are tryptophan residues. In certain embodiments, X of formula (I-I) ₁ Is a tyrosine residue. In certain embodiments, X of formula (I-I) ₁ Is a valine residue.

Another aspect of the invention relates to the coding of formula (I-I) IL-2 protein oligonucleotide sequences. Particular embodiments of such oligonucleotides are described herein in further detail for oligonucleotides encoding IL-2 proteins of formula (I), except that the IL-2 protein of formula (I) is replaced by an IL-2 protein of formula (I-I).

Another aspect of the invention relates to conjugates comprising one or more IL-2 proteins of formula (I-I). Particular embodiments of such conjugates comprising one or more IL-2 proteins of formula (I-I) are described elsewhere herein for conjugates comprising one or more IL-2 proteins of formula (I), except that the IL-2 protein of formula (I) is replaced with an IL-2 protein of formula (I-I).

Another aspect of the invention relates to pharmaceutical compositions comprising at least one IL-2 protein of formula (I-I) or at least one IL-2 conjugate comprising one or more IL-2 proteins of formula (I-I) and at least one excipient. Particular embodiments of such pharmaceutical compositions are described herein in further detail for pharmaceutical compositions comprising at least one IL-2 protein of formula (I) or at least one IL-2 conjugate comprising one or more IL-2 proteins of formula (I), except that the IL-2 protein of formula (I) is replaced by an IL-2 protein of formula (I-I).

Another aspect relates to IL-2 proteins of formula (I-I), IL-2 conjugates comprising at least one IL-2 protein of formula (I-I) or pharmaceutical compositions comprising such IL-2 proteins or IL-2 conjugates for use as a medicament.

Another aspect relates to IL-2 proteins of formula (I-I), IL-2 conjugates comprising at least one IL-2 protein of formula (I-I), or pharmaceutical compositions comprising such IL-2 proteins or IL-2 conjugates for use in the treatment of diseases treatable with IL-2. Specific embodiments of diseases that can be treated with IL-2 are described elsewhere herein.

Another aspect relates to IL-2 proteins of formula (I-I), IL-2 conjugates comprising at least one IL-2 protein of formula (I-I), or pharmaceutical compositions comprising such IL-2 proteins or IL-2 conjugates for use in the manufacture of a medicament for the treatment of a disease treatable with IL-2. Specific embodiments of diseases that can be treated with IL-2 are described elsewhere herein.

Another aspect relates to a method of treating, controlling, delaying or preventing one or more diseases in a mammalian patient, preferably a human patient, in need of treatment, which diseases may be treated with IL-2, the method comprising the steps of: administering to said patient in need thereof a therapeutically effective amount of an IL-2 protein of formula (I-I), an IL-2 conjugate comprising at least one IL-2 protein of formula (I-I), or a pharmaceutical composition comprising such an IL-2 protein or IL-2 conjugate. Specific embodiments of diseases that can be treated with IL-2 are described elsewhere herein.

In certain embodiments, the IL-2 protein of formula (I-I), an IL-2 conjugate comprising at least one IL-2 protein of formula (I-I), or a pharmaceutical composition comprising such IL-2 protein or IL-2 conjugate is administered to a patient prior to, concurrently with, or after administration of one or more additional drugs. Specific embodiments of such one or more additional agents are described elsewhere herein.

Another aspect of the invention relates to IL-2 protein sequences of formula (I-ii)

(Tag ¹ ) _y -(X ₁ ) _x -SEQ A-SEQ D-SEQ C-(Tag ² ) _z (I～ii)，

Wherein the method comprises the steps of

SEQ A and SEQ ID NO:1 has at least 89% sequence identity;

SEQ D and SEQ ID NO:2 has at least 76% sequence identity;

SEQ C and SEQ ID NO:4 has at least 91% sequence identity;

Tag ¹ And Tag ² Independently a label moiety;

x is 0 or 1;

y is 0 or 1; and is also provided with

z is 0 or 1.

SEQ A、SEQ C、Tag ¹ 、Tag ² Specific embodiments of x, y and z are as disclosed in the other sections herein.

In certain embodiments, SEQ D of formula (I-ii) hybridizes to SEQ ID NO:2 has at least 78% sequence identity. In certain embodiments, SEQ D of formula (I-ii) hybridizes to SEQ ID NO:2 has at least 80% sequence identity. In certain embodiments, SEQ D of formula (I-ii) hybridizes to SEQ ID NO:2 has at least 82% sequence identity. In certain embodiments, SEQ D of formula (I-ii) hybridizes to SEQ ID NO:2 has at least 84% sequence identity. In certain embodiments, SEQ D of formula (I-ii) hybridizes to SEQ ID NO:2 has at least 87% sequence identity. In certain embodiments, SEQ D of formula (I-ii) hybridizes to SEQ ID NO:2 has at least 89% sequence identity. In certain embodiments, SEQ D of formula (I-ii) hybridizes to SEQ ID NO:2 has at least 91% sequence identity. In certain embodiments, SEQ D of formula (I-ii) hybridizes to SEQ ID NO:2 has at least 93% sequence identity. In certain embodiments, SEQ D of formula (I-ii) hybridizes to SEQ ID NO:2 has at least 95% sequence identity.

In certain embodiments, SEQ D of formula (I-ii) comprises a sequence that hybridizes to SEQ ID NO:2 compared to eleven amino acid changes. In certain embodiments, SEQ D of formula (I-ii) comprises a sequence that hybridizes to SEQ ID NO:2 compared to ten amino acid changes. In certain embodiments, SEQ D of formula (I-ii) comprises a sequence that hybridizes to SEQ ID NO:2 compared to nine amino acid changes. In certain embodiments, SEQ D of formula (I-ii) comprises a sequence that hybridizes to SEQ ID NO:2 compared to eight amino acid changes. In certain embodiments, SEQ D of formula (I-ii) comprises a sequence that hybridizes to SEQ ID NO:2 compared to seven amino acid changes. In certain embodiments, SEQ D of formula (I-ii) comprises a sequence that hybridizes to SEQ ID NO:2 compared to six amino acid changes. In certain embodiments, SEQ D of formula (I-ii) comprises a sequence that hybridizes to SEQ ID NO:2 compared to five amino acid changes. In certain embodiments, SEQ D of formula (I-ii) comprises a sequence that hybridizes to SEQ ID NO:2 compared to four amino acid changes. In certain embodiments, SEQ D of formula (I-ii) comprises a sequence that hybridizes to SEQ ID NO:2 compared to three amino acid changes. In certain embodiments, SEQ D of formula (I-ii) comprises a sequence that hybridizes to SEQ ID NO:2 compared to two amino acid changes.

In certain embodiments, SEQ D of formula (I-ii) has a sequence selected from the group consisting of:

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in certain embodiments, the SEQ D of formula (I-ii) further comprises a sequence selected from the group consisting of SEQ ID NOs: 2, R8, M9, T11, F12, K13, F14, Y15, E31, E32 and L42 or at least one amino acid mutation present at a corresponding position of a homologue or variant thereof. In certain embodiments, the at least one amino acid mutation comprises a sequence selected from the group consisting of SEQ ID NOs: 2, or a mutation at the amino acid positions of F12, Y15, E31, E32 and L42 or at the corresponding positions of a homologue or variant thereof. In certain embodiments, the at least one amino acid mutation comprises a sequence based on SEQ ID NO:2 or a mutation at the corresponding position of a homologue or variant thereof. In certain embodiments, the at least one amino acid mutation comprises a sequence based on SEQ ID NO:2 or a mutation at the corresponding position of a homologue or variant thereof. In certain embodiments, the at least one amino acid mutation comprises a sequence based on SEQ ID NO:2 or a mutation at the corresponding position of a homologue or variant thereof. In certain embodiments, the at least one amino acid mutation comprises a sequence based on SEQ ID NO:2 or a mutation at the corresponding position of a homologue or variant thereof. In certain embodiments, the at least one amino acid mutation comprises a sequence based on SEQ ID NO:2 or a mutation at the corresponding position of a homologue or variant thereof. In certain embodiments, the at least one amino acid mutation comprises a sequence based on SEQ ID NO:2 or a mutation at the corresponding position of a homologue or variant thereof. In certain embodiments, the at least one amino acid mutation comprises a sequence based on SEQ ID NO:2 or a mutation at the corresponding position of a homologue or variant thereof. In certain embodiments, the at least one amino acid mutation comprises a sequence based on SEQ ID NO:2 or a mutation at the corresponding position of a homologue or variant thereof. In certain embodiments, the at least one amino acid mutation comprises a sequence based on SEQ ID NO:2E31 or a mutation at the corresponding position of a homologue or variant thereof. In certain embodiments, the at least one amino acid mutation comprises a sequence based on SEQ ID NO:2 or a mutation at the corresponding position of a homologue or variant thereof. In certain embodiments, the at least one amino acid mutation comprises a sequence based on SEQ ID NO:2 or a mutation at the corresponding position of a homologue or variant thereof.

In certain embodiments, X of formula (I-ii) ₁ Is an arginine residue. In certain embodiments, X of formula (I-ii) ₁ Is an asparagine residue. In certain embodiments, X of formula (I-ii) ₁ Is an aspartic acid residue. In certain embodiments, X of formula (I-ii) ₁ Are cysteine residues. In certain embodiments, X of formula (I-ii) ₁ Is a glutamine residue. In certain embodiments, X of formula (I-ii) ₁ Is a glutamic acid residue. In certain embodiments, X of formula (I-ii) ₁ Is a glycine residue. In certain embodiments, X of formula (I-ii) ₁ Is a histidine residue. In certain embodiments, X of formula (I-ii) ₁ Is an isoleucine residue. In certain embodiments, X of formula (I-ii) ₁ Is a leucine residue. In certain embodiments, X of formula (I-ii) ₁ Is a lysine residue. In certain embodiments, X of formula (I-ii) ₁ Is a methionine residue. In certain embodiments, X of formula (I-ii) ₁ Is a phenylalanine residue. In certain embodiments, X of formula (I-ii) ₁ Serine residues. In certain embodiments, X of formula (I-ii) ₁ Is a threonine residue. In certain embodiments, X of formula (I-ii) ₁ Are tryptophan residues. In certain embodiments, X of formula (I-ii) ₁ Is a tyrosine residue. In certain embodiments, X of formula (I-ii) ₁ Is a valine residue.

In certain embodiments, the IL-2 protein of formula (II-i) has the sequence

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In certain embodiments, the IL-2 protein of formula (II-i) has the sequence

In certain embodiments, the IL-2 protein of formula (I-ii) may be obtained by enzymatic or chemical removal of N-linked or O-linked glycans from an IL-2 protein of formula (I) or (I-I).

In certain embodiments, the IL-2 protein of formula (I-ii) may be obtained by peptide-N-glycosidase F (PNgaseF) mediated enzymatic removal of one or more N-linked glycans from the IL-2 protein of formula (I) or (I-I).

In certain embodiments, SEQ ID NO:334 can be treated by PNgaseF with SEQ ID NO:9, and the IL-2 protein.

In certain embodiments, SEQ ID NO:340 can be treated by PNgaseF with SEQ ID NO:10, and the IL-2 protein is obtained.

In certain embodiments, SEQ ID NO: the IL-2 protein of 335 can be treated by PNgasef to give the sequence of SEQ ID NO:12, IL-2 protein.

In certain embodiments, SEQ ID NO:341 can be treated by PNgaseF with the IL-2 protein of SEQ ID NO:13, and the IL-2 protein.

In certain embodiments, SEQ ID NO:336 can be treated by PNgaseF with SEQ ID NO:15, and the IL-2 protein of 15.

In certain embodiments, SEQ ID NO:342 can be treated by PNgaseF with SEQ ID NO:16, and the IL-2 protein of 16.

In certain embodiments, SEQ ID NO:337 can be treated by PNgaseF with SEQ ID NO:18, and the IL-2 protein of 18.

In certain embodiments, SEQ ID NO:343 can be treated by PNgaseF with the IL-2 protein of SEQ ID NO:19, and the IL-2 protein of 19.

In certain embodiments, SEQ ID NO:338 can be treated by PNgaseF with SEQ ID NO:21, and the IL-2 protein of 21.

In certain embodiments, SEQ ID NO:344 can be treated by PNgaseF with SEQ ID NO:22, and the IL-2 protein is obtained.

In certain embodiments, SEQ ID NO:339 can be treated by PNgaseF with SEQ ID NO:24, and the IL-2 protein of 24.

In certain embodiments, SEQ ID NO:345 IL-2 protein can be treated by PNgaseF with SEQ ID NO:25, and the IL-2 protein of 25.

In another aspect, the invention relates to an oligonucleotide sequence encoding an IL-2 protein of formula (I-ii). Particular embodiments of such oligonucleotides are the same as the oligonucleotides described elsewhere herein that encode an IL-2 protein of formula (I), except that the IL-2 protein of formula (I) is replaced by an IL-2 protein of formula (I-ii).

In another aspect, the invention relates to conjugates comprising one or more IL-2 proteins of formula (I-ii). Such specific embodiments of conjugates comprising one or more IL-2 proteins of formula (I-ii) are the same as the conjugates comprising one or more IL-2 proteins of formula (I) described elsewhere herein, except that the IL-2 protein of formula (I) is replaced by an IL-2 protein of formula (I-ii).

In another aspect, the invention relates to a pharmaceutical composition comprising at least one IL-2 protein of formula (I-ii) or at least one IL-2 conjugate comprising one or more IL-2 proteins of formula (I-ii) and at least one excipient. Particular embodiments of such pharmaceutical compositions are identical to those described elsewhere herein comprising at least one IL-2 protein of formula (I) or at least one IL-2 conjugate comprising one or more of such IL-2 proteins of formula (I), except that the IL-2 protein of formula (I) is replaced by an IL-2 protein of formula (I-ii).

Another aspect relates to an IL-2 protein of formula (I-ii), an IL-2 conjugate comprising at least one IL-2 protein of formula (I-ii) or a pharmaceutical composition comprising such an IL-2 protein or IL-2 conjugate for use as a medicament.

Another aspect relates to IL-2 proteins of formula (I-ii), IL-2 conjugates comprising at least one IL-2 protein of formula (I-ii) or pharmaceutical compositions comprising such IL-2 proteins or IL-2 conjugates for use in the treatment of diseases treatable with IL-2. Specific embodiments of diseases that can be treated with IL-2 are described elsewhere herein.

Another aspect relates to IL-2 proteins of formula (I-ii), IL-2 conjugates comprising at least one IL-2 protein of formula (I-ii) or pharmaceutical compositions comprising such IL-2 proteins or IL-2 conjugates for the preparation of a medicament for the treatment of a disease treatable with IL-2. Specific embodiments of diseases that can be treated with IL-2 are described elsewhere herein.

Another aspect relates to a method of treating, controlling, delaying or preventing one or more diseases in a mammalian patient, e.g., a human patient, in need of treatment, which diseases may be treated with IL-2, the method comprising the steps of: administering to said patient in need thereof a therapeutically effective amount of an IL-2 protein of formula (I-ii), an IL-2 conjugate comprising at least one IL-2 protein of formula (I-ii), or a pharmaceutical composition comprising such an IL-2 protein or IL-2 conjugate. Specific embodiments of diseases that can be treated with IL-2 are described elsewhere herein.

In certain embodiments, an IL-2 protein of formula (I-ii), an IL-2 conjugate comprising at least one IL-2 protein of formula (I-ii), or a pharmaceutical composition comprising such an IL-2 protein or IL-2 conjugate is administered to a patient prior to, concurrently with, or after the administration of one or more other drugs. Specific embodiments of the one or more other drugs are described elsewhere herein.

Materials and methods

Material

All materials are commercially available unless otherwise indicated.

Method

Determination of protein concentration

Using the manufacturer predefined settings, in alkaline EppendorfThe calculated molecular extinction coefficient and molecular weight were used to determine protein concentration.

