WO2023045977A1 - 白介素2突变体以及其融合蛋白 - Google Patents

白介素2突变体以及其融合蛋白 Download PDF

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WO2023045977A1
WO2023045977A1 PCT/CN2022/120265 CN2022120265W WO2023045977A1 WO 2023045977 A1 WO2023045977 A1 WO 2023045977A1 CN 2022120265 W CN2022120265 W CN 2022120265W WO 2023045977 A1 WO2023045977 A1 WO 2023045977A1
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amino acid
acid sequence
antibody
cells
seq
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PCT/CN2022/120265
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French (fr)
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何开杰
付凤根
伍伟伟
周帅祥
关剑
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信达生物制药(苏州)有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention relates to novel interleukin 2 (IL-2) mutant protein and its application.
  • the present invention relates to IL-2 muteins having improved properties compared to wild-type IL-2, eg, improved IL-2 receptor binding properties and improved druggability.
  • the present invention also provides fusion proteins, dimers, and immunoconjugates comprising the IL-2 mutein, as well as nucleic acids encoding the IL-2 mutein, dimers, and immunoconjugates, vectors comprising the nucleic acid, and host cell. More specifically, the present invention provides an immunoconjugate comprising an IL-2 mutein and an anti-PD-1 antibody.
  • the present invention further provides methods for preparing the IL-2 muteins, fusion proteins, dimers and immunoconjugates, pharmaceutical compositions comprising them and therapeutic uses.
  • Interleukin-2 also known as T cell growth factor (TCGF)
  • TCGF T cell growth factor
  • TCGF T cell growth factor
  • human IL-2 (uniprot: P60568) is synthesized as a 153 amino acid precursor polypeptide that, after removal of the N-terminal 20 amino acids, produces mature, secreted IL-2.
  • sequences of IL-2 from other species have also been published, see NCBI Ref Seq No. NP032392 (mouse), NP446288 (rat) or NP517425 (chimpanzee).
  • Interleukin 2 has 4 antiparallel, amphipathic ⁇ -helices that form a quaternary structure essential to its function (Smith, Science 240, 1169-76 (1988); Bazan, Science 257, 410-413 (1992)). In most cases, IL-2 acts through three different receptors: interleukin 2 receptor alpha (IL-2R ⁇ ; CD25), interleukin 2 receptor beta (IL-2R ⁇ ; CD122) and interleukin 2 receptor gamma ( IL-2R ⁇ ; CD132).
  • IL-2R ⁇ interleukin 2 receptor alpha
  • IL-2R ⁇ interleukin 2 receptor beta
  • IL-2R ⁇ interleukin 2 receptor gamma
  • IL-2R ⁇ and IL-2R ⁇ are essential for IL-2 signaling, while IL-2R ⁇ (CD25) is not essential for signaling but can confer high-affinity binding of IL-2 to the receptor (Krieg et al., Proc Natl Acad Sci 107, 11906-11(2010)).
  • the trimeric receptor formed by IL-2R ⁇ , ⁇ , and ⁇ (IL-2 ⁇ ) is a high-affinity receptor for IL-2 (KD about 10pM), and the dimer receptor composed of ⁇ and ⁇ (IL-2 2 ⁇ ) is an intermediate affinity receptor (KD about 1 nM), and the IL-2 receptor formed solely by the ⁇ subunit is a low affinity receptor.
  • Immune cells express dimeric or trimeric IL-2 receptors. Dimeric receptors are expressed on cytotoxic CD8 + T cells and natural killer cells (NK), whereas trimeric receptors are predominantly expressed on activated lymphocytes and CD4 + CD25 + FoxP3 + suppressive regulatory T cells (Treg) Expression (Byman, O. and Sprent. J. Nat. Rev. Immunol. 12, 180-190 (2012)). Since resting state effector T cells and NK cells do not have CD25 on the cell surface, they are relatively insensitive to IL-2. Treg cells consistently express the highest level of CD25 in vivo, therefore, IL-2 will preferentially stimulate Treg cell proliferation under normal circumstances.
  • IL-2 mediates multiple roles in the immune response by binding to IL-2 receptors on different cells.
  • IL-2 has an immune system stimulatory effect, which can stimulate the proliferation and differentiation of T cells and natural killer (NK) cells. Therefore, IL-2 has been approved as an immunotherapeutic agent for the treatment of cancer and chronic viral infections.
  • IL-2 can also promote the maintenance of immunosuppressive CD4 + CD25 + regulatory T cells (ie, Treg cells) (Fontenot et al., Nature Immunol 6, 1142-51 (2005); D'Cruz and Klein , Nature Immunol 6, 1152-59 (2005); Maloy and Powrie, Nature Immunol 6, 1171-72 (2005)), cause immunosuppression in patients by activated Treg cells.
  • CD4 + CD25 + regulatory T cells ie, Treg cells
  • IL-2 cardiovascular disease 2019
  • vascular leakage Cardiovascular toxicity including syndrome and hypotension.
  • lymphocytes especially T cells and NK cells
  • vascular endothelial cells can shrink, increasing the intercellular space, resulting in an outflow of interstitial fluid, which can cause vascular leakage as a side effect.
  • IL-2 Another limiting problem of clinical use of IL-2 is that its extremely short half-life makes administration difficult. Since the molecular weight of IL-2 is only 15KDa, it will be cleared mainly through the filtration of glomerulus, and the half-life of human body is only about 1 hour. In order to achieve a sufficiently high human exposure, it is clinically necessary to infuse a large dose of IL-2 every 8 hours. However, frequent administration not only brings a heavy burden to patients, but more importantly, high-dose infusion of IL-2 will cause a high peak plasma concentration (Cmax), which is likely to be another cause of drug toxicity. The key factor.
  • Cmax peak plasma concentration
  • IL-2 immunotherapy Several approaches have been taken to overcome these problems associated with IL-2 immunotherapy. For example, it has been found that the combination of IL-2 and certain anti-IL-2 monoclonal antibodies enhances the therapeutic effect of IL-2 in vivo (Kamimura et al., J Immunol 177, 306-14 (2006); Boyman et al., Science 311 , 1924-27(2006)). Some modifications of the IL-2 molecule have also been proposed. For example, Helen R. Mott et al. disclose the mutein F42A of human IL-2 with abolished IL-2R ⁇ binding ability. Rodrigo Vazquez-Lombardi et al.
  • the programmed cell death protein 1 (PD-1 or CD279) is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS and BTLA.
  • PD-1 is a cell surface receptor expressed on activated B cells, T cells, and myeloid cells.
  • the structure of PD-1 is a monomeric type 1 transmembrane protein consisting of an immunoglobulin variable-like extracellular domain and containing an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based inhibitory motif (ITIM). Cytoplasmic domain composition of the switch motif (ITSM).
  • PD-L1 and PD-L2 Two ligands of PD-1 have been identified, PD-L1 and PD-L2, which were shown to downregulate T cell activation upon binding PD-1. Both PD-L1 and PD-L2 are B7 homologues that bind PD-1 but not other CD28 family members.
  • a ligand of PD-1, PD-L1 is abundant in a variety of human cancers. The interaction between PD-1 and PD-L1 leads to a decrease in tumor-infiltrating lymphocytes, a decrease in T cell receptor-mediated proliferation, and immune escape of cancerous cells.
  • PD-1 antibody Various antibodies that bind to PD-1 are known in the art, for example, the PD-1 antibody disclosed in WO2017024465A1.
  • An immunoconjugate comprising (i) an antibody in conjunction with PD-1 and (ii) an IL-2 mutein, the mutein, and wild-type IL-2 (preferably human IL-2, more preferably Comprised of IL-2 comprising the sequence of SEQ ID NO:3), comprising the mutation:
  • a shortened B'C' loop region i.e., the sequence connecting amino acid residues aa72 and aa84
  • said shortened loop region has less than 10, 9, 8, 7, 6, or 5 amino acid length, and preferably 7 amino acid length; preferably, said shortened B'C' loop region results in improved protein expression and/or purity
  • immunoconjugate according to embodiment 1, wherein said mutein, relative to wild-type IL-2, comprises:
  • the immunoconjugate according to embodiment 1, wherein the IL-2 mutein comprises the amino acid sequence of SEQ ID NO: 4, 23, 25, 27, 29 or 31 or has at least 90%, 91%, 92% thereof %, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences or consist thereof.
  • immunoconjugate according to any one of embodiments 1-3, wherein said immunoconjugate comprises:
  • the second monomer comprises an antibody specifically binding to PD-1 or a fragment thereof, preferably, the fragment comprises one heavy chain and one light chain of the anti-PD-1 antibody.
  • immunoconjugate according to any one of embodiments 4-6, wherein the IL-2 mutein fused to an Fc fragment comprises the amino acid sequence of SEQ ID NO: 7, 24, 26, 28, 30 or 32 Or or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.