PNGase F assay

Purified IL-2 variants were mixed with or without PNGase F (Promega, # V4831, lot 0000386354) and processed according to the manufacturer's instructions. Subsequently, the samples were reduced and heat treated, and then loaded onto SDS-PAGE gels. Protein bands were stained with Bio-Safe Coomassie G-250 stain (Bio-Rad, # 161078) according to the manufacturer's instructions. Glycosylated IL-2 variants expressed in HEK293-6E and CHO cells migrate to a double band of approximately 18-20 kDa in SDS-PAGE. After treatment with PNGase F a significant shift down to about 15-17 kDa was observed, indicating efficient N-glycosylation of IL-2 variants expressed in CHO cells.

Peptide profile analysis

10 μg IL-2 variant and 1 μg Pierce were used ^TM trypsin/Lys-C protease (from Thermo Fisher) was mixed in 100mM Hepes buffer pH 8.0. The reaction mixture was incubated at 37.+ -. 1 ℃ for 18.+ -. 1 hour. Undigested sample controls were prepared in the same buffer without any protease.

After incubation, the samples/controls were briefly centrifuged and 20 μl UPLC-MS water was added. Diluted samples were analyzed using an Agilent 1290 UHPLC system connected to mass spectrometer QT. Analysis was performed using 0.075% (v/v) aqueous trifluoroacetic acid (mobile phase A), 0.06% (v/v) aqueous MeCN of trifluoroacetic acid (mobile phase B). The gradient shown in table 1 below was run using a Halo C18, 100×2.1,2.7 μm, 90A column:

Table 1: gradient of

Time [ min]	％B
		0	0.5
1.0	0.5
		9.2	25.7
10.5	33.0
		26.0	61.0
26.2	99.9
		33.0	99.9
34.0	0.5
		40.0	0.5

Note that: flow rate=0.5 mL/min, ct=21°c

The flow rate was set to 0.5 mL/min. The column oven was set to 21 ℃.

Example 1: expression of glycosylated IL2 in HEK293-6E

Vector construction

The IL-2 glycosylation variants were custom made and were derived from an external vendor, where protein expression was performed from 293-6E cells, followed by standard purification strategies known to those skilled in the art. The following proteins were prepared:

corresponding to the DNA sequence SEQ ID NO:7 (SEQ ID NO: 205); corresponding to the DNA sequence SEQ ID NO:8 (SEQ ID NO: 229); corresponding to the DNA sequence SEQ ID NO:202 (SEQ ID NO: 230); and corresponds to the DNA sequence SEQ ID NO:203 protein 1d (SEQ ID NO: 232):

encoding SEQ ID NO: 205. SEQ ID NO: 229. SEQ ID NO:230 and SEQ ID NO:232, comprising an N-terminal human kappa light chain leader sequence and a C-terminal hexahistidine tag, and inserted into the EcoRI and BamHI sites of the pt 5 mammalian expression vector. The constructed plasmids were transformed into E.coli (E.coli) strain (DH 5a/TOP 10) to proliferate on an appropriate scale, respectively. Nucleobond Xtra Maxi Plus EF kit (MN 740426) is used for large scale plasmid production. Purified plasmids were checked by agarose gel and confirmed by sequencing.

Protein expression:

for mammalian cell expression, plasmids for each protein were transfected into HEK293-6E cells obtained from CNRC-NRC ("Conseil National de Recherches Canada") using PEI (Polyscience, catalog No. 23966). Cells were cultured in an incubator shaker under standard cell culture conditions. Conditioned medium was harvested 6-7 days after transfection.

Protein purification and quality control:

conditioned Medium (CM) was recovered by centrifugation to discard cell debris, and then filtered. Clarified CM was loaded onto Ni-excel (GE) column and protein purified according to manufacturer's instructions. The concentrated material was then further purified using a Superdex75 column (GE).

Purified proteins were analyzed by SDS-PAGE (with and without PNGase F (NEB) pretreatment), SEC-HPLC and endotoxin measurement with EndoSafe-PTS kit (Charles River).

Results:

in the construct, protein 1a was expressed well in a similar yield (0.97 mg) to the non-N-glycosylated IL-2 control (protein 1d,1.0 mg). Surprisingly, at least 90% of protein 1a migrates at a higher molecular weight (about 18 kDa) than the control non-N-glycosylated IL-2 protein 1d (15 kDa), suggesting the presence of N-glycans in 1a, which reduces mobility in SDS-PAGE. When protein 1a was treated with protein N-glycosidase F (PNGase F) to remove N-glycans, the treated protein migrated uniformly at about 16 kDa, similar to wild-type IL-2 (protein 1 d), indicating that almost all protein 1a was N-glycosylated. As expected, in PNGase F treated wells there is also another-36 kDa band corresponding to the molecular weight of PGNase F itself.

Example 2: expression of glycosylated IL2 with cleavable HIS tag in CHO cells

And (3) constructing a carrier:

the IL-2 glycosylation variants are custom made and are derived from an external vendor, where expression of the protein is performed from CHO cells, followed by standard purification strategies known to those skilled in the art. The following proteins were prepared:

corresponding to DNA SEQ ID NO:233 (SEQ ID NO: 231).

Protein 1e was then treated with EnteroKinase to yield the final product protein 1f (SEQ ID NO: 10).

For the coding DNA sequence SEQ ID NO:233, which included an N-terminal human kappa light chain leader sequence, an N-terminal hexahistidine tag, and an N-terminal enterokinase cleavage site to remove His tag, and inserted into a pTT5 mammalian expression vector. The constructed plasmids were transformed into E.coli strains (DH 5a/TOP 10) to proliferate on an appropriate scale, respectively. NucleoBond Xtra Maxi Plus EF kit (MN 740426) is used for large scale plasmid production. Purified plasmids were checked by agarose gel and confirmed by sequencing.

Protein expression:

plasmids for each protein were transfected into EXPI-CHO cells obtained from Thermo Fisher using PEI Max (Polyscience, catalog number 24765). Cells were cultured in an incubator shaker under standard cell culture conditions. Conditioned medium was harvested 9 days after transfection.

Protein purification and quality control:

conditioned Medium (CM) was recovered by centrifugation to discard cell debris and then filtered using a Sartopore 2 filter (Sartorius). Clarified CM was loaded onto Ni-excel (GE) column and protein purified according to manufacturer's instructions. The eluted protein was incubated with EK protease (Genscript, Z03004-100) to cleave the His tag. The material was further purified using another nickel column followed by a Superdex 75 column (GE). Purified proteins were analyzed by SDS-PAGE, SEC-HPLC and endotoxin measurements with the EndoSafe-PTS kit (Charles River).

Results:

similar to the expression of N-glycosylated IL2 observed in 293 cells (protein 1 a), at least 90% of the N-glycosylated IL2 expressed in CHO cells (protein 1f derived from protein 1 e) consistently exhibited a higher band with reduced migration of about-17 kDa, indicating a surprisingly effective N-glycosylation of protein 1f, compared to experiments with control IL2 (. About.15 kDa) alone. The migration pattern of CHO-derived His-tag-removed N-glycosylated IL2 (protein 1 f) was slightly smaller (17 kDa) than the His-tag (-18 kDa) -containing 293-expressed construct (protein 1 a) as expected, consistent with MW of the 840Da 6 xHis-tag.

Example 3: biological Activity of glycosylated IL2 produced by 293 in human Primary cell pSTAT5 assay

The biological activity of wild-type IL2 protein 1d and glycosylation-engineered IL2 protein 1a expressed from 293 of example 1 was tested by an external supplier using human blood. After treatment of the samples with protein 1d or protein 1a, phosphorylation of STAT5 (pSTAT 5) (proximal IL2 receptor signaling biomarker) was examined. Heparinized human whole blood was drawn and equilibrated to room temperature, aliquoted to plates and warmed to 37 ℃, then protein 1d or protein 1a was added. Dose titration of protein 1d or protein 1a was prepared and pre-heated to 37 ℃ prior to addition to cells. After addition of protein 1d or protein 1a, the cells were incubated at 37℃for 30 minutes. At the end of the incubation period, the erythrocytes were lysed and the cells were fixed. After washing, the cells were then stained for surface antigens, washed and permeabilized with methanol, then washed and intracellular stained for pSTAT5 and additional lineage markers. The following markers were used to define the cell population of interest: CD8+ T cells are defined as CD3+CD4-CD8+; CD4+ T cells are defined as CD3+CD4+CD8-; regulatory T cells (tregs) are defined as cd3+cd4+cd8-cd25+foxp3+; natural Killer (NK) cells are defined as CD3-CD56 or CD16+.

Results:

the half maximal effective concentration (EC 50) of cd8+ T cells, NK cells and tregs was determined after treatment with wild-type IL-2 control (1 d) or N-glycosylated IL2 (1 a). Importantly, cd8+ T cells and NK cells express high affinity alpha subunits of the IL-2 receptor (IL 2 ra/CD 25) at low resting levels, while tregs express high levels of IL2 ra (CD 25). For this reason, analysis of the change in the relative potency of IL-2 variants between cd8+ T cells or NK cells and tregs is a good measure of the activity on IL-2rβγ (as reflected by cd8+ and NK cell potency) and of IL-2rαβγ (as reflected by Treg potency). We examined the efficacy of these three cell subsets by quantifying the Median Fluorescence Intensity (MFI) of pSTAT5 and the percentage of pSTAT5 positive cells. The summary of pSTAT5 MFI EC50 values is shown in table 2:

table 2: ave EC50 value (ng/ml) based on pSTAT5 MFI value

Using the pSTAT5 MFI metric, N-glycosylated IL-2 protein 1a showed similar potency for CD8+ T cells and NK cells compared to wild type IL-2 control (potency decreased 1.44 and 1.34 fold, respectively, relative to wild type IL-2, table 2). However, N-glycosylated IL-2 protein 1a showed significantly reduced potency (436.56-fold reduced potency, table 2) compared to the wild-type IL-2 control of Treg.

The summary of percent pSTAT5 positive EC50 values is shown in table 3:

table 3: ave EC50 value (ng/ml) based on pSTAT5 percent positive value

Similarly, using the percent pSTAT5 positive metric, N-glycosylated IL-2 protein 1a showed similar potency for cd8+ T cells and NK cells compared to wild-type IL-2 control (1.58 and 2.01 fold decrease in potency, respectively, relative to wild-type IL-2). However, N-glycosylated IL-2 protein 1a showed significantly reduced potency against Treg compared to wild-type IL-2 (1 d) control (598.46-fold reduced potency, table 3).

These data indicate that N-glycosylated IL-2 variant protein 1a shows similar IL-2rβγ activity (as reflected by cd8+ and NK cell potency) but reduced IL-2rβγ activity (as reflected by Treg potency) compared to wild-type IL-2 (1 d). These data demonstrate the "non-IL-2Rα" bias of protein 1 a.

Example 4: biological Activity of CHO-produced glycosylated IL2 in human Primary cell pSTAT5 assay (His-tag removed)

The biological activity of control wild-type IL2 protein 1d and glycosylated altered IL2 protein 1f expressed from CHO cells of example 2 was tested by an external supplier using human blood. After treatment of the samples with protein 1d or protein 1f, phosphorylation of STAT5 (pSTAT 5) (proximal IL2 receptor signaling biomarker) was examined. Heparinized human whole blood was drawn and equilibrated to room temperature, aliquoted to plates and warmed to 37 ℃, then protein 1d or protein 1f was added. Dose titration of protein 1d or protein 1f was prepared and pre-heated to 37 ℃ prior to addition to cells. After addition of protein 1d or protein 1f, the cells were incubated at 37℃for 30 minutes. At the end of the incubation period, the erythrocytes were lysed and the cells were fixed. After washing, the cells were then stained for surface antigens, washed and permeabilized with methanol, then washed and intracellular stained for pSTAT5 and additional lineage markers. The following markers were used to define the cell population of interest: CD8+ T cells are defined as CD3+CD4-CD8+; CD4+ T cells are defined as CD3+CD4+CD8-; regulatory T cells (tregs) are defined as cd3+cd4+cd8-cd25+foxp3+; natural Killer (NK) cells are defined as CD3-CD56 or CD16+.

Results:

the half maximal effective concentration (EC 50) of cd8+ T cells, NK cells and tregs was determined after treatment with wild-type IL-2 control (1 d) or N-glycosylated IL2 (1 f). Importantly, cd8+ T cells and NK cells express high affinity alpha subunits of the IL-2 receptor (IL 2 ra/CD 25) at low resting levels, while tregs express high levels of IL2 ra (CD 25). For this reason, analysis of the change in the relative potency of IL-2 variants between cd8+ T cells or NK cells and tregs is a good measure of the activity of IL-2rβγ (as reflected by cd8+ and NK cell potency) and of IL-2rαβγ (as reflected by Treg potency). We examined the efficacy of these three cell subsets by quantifying the Median Fluorescence Intensity (MFI) of pSTAT5 and the percentage of pSTAT5 positive cells. The summary of pSTAT5 MFI EC50 values is shown in table 4:

table 4: ave EC50 value (ng/ml) based on pSTAT5 MFI value

Using the pSTAT5 MFI metric, N-glycosylated IL-2 protein 1f showed similar potency for CD8+ T cells and NK cells compared to wild type IL-2 control (2.13 and 1.22 fold decrease in potency relative to wild type IL-2, table 4, respectively). However, N-glycosylated IL-2 protein 1f showed significantly reduced potency (371.83-fold reduced potency, table 4) compared to the wild-type IL-2 control of Treg.

These data indicate that N-glycosylated IL-2 variant protein 1f shows similar IL-2rβγ activity (as reflected by cd8+ and NK cell potency) but reduced L-2rαβγ activity (as reflected by Treg potency) compared to wild-type IL-2 (1 d) (supra). These data demonstrate the "non-IL-2Rα" bias of protein 1f expressed in CHO cells.

Example 5: expression of glycosylated IL2 in CHO cells

And (3) constructing a carrier:

the amino acid sequences of protein 2a (SEQ ID NO: 237) and protein 1f (SEQ ID NO: 10) were back translated and codon optimized for expression in CHO cells, each combined with three different N-terminal secretion signal peptides (signal peptide 3a (SEQ ID NO: 238), signal peptide 3b (SEQ ID NO: 239) and signal peptide 3c (SEQ ID NO: 240)) to produce protein 2b (SEQ ID NO:247; signal peptide 3 a+protein 2 a), protein 2c (SEQ ID NO:251; signal peptide 3 c+protein 2 a), protein 2d (SEQ ID NO:249; signal peptide 3 b+protein 2 a), protein 2e (SEQ ID NO:248; signal peptide 3 a+protein 1 f), protein 2f (SEQ ID NO:252; signal peptide 3 c+protein 1 f) and protein 2g (SEQ ID NO:250; signal peptide 3 b+protein 1 f).

The resulting DNA sequences encoding protein 2b (SEQ ID NO: 241), protein 2c (SEQ ID NO: 243), protein 2d (SEQ ID NO: 242), protein 2e (SEQ ID NO: 244), protein 2f (SEQ ID NO: 246) and protein 2g (SEQ ID NO: 245) were synthesized and cloned into the expression vector pPur. The resulting plasmid was maximally prepared from E.coli DH 5. Alpha. Culture (Invitrogen) using the PureLink HiPure Plasmid Maxiprep kit (Invitrogen).

Protein expression:

CHO cells were transfected with the above plasmid and stable cell pools were developed (pool 4a: expressed protein 2b; pool 4b: expressed protein 2c; pool 4c: expressed protein 2d; pool 4d: expressed protein 2e; pool 4e: encoded protein 2f; and pool 4f: expressed protein 2 g). For protein expression, stable libraries were cultured in shake flasks in fed-batch mode. Fed-batch culture was terminated on day 10, and its culture was terminated on day 7 except for pool 4 f. UsingThe easy cell HT (Merck Millipore) assessed cell number and viability. The stable cell banks (banks 4a, 4b, 4c, 4 d),4e and 4 f) were also cultured in Ambr15 bioreactors (Sartorius) in fed-batch mode using commercial growth and feed media for a total of 14 days. By->FLEX2 (Nova) analyzed cell number and viability.