  • the heavy chain can be The variable regions comprise HCDR1, HCDR2, HCDR3 respectively as shown in the following amino acid sequences: SEQ ID NO: 9, 10 and 11.
  • the PD-1 antibody or antigen-binding fragment thereof comprises a light chain comprising a light chain variable region, wherein the light chain
  • the variable region comprises LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 16, 17 and 18, respectively.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises: comprising or at least 90% of the amino acid sequence shown in SEQ ID NO:8 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence or heavy chain variable region consisting of said amino acid sequence, and comprising SEQ ID
  • the light chain variable region consists of amino acid sequences.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises an amino acid sequence comprising SEQ ID NO: 14 or 22 or having a sequence of up to 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence or heavy chain consisting of said amino acid sequence; and comprising SEQ The amino acid sequence shown in ID NO: 20 or an amino acid sequence having up to 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity therewith Or a light chain consisting of said amino acid sequence.
  • An expression vector comprising the polynucleotide of embodiment 13.
  • a host cell comprising the polynucleotide of embodiment 13 or the vector of embodiment 14, preferably the host cell is a yeast cell or a mammalian cell, especially a HEK293 cell or a CHO cell.
  • a method for producing the immunoconjugate of any one of embodiments 1-12 comprising culturing the host cell of embodiment 15 under conditions suitable for expression of said immunoconjugate.
  • a pharmaceutical composition comprising the immunoconjugate according to any one of embodiments 1-12, optionally together with pharmaceutically acceptable excipients.
  • the cancer is a solid tumor or A hematological tumor, such as a gastrointestinal tumor or a melanoma, such as colorectal or colon cancer; for example, the cancer is PD-1 antibody therapy resistant cancer.
  • a method of preventing and/or treating cancer in a subject comprising administering to the subject the immunoconjugate of any one of embodiments 1-12 or the pharmaceutical composition of embodiment 17 , preferably, the cancer is a solid tumor or a hematological tumor, such as a gastrointestinal tumor or a melanoma, such as colorectal cancer or colon cancer; for example, the cancer is a PD-1 antibody treatment-resistant cancer.
  • Figure 1A is the molecular structure of the immunoconjugate of anti-PD-1 and IL-2 mutant of the present invention
  • Figure 1B is the molecular structure of the IL-2-Fc fusion protein of molecules 2124 and 3010.
  • Figure 2 is the crystal structure of IL-2 bound to the receptor (PDB: 2ERJ).
  • Figure 3 is a binding curve of immunoconjugates and control molecules to IL-2R ⁇ .
  • Figure 4 is a binding curve of immunoconjugates or control molecules to IL-2R ⁇ .
  • Figure 5 is a binding curve of an immunoconjugate or a control molecule to human PD1.
  • Figure 6 is an assay of the activity of immunoconjugates or control molecules in CTLL2WT (huPD1-) and CTLL2-hPD-1 (huPD1+).
  • Figure 7 is the activity of immunoconjugates or control molecules in PD-1- and PD-1+ T cell populations (CD4 or CD8), respectively.
  • Figure 8 shows that the immunoconjugates were activated in HEK-Blue TM IL-2 Cells (huPD-1-cells) and overexpressed PD-1 cells (HEK293+hIL2R+hPD-1/SEAP stable cell line (huPD -1+ activity in cells)).
  • Figure 9A shows the effect of 2132 and 2063 on the anti-tumor effect of mice
  • Figure 9B shows the effect of 2132 and 2063 on the body weight of mice.
  • Figure 10A shows the effect of 2063 on the anti-tumor effect of MC38 tumors in mice
  • Figure 10B shows the effect of 2063 on the body weight of mice.
  • Figure 11A shows the effect of 2063 on the anti-tumor effect of B16F10 tumors in mice
  • Figure 11B shows the effect of 2063 on the anti-tumor effect of B16F10 tumors in mice-individual tumor values
  • Figure 11C shows the effect of 2063 on the body weight of mice.
  • Figure 12A shows the effect of 2149 on the anti-tumor effect of mouse MC38 tumor
  • Figure 12B shows the effect of 2149 on the anti-tumor effect of mouse MC38 tumor-survival curve
  • Figure 12C shows the effect of 2149 on the body weight of mice.
  • Figure 13A shows the impact of 2149 on the anti-tumor effect of mouse B16F10 tumors
  • Figure 13B shows the impact of 2149 on the anti-tumor effect of mouse B16F10 tumors-individual tumor values
  • Figure 13C shows the anti-tumor effect of 2149 on mouse B16F10 tumors Effect of Tumor Effects - Survival Curves
  • Figure 13D shows the effect of 2149 on body weight in mice.
  • Figure 14A shows the impact of 2061 and 2149 on the anti-tumor effect of mouse B16F10 tumors-individual tumor values
  • Figure 14B shows the impact of 2061 and 2149 on the anti-tumor effects of mouse B16F10 tumors-survival curve
  • Figure 14C shows Effects of 2061 and 2149 on body weight in mice.
  • Figure 15A shows the effect of 2214 on the anti-tumor effect of mice
  • Figure 15B shows the effect of 2214 on the anti-tumor effect of mice-survival curve
  • Figure 15C shows the effect of 2214 on the body weight of mice.
  • Figure 16A shows the impact of 2214 on the anti-tumor effect of mouse B16F10 tumors-survival curve
  • Figure 16B shows the impact of 2214 on the anti-tumor effect of mouse B16F10 tumors-survival curve
  • Figure 16C shows the effect of 2214 on the body weight of mice Influence.
  • the term “comprising” or “comprising” means including stated elements, integers or steps, but not excluding any other elements, integers or steps.
  • the term “comprising” or “comprises” is used, unless otherwise specified, it also covers the situation consisting of the mentioned elements, integers or steps.
  • an IL-2 mutein that "comprises” or “comprises” a certain mutation or combination of mutations, it is also intended to encompass an IL-2 mutein that only has said mutation or combination of mutations.
  • wild-type "interleukin-2” or “IL-2” refers to the parental IL-2 protein, preferably a naturally occurring IL-2 protein, e.g. derived from human, Native IL-2 proteins of mice, rats, non-human primates, including unprocessed (eg, signal peptide not removed) forms and processed (eg, signal peptide removed) forms.
  • a full-length native human IL-2 sequence including the signal peptide is shown in SEQ ID NO:1, and the sequence of its mature protein is shown in SEQ ID NO:2.
  • the expression also includes naturally occurring IL-2 allelic and splice variants, isoforms, homologs, and species homologues.
  • the expression also includes variants of native IL-2, which may, for example, be at least 95%-99% or more identical to native IL-2 or have no more than 1-10 or 1-5 amino acids mutations (eg, conservative substitutions), and preferably have substantially the same binding affinity for IL-2R ⁇ and/or binding affinity for IL2R ⁇ as the native IL-2 protein. Therefore, in some embodiments, wild-type IL-2 may contain amino acid mutations that do not affect its binding to the IL-2 receptor compared to native IL-2 protein, for example, natural human IL with a mutation C125S introduced at position 125 -2 protein (uniprot: P60568) belongs to the wild-type IL-2 of the present invention.
  • native IL-2 may, for example, be at least 95%-99% or more identical to native IL-2 or have no more than 1-10 or 1-5 amino acids mutations (eg, conservative substitutions), and preferably have substantially the same binding affinity for IL-2R ⁇ and/or binding affinity for IL2R ⁇ as the native IL
  • wild-type human IL-2 protein comprising the C125S mutation is shown in SEQ ID NO:3.
  • the wild-type IL-2 sequence may have at least 85%, 95%, or even at least 96%, 97%, 98%, or 99% or more of the amino acid sequence of SEQ ID NO: 1 or 2 or 3 High amino acid sequence identity.
  • amino acid mutations may be amino acid substitutions, deletions, insertions and additions. Any combination of substitutions, deletions, insertions and additions can be made to obtain a final mutein construct with desired properties such as reduced IL-2R ⁇ binding affinity and/or improved druggability and/or attenuated IL-2R ⁇ .
  • Amino acid deletions and insertions include deletions and insertions at the amino and/or carboxyl termini of the polypeptide sequence, as well as deletions and insertions within the polypeptide sequence. For example, an alanine residue may be deleted at position 1 of full-length human IL-2, or one or several amino acids may be deleted in the B'C' loop region to shorten the length of the loop region.
  • preferred amino acid mutations are amino acid substitutions, such as combinations of single amino acid substitutions or substitutions of segments of amino acid sequence.
  • amino acid substitutions such as combinations of single amino acid substitutions or substitutions of segments of amino acid sequence.
  • the whole or part of the sequence of the B'C' loop region of wild-type IL-2 may be replaced by a different sequence (such as the B'C' loop of IL-15), preferably to obtain a shortened B'C' loop sequence.
  • the amino acid positions of the IL-2 protein or IL-2 sequence segment when referring to the amino acid position in the IL-2 protein or IL-2 sequence segment, by reference to the amino acid sequence SEQ ID NO of the wild-type human IL-2 protein (also called IL-2 WT ): 3, to be determined.