The analysis method comprises the following steps:

the supernatant was quantified for IL-2 protein by ELISA (IL-2 human kit, abcam#ab100566, lot GR 3344842) using rabbit anti-human IL-2 (Abcam, catalog Ab 9618) as primary antibody and donkey anti-rabbit IgG-HRP (Jackson catalog 711-036-152) as secondary antibody and analyzed qualitatively by Western blotting. Western blot detection was performed by using the absorbance of TMB (3, 3', 5' -tetramethylbenzidine) substrate. As a molecular size reference in Western blot analysis, a molecular weight ladder was used Sharp pre-stained protein standard, invitrogen, catalog LC 5801) and recombinant E.coli-derived hIL-2 (Abcam, catalog ab 9619).

Protein purification:

method 1: IL-2 variants were purified by loading clarified CM on Superdex 20016/600 size exclusion chromatography column (GE Hea1 thcare). Fractions were collected and analyzed by SDS-PAGE and Coomassie blue staining. The bands corresponding to monomer IL2 were pooled and buffer exchanged for loading onto a HiScreen Capto Q anion exchange chromatography column (GE Healthcare). Proteins were eluted by applying a linear NaCl gradient followed by a second linear NaCl gradient. Fractions were collected and analyzed by SDS-PAGE and Coomassie blue staining to identify bands corresponding to monomeric IL-2. Purified IL-2 is concentrated and buffer exchanged into storage and/or assay buffer for subsequent experiments.

Method 2: alternatively, IL-2 variants were purified using a method consisting of 4 column steps. Herein, the pH of clarified CM was adjusted to pH 6.0 and concentrated. The cell supernatant was then loaded onto a HiScreen Capto MMC column (GE Healthcare). After washing the column, the IL-2 protein was eluted, followed by a linear NaCl gradient. Fractions were collected and analyzed by SDS-PAGE and Coomassie blue staining. The bands corresponding to monomer IL2 were combined and pH adjusted to pH7.4 for loading onto a HiScreen Capto Blue chromatography column (GE Healthcare). Proteins were eluted by applying a fractionated NaC1 gradient, and then linear NaCl fractions were collected and analyzed by SDS-PAGE and Coomassie blue staining to identify fractions containing monomeric IL-2. Pooled IL-2 fractions from Capto Blue runs were loaded onto HiScreen Capto Adhere column (GE Healthcare) and purified in a flow-through chromatography step. Monomeric IL-2 fractions were identified by SDS-PAGE and Coomassie staining, buffer exchanged and concentrated. The IL-2 protein samples were then loaded onto HiScreen Capto MMC columns (GE Healthcare) and purified as described above. Purified IL-2 was concentrated to-0.5 mg/mL and the buffer was replaced with storage and/or assay buffer for subsequent experiments.

Results:

for expression in shake flasks, the concentration of recombinant IL-2 protein in culture supernatants of cell lines expressing protein 2a (4 a, 4b and 4 c) and protein 1f (4 d, 4e and 4 f) was determined by ELISA on days 8 and 10 of fed-batch culture (Table 5). On days 8 and 10, the concentration of protein 1f was several times higher than the concentration of protein 2 a. For constructs with signal peptide 3a, the expression level of protein 1f was 11.0 times higher than protein 2a on day 8 and 10.0 times higher on day 10; for the construct with signal peptide 3c, the fold change was 3.7 fold at day 8 and 6.0 fold at day 10, and for the construct with signal peptide 3b, the expression level of protein 1f was 12.9 fold higher on day 7 than on day 8.

Table 5. Cell concentration, viability and titre of IL-2 muteins on day 8 and day 10 of fed-batch culture. *4f terminated on day 7, thus presenting the construct's day 7 value.

Western blots of non-reduced samples from culture supernatants of 4a, 4b, and 4c on day 10 of fed-batch culture showed double bands of 14-15kDa in size, consistent with the expectation that CHO cells produced non-glycosylated and O-glycosylated forms of IL-2. The lower product band corresponding to the non-glycosylated protein 2a product has the same size as the non-glycosylated E.coli derived wt hIL-2 reference standard loaded on the same gel. In contrast, western blots from non-reduced samples of 4d (day 10), 4e (day 10) and 4f (day 7) showed a significant upward shift in product size compared to the 4a, 4b and 4c samples and the hll-2 reference standard, indicating efficient N-glycosylation of protein 1 f. The shift is almost complete, since only a weak, almost invisible band is present at the size corresponding to non-glycosylated hIL-2. The product bands of 4d, 4e and 4f also have the form of double bands on western blots, indicating that the N-glycosylated product comprises a fraction of the O-glycosylated product. In addition to the bands, weaker product bands, approximately twice the size of the major product bands, were also detected on western blots of proteins 2a (4 a, 4b and 4 c) and 1f (4 d, 4e and 4 f), indicating some dimer formation.

For expression in Ambr15 bioreactor, the concentration of recombinant IL-2 muteins in the culture supernatant of the cell lines expressing proteins 2a (4 a, 4b and 4 c) and 1f (4 d, 4e and 4 f) was determined by ELISA on days 10 and 14 of fed-batch culture (table 6). On days 10 and 14, the concentration of protein 1f was several times higher than the concentration of protein 2 a. For the construct with signal peptide 3a, the expression level of protein 1f was 10.1 times higher than protein 2a on day 10, 10.9 times higher on day 14, and for the construct with signal peptide 3c, the fold change was 4.5 times on day 10, and 2.8 times on day 14. For the construct with signal peptide 3b, protein 2a titers were very low at day 10 (0.03 g/L) and undetectable at day 14, while for protein 1f were significant at days 10 and 14 (0.56 g/L and 1.1g/L, respectively).

TABLE 6 cell concentration, viability and titre of IL-2 muteins on day 8 and day 10 of fed-batch culture in AmBr15 bioreactor

Example 6: identity analysis of glycosylated IL-2 expressed in CHO cells

The identity of protein 1f (SEQ ID NO: 10) was confirmed by performing peptide mapping. Wild-type species with O-glycosylation sites are identified and heterogeneous glycosylation patterns are characterized. The introduced N-glycosylation sites were identified and heterogeneous glycosylation patterns characterized. No "free" LTFGNSTK was detected (within the detection limit), indicating that all IL-2 variants carry N-glycosylation at the designated introduced N-glycosylation site.

Example 7: SPR analysis of IL-2 Compounds

The binding kinetics of protein 5a (IL-2 of SEQ ID NO: 213; peprotech, catalog number AF-200-02) and protein 1f (SEQ ID NO: 10) were assessed using Surface Plasmon Resonance (SPR) spectroscopy with a Biacore instrument (T200, GE Healthcare) using the immobilized extracellular domain of the IL-2 receptor subunit. Briefly, according to the manufacturer's instructions, carboxymethylated dextran biosensor chips (CM 5, GE Healthcare) were activated with N- (3-dimethylaminopropyl) -N' -Ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS). Immobilization of monoclonal mouse anti-human IgG (Fc) antibodies was also performed according to the manufacturer's instructions (GE Healthcare, catalog number BR-1008-39).

To determine the binding kinetics to IL-2Rα, the following chip preparation was used: human IL-2 receptor alpha, fc-Tag (Symantis, new Zealand) was diluted to about 0.67. Mu.g/ml in HBS-EP running buffer (GE Healthcare,10mM HEPES,150mM NaCl,3mM EDTA,0.05% surfactant P20, pH 7.4) and fixed at a flow rate of 50. Mu.L/min for 60 seconds to reach 80-100 Response Units (RU).

To determine the binding kinetics to IL-2Rβ, the following chip preparation was used: human IL-2 receptor beta, fc-Tag (Symantis, new Zealand) was diluted to about 0.80. Mu.g/ml in HBS-EP running buffer (GE Healthcare,10mM HEPES,150mM NaCl,3mM EDTA,0.05% surfactant P20, pH 7.4) and fixed at a flow rate of 50. Mu.L/min for 60 seconds to reach 140-180 Response Units (RU).

For both receptorsKinetic measurements of body subunit settings protein 5a or protein 1f was analyzed in multicyclic kinetics and thus injected in HBS-EP running buffer at least five different concentrations at 25 ℃ (50 μl/min flow rate, 60s contact time, 300s dissociation time). Double reference data (subtracted from reference flow cell and buffer only sample) were analyzed by kinetic fitting (1:1 binding model) to determine K _D 、k _a And k _d . With 3M MgCl ₂ Regeneration after each cycle is performed.

The data obtained are summarized in table 7.

Table 7: summary of SPR binding data to IL-2 receptor subunits

These results show that protein 1f has a strongly reduced affinity for IL-2Rα compared to protein 5a, while the affinity for IL-2Rβ is within a similar range.

Example 8: calculation of the deflection ratio

To test whether protein 1f is biased IL-2, the following requirements were met:

wherein the method comprises the steps of

And (3) with

“K _D Protein 1f vs. IL-2Rα "is K for protein 1f vs. IL-2Rα _D ，

“K _D Protein 1f vs. IL-2Rβ "is protein 1f vs. IL-2RβK _D ，

“K _D Protein 5a vs L-2Rα "is K for protein 5a vs IL-2Rα _D And (2) and

“K _D protein 5a vs. IL-2Rβ "is the K of protein 5a vs. IL-2Rβ _D 。

Using the above formula, protein 1f is clearly biased towards IL-2, because of the calculated ratio _{Protein 1r} Ratio to (V/V) _{Protein 5a} The ratio of (2) is greater than 200.

Example 9: expression of glycosylated IL-2 protein 6 in a clone-stable CHO cell line

To establish a clonally stable CHO cell line expressing protein 6 (SEQ ID NO: 10), cells from stable cell bank 4d (example 5) were supertransfected with a second expression vector comprising the same IL-2 encoding DNA sequence (SEQ ID NO:244, see example 5) as the expression vector used to construct cell bank 4d, but carrying a different selectable marker. Following supertransfection, monoclonal candidates were isolated using a ClonePix-2 instrument (Molecular Devices) and screened for monoclonal growth (Celigo S, nexcelom) and protein 6 titers in the supernatant (ELISA as described in example 5). Selected monoclonal clones were evaluated by fed-batch culture in Ambr15 Bioreactors (Sartorius) using commercial growth and feed media for a total of 14 days. Cell number, viability and titer of protein 6 were measured during the time of fed-batch culture as described in example 5. Based on cell culture performance and titer of protein 6, stable clonal cell line 9 was selected as the best clone for further material production. To verify the stability of clone 9, cells were passaged in 5mL spin tubes for a total of 60 passages and the performance of the cells stored at the beginning, middle and end of the study were compared in fed-batch culture. The results showed that the cell line was stable and that the final protein 6 titer was not reduced during the 60 passages.

Stable clonal cell line 9 expressing protein 6 was cultured in fed-batch mode in shake flasks using commercial growth and feed medium and harvested on day 10. Cell number and viability were analyzed by a Vicell BLU cell counter (Beckman Coulter).

Example 10: purification of protein 6 from a clone-stable CHO cell line

Protein 6 was purified as described in method 2 of example 5. The purified protein buffer was exchanged into storage and/or assay buffer and concentrated to 1mg/mL for subsequent experiments.

Example 11: preparation of conjugate 7

The dotted line represents the attachment of primary amine nitrogen to the N-terminus or lysine side chain of protein 6 and each N is an integer from 200 to 250.

Using connections toThe HiPrep 26/10 desalting column of the system exchanged 2.5mL of 2.5mL protein 6 buffer in 20mM sodium phosphate, 140mM NaCl (pH 7.4) at 3.47mg/mL with 100mM borate (pH 9.0). The collected protein solution was concentrated using a centrifugal filter (MWCO 3 kDa) to give a final volume of 1.55mL of solution at a concentration of 4.80mg/mL, for example by using 0.662 mL. Mg ^-1 ·cm ^-1 Is determined by photometric concentration measurement at 280 nm. 0.0932g 40kDa mPEG-linker reagent (which can be synthesized as described in compound 17ca in example 7 of WO2009/133137 using compounds 16c and 1A from the same patent) was dissolved in 1.07mL cold water to give 2.1×10 ^- ³ A stock solution of mol/L. The solution was stored on ice. 1.55mL of the protein solution was diluted to 4mg/mL by adding 100mM borate (pH 9.0). Then 933 μl of cooled 40kDa mPEG-linker reagent stock solution (corresponding to 4mol eq. Of protein) was added. The conjugation mixture was placed in a water bath at 14 ℃ for 2h. The pH was adjusted to pH 4 by adding 933. Mu.L of water and 3.732mL of 200mM sodium acetate (pH 3.6). After incubation overnight at 25℃use with +.>HiScreen Cap for system connectionthe to MMC ImpResc column separates from the reaction mixture the conjugate (single conjugate) attached to one single 40kDa mPEG-linker. A linear salt gradient of 12 column volumes of 10mM succinic acid pH5.0 to 10mM succinic acid, 800mM NaCl, pH5.0 was applied at 1.2 mL/min. Peaks eluted during the gradient, mainly comprising single conjugates, were collected. The NaCl content of this fraction was adjusted to 150mM by adding 10mM succinic acid pH 5.0. After concentration to 2.18mL at 1.49mg/mL using a centrifugal filter (MWCO 10 kDa), the sample was diluted to a final concentration of 6 equivalents of 1mg/mL protein (based on the molecular weight of the protein without regard to glycosylation) with 115. Mu.L of 10mM succinic acid, 150mM NaCl, 1% Tween20 pH5.0, and 963. Mu.L of 10mM succinic acid, 150mM NaCl, 0.05%Tween20 pH5.0, yielding conjugate 7.

Example 12: preparation of conjugate release mixture 8

The pH of 150. Mu.L of conjugate 7 was shifted to pH 9.0 by dilution with 62. Mu.L of 50mM boric acid pH 10.0. The samples were incubated at 37℃for 24 hours in an incubator, yielding conjugate release mixture (conjugate release mixture) 8. After incubation, the acquisition UPLC peptide BEH C18 column on the HPLC system (Waters,2.1X50 mm,1.7 μm), the percentage of protein 6 released was determined by RP-HPLC. The column temperature was maintained at 30℃and the flow rate was set to 0.25mL/min. UV detection was performed at 215 and 280 nm. Analysis was performed using 0.05% (v/v) aqueous trifluoroacetic acid (eluent A) and 0.04% (v/v) acetonitrile trifluoroacetic acid (eluent B). HPLC gradients are described in table 8.

Table 8: HPLC gradient

The amount of protein 6 released was determined using calibration curves of six different concentrations of purified reference material using the same RP-HPLC conditions. UV detection was performed at 280 nm. The conjugate release mixture 8 was used without purification and therefore comprised mainly protein 6 and cleaved 40kDa mPEG-linker with >10% residual conjugate 7.

Example 13: in vitro release kinetics of protein 6 from conjugate 7

The in vitro release kinetics of protein 6 from conjugate 7 was determined at pH 7.4 and 37 ℃ to mimic physiological pH and temperature conditions. For this purpose use is made of The system and two attached HiTrap desalting columns (Cytiva) were buffer exchanged into 25mM HEPES, 135mM NaCl, 1mM EDTA, 10mM L-methionine, 2mg/mL Pluronic F-68, pH 7.4 using a flow rate of 2mL/min and UV detection at 215 and 280 nm. The buffer exchanged samples were incubated in a water bath for up to 1 week at 37 ℃ under temperature controlled conditions (168 h). After acidification of the sample, the sample was purified by reverse phase high pressure liquid chromatography on a column equipped with the Acquity UPLC peptide BEH C18 (/ -)>1.7 μm,2.1mm x 50 mm) on the 1260Infinity II system (Agilent Technologies) and the determination of the cleavage of the linker and the release of protein 6. Analysis was performed using 0.05% (v/v) aqueous trifluoroacetic acid (eluent A) and 0.04% (v/v) MeCN solution of trifluoroacetic acid (eluent B). The HPLC gradient is described in table 8 of example 12.