  • SEQ ID NO of the wild-type human IL-2 protein also called IL-2 WT
  • the corresponding amino acid positions on other IL-2 proteins or polypeptides can be identified by amino acid sequence alignment with SEQ ID NO:3. Therefore, in the present invention, unless otherwise specified, the amino acid positions of the IL-2 protein or polypeptide are the amino acid positions numbered according to SEQ ID NO:3.
  • F42 refers to the 42nd phenylalanine residue F of SEQ ID NO: 3, or the amino acid residue at the corresponding position in other IL-2 polypeptide sequences after alignment.
  • the mutation of the present invention involves truncation or deletion of some specific segments (for example, the sequence of the B'C' loop region, that is, the 73-83 positions of SEQ ID NO:3 11 amino acid residues in total), given the specific mutation region and its mutation method, the numbering of amino acid residues outside the region remains unchanged, for example, the sequence of the B'C' loop region, That is, the 73-83 positions of SEQ ID NO: 3 have a total of 11 amino acid residues.
  • single amino acid substitutions are described in the following manner: [original amino acid residue/position/substituted amino acid residue].
  • K35E a substitution of lysine at position 35 to glutamic acid
  • the amino acid substitution can be expressed as: K35D/E.
  • single amino acid substitutions can be linked by a plus sign "+" or "-" to indicate a combined mutation at multiple given positions.
  • the combined mutation of F42A, N88R and S127E can be expressed as: F42A+N88R+S127E, or F42A-N88R-S127E.
  • percent sequence identity can be determined by comparing two optimally aligned sequences over a comparison window.
  • sequence identity is determined over the full length of the reference sequence (eg SEQ ID NO: 3).
  • Methods of alignment of sequences for comparison are well known in the art. Algorithms suitable for determining percent sequence identity include, for example, the BLAST and BLAST 2.0 algorithms (see Altschul et al., Nuc. Acids Res. 25:3389-402, 1977 and Altschul et al. J. Mol. Biol. 215:403-10, 1990) .
  • Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information. For purposes of this application, percent identity was determined using the Basic Local Alignment Search Tool available at https://blast.ncbi.nlm.nih.gov/Blast.cgi with default parameters.
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the biological function of the protein/polypeptide comprising the amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Typical conservative amino acid substitutions refer to the substitution of one amino acid for another amino acid with similar chemical properties such as charge or hydrophobicity.
  • Conservative substitution tables for functionally similar amino acids are well known in the art.
  • the conservative substitution residues are from the following conservative substitution table X, especially the preferred conservative amino acid substitution residues in Table X.
  • the wild-type IL-2 protein may have conservative amino acid substitutions relative to one of SEQ ID NO: 1-3, or only conservative amino acid substitutions, and in a preferred embodiment, the conservative substitutions are no more than 10 amino acid residues, Such as 1,2,3,4,5,6,7,8,9,10 residues.
  • the mutant IL-2 protein of the present invention may have conservative amino acids relative to the IL-2 mutein sequence (such as any of SEQ ID NO: 4, 23, 25, 27, 29 and 31) specified herein Substitutions, or only with conservative amino acid substitutions and in a preferred embodiment, conservative substitutions of no more than 10 amino acid residues, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 residues.
  • Binding affinity can be used to reflect the intrinsic binding ability of interactions between members of a binding pair.
  • the affinity of a molecule X for its binding partner Y can be expressed by the equilibrium dissociation constant (K D ), which is the ratio of the dissociation rate constant and the association rate constant (k dis and k on , respectively).
  • K D equilibrium dissociation constant
  • Binding affinity can be measured by common methods known in the art. One specific method for measuring affinity is the SPR affinity assay technique or the BLI assay technique herein.
  • an antigen-binding molecule is a polypeptide molecule that can specifically bind an antigen, for example, an immunoglobulin molecule, an antibody or an antibody fragment, such as a Fab fragment and a scFv fragment.
  • the antigen binding molecule of the invention is a binding molecule, such as an antibody, eg a monoclonal antibody, directed against an immune checkpoint molecule as an antigen.
  • the immune checkpoint molecule is PD-1 or PD-L1 or PD-L2.
  • antibody Fc fragment refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region, and may include native sequence Fc fragments and variant Fc fragments.
  • Native sequence Fc fragments cover various immunoglobulin Fc sequences that occur in nature, such as the Fc regions of various Ig subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi : 10.3389/fimmu.2014.00520.)
  • the human IgG heavy chain Fc fragment extends from Cys226 or from Pro230 of the heavy chain to the carboxyl terminus.
  • the C-terminal lysine (Lys447) of the Fc-fragment may or may not be present.
  • the Fc fragment is a variant Fc fragment comprising a mutation, for example comprising the L234A-L235A mutation.
  • the numbering of amino acid residues in the Fc fragment is according to the EU numbering system, also known as the EU index, such as Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.
  • the Fc fragment of an antibody may carry an IgG1 hinge sequence or a partial IgG1 hinge sequence at the N-terminus, eg, a sequence from E216 to T225 or a sequence from D221 to T225 according to EU numbering. Mutations may be contained in the hinge sequence.
  • IL-2 protein belongs to the family of short-chain type I cytokines with four ⁇ -helical bundles (A, B, C, D) structure.
  • B'C'Loop or "B'C' loop region” or “B'C' loop sequence” are used interchangeably and refer to the gap between the B and C helices of the IL-2 protein. link sequence.
  • the B'C' loop sequence of an IL-2 protein can be determined.
  • the B'C' loop sequence refers to the sequence connecting the residue at position 72 and the residue at position 84 in the IL-2 polypeptide according to the numbering of SEQ ID NO:3.
  • the connecting sequence includes a total of 11 amino acids of A73-R83.
  • the term "shortened loop region” or “shortened B'C' loop region” means that the mutein has a B'C' loop sequence of reduced length relative to the wild-type IL-2 protein, That is, according to the numbering of SEQ ID NO: 3, the linking sequence length between amino acid residues aa72 and aa84 is shortened.
  • a "shortened loop region” can be achieved by substitution or truncation of the loop sequence. The substitution or truncation may occur in any region or part of the B'C' loop sequence.
  • the substitution or truncation may be a substitution of the loop region A73-R83 sequence (e.g. replacement with the IL-15B' C loop region) or a truncation of one or more amino acid residues from the C-terminus of the sequence.
  • the substitution or truncation may be the substitution of the Q74-R83 sequence of the loop region or the truncation of one or more amino acid residues from the C-terminus of this sequence.
  • a single amino acid substitution can be further introduced in the loop region sequence, such as an amino acid substitution for eliminating glycosylation, and/or a back mutation to further improve the performance of the mutant protein, Such as medicinal properties.
  • the mutated shortened B'C' loop region can be described by the sequence connecting the residue at position 72 and the residue at position 84 after introducing the mutation.
  • the "IL-2R ⁇ binding interface” mutation refers to the mutation occurring at the amino acid site where IL-2 interacts with IL-2R ⁇ (ie, CD25). These interaction sites can be determined by analyzing the crystal structure of IL-2 in complex with its receptor (eg PDB: 1Z92).
  • the mutation especially refers to a mutation in the region of amino acid residues 35-72 of IL-2, especially a mutation at the following amino acid positions: 35; 37; 38; 41; 42; 43; 45; 61; 62; 68; 72.
  • the IL-2 protein comprising said mutation has reduced or eliminated IL-2R ⁇ binding compared to the corresponding protein before introduction of said mutation.
  • the "IL-2 ⁇ binding interface” mutation refers to the mutation occurring at the amino acid site where IL-2 interacts with IL-2R ⁇ (ie, CD122 and CD132). These interacting amino acid sites can be determined by analysis of the crystal structure of IL-2 in complex with its receptor (eg PDB:2ERJ).
  • the mutation especially refers to a mutation in the region of amino acid residues 12-20, amino acid residues 84-95, and amino acid residues 126-130 of IL-2, especially at the following amino acid positions Mutations: 12, 15, 16, 19, 20, 84, 87, 88, 91, 92, 95, 126, 127, 130.
  • the IL-2 protein comprising said mutation has reduced IL-2R ⁇ binding compared to the corresponding protein before introducing said mutation.
  • IL-2R ⁇ receptor binding refers to the introduction of a mutation at the IL-2R ⁇ binding interface, wherein relative to the corresponding IL-2 protein before the introduction of the mutation, the Mutations result in reduced binding affinity for the IL-2R ⁇ receptor. It is further preferred that the weakened molecule has reduced T cell (eg CD8+ T cell or CD4+ T cell) and/or NK cell activation activity relative to the corresponding protein.
  • T cell eg CD8+ T cell or CD4+ T cell
  • the reduction factor can reach, for example, more than 5 times, such as more than 10 times, or more than 50 times, or more than 100 times, or even more than 1000 times.
  • the T cell activation activity of the weakened molecule can be reduced by 10-50 times, or 50-100 times, or 100-1000 times, or higher.
  • the weakened molecules of the invention have "weakened" binding affinity for the IL-2R ⁇ receptor and "weakened" T cell activation activity.
  • Antigen-binding fragment refers to a molecule, distinct from an intact antibody, that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; dAb (domain antibody); linear antibodies; single chain antibodies (e.g. scFv); ; a diabody or a fragment thereof; or a camelid antibody.
  • antigen refers to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immune cells, or both.
  • antigens can be derived from recombinant or genomic DNA.
  • the antigen described in the present invention is a tumor-associated antigen, that is, an antigen associated with the occurrence, development or progression of a tumor, such as PD-1, PD-L1 or PD-L2.
  • a “complementarity determining region” or “CDR region” or “CDR” is an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and/or contains antigen-contacting residues ( "antigen contact point”).
  • the CDRs are primarily responsible for binding to antigenic epitopes.
  • the CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially starting from the N-terminus.
  • the CDRs located within the variable domain of an antibody heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the variable domain of an antibody light chain are referred to as LCDR1, LCDR2, and LCDR3.
  • each CDR can be determined using any one or combination of a number of well-known antibody CDR assignment systems, including For example: Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., U.S.
  • the residues of each CDR are as follows.
  • a CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (eg, any of the exemplary CDRs of the invention).
  • the heavy chain variable region and light chain variable region CDRs of an antibody of the invention are CDR sequences defined according to the North numbering scheme.
  • linker refers to any molecule that enables the direct linking of different parts of a fusion protein.
  • linkers that establish covalent linkages between different parts of fusion proteins include peptide linkers and non-proteinaceous polymers including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene or polyethylene glycol, polypropylene glycol of copolymers.
  • PEG polyethylene glycol
  • peptide linker refers to a sequence of amino acids, wherein said sequence links the amino acid sequence of the first part of the fusion protein to the second part of the fusion protein.
  • a peptide linker can link the IL-2 portion of the fusion protein to the Fc domain or a fragment thereof.
  • a peptide linker can also link the antibody to IL-2, such as linking the C-terminus of an antibody heavy chain to IL-2.
  • the peptide linker is of a length sufficient to link the two entities in such a way that they maintain their conformation relative to each other so as not to interfere with the desired activity.
  • the peptide linker may or may not consist essentially of the following amino acid residues: Gly, Ser, Ala or Thr.
  • Useful linkers include glycine-serine polymers including, for example, (GS) n , (GSGGS) n , (GGGGS) n , (GGGS) n, and (GGGGS) nG , where n is at least 1 (and preferably 2, 3, 4, 5, 6, 7, 8, 9, 10) integers.
  • Useful linkers also include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers.
  • the linker of the invention is SEQ ID NO:5.
  • an antibody in IgG form refers to the IgG form to which the heavy chain constant region of the antibody belongs.
  • the heavy chain constant regions of all antibodies of the same type are the same, and the heavy chain constant regions of antibodies of different types are different.
  • an antibody in IgG4 form means that its heavy chain constant region is from IgG4
  • an antibody in IgGl form means that its heavy chain constant region is from IgGl.
  • a “humanized” antibody is one that comprises amino acid residues from non-human CDRs and amino acid residues from human FRs.
  • a humanized antibody will comprise substantially all of at least one, usually two, variable domains, wherein all or substantially all of the CDRs (e.g., CDRs) correspond to those of a non-human antibody, and all Or substantially all of the FRs correspond to those of a human antibody.
  • a humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has been humanized.
  • Human antibody or “fully human antibody” or “fully human antibody” are used interchangeably and refer to an antibody having an amino acid sequence corresponding to that of an antibody derived from a human Or human cells are produced or derived from non-human sources that utilize human antibody repertoires or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.
  • the antibody moiety in the immunoconjugates of the invention may be a humanized antibody, a human antibody or a chimeric antibody.
  • fusion refers to a fusion formed by linking two or more originally separate proteins/genes/compounds. If the entities making up the fusion are proteins, it is called a fusion protein. Fusion proteins are encompassed within the scope of fusions of this application.
  • an IL-2 fusion protein can be constructed by linking IL-2 to an Fc dimer. The link between the two entities constituting the fusion may or may not be achieved through a linker.
  • immunoconjugate refers to a polypeptide molecule comprising at least one IL-2 molecule and at least one antibody or antibody fragment.
  • IL-2 molecules can be linked to antibodies through a variety of interactions and in a variety of configurations.
  • a fusion protein of IL-2 and Fc and a fragment of an antibody molecule comprising a heavy chain and a light chain can form an immunoconjugate through dimerization.
  • the immunoconjugate of the present invention has the structure shown in Figure 1A, or the structure shown in Figure 1A in which the IL-2 part and the PD-1 antibody part are interchanged.
  • first and second are used with respect to Fc domains or monomers etc. for ease of distinction when there is more than one module of each type. Use of these terms is not intended to confer a particular order or orientation of the immunoconjugates unless expressly so stated.
  • therapeutic agent encompasses any substance effective in the prevention or treatment of tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (such as immunosuppressants).
  • an “effective amount” refers to such an amount or dose of the antibody or fragment or composition or combination of the present invention, which produces the desired effect in a patient in need of treatment or prevention after being administered to the patient in single or multiple doses.
  • An “effective amount” may encompass a “therapeutically effective amount” or a “prophylactically effective amount”.
  • a “therapeutically effective amount” refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired therapeutic result.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody fragment or composition or combination are outweighed by the therapeutically beneficial effects.
  • a “therapeutically effective amount” preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 40%, even more preferably at least about 50%, 55%, 60%, 65%, 70%, 75%, relative to an untreated subject. %, 80%, 85%, 90% or even 100%.
  • a “prophylactically effective amount” refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired prophylactic result. Typically, a prophylactically effective amount will be less than a therapeutically effective amount because the prophylactic dose is administered in the subject before or at an earlier stage of the disease.
  • host cell refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parental cell, but may contain mutations. Mutant progeny screened or selected for the same function or biological activity in originally transformed cells are included herein.
  • label refers to a compound or composition that is directly or indirectly conjugated or fused to an agent, such as a polynucleotide probe or antibody, and facilitates detection of the agent to which it is conjugated or fused.
  • Labels can themselves be detectable (eg, radioisotopic or fluorescent labels) or, in the case of enzymatic labels, can catalyze the chemical alteration of a detectable substrate compound or composition.
  • the term is intended to encompass both direct labeling of a probe or antibody by conjugating (ie, physically linking) a detectable substance to the probe or antibody as well as indirect labeling of a probe or antibody by reacting with another reagent that is directly labeled.
  • “Individual” or “subject” includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., , mice and rats). In some embodiments, the individual or subject is a human.
  • anti-tumor effect refers to a biological effect that can be exhibited by various means, including but not limited to, for example, reduction in tumor volume, reduction in tumor cell number, reduction in tumor cell proliferation, or reduction in tumor cell survival. In some embodiments, the anti-tumor effect also involves an anti-tumor effect without reducing the body weight of the subject.
  • tumor and cancer are used interchangeably herein to encompass both solid tumors and hematological tumors.
  • cancer refers to or describes a physiological disorder in mammals that is typically characterized by unregulated cell growth.
  • cancers suitable for treatment by the antibodies of the invention include solid tumors or hematological tumors, etc., including cancers. metastatic form.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues.
  • cancer cancer, “cancerous” and “tumor” are not mutually exclusive when referred to herein.
  • pharmaceutical excipient refers to a diluent, adjuvant (such as Freund's adjuvant (complete and incomplete)), excipient, carrier or stabilizer, etc., which are administered together with the active substance.
  • adjuvant such as Freund's adjuvant (complete and incomplete)
  • excipient carrier or stabilizer, etc.
  • composition refers to a composition that is present in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain additional substances that are unacceptably toxic to the subject to which the composition is administered. ingredients.
  • non-fixed combination means that the active ingredients (e.g., (i) muteins or fusions of the invention, and (ii) other therapeutic agents) are combined as separate entities simultaneously, without specific time limitation, or in the same or different Time-spaced, sequential administration to a patient, wherein such administration provides prophylactically or therapeutically effective levels of two or more active agents in the patient.
  • fixed combination means that two or more active agents are administered to a patient simultaneously as a single entity.
  • Dosages and/or intervals of two or more active agents are preferably selected such that the combined use of the parts produces a greater effect in treating a disease or condition than can be achieved by any one component alone.
  • the components may each be in the form of separate formulations, which may be the same or different.
  • combination therapy refers to the administration of two or more therapeutic agents or treatment modalities, such as radiation therapy or surgery, to treat a disease described herein.
  • administration includes co-administration of the therapeutic agents in a substantially simultaneous manner, eg, in a single capsule with fixed ratios of the active ingredients.
  • administration includes co-administration for each active ingredient in multiple or in separate containers (eg tablets, capsules, powders and liquids). Powders and/or liquids can be reconstituted or diluted to the desired dosage before administration.