In a corresponding RP-HPLC chromatogram at 215nm, the release was quantified by integrating the peaks of conjugate 7 and the release-related species (protein 6 and cleaved 40kDa mPEG-linker species) at 5 different time points. The percentage of released species was plotted against incubation time and a non-linear single phase binding fit (plateau set at 95%) was applied using curve fitting software to determine the half-life of the linker cleavage kinetics. In vitro linker half-life was determined at pH 7.4 and 37 ℃ for 65 hours (95% confidence interval = 52-83 hours).

Example 14: control IL-2 or clone-stable CHO-produced protein 6 bioactivity in a human peripheral blood mononuclear cell lymphocyte subpopulation.

To evaluate the control IL-2 (protein 1 d) or cloning-stable CHO-produced protein 6 in situCell type specific immunostimulation in human cells human Peripheral Blood Mononuclear Cells (PBMC) from two donors were incubated for 30 min with different concentrations of protein 1d or protein 6 and intracellular levels of phosphorylated STAT5 (pSTAT 5) in unique cell subsets such as tregs expressing mainly IL-2rα/β/γ and cd8+ T cells (CD 8) expressing mainly IL-2rβ/γ and NK cells were analyzed by flow cytometry. Median fluorescence intensity of pSTAT5 was analyzed for all subpopulations and EC was calculated for each compound in all cell types ₅₀ Values.

Protein 6 showed significantly reduced potency in Treg compared to protein 1d while maintaining the same potency as CD8 ⁺ T cells and NK cells have almost the same potency. For example, in human blood, protein 1d showed an EC of 0.02ng/mL pSTAT5 in Treg ₅₀ Value, whereas protein 6 showed a significantly higher EC of 38.01 ng/mL ₅₀ Values. In contrast, protein 1d shows the EC of pSTAT5 in human NK cells ₅₀ A value of 15.91ng/mL, whereas protein 6 showed nearly identical EC of 11.66ng/mL ₅₀ Values. Similar to the results observed for NK cells, protein 1d demonstrated human CD8 ⁺ EC of pSTAT5 in T cell ₅₀ A value of 82.98ng/mL, while protein 6 showed almost the same EC of 78.82ng/mL ₅₀ Values (table 9). These results suggest that protein 6 shows a response to IL-2Rα/β/γ ⁺ Significantly reduced (about 1900 fold lower) potency of cells, e.g., tregs, but for IL-2rβ/γ ⁺ Cells, e.g. NK cells and CD8 ⁺ T cells, maintain potency.

Table 9: generalization of pSTAT5 efficacy values in human PBMC

EC ₅₀ (ng/mL)	Treg	NK	CD8
				Protein 1d	0.02	15.91	82.98
Protein 6	38.01	11.66	78.82

Example 15: calculation of receptor binding and bias for protein 6 in IL-2Rα and IL-2Rβ binding experiments

The binding kinetics of protein 5a (IL-2 of SEQ ID NO: 213; peprotech, catalog number AF-200-02) and protein 6 (SEQ ID NO: 10) were assessed using Surface Plasmon Resonance (SPR) spectroscopy with a Biacore instrument (T200, GE Healthcare) using the immobilized extracellular domain of the IL-2 receptor subunit. Briefly, according to the manufacturer's instructions, carboxymethylated dextran biosensor chips (CM 5, GE Healthcare) were activated with N- (3-dimethylaminopropyl) -N' -Ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS). Immobilization of monoclonal mouse anti-human IgG (Fc) antibodies was also performed according to the manufacturer's instructions (GE Healthcare, catalog number BR-1008-39).

To determine the binding kinetics to IL-2Rα, the following chip preparation was used: human IL-2 receptor alpha, fc-tag (Symantis, new Zealand) was diluted to about 0.25. Mu.g/mL in HBS-EP running buffer (GE Healthcare,10mM HEPES,150mM NaCl,3mM EDTA,0.05% surfactant P20, pH 7.4) and fixed at a flow rate of 50. Mu.L/min for 60s to reach 90-110 Response Units (RU).

To determine the binding kinetics to IL-2Rβ, the following chip preparation was used: human IL-2 receptor beta, fc-tag (Symantis, new Zealand) was diluted to about 0.25. Mu.g/ml in HBS-EP running buffer (GE Healthcare,10mM HEPES,150mM NaCl,3mM EDTA,0.05% surfactant P20, pH 7.4) and fixed at a flow rate of 50. Mu.L/min for 60s to achieve 300-330 Response Units (RU).

For kinetic measurements of two receptor subunit settings, protein 5a or protein 1f was analyzed in multicycle kinetics, thus injected in HBS-EP running buffer at least five different concentrations at 25 ℃ (50 μl/min flow rate, 60s contact time, 300s dissociation time). Double reference data (subtracted from reference flow cell and buffer only sample) were analyzed by kinetic fitting (1:1 binding model) to determine K _D 、k _a And k _d . With 3M MgCl ₂ Regeneration after each cycle is performed.

Table 10: summarization of SPR binding data to IL-2 receptor subunits

The results show that protein 6 has a strongly reduced affinity for IL-2Rα compared to protein 5a, while the affinity for IL-2Rβ is within a similar range (Table 10).

To test whether protein 6 is biased IL-2, the following requirements were met:

wherein the method comprises the steps of

And (3) with

“K _D Protein 6 vs. IL-2Rα "is K for protein 6 vs. IL-2Rα _D ，

“K _D Protein 6 vs. IL-2Rβ "is the K of protein 6 vs. IL-2Rβ _D ，

“K _D Protein 5a vs. IL-2Rα "is K for protein 5a vs. IL-2Rα _D And (2) and

“K _D protein 5a vs. IL-2Rβ "is the K of protein 5a vs. IL-2Rβ _D 。

Using the above formula, protein 6 is clearly biased towards IL-2, because of the calculated ratio _{Protein 6} Ratio to (V/V) _{Protein 5a} The ratio of (2) is greater than 100.

Example 16: cloning of the biological Activity of stable CHO-produced protein 6 in HH pSTAT5 and CTLL-2pSTAT5 assays

The biological activity of wild-type IL2 protein 5a as well as glycosylated IL2 protein 6 was tested by an external supplier using the STAT5 phosphorylation assay (pSTAT 5) and the HH and CTLL-2 cell lines. The HH cell line is IL-2-Rβ/γ expressing human T-lymphoblastic cells derived from a patient with cutaneous T-cell lymphoma. The CTLL-2 cell line is a murine T lymphocyte cell line expressing IL-2-Rα/β/γ. Half maximal effective concentrations (EC 50) of STAT5 phosphorylation of HH and CTLL-2 cells were measured in duplicate and analyzed using a 4-parameter logistic fit.

Table 11: generalization of pSTAT5 EC50 data using HH cells and CTLL-2 cells

The results show that protein 6 vs IL-2Rα/β/γ compared to protein 5a ⁺ (CTLL-2 cells) have a strongly reduced potency against IL-2Rβ/γ ⁺ The potency of (HH cells) is in a similar range (table 11). Similar results were obtained for conjugate release mixture 8, which also was for IL-2Rα/β/γ compared to protein 5a ⁺ (CTLL-2 cells) show strongly reduced potency against IL-2Rβ/γ ⁺ The potency of (HH cells) is in a similar range (table 11). Thus, conjugate release mixture 8 showed no significant loss in potency and IL-2R bias compared to protein 6.

To test whether protein 6 is biased IL-2, the following requirements were met:

wherein the method comprises the steps of

And (3) with

"EC50 protein 6 versus CTLL-2 cell" is the EC50 of protein 6 versus CTLL-2 cell,

"EC50 protein 6 versus HH cell" is the EC50 of protein 6 versus HH cell,

"EC50 protein 5a and CTLL-2 cells" are the EC50 of protein 5a for CTLL-2 cells, an

"EC50 protein 5a and HH cell" is the EC50 of protein 5a on HH cell

Using the above formula, protein 6 is clearly biased towards IL-2, because of the calculated ratio _{Protein 6} Ratio to (V/V) _{Protein 5a} Is greater than 250. Conjugate release mixture 8 also showed a bias towards IL-2rβ/γ.

Abbreviations (abbreviations)

Degree centigrade

bp base pair

CAR chimeric antigen receptor

cDNA complementary DNA

CHO chinese hamster ovary

DNA deoxyribonucleic acid

DO dissolved oxygen

h hours

HEK human embryo kidney

IL-2 interleukin-2

IL-2R interleukin-2 receptor

kDa kilodaltons

MES 4-morpholinoethanesulfonic acid

molar equivalent of

mPEG methoxy polyethylene glycol

MWCO molecular weight cut-off value

NaCl sodium chloride

NK cell natural killer cells

P. pastoris Pichia pastoris

PBS phosphate buffered saline

RNA ribonucleic acid

RP inversion

RP-HPLC reversed phase high performance liquid chromatography

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

SEC size exclusion chromatography

TBS Tris-buffered saline

TCEP (tris (2-carboxyethyl) phosphine hydrochloride)

UPLC ultra performance liquid chromatography

VCD viable cell Density

Sequence listing

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atggatatgc gggtccctgc tcagctgctg ggcctgctgc tgctgtggtt tcctgggtca 60

aggtgcgccc ccacatctag ttctacaaag aagacacaat tacaattaga gcacttatta 120

ttagacttac aaatgatatt aaatggcata aataattata agaatcccaa gttaactttc 180

ggcaacagca ccaagtttta tatgcctaag aaggctacag agttaaagca cttacaatgt 240

ttagaggagg agttaaagcc attagaggag gtattaaatt tagcccaatc aaagaatttt 300

cacttacggc cccgggactt aatttccaat attaatgtta ttgtgttaga gttaaagggc 360

tctgagacaa cttttatgtg tgagtatgcc gacgagacag ccacaattgt tgagttctta 420

aatcggtgga ttactttttc acaatccatt atctccactt taactcatca tcaccatcac 480

cactaa 486

<210> 8

<211> 486

<212> DNA

<213> artificial sequence

<220>

<223> DNA sequence, protein 1b

<400> 8

atggatatgc gggtccctgc tcagctgctg ggcctgctgc tgctgtggtt tcctgggtca 60

aggtgcgccc ccacatctag ttctacaaag aagacacaat tacaattaga gcacttatta 120

ttagacttac aaatgatatt aaatggcata aataattata agaatcccaa gttaactaga 180

atgttattcg gcaacagcac catgcctaag aaggctacag agttaaagca cttacaatgt 240

ttagaggagg agttaaagcc attagaggag gtattaaatt tagcccaatc aaagaatttt 300

cacttacggc cccgggactt aatttccaat attaatgtta ttgtgttaga gttaaagggc 360

tctgagacaa cttttatgtg tgagtatgcc gacgagacag ccacaattgt tgagttctta 420

aatcggtgga ttactttttc acaatccatt atctccactt taactcatca tcaccatcac 480

cactaa 486

<210> 9

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 9

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 10

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 10

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 11

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 11

Tyr Lys Asn Pro Lys Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 12

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 12

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Phe Ala Asn Ser Thr Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 13

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 13

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Phe Ala Asn Ser Thr Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 14

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 14

Tyr Lys Asn Pro Lys Leu Thr Phe Ala Asn Ser Thr Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 15

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 15

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Phe Gly Asn Ser Thr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 16

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 16

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Phe Gly Asn Ser Thr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 17

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 17

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Phe Gly Asn Ser Thr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 18

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 18

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Phe Ala Asn Ser Thr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 19

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 19

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Phe Ala Asn Ser Thr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 20

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 20

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Phe Ala Asn Ser Thr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 21

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 21

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Phe Gly Asn Ser Thr

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 22

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 22

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Phe Gly Asn Ser

50 55 60

Thr Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 23

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 23

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Phe Gly

20 25 30

Asn Ser Thr Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 24

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 24

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Phe Ala Asn Ser Thr

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 25

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 25

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Phe Ala Asn Ser

50 55 60

Thr Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 26

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 26

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Phe Ala

20 25 30

Asn Ser Thr Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 27

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 27

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Glu Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 28

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 28

Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Glu Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 29

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 29

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Glu Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 30

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 30

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Phe Ala Asn Ser Thr Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 31

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 31

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Phe Ala Asn Ser Thr Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 32

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 32

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Phe Ala Asn Ser Thr Gln Ser Lys

35 40 45

<210> 33

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 33

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Phe Gly Asn Ser Thr Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 34

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 34

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Phe Gly Asn Ser Thr Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 35

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 35

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Phe Gly Asn Ser Thr Gln Ser Lys

35 40 45

<210> 36

<211> 29

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 36

Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn

20 25

<210> 37

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 37

Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 38

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 38

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 39

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 39

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 40

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 40

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 41

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 41

Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 42

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 42

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 43

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 43

Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Phe Ala Asn Ser Thr Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 44

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 44

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Phe Ala Asn Ser Thr Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 45

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 45

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Phe Ala Asn Ser Thr Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 46

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 46

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Phe Ala Asn Ser Thr Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 47

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 47

Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Phe Ala Asn Ser Thr Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 48

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 48

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Phe Ala Asn Ser Thr Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 49

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 49

Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Phe Gly Asn Ser Thr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 50

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 50

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Phe Gly Asn Ser Thr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 51

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 51

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Phe Gly Asn Ser Thr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 52

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 52

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Phe Gly Asn Ser Thr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 53

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 53

Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Phe Gly Asn Ser Thr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 54

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 54

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Phe Gly Asn Ser Thr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 55

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 55

Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Phe Ala Asn Ser Thr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 56

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 56

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Phe Ala Asn Ser Thr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 57

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 57

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Phe Ala Asn Ser Thr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 58

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 58

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Phe Ala Asn Ser Thr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 59

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 59

Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Phe Ala Asn Ser Thr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 60

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 60

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Phe Ala Asn Ser Thr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 61

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 61

Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Phe Gly Asn Ser Thr

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 62

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 62

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Phe Gly Asn Ser

50 55 60

Thr Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 63

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 63

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Phe Gly Asn Ser Thr

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 64

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 64

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Phe Gly Asn Ser

50 55 60

Thr Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 65

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 65

Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Phe Gly Asn Ser Thr

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 66

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 66

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Phe Gly Asn Ser

50 55 60

Thr Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 67

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 67

Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Phe Ala Asn Ser Thr

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 68

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 68

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Phe Ala Asn Ser

50 55 60

Thr Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 69

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 69

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Phe Ala Asn Ser Thr

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 70

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 70

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Phe Ala Asn Ser

50 55 60

Thr Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 71

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 71

Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Phe Ala Asn Ser Thr

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 72

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 72

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Phe Ala Asn Ser

50 55 60

Thr Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 73

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 73

Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Phe Ala Asn Ser Thr Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 74

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 74

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Phe Ala Asn Ser Thr Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 75

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 75

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Phe Ala Asn Ser Thr Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 76

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 76

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Phe Ala Asn Ser Thr Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 77

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 77

Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Phe Ala Asn Ser Thr Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 78

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 78

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Phe Ala Asn Ser Thr Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 79

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 79

Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Phe Gly Asn Ser Thr Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 80

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 80

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Phe Gly Asn Ser Thr Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 81

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 81

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Phe Gly Asn Ser Thr Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 82

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 82

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Phe Gly Asn Ser Thr Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 83

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 83

Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Phe Gly Asn Ser Thr Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 84

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 84

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Phe Gly Asn Ser Thr Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 85

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> T, S or C

<400> 85

Asn Xaa Xaa Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 86

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> T, S or C

<400> 86

Tyr Asn Xaa Xaa Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 87

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> T, S or C

<400> 87

Tyr Lys Asn Xaa Xaa Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 88

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> misc_feature

<222> (5)..(5)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> misc_feature

<222> (6)..(6)

<223> T, S or C

<400> 88

Tyr Lys Asn Asn Xaa Xaa Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 89

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> T, S or C

<400> 89

Tyr Lys Asn Pro Asn Xaa Xaa Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 90