  • administration also includes using each type of therapeutic agent in a sequential manner at about the same time or at different times. In either case, the treatment regimen will provide for the beneficial effect of the drug combination in treating the disorders or conditions described herein.
  • treating means slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.
  • prevention includes the inhibition of the occurrence or development of a disease or disorder or a symptom of a particular disease or disorder.
  • subjects with a family history of cancer are candidates for prophylactic regimens.
  • prevention refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it has been linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors.”
  • Subject/patient/individual sample refers to a collection of cells or fluid obtained from a patient or subject.
  • the source of the tissue or cell sample can be solid tissue like from fresh, frozen and/or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood components; body fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid ), peritoneal fluid (ascites), or interstitial fluid; cells from any time during pregnancy or development of a subject.
  • Tissue samples may contain compounds that are not naturally intermingled with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.
  • the inventors have found through long-term research that the following molecular mutations and transformations or a combination of one or more of the mutations and transformations can be implemented as follows to simultaneously improve the efficacy of IL-2, reduce the toxic and side effects of IL-2, and Achieve good production performance:
  • the IL-2 mutein of the present invention can activate lymphocytes to kill tumors while avoiding the release of a large number of inflammatory factors caused by excessive activation of lymphocytes, and the resulting drug-related toxicity.
  • the weakening mutation can also reduce the IL-2 mutein clearance mediated by the IL-2 receptor by reducing the binding affinity of the IL-2 mutein of the present invention to the IL-2 receptor widely present on lymphocytes, Delay the action time of IL-2 mutein;
  • (iii) Modify the B'C'loop structure of IL-2, for example, replace the IL-15 molecule loop or truncate the IL-2 molecule B'C'loop.
  • the B'C'loop mutation can significantly enhance the stability of the B'C'loop structure in the IL-2 mutein of the present invention, and significantly improve the IL-2 mutein and the IL-2-Fc dimer constructed therefrom Molecular manufacturing properties, such as significantly improved expression and purity;
  • the IL2-Fc series molecules of the present invention weaken the binding affinity with its receptor IL2R ⁇ / ⁇ , and achieve better pharmacokinetic and pharmacodynamic results; on the other hand, Significant improvements have been made in druggability such as protein expression and purity.
  • the present invention provides IL-2 muteins with improved druggability properties and improved IL-2 receptor binding properties.
  • the IL-2-Fc molecule containing the IL-2 mutant protein of the present invention can effectively avoid the excessive release of inflammatory factors caused by strong stimulation of lymphocytes, and has more stable and long-acting pharmacokinetic properties. Therefore, a lower single dose can be used to achieve a sufficiently high human drug exposure and avoid drug-related toxicity caused by excessive Cmax.
  • the long-acting IL-2-Fc molecule of the present invention has weakened lymphocyte immunostimulatory activity relative to natural IL-2, but because of the significant improvement of pharmacokinetic properties, the molecule of the present invention
  • the effective drug concentration in the body lasts longer, can exert a longer period of continuous stimulation of lymphocytes, achieve pharmacodynamic effects comparable to or even better than natural IL-2 molecules, and achieve better anti-tumor effects in animals Drug efficacy and tolerability.
  • mutant IL-2 protein with favorable biological properties of the present invention can also be used to form immunoconjugates with antigen-binding molecules (such as antibodies or fragments thereof) to activate and expand T cells or NK cells while enhancing Immunological or immunotherapeutic effects of antigen binding molecules.
  • antigen-binding molecules such as antibodies or fragments thereof
  • the IL-2 muteins of the invention have improved druggability properties, for example, when expressed in mammalian cells such as HEK293 or CHO cells, especially as Fc fusion proteins, have a selected from One or more properties: (i) superior expression of wild-type IL-2 protein; and (ii) ease of purification to higher protein purity.
  • the IL-2 muteins of the invention exhibit increased expression levels compared to wild-type IL-2.
  • the increased expression occurs in a mammalian cell expression system.
  • Expression levels can be determined by any suitable method that allows quantitative or semi-quantitative analysis of the amount of recombinant IL-2 protein in cell culture supernatants, preferably after one-step affinity chromatography purification.
  • the amount of recombinant IL-2 protein in a sample can be assessed by Western blot or ELISA.
  • the expression level of the IL-2 mutein of the present invention in mammalian cells is increased by at least 1.1 times, or at least 1.5 times, or at least 2 times, 3 times or 4 times compared with wild-type IL-2. times, or at least 5, 6, 7, 8 or 9 times, or even 10 times more, such as about 10, 15, 20, 25, 30 and 35 times.
  • the IL-2 mutein-Fc fusions of the invention exhibit greater high purity.
  • protein purity is tested by SEC-HPLC techniques.
  • the purity of the IL-2 mutein-Fc fusion of the present invention can reach 70%, or 80%, or more than 90%, preferably 92% , 93%, 94%, 95%, 98% or more than 99%.
  • an IL-2-Fc dimer protein of the invention relative to the corresponding IL-2 Fc formed from wild-type IL-2 protein, as shown by determining the purity of the purified protein after protein A affinity chromatography. 2-Fc dimer protein, showing higher purity.
  • protein purity is tested by SEC-HPLC techniques.
  • the purity of the IL-2-Fc dimer protein of the present invention can reach 70%, or 80%, or more than 90%, preferably 92% , 93%, 94%, 95%, 98% or more than 99%.
  • an IL-2 mutein of the invention has a weakened IL-2 ⁇ binding affinity relative to the corresponding protein before introducing said mutation.
  • IL-2 muteins of the invention have reduced binding affinity for IL-2R ⁇ and/or IL-2R ⁇ receptors relative to prior to attenuation by introduction of IL-2R ⁇ binding interface mutations.
  • the binding affinity of the IL-2 mutein of the present invention to the IL-2R ⁇ receptor is reduced, for example, by 1-20 times or more, compared to before attenuation, including, in some embodiments, removing the binding affinity for the IL-2R ⁇ receptor. Binding of the IL-2R ⁇ receptor.
  • the binding affinity of the IL-2 mutein of the present invention to the IL-2R ⁇ receptor is reduced, for example by 1-100 times or more, compared to before attenuation.
  • the IL-2 muteins of the invention do not bind to the IL-2R ⁇ receptor, but are still capable of binding to the IL-2R ⁇ receptor, preferably wherein said binding to the IL-2R ⁇ receptor is comparable to Compared with before weakening, it can be reduced by 1-100 times, such as about 20-80 times, for example.
  • the IL-2 mutein of the present invention for example, the IL-2 mutein of the present invention fused with an Fc fragment or its dimer molecule, and the receptor IL-2R ⁇ or IL-2R ⁇ receptor can be determined by SPR affinity assay technology. The equilibrium dissociation constant (K D ) was used to determine binding affinity.
  • the IL-2 mutein of the present invention has weakened IL-2 activity relative to the corresponding protein before introducing the mutation, for example, selected from at least one of the following Items for IL-2 activity:
  • T cells e.g. CD4+ and CD8+ T cells, e.g. CD4+/CD8+CD25- T cells, CD4+CD25+ T cells
  • T cells e.g. CD4+ and CD8+ T cells, e.g. CD4+/CD8+CD25- T cells, CD4+CD25+ T cells
  • the IL-2 muteins of the invention result in reduced IL-2 mediated activation and/or proliferation of lymphocytes (eg T cells and/or NK cells) relative to before attenuation.
  • the lymphocytes are CD4+ and CD8+ T cells, such as CD25 ⁇ T cells.
  • IL-2 muteins activate CD4+ and CD8 by detecting activation of STAT5 phosphorylation signaling by IL-2 muteins in lymphocytes, such as T cells or NK cells, in a STAT5 phosphorylation assay. + T cell capacity.
  • STAT5 phosphorylation in cells can be analyzed by flow cytometry to determine the half-maximal effective concentration (EC50).
  • EC50 half-maximal effective concentration
  • the IL-2 mutant molecule of the present invention has "weakened" T cell activation activity.
  • the T cell activation activity of the IL-2 mutant molecule of the present invention can be reduced by, for example, more than 5 times, such as more than 10 times, or more than 50 times, or more than 100 times, or even more than 1000 times.
  • the T cell activation activity of the IL-2 mutant molecules of the present invention can be reduced by 10-50 times, or 50-100 times, or 100-1000 times, or higher.
  • the IL-2 muteins of the invention have reduced IL-2 clearance mediated by the cell surface IL-2 receptor and exhibit increased half-life in vivo relative to wild-type IL-2.
  • the IL-2 muteins of the invention have reduced in vivo toxicity mediated by IL-2 and its receptor relative to wild-type IL-2.
  • IL-2 proteins initiate signaling and function by interacting with IL-2 receptors.
  • Wild-type IL-2 shows different affinities for different IL-2 receptors.