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> T, S or C

<400> 90

Tyr Lys Asn Pro Lys Asn Xaa Xaa Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 91

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> T, S or C

<400> 91

Tyr Lys Asn Pro Lys Leu Asn Xaa Xaa Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 92

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> T, S or C

<400> 92

Tyr Lys Asn Pro Lys Leu Thr Asn Xaa Xaa Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 93

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (11)..(11)

<223> T, S or C

<400> 93

Tyr Lys Asn Pro Lys Leu Thr Arg Asn Xaa Xaa Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 94

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> misc_feature

<222> (11)..(11)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> misc_feature

<222> (12)..(12)

<223> T, S or C

<400> 94

Tyr Lys Asn Pro Lys Leu Thr Arg Met Asn Xaa Xaa Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 95

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (12)..(12)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (13)..(13)

<223> T, S or C

<400> 95

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Asn Xaa Xaa Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 96

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (13)..(13)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (14)..(14)

<223> T, S or C

<400> 96

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Asn Xaa Xaa Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 97

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (14)..(14)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (15)..(15)

<223> T, S or C

<400> 97

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Asn Xaa Xaa Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 98

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (15)..(15)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (16)..(16)

<223> T, S or C

<400> 98

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Asn Xaa Xaa

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 99

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (16)..(16)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (17)..(17)

<223> T, S or C

<400> 99

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Asn Xaa

1 5 10 15

Xaa Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 100

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (17)..(17)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (18)..(18)

<223> T, S or C

<400> 100

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Asn

1 5 10 15

Xaa Xaa Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 101

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (18)..(18)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (19)..(19)

<223> T, S or C

<400> 101

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Asn Xaa Xaa Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 102

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (19)..(19)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (20)..(20)

<223> T, S or C

<400> 102

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Asn Xaa Xaa Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 103

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (20)..(20)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (21)..(21)

<223> T, S or C

<400> 103

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Asn Xaa Xaa Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 104

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (21)..(21)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (22)..(22)

<223> T, S or C

<400> 104

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Asn Xaa Xaa Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 105

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (22)..(22)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (23)..(23)

<223> T, S or C

<400> 105

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Asn Xaa Xaa Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 106

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (23)..(23)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (24)..(24)

<223> T, S or C

<400> 106

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Asn Xaa Xaa His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 107

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (24)..(24)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (25)..(25)

<223> T, S or C

<400> 107

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Asn Xaa Xaa Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 108

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (25)..(25)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (26)..(26)

<223> T, S or C

<400> 108

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Asn Xaa Xaa Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 109

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (26)..(26)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (27)..(27)

<223> T, S or C

<400> 109

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys Asn Xaa Xaa Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 110

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (27)..(27)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (28)..(28)

<223> T, S or C

<400> 110

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Asn Xaa Xaa Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 111

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (28)..(28)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (29)..(29)

<223> T, S or C

<400> 111

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Asn Xaa Xaa Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 112

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 112

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Asn Ser

20 25 30

Thr Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 113

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (29)..(29)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (30)..(30)

<223> T, S or C

<400> 113

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Asn Xaa Xaa Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 114

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (30)..(30)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (31)..(31)

<223> T, S or C

<400> 114

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Asn Xaa Xaa Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 115

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (31)..(31)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (32)..(32)

<223> T, S or C

<400> 115

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Asn Xaa Xaa

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 116

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (32)..(32)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (33)..(33)

<223> T, S or C

<400> 116

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Asn Xaa

20 25 30

Xaa Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 117

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (33)..(33)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (34)..(34)

<223> T, S or C

<400> 117

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Asn

20 25 30

Xaa Xaa Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 118

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (34)..(34)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (35)..(35)

<223> T, S or C

<400> 118

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Asn Xaa Xaa Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 119

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (35)..(35)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (36)..(36)

<223> T, S or C

<400> 119

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Asn Xaa Xaa Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 120

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (36)..(36)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (37)..(37)

<223> T, S or C

<400> 120

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Asn Xaa Xaa Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 121

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (37)..(37)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (38)..(38)

<223> T, S or C

<400> 121

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Asn Xaa Xaa Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 122

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (38)..(38)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (39)..(39)

<223> T, S or C

<400> 122

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Asn Xaa Xaa Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 123

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (39)..(39)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (40)..(40)

<223> T, S or C

<400> 123

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Asn Xaa Xaa Asn Leu Ala Gln Ser Lys

35 40 45

<210> 124

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (40)..(40)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (41)..(41)

<223> T, S or C

<400> 124

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Asn Xaa Xaa Leu Ala Gln Ser Lys

35 40 45

<210> 125

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (41)..(41)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (42)..(42)

<223> T, S or C

<400> 125

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Asn Xaa Xaa Ala Gln Ser Lys

35 40 45

<210> 126

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (42)..(42)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (43)..(43)

<223> T, S or C

<400> 126

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Xaa Xaa Gln Ser Lys

35 40 45

<210> 127

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (43)..(43)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (44)..(44)

<223> T, S or C

<400> 127

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Asn Xaa Xaa Ser Lys

35 40 45

<210> 128

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (44)..(44)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (45)..(45)

<223> T, S or C

<400> 128

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Asn Xaa Xaa Lys

35 40 45

<210> 129

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (45)..(45)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (46)..(46)

<223> T, S or C

<400> 129

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Asn Xaa Xaa

35 40 45

<210> 130

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> T, S or C

<400> 130

Xaa Xaa Asn Xaa Xaa Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 131

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> T, S or C

<400> 131

Tyr Xaa Xaa Asn Xaa Xaa Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 132

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> T, S or C

<400> 132

Tyr Lys Xaa Xaa Asn Xaa Xaa Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 133

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> T, S or C

<400> 133

Tyr Lys Asn Xaa Xaa Asn Xaa Xaa Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 134

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> T, S or C

<400> 134

Tyr Lys Asn Pro Xaa Xaa Asn Xaa Xaa Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 135

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> T, S or C

<400> 135

Tyr Lys Asn Pro Lys Xaa Xaa Asn Xaa Xaa Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 136

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (11)..(11)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 136

Tyr Lys Asn Pro Lys Leu Xaa Xaa Asn Xaa Xaa Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 137

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (11)..(11)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (12)..(12)

<223> T, S or C

<400> 137

Tyr Lys Asn Pro Lys Leu Thr Xaa Xaa Asn Xaa Xaa Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 138

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (12)..(12)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (13)..(13)

<223> T, S or C

<400> 138

Tyr Lys Asn Pro Lys Leu Thr Arg Xaa Xaa Asn Xaa Xaa Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 139

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (11)..(11)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (13)..(13)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (14)..(14)

<223> T, S or C

<400> 139

Tyr Lys Asn Pro Lys Leu Thr Arg Met Xaa Xaa Asn Xaa Xaa Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 140

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (11)..(11)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (12)..(12)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (14)..(14)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> T, S or C

<222> (15)..(15)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 140

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Xaa Xaa Asn Xaa Xaa Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 141

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (12)..(12)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (13)..(13)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (15)..(15)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (16)..(16)

<223> T, S or C

<400> 141

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Xaa Xaa Asn Xaa Xaa

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 142

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (13)..(13)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (14)..(14)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (16)..(16)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (17)..(17)

<223> T, S or C

<400> 142

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Xaa Xaa Asn Xaa

1 5 10 15

Xaa Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 143

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (14)..(14)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (15)..(15)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (17)..(17)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (18)..(18)

<223> T, S or C

<400> 143

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Xaa Xaa Asn

1 5 10 15

Xaa Xaa Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 144

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (15)..(15)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (16)..(16)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (18)..(18)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (19)..(19)

<223> T, S or C

<400> 144

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Xaa Xaa

1 5 10 15

Asn Xaa Xaa Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 145

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (16)..(16)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (17)..(17)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (19)..(19)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (20)..(20)

<223> T, S or C

<400> 145

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Xaa

1 5 10 15

Xaa Asn Xaa Xaa Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 146

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (17)..(17)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (18)..(18)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (20)..(20)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (21)..(21)

<223> T, S or C

<400> 146

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Xaa Xaa Asn Xaa Xaa Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 147

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (18)..(18)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (19)..(19)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (21)..(21)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (22)..(22)

<223> T, S or C

<400> 147

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Xaa Xaa Asn Xaa Xaa Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 148

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (19)..(19)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (20)..(20)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (22)..(22)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (23)..(23)

<223> T, S or C

<400> 148

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Xaa Xaa Asn Xaa Xaa Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 149

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (20)..(20)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (21)..(21)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (23)..(23)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (24)..(24)

<223> T, S or C

<400> 149

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Xaa Xaa Asn Xaa Xaa His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 150

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (21)..(21)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (22)..(22)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (24)..(24)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (25)..(25)

<223> T, S or C

<400> 150

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Xaa Xaa Asn Xaa Xaa Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 151

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (22)..(22)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (23)..(23)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (25)..(25)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (26)..(26)

<223> T, S or C

<400> 151

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Xaa Xaa Asn Xaa Xaa Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 152

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (23)..(23)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (24)..(24)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (26)..(26)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (27)..(27)

<223> T, S or C

<400> 152

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Xaa Xaa Asn Xaa Xaa Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 153

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (24)..(24)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (25)..(25)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (27)..(27)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (28)..(28)

<223> T, S or C

<400> 153

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Xaa Xaa Asn Xaa Xaa Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 154

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (25)..(25)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (26)..(26)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (28)..(28)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (29)..(29)

<223> T, S or C

<400> 154

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys Xaa Xaa Asn Xaa Xaa Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 155

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (26)..(26)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (27)..(27)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (29)..(29)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (30)..(30)

<223> T, S or C

<400> 155

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Xaa Xaa Asn Xaa Xaa Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 156

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (27)..(27)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (28)..(28)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (30)..(30)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (31)..(31)

<223> T, S or C

<400> 156

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Xaa Xaa Asn Xaa Xaa Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 157

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (28)..(28)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (29)..(29)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (31)..(31)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (32)..(32)

<223> T, S or C

<400> 157

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Xaa Xaa Asn Xaa Xaa

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 158

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (29)..(29)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (30)..(30)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (32)..(32)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (33)..(33)

<223> T, S or C

<400> 158

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Xaa Xaa Asn Xaa

20 25 30

Xaa Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 159

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (30)..(30)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (31)..(31)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (33)..(33)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (34)..(34)

<223> T, S or C

<400> 159

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Xaa Xaa Asn

20 25 30

Xaa Xaa Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 160

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (31)..(31)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (32)..(32)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (34)..(34)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (35)..(35)

<223> T, S or C

<400> 160

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Xaa Xaa

20 25 30

Asn Xaa Xaa Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 161

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (32)..(32)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (33)..(33)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (35)..(35)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (36)..(36)

<223> T, S or C

<400> 161

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Xaa

20 25 30

Xaa Asn Xaa Xaa Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 162

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (33)..(33)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (34)..(34)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (36)..(36)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (37)..(37)

<223> T, S or C

<400> 162

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Xaa Xaa Asn Xaa Xaa Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 163

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (34)..(34)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (35)..(35)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (37)..(37)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (38)..(38)

<223> T, S or C

<400> 163

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Xaa Xaa Asn Xaa Xaa Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 164

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (35)..(35)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (36)..(36)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (38)..(38)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (39)..(39)

<223> T, S or C

<400> 164

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Xaa Xaa Asn Xaa Xaa Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 165

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (36)..(36)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (37)..(37)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (39)..(39)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (40)..(40)

<223> T, S or C

<400> 165

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Xaa Xaa Asn Xaa Xaa Asn Leu Ala Gln Ser Lys

35 40 45

<210> 166

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (37)..(37)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (38)..(38)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (40)..(40)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (41)..(41)

<223> T, S or C

<400> 166

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Xaa Xaa Asn Xaa Xaa Leu Ala Gln Ser Lys

35 40 45

<210> 167

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (38)..(38)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (39)..(39)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (41)..(41)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (42)..(42)

<223> T, S or C

<400> 167

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Xaa Xaa Asn Xaa Xaa Ala Gln Ser Lys

35 40 45

<210> 168

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (39)..(39)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (40)..(40)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (42)..(42)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (43)..(43)

<223> T, S or C

<400> 168

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Xaa Xaa Asn Xaa Xaa Gln Ser Lys

35 40 45

<210> 169

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (40)..(40)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (41)..(41)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (43)..(43)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (44)..(44)

<223> T, S or C

<400> 169

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Xaa Xaa Asn Xaa Xaa Ser Lys

35 40 45

<210> 170

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (41)..(41)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (42)..(42)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (44)..(44)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (45)..(45)

<223> T, S or C

<400> 170

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Xaa Xaa Asn Xaa Xaa Lys

35 40 45

<210> 171

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<220>

<221> MISC_FEATURE

<222> (42)..(42)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (43)..(43)

<223> any Proteinogenic or non-Proteinogenic amino acid

<220>

<221> MISC_FEATURE

<222> (45)..(45)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (46)..(46)

<223> T, S or C

<400> 171

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Xaa Xaa Asn Xaa Xaa

35 40 45

<210> 172

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 172

Tyr Lys Asn Pro Asn Ser Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 173

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 173

Tyr Lys Asn Pro Lys Leu Thr Asn Ser Thr Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 174

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 174

Tyr Lys Asn Pro Lys Leu Thr Arg Asn Ser Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 175

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 175

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Asn Ser Thr Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 176

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 176

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Asn Ser Thr Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 177

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 177

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Asn Ser Thr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 178

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 178

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Asn Ser Thr

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 179

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 179

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Asn Ser

1 5 10 15

Thr Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 180

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 180

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Asn

20 25 30

Ser Thr Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 181

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 181

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Asn Ser Thr Ser Lys

35 40 45

<210> 182

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 182

Tyr Lys Asn Pro Phe Ala Asn Ser Thr Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 183

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 183

Tyr Lys Asn Pro Lys Leu Thr Arg Phe Ala Asn Ser Thr Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 184

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 184

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Ala Asn Ser Thr

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 185

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 185

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Phe Ala Asn Ser

1 5 10 15

Thr Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 186

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 186

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Ala Asn

1 5 10 15

Ser Thr Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 187

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 187

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Phe Ala

1 5 10 15

Asn Ser Thr Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 188

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 188

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Phe

20 25 30

Ala Asn Ser Thr Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 189

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 189

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Phe Ala Asn Ser Thr

35 40 45

<210> 190

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 190

Tyr Lys Asn Pro Phe Gly Asn Ser Thr Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 191

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 191

Tyr Lys Asn Pro Lys Leu Thr Arg Phe Gly Asn Ser Thr Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 192

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 192

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Gly Asn Ser Thr

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 193

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 193

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Phe Gly Asn Ser

1 5 10 15

Thr Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 194

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 194

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Gly Asn

1 5 10 15

Ser Thr Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 195

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 195

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Phe Gly

1 5 10 15

Asn Ser Thr Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 196

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 196

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Phe

20 25 30

Gly Asn Ser Thr Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 197

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 197

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Phe Gly Asn Ser Thr

35 40 45

<210> 198

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 198

Tyr Lys Asn Ser Thr Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 199

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 199

Phe Ala Asn Ser Thr Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 200

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 200

Phe Gly Asn Ser Thr Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 201

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 201

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Ser Thr Gln Ser Lys

35 40 45

<210> 202

<211> 486

<212> DNA

<213> artificial sequence

<220>

<223> DNA sequence, protein 1c

<400> 202

atggatatgc gggtccctgc tcagctgctg ggcctgctgc tgctgtggtt tcctgggtca 60

aggtgcgccc ccacatctag ttctacaaag aagacacaat tacaattaga gcacttatta 120

ttagacttac aaatgatatt aaatggcata aataattata agaatcccaa gttaactaga 180

atgttaacat tcaagtttta tatgcctaag aaggctacag agttaaagca cttacaatgt 240

ttagagttcg gcaacagcac cttagaggag gtattaaatt tagcccaatc aaagaatttt 300

cacttacggc cccgggactt aatttccaat attaatgtta ttgtgttaga gttaaagggc 360

tctgagacaa cttttatgtg tgagtatgcc gacgagacag ccacaattgt tgagttctta 420

aatcggtgga ttactttttc acaatccatt atctccactt taactcatca tcaccatcac 480

cactaa 486

<210> 203

<211> 486

<212> DNA

<213> artificial sequence

<220>

<223> DNA sequence, protein 1d

<400> 203

atggatatgc gggtccctgc tcagctgctg ggcctgctgc tgctgtggtt tcctgggtca 60

aggtgcgccc ccacatctag ttctacaaag aagacacaat tacaattaga gcacttatta 120

ttagacttac aaatgatatt aaatggcata aataattata agaatcccaa gttaactaga 180

atgttaacat tcaagtttta tatgcctaag aaggctacag agttaaagca cttacaatgt 240

ttagaggagg agttaaagcc attagaggag gtattaaatt tagcccaatc aaagaatttt 300

cacttacggc cccgggactt aatttccaat attaatgtta ttgtgttaga gttaaagggc 360

tctgagacaa cttttatgtg tgagtatgcc gacgagacag ccacaattgt tgagttctta 420

aatcggtgga ttactttttc acaatccatt atctccactt taactcatca tcaccatcac 480

cactaa 486

<210> 204

<211> 138

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variant with glycosylation motif and His tag