  • IL-2 ⁇ and ⁇ receptors with lower affinity to wild-type IL-2 are expressed on resting effector cells, including CD8 + T cells and NK cells.
  • IL-2R ⁇ which has a high affinity for wild-type IL-2, is expressed on regulatory T cell (Treg) cells and activated effector cells. Due to the high affinity, wild-type IL-2 will preferentially bind to IL-2R ⁇ on the cell surface, then recruit IL-2R ⁇ , release downstream p-STAT5 signal through IL-2R ⁇ , and stimulate Treg cells and activated effector cells.
  • altering the affinity of IL-2 for the IL-2R ⁇ receptor will alter the bias of IL-2 to preferentially activate CD25 + cells and alter the immune downregulation of Treg cells mediated by IL-2.
  • the IL-2 muteins of the invention have maintained or altered IL-2R ⁇ receptor binding ability relative to wild-type IL-2.
  • the IL-2 muteins of the invention retain binding to the IL-2R ⁇ receptor relative to wild-type IL-2.
  • the expression “maintains binding to IL-2R ⁇ receptor” means that IL-2 mutein has comparable binding activity to IL-2R ⁇ receptor as compared to wild-type IL-2 protein.
  • “comparable binding activity” means that when the same measurement method is used to determine, the ratio between the binding activity values (for example, binding affinity K D ) of the IL-2 mutein and the wild-type IL-2 protein is 1: Between 20 and 20:1, preferably between 1:10 and 10:1.
  • the IL-2 mutein does not have an IL-2R ⁇ binding interface mutation relative to wild-type IL-2.
  • the IL-2 muteins of the invention are attenuated IL-2 muteins that retain binding to the IL-2R ⁇ receptor. In yet other embodiments, the attenuated IL-2 muteins of the invention have no IL-2R ⁇ binding interface mutations relative to wild-type IL-2. Preferably, the attenuated IL-2 mutant molecule has improved Treg selectivity and/or improved NK cell (eg, CD3 ⁇ CD56 + NK cell) selectivity.
  • NK cell eg, CD3 ⁇ CD56 + NK cell
  • IL-2 muteins are identified by detecting the activation of STAT5 phosphorylation signaling by IL-2 muteins in different lymphocytes such as Treg cells, NK cells, CD4+ and CD8+ effector T cells in a STAT5 phosphorylation assay assay. Selectivity of the -2 mutein for lymphocytes. In one embodiment, IL-2 mutein selectivity can be achieved by selectively activating IL-2 in specific lymphocyte(s) without substantially activating other lymphocytes in a STAT5 phosphorylation assay. Mutant protein dose window to reflect.
  • Attenuated IL-2 muteins of the invention may exhibit improved Treg selectivity and/or improved NK cell (CD3 ⁇ CD56 + NK cell) selectivity relative to effector T cells, such as CD25 -/low CD4+ and/or CD8+ effector T lymphocytes.
  • improved selectivity may be reflected in low drug-related toxicity of IL-2 muteins.
  • the IL-2 muteins of the invention relative to wild-type IL-2, introduce mutations in the IL-2R ⁇ binding interface that result in IL-2 muteins with reduced or eliminated IL-2R ⁇ receptors. Body combination.
  • the IL-2 muteins of the invention reduce the bias of IL-2 to preferentially activate CD25 + cells relative to wild-type IL-2. In some other embodiments, the IL-2 mutein of the present invention, compared with wild-type IL-2, reduces IL-2-mediated immune downregulation of Treg cells.
  • the IL-2 muteins of the present invention have immune downregulation effects. In still other embodiments, the IL-2 muteins of the present invention can be used in the treatment of autoimmune diseases.
  • the IL-2 muteins of the invention have improved properties selected from, for example, one or more of the following:
  • IL-2R receptors IL-2R ⁇ , IL-2R ⁇ and/or IL2R ⁇
  • CD25+ cells e.g. CD8+ T cells and Treg cells
  • CD25+ cells e.g. Treg cells
  • binding of an IL-2 mutein of the invention to the IL-2R ⁇ receptor relative to wild-type IL-2 e.g., IL-2 WT set forth in SEQ ID NO: 1 or SEQ ID NO: 3
  • the affinity is reduced by at least 5-fold, at least 10-fold, or at least 25-fold, especially at least 30-fold, 50-fold or more than 100-fold.
  • the muteins of the invention do not bind IL-2 receptor alpha.
  • the equilibrium dissociation constant of the IL-2 mutein of the present invention such as the IL-2 mutein of the present invention fused to an Fc fragment or its dimer molecule, and the receptor IL-2R ⁇ receptor can be determined by SPR affinity assay technology (K D ) to determine the binding affinity.
  • the IL-2 muteins of the invention result in reduced IL-2-mediated activation and/or proliferation of CD25 + cells relative to wild-type IL-2.
  • the CD25 + cells are CD25 + CD8 + T cells.
  • the CD25 + cells are Treg cells.
  • the ability of the IL-2 mutein to activate CD25 + cells is identified by detecting activation of the STAT5 phosphorylation signal by the IL-2 mutein in the CD25+ cells in a STAT5 phosphorylation assay. For example, as described in the Examples of this application, STAT5 phosphorylation in cells can be analyzed by flow cytometry to determine the half-maximal effective concentration (EC50).
  • the IL-2 mutein of the present invention removes or reduces the preference of IL-2 for preferential activation of CD25 + cells relative to wild-type IL-2.
  • the CD25 + cells are CD25 + CD8 + T cells.
  • the CD25 + cells are Treg cells.
  • IL-2 mutein activation is identified by detecting the EC50 value of the IL-2 mutein for activating STAT5 phosphorylation signaling in CD25- cells and in CD25 + cells, respectively, in a STAT5 phosphorylation assay. CD25 - cell capacity.
  • the activation bias of IL-2 muteins on CD25+ cells was determined by calculating the ratio of EC50 values for activating STAT5 phosphorylation signaling on CD25- and CD25+ T cells .
  • the mutant protein is at least 10-fold, preferably at least 100-fold, 150-fold, 200-fold, 300-fold or more reduced in its preference for CD25 + relative to the wild-type protein.
  • the IL-2 muteins of the present invention have the properties of the muteins shown in PCT/CN2021/081840, which is incorporated herein in its entirety.
  • the present invention provides an IL-2 mutein, wherein said mutein, compared with wild-type IL-2 (preferably human IL-2, more preferably IL-2 comprising the sequence of SEQ ID NO: 3), Include the mutation:
  • a shortened B'C' loop region i.e., the sequence connecting amino acid residues aa72 and aa84
  • said shortened loop region has less than 10, 9, 8, 7, 6, or 5 amino acid length, and preferably 7 amino acid length; preferably, said shortened B'C' loop region results in improved protein expression and/or purity
  • amino acid positions are numbered according to SEQ ID NO:3.
  • the mutein comprises mutations (i) and (iii) or comprises mutations (ii) and (iii) or comprises (i), (ii) and (iii).
  • the IL-2R ⁇ binding interface mutations suitable for the muteins of the present invention can be any that can be combined with other mutations of the present invention and result in weakened or reduced IL-2R ⁇ binding affinity and/or weakened lymphocytes (e.g. T cells/NK cells) Activating mutations.
  • Such mutations include, but are not limited to: at least one position selected from positions 88, 127 and 130 at the IL-2 binding interface with IL-2R ⁇ , resulting in a weakened or reduced IL-2R ⁇ receptor combined mutations.
  • the IL-2R ⁇ binding interface mutation comprises one or more or a combination of the following mutations selected from:
  • an IL-2 mutein of the invention comprising an IL-2R ⁇ binding interface mutation of the invention has attenuated or reduced IL-2R ⁇ binding, e.g., attenuated or reduced IL-2R ⁇ binding affinity as determined by SPR affinity .
  • the IL-2 muteins of the invention comprise a mutation of the B'C' loop region relative to wild-type IL-2, preferably said mutation results in a B'C' loop region with increased stability; more preferably , said mutations lead to improved druggability of the IL-2 mutein of the present invention, for example, increased expression and/or purity.
  • the mutation introduced results in the mutein comprising a shortened B'C as compared to wild-type IL-2 (preferably human IL-2, more preferably IL-2 comprising the sequence of SEQ ID NO:3).
  • wild-type IL-2 preferably human IL-2, more preferably IL-2 comprising the sequence of SEQ ID NO:3
  • 'loop region ie, shortened length of linking sequence between amino acid residues aa72 and aa84.
  • said shortened loop region is less than 10, 9, 8, 7, 6 or 5 amino acids in length, and preferably 7 amino acids in length, wherein the amino acid residues are numbered according to SEQ ID NO:3.
  • mutations of the B'C' loop region suitable for use in the present invention include truncation and substitution of the B'C' loop region.
  • the mutation comprises a truncation (eg, 1, 2, 3, or 4 amino acids of the B'C' loop) or substitution of amino acid residues aa73 to aa83 of the B'C' loop, For example truncated to A(Q/G)SKN(F/I)H, preferably AQSKNFH, or replaced by SGDASIH.