<400> 204

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr His His His His His His

130 135

<210> 205

<211> 139

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variant with glycosylation motif and His tag

<400> 205

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr His His His His His His

130 135

<210> 206

<211> 138

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variant with glycosylation motif and His tag

<400> 206

Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr His His His His His His

130 135

<210> 207

<211> 139

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variant with glycosylation motif and His tag

<400> 207

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr His His His His His His

130 135

<210> 208

<211> 138

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variant with glycosylation motif and His tag

<400> 208

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr His His His His His His

130 135

<210> 209

<211> 139

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variant with glycosylation motif and His tag

<400> 209

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr His His His His His His

130 135

<210> 210

<211> 138

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variant with glycosylation motif and His tag

<400> 210

Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr His His His His His His

130 135

<210> 211

<211> 139

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variant with glycosylation motif and His tag

<400> 211

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr His His His His His His

130 135

<210> 212

<211> 57

<212> PRT

<213> artificial sequence

<220>

<223> SEQ C, with C49A

<400> 212

Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile

1 5 10 15

Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala

20 25 30

Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe

35 40 45

Ala Gln Ser Ile Ile Ser Thr Leu Thr

50 55

<210> 213

<211> 134

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants

<400> 213

Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu

1 5 10 15

His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr

20 25 30

Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro

35 40 45

Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu

50 55 60

Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His

65 70 75 80

Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu

85 90 95

Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr

100 105 110

Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser

115 120 125

Ile Ile Ser Thr Leu Thr

130

<210> 214

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 214

Tyr Lys Asn Pro Asp Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 215

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, having glycosylation motif

<400> 215

Tyr Lys Asn Pro Glu Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 216

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 216

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Asp Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 217

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 217

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Asp Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 218

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 218

Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Asp Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 219

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 219

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Asp Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 220

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 220

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Asp Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 221

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 221

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Asp Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 222

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 222

Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Asp Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 223

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 223

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Asp Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 224

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 224

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Glu Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 225

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 225

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Glu Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 226

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 226

Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Glu Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 227

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 227

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Glu Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 228

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motifs

<400> 228

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Glu Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 229

<211> 139

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variant with glycosylation motif and His tag

<400> 229

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Phe Gly Asn Ser Thr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr His His His His His His

130 135

<210> 230

<211> 139

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variant with glycosylation motif and His tag

<400> 230

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Phe Gly Asn Ser

50 55 60

Thr Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr His His His His His His

130 135

<210> 231

<211> 147

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variants with glycosylation motif and cleavable His tag

<400> 231

Ala His His His His His His Gly Ser Asp Asp Asp Asp Lys Ala Pro

1 5 10 15

Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu

20 25 30

Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro

35 40 45

Lys Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr Met Pro Lys Lys Ala

50 55 60

Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu

65 70 75 80

Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro

85 90 95

Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly

100 105 110

Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile

115 120 125

Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser

130 135 140

Thr Leu Thr

145

<210> 232

<211> 139

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 variant with His tag

<400> 232

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr His His His His His His

130 135

<210> 233

<211> 510

<212> DNA

<213> artificial sequence

<220>

<223> DNA sequence encoding protein 1e

<400> 233

atggatatgc gggtccctgc tcagctgctg ggcctgctgc tgctgtggtt tcctgggtca 60

aggtgcgccc atcatcacca tcaccacggc agcgacgacg acgacaaggc ccccacatct 120

agttctacaa agaagacaca attacaatta gagcacttat tattagactt acaaatgata 180

ttaaatggca taaataatta taagaatccc aagttaactt tcggcaacag caccaagttt 240

tatatgccta agaaggctac agagttaaag cacttacaat gtttagagga ggagttaaag 300

ccattagagg aggtattaaa tttagcccaa tcaaagaatt ttcacttacg gccccgggac 360

ttaatttcca atattaatgt tattgtgtta gagttaaagg gctctgagac aacttttatg 420

tgtgagtatg ccgacgagac agccacaatt gttgagttct taaatcggtg gattactttt 480

tcacaatcca ttatctccac tttaacttaa 510

<210> 234

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> His tag

<400> 234

Ala His His His His His His Gly Ser Asp Asp Asp Asp Lys

1 5 10

<210> 235

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, FANST with glycosylation motif

<400> 235

Tyr Lys Asn Pro Asp Leu Thr Phe Ala Asn Ser Thr Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 236

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ B, FANST with glycosylation motif

<400> 236

Tyr Lys Asn Pro Glu Leu Thr Phe Ala Asn Ser Thr Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 237

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> protein 2a

<400> 237

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Cys Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 238

<211> 24

<212> PRT

<213> artificial sequence

<220>

<223> Signal peptide 3a

<400> 238

Met Ala Trp Lys Thr Leu Pro Ile Tyr Leu Leu Leu Leu Leu Ser Val

1 5 10 15

Phe Val Ile Gln Gln Val Ser Ser

20

<210> 239

<211> 19

<212> PRT

<213> artificial sequence

<220>

<223> Signal peptide 3b

<400> 239

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Ile Lys Gly

1 5 10 15

Val Gln Cys

<210> 240

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Signal peptide 3c

<400> 240

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser

20

<210> 241

<211> 471

<212> DNA

<213> artificial sequence

<220>

<223> DNA sequence encoding protein 2b

<400> 241

atggcctgga agactctccc catctacctc ctgctgctcc tgtcggtctt cgtcatccag 60

caggtgtcct ccgcccccac gtcgtcctcc accaagaaga cccagctgca gctcgagcac 120

ctcctgctgg acctgcagat gatcctgaac gggatcaaca actacaagaa cccgaagctg 180

acttgcatgc tgaccttcaa gttctacatg ccgaagaagg cgaccgagct gaagcacctg 240

cagtgcctgg aggaggagct caagccgctc gaggaggtgc tgaatctggc ccagtccaag 300

aacttccacc tgcgcccgcg ggacctcatc agcaacatca acgtcatcgt gctggagctg 360

aagggcagcg agacgacgtt tatgtgcgag tacgcagacg agacggccac catcgtcgag 420

ttcttgaacc ggtggatcac cttcagccag tccatcatct ccaccctgac c 471

<210> 242

<211> 456

<212> DNA

<213> artificial sequence

<220>

<223> DNA sequence encoding protein 2d

<400> 242

atggagttcg gtctgtcctg ggtgttcctg gtcgccatca tcaagggcgt gcagtgcgcc 60

cccacgtcgt cctccaccaa gaagacccag ctgcagctcg agcacctcct gctggacctg 120

cagatgatcc tgaacgggat caacaactac aagaacccga agctgacttg catgctgacc 180

ttcaagttct acatgccgaa gaaggcgacc gagctgaagc acctgcagtg cctggaggag 240

gagctcaagc cgctcgagga ggtgctgaat ctggcccagt ccaagaactt ccacctgcgc 300

ccgcgggacc tcatcagcaa catcaacgtc atcgtgctgg agctgaaggg cagcgagacg 360

acgtttatgt gcgagtacgc agacgagacg gccaccatcg tcgagttctt gaaccggtgg 420

atcaccttca gccagtccat catctccacc ctgacc 456

<210> 243

<211> 459

<212> DNA

<213> artificial sequence

<220>

<223> DNA sequence encoding protein 2c

<400> 243

atgtaccgta tgcagctgct ctcgtgcatc gcactgagcc tcgccctggt gaccaactca 60

gcccccacgt cgtcctccac caagaagacc cagctgcagc tcgagcacct cctgctggac 120

ctgcagatga tcctgaacgg gatcaacaac tacaagaacc cgaagctgac ttgcatgctg 180

accttcaagt tctacatgcc gaagaaggcg accgagctga agcacctgca gtgcctggag 240

gaggagctca agccgctcga ggaggtgctg aatctggccc agtccaagaa cttccacctg 300

cgcccgcggg acctcatcag caacatcaac gtcatcgtgc tggagctgaa gggcagcgag 360

acgacgttta tgtgcgagta cgcagacgag acggccacca tcgtcgagtt cttgaaccgg 420

tggatcacct tcagccagtc catcatctcc accctgacc 459

<210> 244

<211> 471

<212> DNA

<213> artificial sequence

<220>

<223> DNA sequence encoding protein 2e

<400> 244

atggcctgga agactctccc catctacctc ctgctgctcc tgtcggtctt cgtcatccag 60

caggtgtcct ccgcgcccac ttcctcgtcc accaagaaga cccagctgca gctcgaacac 120

ctgctcctcg acctccagat gatcctcaac ggaatcaaca actacaagaa ccccaagctg 180

acgttcggca actccaccaa gttctacatg cccaagaagg ccaccgagct gaagcatctg 240

cagtgcctgg aagaggagct caagccgctc gaggaagtgc tgaacctggc ccagagcaag 300

aacttccacc tccgcccgag ggacctgatc tcgaacatca acgtcatcgt gctggagctg 360

aagggatcgg agaccacgtt catgtgcgaa tacgccgacg agacggccac catcgtggag 420

ttcctcaacc gctggatcac cttctcccag agcataatct ccaccctgac c 471

<210> 245

<211> 456

<212> DNA

<213> artificial sequence

<220>

<223> DNA sequence, encoding protein 2g

<400> 245

atggagttcg gtctgtcctg ggtgttcctg gtcgccatca tcaagggcgt gcagtgcgcg 60

cccacttcct cgtccaccaa gaagacccag ctgcagctcg aacacctgct cctcgacctc 120

cagatgatcc tcaacggaat caacaactac aagaacccca agctgacgtt cggcaactcc 180

accaagttct acatgcccaa gaaggccacc gagctgaagc atctgcagtg cctggaagag 240

gagctcaagc cgctcgagga agtgctgaac ctggcccaga gcaagaactt ccacctccgc 300

ccgagggacc tgatctcgaa catcaacgtc atcgtgctgg agctgaaggg atcggagacc 360

acgttcatgt gcgaatacgc cgacgagacg gccaccatcg tggagttcct caaccgctgg 420

atcaccttct cccagagcat aatctccacc ctgacc 456

<210> 246

<211> 459

<212> DNA

<213> artificial sequence

<220>

<223> DNA sequence encoding protein 2f

<400> 246

atgtaccgca tgcagctgct gtcgtgcatc gcgctcagcc tggcgctggt cacgaactcg 60

gcgcccactt cctcgtccac caagaagacc cagctgcagc tcgaacacct gctcctcgac 120

ctccagatga tcctcaacgg aatcaacaac tacaagaacc ccaagctgac gttcggcaac 180

tccaccaagt tctacatgcc caagaaggcc accgagctga agcatctgca gtgcctggaa 240

gaggagctca agccgctcga ggaagtgctg aacctggccc agagcaagaa cttccacctc 300

cgcccgaggg acctgatctc gaacatcaac gtcatcgtgc tggagctgaa gggatcggag 360

accacgttca tgtgcgaata cgccgacgag acggccacca tcgtggagtt cctcaaccgc 420

tggatcacct tctcccagag cataatctcc accctgacc 459

<210> 247

<211> 157

<212> PRT

<213> artificial sequence

<220>

<223> protein 2b

<400> 247

Met Ala Trp Lys Thr Leu Pro Ile Tyr Leu Leu Leu Leu Leu Ser Val

1 5 10 15

Phe Val Ile Gln Gln Val Ser Ser Ala Pro Thr Ser Ser Ser Thr Lys

20 25 30

Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile

35 40 45

Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Cys Met Leu

50 55 60

Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu

65 70 75 80

Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu

85 90 95

Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn

100 105 110

Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met

115 120 125

Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg

130 135 140

Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr

145 150 155

<210> 248

<211> 157

<212> PRT

<213> artificial sequence

<220>

<223> protein 2e

<400> 248

Met Ala Trp Lys Thr Leu Pro Ile Tyr Leu Leu Leu Leu Leu Ser Val

1 5 10 15

Phe Val Ile Gln Gln Val Ser Ser Ala Pro Thr Ser Ser Ser Thr Lys

20 25 30

Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile

35 40 45

Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Phe Gly Asn

50 55 60

Ser Thr Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu

65 70 75 80

Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu

85 90 95

Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn

100 105 110

Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met

115 120 125

Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg

130 135 140

Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr

145 150 155

<210> 249

<211> 152

<212> PRT

<213> artificial sequence

<220>

<223> protein 2d

<400> 249

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Ile Lys Gly

1 5 10 15

Val Gln Cys Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln

20 25 30

Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn

35 40 45

Asn Tyr Lys Asn Pro Lys Leu Thr Cys Met Leu Thr Phe Lys Phe Tyr

50 55 60

Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu

65 70 75 80

Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn

85 90 95

Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val

100 105 110

Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp

115 120 125

Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser

130 135 140

Gln Ser Ile Ile Ser Thr Leu Thr

145 150

<210> 250

<211> 152

<212> PRT

<213> artificial sequence

<220>

<223> protein 2g

<400> 250

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Ile Lys Gly

1 5 10 15

Val Gln Cys Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln

20 25 30

Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn

35 40 45

Asn Tyr Lys Asn Pro Lys Leu Thr Phe Gly Asn Ser Thr Lys Phe Tyr

50 55 60

Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu

65 70 75 80

Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn

85 90 95

Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val

100 105 110

Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp

115 120 125

Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser

130 135 140

Gln Ser Ile Ile Ser Thr Leu Thr

145 150

<210> 251

<211> 153

<212> PRT

<213> artificial sequence

<220>

<223> protein 2c

<400> 251

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu

20 25 30

Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile

35 40 45

Asn Asn Tyr Lys Asn Pro Lys Leu Thr Cys Met Leu Thr Phe Lys Phe

50 55 60

Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu

65 70 75 80

Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

85 90 95

Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile

100 105 110

Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala

115 120 125

Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe

130 135 140

Ser Gln Ser Ile Ile Ser Thr Leu Thr

145 150

<210> 252

<211> 153

<212> PRT

<213> artificial sequence

<220>

<223> protein 2f

<400> 252

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu

20 25 30

Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile

35 40 45

Asn Asn Tyr Lys Asn Pro Lys Leu Thr Phe Gly Asn Ser Thr Lys Phe

50 55 60

Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu

65 70 75 80

Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

85 90 95

Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile

100 105 110

Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala

115 120 125

Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe

130 135 140

Ser Gln Ser Ile Ile Ser Thr Leu Thr

145 150

<210> 253

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> peptide sequence example

<400> 253

Ala Leu Met Gly Arg

1 5

<210> 254

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X1X2NX3X4

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> threonine, serine or cysteine

<400> 254

Xaa Xaa Asn Xaa Xaa

1 5

<210> 255

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X2NX3X4

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> threonine, serine or cysteine

<400> 255

Xaa Asn Xaa Xaa

1

<210> 256

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> X2NX3T

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> misc_feature

<222> (3)..(3)