  • the mutation comprises a truncation or substitution of amino acid residues aa74 to aa83 of the B'C' loop, for example a truncation to (Q/G)SKN(F/I)H, or a substitution to GDASIH or AGDASIH.
  • the IL-2 muteins of the invention comprise a B'C' loop chimeric mutation.
  • the mutein comprises a total or partial substitution of the sequence connecting aa72 to aa84, for example a short B'C' loop sequence from other four-helix short-chain cytokine family members. It can be identified from other four-helix short-chain cytokine IL family members, such as IL-15, IL-4, IL-21, or from non-human species (such as mice), by superpose of crystal structure.
  • the sequence used for substitution is the B'C' loop sequence from interleukin IL-15, especially human IL-15.
  • the substitution comprises a substitution of amino acid residues aa73 to aa83 of the B'C' loop region.
  • the substitution comprises a substitution of amino acid residues aa74 to aa83 of the B'C' loop region.
  • the IL-2 mutein of the present invention has a B'C' loop sequence (ie, the sequence connecting aa72 to aa84) selected from: SGDASIH or AGDASIH, preferably AGDASIH.
  • the IL-2 muteins of the invention comprise a B'C' loop truncating mutation.
  • the mutein comprises a truncation of the sequence connecting aa72 to aa84 relative to wild-type IL-2.
  • the truncation comprises a truncation of amino acid residues aa73 to aa83 of the B'C' loop region.
  • said truncation comprises a truncation of amino acid residues aa74 to aa83 of the B'C' loop region.
  • 1, 2, 3 or 4 amino acids may be truncated from the C-terminus.
  • the B'C' loop region of the IL-2 mutein of the present invention has the sequence A(Q/G)SKN(F/I)H, preferably AQSKNFH.
  • the IL-2 mutein of the present invention has a B'C' loop sequence (i.e., the sequence connecting aa72 to aa84) selected from the following:
  • the IL-2 mutein of the invention comprises a B'C' loop region sequence (ie, the sequence connecting aa72 to aa84) selected from: AQSKNFH or AGDASIH.
  • the IL-2 muteins of the invention comprise one or more mutations at the IL-2R ⁇ binding interface, preferably at positions 35 and/or 42, relative to wild-type IL-2.
  • the mutation abolishes or reduces binding affinity for the IL-2R ⁇ receptor.
  • the IL-2R ⁇ binding interface mutations of the invention comprise mutations K35E and/or F42A.
  • IL-2 muteins of the invention comprising IL-2R ⁇ binding interface mutations of the invention have altered IL-2R ⁇ binding, e.g., altered (preferably reduced or eliminated) IL-2R ⁇ binding as determined by SPR affinity. 2R ⁇ binding.
  • IL-2 mutein of the present invention can also have 1 or 2 in other regions or positions multiple mutations, as long as they retain one or more of the aforementioned beneficial properties of the IL-2 muteins of the present invention.
  • IL-2 muteins of the invention may also comprise a substitution at position 125, such as C125S, C125A, C125T, or C125V, to provide additional advantages, such as improved expression or homogeneity or stability (see, e.g., U.S. Patent No. 4,518,584).
  • the IL-2 mutein of the present invention may also contain a substitution at position 3, such as T3A, to remove the O-sugar modification at the N-terminus of IL2.
  • a substitution at position 3 such as T3A
  • the IL-2 muteins of the invention have reduced IL-2R ⁇ binding and have improved properties selected from one or both of the following: (i) reduced (or eliminated) IL-2R ⁇ binding; and (ii) improved expression levels and purity. In some embodiments, the IL-2 mutein retains IL-2R ⁇ receptor binding relative to wild-type IL-2 protein.
  • the invention provides IL-2 muteins, wherein said muteins, relative to wild-type IL-2, comprise:
  • the invention provides IL-2 muteins, wherein said muteins, relative to wild-type IL-2, comprise:
  • the IL-2 muteins of the invention are IL-2 muteins of the invention.
  • (i) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 4 or consists of said amino acid sequence;
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO:4;
  • said mutein comprises a N88D substitution and the B'C' loop sequence AGDASIH, and optionally T3A.
  • said IL-2 muteins of the invention comprises a N88D substitution and the B'C' loop sequence AGDASIH, and optionally T3A.
  • (i) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 23 or consists of said amino acid sequence;
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 23;
  • said mutein comprises a N88R substitution and the B'C' loop sequence AGDASIH, and optionally T3A.
  • said IL-2 muteins of the invention comprises a N88R substitution and the B'C' loop sequence AGDASIH, and optionally T3A.
  • (i) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 25 or consists of said amino acid sequence;
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 25;
  • said mutein comprises N88R+S130R substitution and B'C' loop region sequence AGDASIH, and optionally T3A.
  • the IL-2 muteins of the invention are IL-2 muteins of the invention.
  • (i) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 27 or consists of said amino acid sequence; or
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 27;
  • said mutein comprises a F42A+N88R+S127E substitution and a B'C' loop sequence AQSKNFH, and optionally T3A.
  • the IL-2 muteins of the invention are IL-2 muteins of the invention.
  • (i) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 29 or consists of said amino acid sequence; or
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 29;
  • said mutein comprises a F42A+N88R+S127E substitution and a B'C' loop region sequence AGDASIH, and optionally T3A.
  • the IL-2 muteins of the invention are IL-2 muteins of the invention.
  • (i) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 31 or consists of said amino acid sequence; or
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 31;
  • said mutein comprises a K35E+N88R+S127E substitution and a B'C' loop sequence AQSKNFH, and optionally T3A.
  • the sequence difference between the IL-2 mutein and the wild-type protein can be expressed by sequence identity, and can also be expressed by the number of different amino acids between the two.
  • the IL-2 mutein has at least 85%, 86%, 87%, 88%, 89% identity, preferably more than 90% identity, preferably 95%, but preferably not More than 97%, more preferably no more than 96% identity.
  • there may be no more than 15 mutations between the IL-2 mutein and the wild-type protein such as 1-10, or 1-5 mutations, for example, 0 ,1,2,3,4 mutations.
  • the remaining mutations may be conservative substitutions.
  • the invention also provides a fusion protein comprising an IL-2 mutein of the invention.
  • the IL-mutein of the invention is fused to another polypeptide that can confer improved pharmacokinetic properties, such as albumin, more preferably an antibody Fc fragment.
  • the present invention provides an IL-2 mutein fusion protein comprising an IL-2 mutein of the present invention fused to an Fc fragment of an antibody.
  • Fc fragments used in the present invention may contain mutations that reduce or remove effector function.
  • the Fc fragment has reduced effector functions mediated by the Fc region, eg reduced or eliminated ADCC and/or ADCP and/or CDC effector functions.
  • the Fc fragment used in the present invention has the L234A/L235A mutation or L234A/L235E/G237A that reduces binding to the Fc ⁇ receptor.
  • the Fc fragment may have mutations that result in increased serum half-life, such as mutations that improve binding of the Fc fragment to FcRn.
  • the Fc fragment fused to the IL-2 mutein is human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, or human IgG4 Fc.
  • the Fc fragment comprises or is at least 90% identical to the amino acid sequence SEQ ID NO: 6 or 12 or 42 or 43, such as 95%, 96%, 97%, 99% or more identical or consists of it.
  • the IL-2 mutein is fused to Fc directly or through a linker.
  • the linker can be selected to enhance the activation of CD25 ⁇ T cells by the Fc fusion protein.
  • the linker is (GGGGS) n or GSGS, more preferably (GGGGS) 2 .
  • the invention also provides a dimeric molecule comprising an IL-2 mutein of the invention fused to an Fc fragment.
  • dimer molecules increase the molecular weight to 60-80KDa, which greatly reduces renal clearance, and can further prolong the half-life of IL2-Fc fusion protein through FcRn-mediated recycling in vivo.
  • the present invention provides IL-2-Fc dimer protein, which is a homodimer, wherein the first monomer and the second monomer respectively comprise from N-terminus to C-terminus: i) IL- 2 mutein; ii) linker; and iii) Fc fragment.
  • the invention provides an IL-2-Fc dimer protein that is a heterodimer comprising:
  • the first Fc fragment and the second Fc fragment comprise first and second heterodimerization mutations, respectively, that promote heterodimerization of the first monomer with the second monomer.
  • the first and second heterodimerization mutations comprise a Knob:Hole mutation combination, for example the mutation combination T366W/S354C:Y349C/T366S/L368A/Y407V.
  • the first heterodimer mutation on the first Fc fragment comprises a Knob mutation and the second heterodimer mutation on the second Fc fragment comprises a Hole mutation; alternatively, the first heterodimer mutation on the first Fc fragment comprises a Hole mutation; The heterodimer mutation comprises a Hole mutation and the second heterodimer mutation of the second Fc fragment comprises a Knob mutation.