<223> Xaa can be any naturally occurring amino acid

<400> 256

Xaa Asn Xaa Thr

1

<210> 257

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X2NX3S

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 257

Xaa Asn Xaa Ser

1

<210> 258

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X2NX3C

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 258

Xaa Asn Xaa Cys

1

<210> 259

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X2NSX4

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> threonine, serine or cysteine

<400> 259

Xaa Asn Ser Xaa

1

<210> 260

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X2NST

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<400> 260

Xaa Asn Ser Thr

1

<210> 261

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X2NSS

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<400> 261

Xaa Asn Ser Ser

1

<210> 262

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X2NSC

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<400> 262

Xaa Asn Ser Cys

1

<210> 263

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif GNX3X4

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> threonine, serine and cysteine

<400> 263

Gly Asn Xaa Xaa

1

<210> 264

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif GNX3T

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 264

Gly Asn Xaa Thr

1

<210> 265

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif GNX3S

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 265

Gly Asn Xaa Ser

1

<210> 266

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif GNX3C

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 266

Gly Asn Xaa Cys

1

<210> 267

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif GNSX4

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> threonine, serine or cysteine

<400> 267

Gly Asn Ser Xaa

1

<210> 268

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif GNST

<400> 268

Gly Asn Ser Thr

1

<210> 269

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif GNSS

<400> 269

Gly Asn Ser Ser

1

<210> 270

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif GNSC

<400> 270

Gly Asn Ser Cys

1

<210> 271

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif ANX3X4

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> threonine, serine or cysteine

<400> 271

Ala Asn Xaa Xaa

1

<210> 272

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif ANX3T

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 272

Ala Asn Xaa Thr

1

<210> 273

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif ANX3S

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 273

Ala Asn Xaa Ser

1

<210> 274

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif ANX3C

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 274

Ala Asn Xaa Cys

1

<210> 275

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif ANSX4

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> threonine, serine or cysteine

<400> 275

Ala Asn Ser Xaa

1

<210> 276

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif ANSC

<400> 276

Ala Asn Ser Cys

1

<210> 277

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif ANST

<400> 277

Ala Asn Ser Thr

1

<210> 278

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif ANSS

<400> 278

Ala Asn Ser Ser

1

<210> 279

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X1X2NX3T

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 279

Xaa Xaa Asn Xaa Thr

1 5

<210> 280

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X1X2NX3S

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 280

Xaa Xaa Asn Xaa Ser

1 5

<210> 281

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X1X2NX3C

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 281

Xaa Xaa Asn Xaa Cys

1 5

<210> 282

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X1X2NSX4

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> threonine, serine or cysteine

<400> 282

Xaa Xaa Asn Ser Xaa

1 5

<210> 283

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X1X2NST

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> any Pron or non-Pron amino acids or absence

<400> 283

Xaa Xaa Asn Ser Thr

1 5

<210> 284

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X1X2NSS

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> any Pron or non-Pron amino acids or absence

<400> 284

Xaa Xaa Asn Ser Ser

1 5

<210> 285

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X1X2NSC

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> any Pron or non-Pron amino acids or absence

<400> 285

Xaa Xaa Asn Ser Cys

1 5

<210> 286

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X1GNX3X4

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> threonine, serine or cysteine

<400> 286

Xaa Gly Asn Xaa Xaa

1 5

<210> 287

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X1GNX3T

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 287

Xaa Gly Asn Xaa Thr

1 5

<210> 288

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X1GNX3S

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 288

Xaa Gly Asn Xaa Ser

1 5

<210> 289

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X1GNX3C

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 289

Xaa Gly Asn Xaa Cys

1 5

<210> 290

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X1GNSX4

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> threonine, serine or cysteine

<400> 290

Xaa Gly Asn Ser Xaa

1 5

<210> 291

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X1GNST

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<400> 291

Xaa Gly Asn Ser Thr

1 5

<210> 292

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X1GNSS

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<400> 292

Xaa Gly Asn Ser Ser

1 5

<210> 293

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X1GNSC

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<400> 293

Xaa Gly Asn Ser Cys

1 5

<210> 294

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FX2NX3X4

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> threonine, serine or cysteine

<400> 294

Phe Xaa Asn Xaa Xaa

1 5

<210> 295

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FX2NX3T

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 295

Phe Xaa Asn Xaa Thr

1 5

<210> 296

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FX2NX3S

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 296

Phe Xaa Asn Xaa Ser

1 5

<210> 297

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FX2NX3C

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 297

Phe Xaa Asn Xaa Cys

1 5

<210> 298

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FX2NSX4

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> threonine, serine or cysteine

<400> 298

Phe Xaa Asn Ser Xaa

1 5

<210> 299

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FX2NST

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> any Pron or non-Pron amino acids or absence

<400> 299

Phe Xaa Asn Ser Thr

1 5

<210> 300

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FX2NSS

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> any Pron or non-Pron amino acids or absence

<400> 300

Phe Xaa Asn Ser Ser

1 5

<210> 301

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FX2NSC

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> any Pron or non-Pron amino acids or absence

<400> 301

Phe Xaa Asn Ser Cys

1 5

<210> 302

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FGNX3X4

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> threonine, serine or cysteine

<400> 302

Phe Gly Asn Xaa Xaa

1 5

<210> 303

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FGNX3T

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 303

Phe Gly Asn Xaa Thr

1 5

<210> 304

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FGNX3S

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 304

Phe Gly Asn Xaa Ser

1 5

<210> 305

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FGNX3C

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 305

Phe Gly Asn Xaa Cys

1 5

<210> 306

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FGNSX4

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> threonine, serine or cysteine

<400> 306

Phe Gly Asn Ser Xaa

1 5

<210> 307

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FGNST

<400> 307

Phe Gly Asn Ser Thr

1 5

<210> 308

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FGNSS

<400> 308

Phe Gly Asn Ser Ser

1 5

<210> 309

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FGNSC

<400> 309

Phe Gly Asn Ser Cys

1 5

<210> 310

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X1ANX3X4

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> threonine, serine or cysteine

<400> 310

Xaa Ala Asn Xaa Xaa

1 5

<210> 311

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X1ANX3T

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 311

Xaa Ala Asn Xaa Thr

1 5

<210> 312

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X1ANX3S

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 312

Xaa Ala Asn Xaa Ser

1 5

<210> 313

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X1ANX3C

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> absence of any proteinogenic or non-proteinogenic amino acids or is

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 313

Xaa Ala Asn Xaa Cys

1 5

<210> 314

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X1ANSX4

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> threonine, serine or cysteine

<220>

<221> misc_feature

<222> (5)..(5)

<223> Xaa can be any naturally occurring amino acid

<400> 314

Xaa Ala Asn Ser Xaa

1 5

<210> 315

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X1ANST

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<400> 315

Xaa Ala Asn Ser Thr

1 5

<210> 316

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X1ANSS

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> absence of any proteinogenic or non-proteinogenic amino acids or is

<400> 316

Xaa Ala Asn Ser Ser

1 5

<210> 317

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif X1ANSC

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any Pron or non-Pron amino acids or absence

<400> 317

Xaa Ala Asn Ser Cys

1 5

<210> 318

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FX2NX3X4

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> threonine, serine or cysteine

<400> 318

Phe Xaa Asn Xaa Xaa

1 5

<210> 319

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FX2NX3T

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 319

Phe Xaa Asn Xaa Thr

1 5

<210> 320

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FX2NX3S

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 320

Phe Xaa Asn Xaa Ser

1 5

<210> 321

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FX2NX3C

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 321

Phe Xaa Asn Xaa Cys

1 5

<210> 322

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FX2NSX4

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> any Pron or non-Pron amino acids or absence

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> threonine, serine or cysteine

<400> 322

Phe Xaa Asn Ser Xaa

1 5

<210> 323

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FX2NST

<220>

<221> MISC_FEATURE

<223> any Pron or non-Pron amino acids or absence

<220>

<221> misc_feature

<222> (2)..(2)

<223> Xaa can be any naturally occurring amino acid

<400> 323

Phe Xaa Asn Ser Thr

1 5

<210> 324

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FX2NSS

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> any Pron or non-Pron amino acids or absence

<400> 324

Phe Xaa Asn Ser Ser

1 5

<210> 325

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FX2NSC

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> absence of any proteinogenic or non-proteinogenic amino acids or is

<400> 325

Phe Xaa Asn Ser Cys

1 5

<210> 326

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FANX3X4

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> threonine, serine or cysteine

<400> 326

Phe Ala Asn Xaa Xaa

1 5

<210> 327

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FANX3T

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 327

Phe Ala Asn Xaa Thr

1 5

<210> 328

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FANX3S

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 328

Phe Ala Asn Xaa Ser

1 5

<210> 329

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FANX3C

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> any proteinogenic or nonproteinogenic amino acid other than proline

<400> 329

Phe Ala Asn Xaa Cys

1 5

<210> 330

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FANSX4

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> threonine, serine or cysteine

<400> 330

Phe Ala Asn Ser Xaa

1 5

<210> 331

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FANST

<400> 331

Phe Ala Asn Ser Thr

1 5

<210> 332

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FANSS

<400> 332

Phe Ala Asn Ser Ser

1 5

<210> 333

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> glycosylation motif FANSC

<400> 333

Phe Ala Asn Ser Cys

1 5

<210> 334

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 protein, formula (I-ii)

<400> 334

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Phe Gly Asp Ser Thr Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 335

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 protein, formula (I-ii)

<400> 335

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Phe Ala Asp Ser Thr Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 336

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 protein, formula (I-ii)

<400> 336

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Phe Gly Asp Ser Thr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 337

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 protein, formula (I-ii)

<400> 337

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Phe Ala Asp Ser Thr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 338

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 protein, formula (I-ii)

<400> 338

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Phe Gly Asp Ser Thr

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 339

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 protein, formula (I-ii)

<400> 339

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Phe Ala Asp Ser Thr

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 340

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 protein, formula (I-ii)

<400> 340

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Phe Gly Asp Ser Thr Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 341

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 protein, formula (I-ii)

<400> 341

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Phe Ala Asp Ser Thr Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 342

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 protein, formula (I-ii)

<400> 342

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Phe Gly Asp Ser Thr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 343

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 protein, formula (I-ii)

<400> 343

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Phe Ala Asp Ser Thr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 344

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 protein, formula (I-ii)

<400> 344

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Phe Gly Asp Ser

50 55 60

Thr Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 345

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> IL-2 protein, formula (I-ii)

<400> 345

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Phe Ala Asp Ser

50 55 60

Thr Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 346

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ D

<400> 346

Tyr Lys Asn Pro Phe Ala Asp Ser Thr Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 347

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ D

<400> 347

Tyr Lys Asn Pro Lys Leu Thr Phe Ala Asp Ser Thr Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 348

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ D

<400> 348

Tyr Lys Asn Pro Lys Leu Thr Arg Phe Ala Asp Ser Thr Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 349

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ D

<400> 349

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Phe Ala Asp Ser Thr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 350

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ D

<400> 350

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Ala Asp Ser Thr

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 351

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ D

<400> 351

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Phe Ala Asp Ser

1 5 10 15

Thr Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 352

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ D

<400> 352

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Ala Asp

1 5 10 15

Ser Thr Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 353

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ D

<400> 353

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Phe Ala

1 5 10 15

Asp Ser Thr Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 354

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ D

<400> 354

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Phe Ala

20 25 30

Asp Ser Thr Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 355

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ D

<400> 355

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Phe

20 25 30

Ala Asp Ser Thr Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 356

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ D

<400> 356

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Phe Ala Asp Ser Thr

35 40 45

<210> 357

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ D

<400> 357

Tyr Lys Asn Pro Phe Gly Asp Ser Thr Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 358

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ D

<400> 358

Tyr Lys Asn Pro Lys Leu Thr Phe Gly Asp Ser Thr Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 359

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ D

<400> 359

Tyr Lys Asn Pro Lys Leu Thr Arg Phe Gly Asp Ser Thr Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 360

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ D

<400> 360

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Phe Gly Asp Ser Thr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 361

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ D

<400> 361

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Gly Asp Ser Thr

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 362

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ D

<400> 362

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Phe Gly Asp Ser

1 5 10 15

Thr Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 363

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ D

<400> 363

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Gly Asp

1 5 10 15

Ser Thr Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 364

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ D

<400> 364

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Phe Gly

1 5 10 15

Asp Ser Thr Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 365

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ D

<400> 365

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Phe Gly

20 25 30

Asp Ser Thr Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 366

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ D

<400> 366

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Phe

20 25 30

Gly Asp Ser Thr Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

35 40 45

<210> 367

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> SEQ D

<400> 367

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

1 5 10 15

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

20 25 30

Leu Lys Pro Leu Glu Glu Val Leu Asn Phe Gly Asp Ser Thr

35 40 45

<210> 368

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> sequence example

<400> 368

Phe Gly Asp Ser Thr

1 5

Claims

1. IL-2 protein sequence of formula (I)

(Tag ¹ ) _y -(Ala) _x -SEQ A-SEQ B-SEQ C-(Tag ² ) _z (I)，

Wherein the method comprises the steps of

SEQ A and SEQ ID NO:1 has at least 89% sequence identity;

SEQ C and SEQ ID NO:4 has at least 91% sequence identity;

Tag ¹ And Tag ² Independently a label moiety;

ala is an alanine residue;

x is 0 or 1;

y is 0 or 1; and is also provided with

z is 0 or 1.

2. The IL-2 protein of claim 1, wherein SEQ a is selected from the group consisting of SEQ ID NOs: 1 and SEQ ID NO:36.

3. the IL-2 protein of claim 1 or 2, wherein at least one glycosylation motif in SEQ B is an N-glycosylation motif or an O-glycosylation motif.

4. The IL-2 protein of any one of claims 1-3, wherein at least one N-glycosylation motif in SEQ B has an amino acid sequence X ₁ X ₂ NX ₃ X ₄ (SEQ ID NO: 254), wherein

X ₁ Is any proteinogenic or non-proteinogenic amino acid or is absent;

X ₂ is any proteinogenic or non-proteinogenic amino acid or is absent;

n is asparagine;

X ₃ is any proteinogenic or non-proteinogenic amino acid other than proline; and is also provided with

X ₄ Selected from threonine, serine, and cysteine.

5. The IL-2 protein of any one of claims 1-4, wherein at least one N-glycosylation motif in SEQ B is FGNST (SEQ ID NO: 307) or fasst (SEQ ID NO: 331).

6. The IL-2 protein of any one of claims 1-5, wherein SEQ B is selected from the group consisting of SEQ ID NOs: 11. SEQ ID NO: 14. SEQ ID NO: 17. SEQ ID NO: 20. SEQ ID NO: 23. SEQ ID NO: 26. SEQ ID NO: 32. SEQ ID NO: 35. SEQ ID NO:214 and SEQ ID NO:215.

7. The IL-2 protein of any one of claims 1-6, wherein SEQ B has the amino acid sequence of SEQ ID NO: 11.

8. The IL-2 protein of any one of claims 1-7, wherein SEQ C is selected from the group consisting of SEQ ID NOs: 3. SEQ ID NO:4 and SEQ ID NO:212.

9. the IL-2 protein of any one of claims 1-8, wherein x of formula (I) is 1.

10. The IL-2 protein of any one of claims 1-9, wherein y and z of formula (I) are 0.

11. The IL-2 protein of any one of claims 1-10, wherein the IL-2 protein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 10; SEQ ID NO:13; SEQ ID NO:16; SEQ ID NO:19; SEQ ID NO:22; SEQ ID NO:25, a step of selecting a specific type of material; SEQ ID NO:31; SEQ ID NO:34; SEQ ID NO:217; and SEQ ID NO: 225.

12. The IL-2 protein of any one of claims 1-11, wherein the IL-2 protein has the amino acid sequence of SEQ ID NO: 10.

13. The IL-2 protein of any one of claims 1-12, wherein the IL-2 protein is a biased IL-2.

14. A conjugate comprising one or more IL-2 proteins of any one of claims 1-13.

15. The conjugate of claim 14, wherein the conjugate is an IL-2 conjugate of formula (Ia) or (Ib) or a pharmaceutically acceptable salt thereof

Wherein the method comprises the steps of

-D comprises an IL-2 protein of formula (I);

-L ¹ -a linker moiety covalently and reversibly linked to-D;

-L ² -a chemical bond or a spacer moiety;

-Z is a polymer moiety or a substituted fatty acid moiety;

y is an integer selected from 2, 3, 4 and 5.

16. The conjugate of claim 14 or 15, or a pharmaceutically acceptable salt thereof, wherein-Z is a polymeric moiety.

17. The conjugate of any one of claims 14-16, wherein-Z is a polymer moiety having a molecular weight in the range of 1kDa-1000kDa, or a pharmaceutically acceptable salt thereof.

18. The conjugate of any one of claims 14-17, wherein-Z is a PEG-based polymer moiety, or a pharmaceutically acceptable salt thereof.

19. The conjugate of any one of claims 14-18, or a pharmaceutically acceptable salt thereof, wherein-Z comprises a moiety of formula (a)

Wherein the method comprises the steps of

R ⁸ Selected from H, C _1-6 Alkyl, C _2-6 Alkenyl and C _2-6 Alkynyl;

-P ¹ 、-P ² 、-P ³ 、-P ⁴ are, independently of each other, PEG-based chains comprising at least 40% PEG and having a molecular weight range of 3-40 kDa;

-C ¹ -、-C ² -are independently from each other selected from C _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl; wherein C is _1-50 Alkyl, C ₂ - ₅₀ Alkenyl and C _2-50 Alkynyl groups optionally being substituted by one or more R, which may be the same or different ⁹ Substituted, and wherein C _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl groups are optionally interrupted by one or more groups selected from the group consisting of-T-, -C (O) O-, -C (O) N (R) ¹⁰ )-、-S(O) ₂ N(R ¹⁰ )-、-S(O)N(R ¹⁰ )-、-S(O) ₂ -、-S(O)-、-N(R ¹⁰ )S(O) ₂ N(R ^10ax )-、-S-、-N(R ¹⁰ )-、-OC(OR ¹⁰ )(R ^10a )-、-N(R ¹⁰ )C(O)N(R ^10a ) -and-OC (O) N (R) ¹⁰ )-；

T is each independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C _3-10 Cycloalkyl, 3-to 10-membered heterocyclyl, 8-to 11-membered heterobicyclo, 8-to 30-membered carbopolycyclic and 8-to 30-membered heteropolycyclic; wherein T are each independently optionally substituted with one or more R's, which may be the same or different ⁹ Substitution;

R ⁹ each independently selected from halogen, -CN, oxo (= O), -COOR ¹¹ 、-OR ¹¹ 、-C(O)R ¹¹ 、-C(O)N(R ¹¹ R ^11a )、-S(O) ₂ N(R ¹¹ R ^11a )、-S(O)N(R ¹¹ R ^11a )、-S(O) ₂ R ¹¹ 、-S(O)R ¹¹ 、-N(R ¹¹ )S(O) ₂ N(R ^11a R ^11b )、-SR ¹¹¹¹ 、-N(R ¹¹ R ^11a )、-NO ₂ 、-OC(O)R ¹¹ 、-N(R ¹¹ )C(O)R ^11a 、-N(R ¹¹ )S(O) ₂ R ^11a 、-N(R ¹¹ )S(O)R ^11a 、-N(R ¹¹ )C(O)OR ^11a 、-N(R ¹¹ )C(O)N(R ^11a R ^11b )、-OC(O)N(R ¹¹ R ^1la ) And C _1-6 An alkyl group; wherein C is _1-6 Alkyl is optionally substituted with one or more halogen, the same or different; and is also provided with

R ¹⁰ 、R ^10a 、R ¹¹ 、R ^11a And R is ^11b Each independently selected from-H and C _1-6 Alkyl, wherein C _1-6 The alkyl groups are optionally substituted with one or more halogen groups, which may be the same or different.

20. The conjugate of claim 19, or a pharmaceutically acceptable salt thereof, wherein C ¹ And C ² Having (A-a)

Wherein the method comprises the steps of

The dotted line marked with an asterisk indicates the connection to BP ¹ ；

The unlabeled dashed lines represent the respective links to BP ² Or Bp ³ ；

q1 is selected from 1, 2, 3, 4, 5, 6, 7 and 8;

q2 is selected from 1, 2, 3, 4 and 5;

q3 is selected from 1, 2,3, 4, 5, 6, 7 and 8; and is also provided with

q4 is selected from 1, 2 and 3.

21. The conjugate of claim 19 or 20, or a pharmaceutically acceptable salt thereof, wherein P ¹ 、P ² 、P ³ And P ⁴ Independently of one another, of the formula (A-b)

Wherein the method comprises the steps of

The dashed line represents the remainder attached to-Z;

m is 0 or 1;

p is an integer of 70 to 900; and is also provided with

q is selected from 1, 2,3, 4, 5 and 6.

22. The conjugate of any one of claims 19-21, or a pharmaceutically acceptable salt thereof, wherein BP ¹ is-N <.

23. The conjugate of any one of claims 19-22, or a pharmaceutically acceptable salt thereof, wherein BP ² And BP ² Are all-CH <.

24. The conjugate of any one of claims 14-23, or a pharmaceutically acceptable salt thereof, wherein-Z comprises a moiety of formula (a-c):

wherein the method comprises the steps of

p1, p2, p3, p4 are independently integers from 70 to 900.

25. The conjugate of any one of claims 14-24, or a pharmaceutically acceptable salt thereof, wherein-L ¹ -having formula (IX-a):

wherein the method comprises the steps of

Dotted lines marked with asterisks indicate the attachment to-DNitrogen, and the unlabeled dotted line indicates the attachment to-L ² -Z；

n is 0, 1, 2,3 or 4;

＝Y ₁ selected from = O and = S;

-Y ₂ -selected from-O-and-S-;

-Y ₃ -selected from-O-and-S-;

-Y ₄ -is selected from-O-, -NR ⁵ -and-C (R) ⁶ R ^6a )-；

＝Y ₅ Selected from = O and = S;

-R ³ 、-R ⁵ 、-R ⁶ 、-R ^6a independently of each other, selected from the group consisting of-H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2-dimethylbutyl, 2, 3-dimethylbutyl and 3, 3-dimethylpropyl;

-W-is selected from C _1-20 Alkyl optionally interrupted by one or more groups selected from C _3-10 Cycloalkyl, 8-to 30-membered carbocyclyl, 3-to 10-membered heterocyclyl, -C (O) -, -C (O) N (R) ⁷ ) -, -O-, -S-and-N (R) ⁷ )-；

-Nu is a nucleophile selected from the group consisting of-N (R ⁷ R ^7a )、-N(R ⁷ OH)、-N(R ⁷ )-N(R ^7a R ^7b )、-S(R ⁷ )，-COOH、

Ar-is selected from

Wherein the method comprises the steps of

-Z ¹ -is selected from-O-, -S-and-N (R) ⁷ ) -, and

-Z ² -is-N (R) ⁷ ) -; and is also provided with

wherein-L ¹ Optionally further substituted.

26. The conjugate of any one of claims 14-25, or a pharmaceutically acceptable salt thereof, wherein-L ¹ -having the formula (IX-c)

Wherein the method comprises the steps of

the unlabeled dashed line indicates the attachment to-L ² -Z; and is also provided with

s1 is an integer selected from 1, 2,3, 4, 5, 6, 7, 8, 9 and 10.

27. The conjugate of claim 26, or a pharmaceutically acceptable salt thereof, wherein s1 is 3.

28. The conjugate of any one of claims 14-27, or a pharmaceutically acceptable salt thereof, wherein-L ² -a spacer.

29. The conjugate of any one of claims 14-28, or a pharmaceutically acceptable salt thereof, wherein-L ² -selected from-T-, -C (O) O-, -C (O) N (R) ^y1 )-、-S(O) ₂ N(R ^y1 )-、-S(O)N(R ^y1 )-、-S(O) ₂ -、-S(O)-、-N(R ^y1 )S(O) ₂ N(Ry ^1a )-、-S-、-N(R ^y1 )-、-OC(OR ^y1 )(R ^y1a )-、-N(R ^y1 )C(O)N(R ^y1a )-、-OC(O)N(R ^y1 )-，C _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl; wherein-T-, C _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl is optionally substituted with one or more of the same or different-R ^y2 Substituted, and wherein C _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl groups are optionally interrupted by one or more groups selected from the group consisting of-T-, -C (O) O-, -C (O) N (R) ^y3 )-、-S(O) ₂ N(R ^y3 )-、-S(O)N(R ^y3 )-、-S(O) ₂ -、-S(O)-、-N(R ^y3 )S(O) ₂ N(R ^y3a )-，

-S-、-N(R ^y3 )-、-OC(OR ^y3 )(R ^y3a )-、-N(R ^y3 )C(O)N(R ^y3a ) -and-OC (O) N (R) ^y3 )-；

-R ^y1 and-Ry ^1a Independently of one another, from-H, -T, C _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl; wherein-T, C _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl is optionally substituted with one or more of the same or different-R ^y2 Substituted, and wherein C _1-50 Alkyl, C _2-50 Alkenyl and C _2-50 Alkynyl groups are optionally interrupted by one or more groups selected from the group consisting of-T-, -C (O) O-, -C (O) N (R) ^y4 )-、-S(O) ₂ N(R ^y4 )-、-S(O)N(R ^y4 )-、-S(O) ₂ -、-S(O)-、-N(R ^y4 )S(O) ₂ N(R ^y4a )-、-S-、-N(R ^y4 )-、-OC(OR ^y4 )(R ^y4a )-、-N(R ^y4 )C(O)N(R ^y4a ) -and-OC (O) N (R) ^y4 )-；

T is each independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C _3-10 Cycloalkyl, 3-to 10-membered heterocyclyl, 8-to 11-membered heterobicycloyl, 8-to 30-membered carbopolycycloyl and 8-to 30-membered heteropolycycloyl; wherein T is each independently optionally substituted with one or more of the same or different-R ^y2 Substitution;

-R ^y2 each independently selected from halogen, -CN, oxo (= O), -COOR ^y5 、-OR ^y5 、-C(O)R ^y5 、-C(O)N(R ^y5 R ^y5a )、-S(O) ₂ N(R ^y5 R ^y5a )、-S(O)N(R ^y5 R ^y5a )、-S(O) ₂ R ^y5 、-S(O)R ^y5 、-N(R ^y5 )S(O) ₂ N(R ^y5a R ^y5b )、-SR ^y5 、-N(R ^y5 R ^y5a )、-NO ₂ 、-OC(O)R ^y5 、-N(R ^y5 )C(O)R ^y5a 、-N(R ^y5 )S(O) ₂ R ^y5a 、-N(R ^y5 )S(O)R ^y5a 、-N(R ^y5 )C(O)OR ^y5a 、-N(R ^y5 )C(O)N(R ^y5a R ^y5b )、-OC(O)N(R ^y5 R ^y5a ) And C _1-6 An alkyl group; wherein C is _1-6 Alkyl is optionally substituted with one or more halogen, the same or different; and is also provided with

-R ^y3 、-R ^y3a 、-R ^y4 、-R ^y4a 、-R ^y5 、-R ^y5a and-R ^y5b Each independently selected from-H and C _1-6 Alkyl, wherein C _1-6 The alkyl groups are optionally substituted with one or more halogen groups, which may be the same or different.

30. The conjugate of any one of claims 14-29, or a pharmaceutically acceptable salt thereof, wherein-L ² -is C _1-20 Alkyl chains, optionally interrupted by one or more groups independently selected from-O-, -T-and-C (O) N (R ^y1 ) -; and the C is _1-20 The alkyl chain is optionally substituted with one or more groups independently selected from-OH, -T and-C (O) N (R) ^y6 R ^y6a ) The method comprises the steps of carrying out a first treatment on the surface of the wherein-R ^y1 、-R ^y6 、-R ^y6a Independently selected from H and C _1-4 Alkyl, and wherein T is selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C _3-10 Cycloalkyl, 3-and 10-membered heterocyclyl, 8-to 11-membered heterobicyclo8-to 30-membered carbon polycyclic group and 8-to 30-membered hetero polycyclic group.

31. The conjugate of any one of claims 14-30, or a pharmaceutically acceptable salt thereof, wherein-L ² Having the formula (IX-e)

Wherein the method comprises the steps of

The dotted line marked with an asterisk indicates the attachment to-L ¹ -；

The unlabeled dashed line represents a link to-Z; and is also provided with

32. The conjugate of claim 31, or a pharmaceutically acceptable salt thereof, wherein s2 is 3.

33. A pharmaceutical composition comprising at least one IL-2 protein of any one of claims 1-13 or at least one conjugate of any one of claims 14-32, or a pharmaceutically acceptable salt thereof, and at least one excipient.

34. The IL-2 protein of any one of claims 1-13, the conjugate of any one of claims 14-32, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 33, for use as a medicament.

35. The IL-2 protein of any one of claims 1-13, the conjugate of any one of claims 14-32, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 33, for use in a method of treating cancer.

36. The IL-2 protein, conjugate thereof or pharmaceutically acceptable salt or pharmaceutical composition for use according to claim 35, wherein the cancer is selected from sarcoma, chordoma, colon cancer, rectal cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, wilms 'tumor, cervical cancer, testicular cancer, gastric cancer, non-small cell lung cancer, bladder cancer, renal cell carcinoma, urothelial carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyoma, ependymoma, pineal tumor, angioblastoma, auditory neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, non-hodgkin's lymphoma, skin T cell leukemia, and acute leukemia.

37. The IL-2 protein, conjugate or pharmaceutically acceptable salt thereof or pharmaceutical composition for use according to claim 34 or 35, wherein the IL-2 protein, the conjugate or pharmaceutically acceptable salt thereof or the pharmaceutical composition is administered to the patient before, simultaneously with or after administration of one or more additional drugs.

38. The IL-2 protein, conjugate thereof or pharmaceutically acceptable salt thereof or pharmaceutical composition for use according to claim 37, wherein the one or more additional drugs are selected from the group consisting of Pattern Recognition Receptor Agonists (PRRA), cytotoxic/chemotherapeutic agents, immune checkpoint inhibitors or antagonists, immune checkpoint agonists, immune activated receptor agonists, multispecific drugs, antibody-drug conjugates (ADCs), antibody-adjuvant conjugates (AACs), radionuclide or targeted radionuclide therapeutics, DNA damage repair inhibitors, tumor metabolism inhibitors, pattern recognition receptor agonists, protein kinase inhibitors, chemokines and chemoattractant receptor agonists, chemokines or chemokine receptor antagonists. Cytokine receptor agonists, death receptor agonists, CD47 or sirpa antagonists, oncolytic drugs, signaling proteins, epigenetic modifiers, tumor peptides or tumor vaccines, heat Shock Protein (HSP) inhibitors, proteolytic enzymes, ubiquitin and proteasome inhibitors, adhesion molecule antagonists, hormones including hormone peptides and synthetic hormones, and adoptive cell therapies, such as Tumor Infiltrating Lymphocyte (TIL) therapies, chimeric Antigen Receptor (CAR) therapies, T cell therapies, natural Killer (NK) cell therapies, CAR-T therapies, CAR-NK therapies, CAR- γδ therapies, CAR-macrophage therapies, or any other cell therapies with genetically modified or genetically unmodified immune cell types.

39. An oligonucleotide encoding the IL-2 protein of claim 1.

40. The oligonucleotide according to claim 39, wherein the oligonucleotide is selected from the group consisting of DNA, RNA and cDNA.

41. According to claim 39 or 40 of the oligonucleotide, for the treatment of can be used with IL-2, especially with IL-2 biased treatment of diseases.

42. The oligonucleotide of claim 39 or 40 for use of claim 41, wherein the oligonucleotide is delivered into a host cell in vivo or ex vivo.

43. The oligonucleotide for use according to claim 42, wherein said host cell is an immune cell.