  • the Fc fragment suitable for use in the fusion proteins and dimeric molecules of the present invention can be any antibody Fc fragment.
  • the Fc fragments of the invention are effector function silenced.
  • the Fc fragment is modified in one or more properties selected from the group consisting of effector function of the Fc region and complement activation function of the Fc region.
  • said effector function or complement activation function has been reduced or eliminated relative to a wild-type Fc region of the same isotype.
  • effector function is reduced or eliminated by a method selected from the group consisting of reducing glycosylation of the Fc region, using an Fc isotype that naturally has reduced or eliminated effector function, and Fc region modification.
  • effector function is reduced or eliminated by reducing glycosylation of the Fc region.
  • the glycosylation of the Fc region is reduced by a method selected from: producing the fusion protein or dimeric molecule of the invention in an environment that does not allow wild-type glycosylation; carbohydrate groups; and modifying the Fc region such that wild-type glycosylation does not occur.
  • the glycosylation of the Fc region is reduced by modifying such that wild-type glycosylation does not occur, such as comprising a mutation at position 297 of the Fc region such that the wild-type asparagine residue at this position is replaced by another An amino acid substitution that interferes with glycosylation at this position, such as the N297A mutation.
  • effector function is reduced or eliminated by at least one Fc region modification.
  • at least one Fc region modification is selected from: Fc region point mutations that impair binding to one or more Fc receptors at positions selected from: 238, 239, 248, 249, 252, 254, 265 , 268, 269, 270, 272, 278, 289, 292, 293, 294, 295, 296, 297, 298, 301, 303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376 , 382, 388, 389, 414, 416, 419, 434, 435, 437, 438, and 439; point mutations in the Fc region that impair C1q binding at positions selected from: 270, 322, 329, and 321; and of the CH1 domain Point mutation at position 132.
  • effector function is reduced or eliminated by point mutations L234A & L235A (ie LALA mutation) in the Fc region.
  • the modification is an Fc region point mutation that impairs CIq binding at a position selected from: 270, 322, 329 and 321.
  • the modification is the elimination of some Fc regions.
  • the Fc fragment of the dimer molecule of the present invention may contain mutations that facilitate dimerization of the first monomer with the second monomer.
  • mutations that facilitate dimerization of the first monomer with the second monomer Preferably, based on the Knob-in-Hole technique, corresponding Knob mutations and Hole mutations are introduced into the first monomer and the second monomer.
  • the dimeric molecule of the invention comprises:
  • a homodimeric Fc-region of human IgG1 subclass optionally with mutations P329G, L234A and L235A or with mutations L234A and L235A, or
  • a homodimeric Fc-region of human IgG4 subclass optionally with mutations P329G, S228P and L235E, or
  • one Fc-region polypeptide comprises mutation T366W and the other Fc-region polypeptide comprises mutations T366S, L368A and Y407V, or
  • one Fc-region polypeptide comprises mutations T366W and Y349C and the other Fc-region polypeptide comprises mutations T366S, L368A, Y407V and S354C, or
  • one Fc-region polypeptide comprises mutations T366W and S354C and the other Fc-region polypeptide comprises mutations T366S, L368A, Y407V and Y349C,
  • Fc-region polypeptides comprise the mutations P329G, L234A and L235A, and
  • one Fc-region polypeptide comprises mutation T366W and the other Fc-region polypeptide comprises mutations T366S, L368A and Y407V, or
  • one Fc-region polypeptide comprises mutations T366W and Y349C and the other Fc-region polypeptide comprises mutations T366S, L368A, Y407V and S354C, or
  • one Fc-region polypeptide comprises mutations T366W and S354C and the other Fc-region polypeptide comprises mutations T366S, L368A, Y407V and Y349C,
  • one Fc-region polypeptide comprises mutation T366W and the other Fc-region polypeptide comprises mutations T366S, L368A and Y407V, or
  • one Fc-region polypeptide comprises mutations T366W and Y349C and the other Fc-region polypeptide comprises mutations T366S, L368A, Y407V and S354C, or
  • Fc-region polypeptide comprises mutations T366W and S354C and the other Fc-region polypeptide comprises mutations T366S, L368A, Y407V and Y349C.
  • the Fc region further comprises other mutations that facilitate heterodimer purification.
  • the H435R mutation Eric J. Smith,, Scientific Reports
  • the H435R mutation can be introduced into one of the Fc regions of the heterodimer (eg, the Fc region with the Hole mutation) to facilitate Protein A was used to purify heterodimers.
  • mutations such as C220S can also be introduced in the hinge region to facilitate the formation of heterodimers.
  • Fc regions suitable for use in fusion proteins of the invention may also be used as Fc portions in immunoconjugates of the invention.
  • the IL-2 mutein fused to the Fc region is fused to the Fc region
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 7, 24, 26, 28, 30 or 32; or
  • said IL-2 mutein comprises the mutations described herein.
  • the Fc-fused IL-2 muteins of the present invention are present invention.
  • (i) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 7 or consists of said amino acid sequence;
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 7;
  • said mutein comprises a N88D substitution and the B'C' loop sequence AGDASIH, and optionally T3A.
  • the Fc-fused IL-2 muteins of the present invention are present invention.
  • (i) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 24 or consists of said amino acid sequence; or
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 24;
  • said mutein comprises a N88R substitution and the B'C' loop sequence AGDASIH, and optionally T3A.
  • the Fc-fused IL-2 muteins of the present invention are present invention.
  • (i) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 26 or consists of said amino acid sequence;
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 26;
  • said mutein comprises N88R+S130R substitution and B'C' loop region sequence AGDASIH, and optionally T3A.
  • the Fc-fused IL-2 muteins of the present invention are present invention.
  • (i) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 28 or consists of said amino acid sequence; or
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 28;
  • said mutein comprises a F42A+N88R+S127E substitution and a B'C' loop sequence AQSKNFH, and optionally T3A.
  • the Fc-fused IL-2 muteins of the present invention are present invention.
  • (i) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 30 or consists of said amino acid sequence;
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 30;
  • said mutein comprises a F42A+N88R+S127E substitution and a B'C' loop region sequence AGDASIH, and optionally T3A.
  • the Fc-fused IL-2 muteins of the present invention are present invention.
  • (i) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 32 or consists of said amino acid sequence; or
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 32;
  • said mutein comprises a K35E+N88R+S127E substitution and a B'C' loop sequence AQSKNFH, and optionally T3A.
  • the linker suitable for linking the IL-2 mutein and the Fc fragment in the fusion protein and dimer molecule of the present invention may be any linker known in the art.
  • the linker may comprise an IgG1 hinge, or may comprise a linker sequence selected from: (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is an integer of at least 1 .
  • the linker comprises (G 4 S) 2 , ie GGGGSGGGGS (SEQ ID NO: 5).
  • the invention also provides an immunoconjugate comprising an IL2 mutein or IL-2 mutein fusion protein (eg, a fusion protein fused to an Fc fragment) of the invention and an antigen-binding molecule.
  • the antigen binding molecules are immunoglobulin molecules, especially IgG molecules, or antibodies or antibody fragments, especially Fab molecules and scFv molecules or half antibodies (comprising a heavy chain and a light chain, or consisting of a heavy chain and a light chain composition).
  • the antigen binding molecule specifically binds to an antigen presented on a tumor cell or in the tumor environment, particularly preferably PD-1, PD-L1 and/or PD-L2.
  • the immunoconjugates of the invention can be targeted to tumor cells or the tumor environment after administration to a subject, thereby providing further therapeutic benefits, such as the feasibility of treatment with lower doses and the resulting Low side effects; enhanced immunotherapy effect or anti-tumor effect, etc.
  • the IL-2 muteins of the invention may be linked directly or via a linker to another molecule or antigen-binding molecule, and in some embodiments, contain a proteolytic cleavage site therebetween .
  • the IL-2 mutein or fusion protein thereof of the present invention can also be linked to another molecule or an antigen-binding molecule through dimerization.
  • the antibody is an antibody against a tumor-associated antigen, such as PD-1, PD-L1, or PD-L2.
  • Antibodies suitable for conjugation to IL-2 muteins can be whole antibodies, or antigen-binding fragments thereof.
  • the antibody of the invention is an antibody in the IgG1 format or an IgG2 format antibody or an IgG3 antibody format or an IgG4 format antibody, preferably, an IgG1 format antibody.
  • antibodies of the invention are monoclonal antibodies.
  • antibodies of the invention are humanized.
  • antibodies of the invention are human antibodies.
  • antibodies of the invention are chimeric antibodies.
  • the antigen-binding fragment of an antibody of the invention is an antibody fragment selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single chain antibody (e.g. scFv), (Fab')2, single domain Antibody such as VHH, dAb (domain antibody) or linear antibody or half antibody.
  • the immunoconjugate of the present invention comprises an IL-2 mutein or a fusion protein thereof and an anti-PD-1 antibody or an antigen-binding fragment thereof.
  • the immunoconjugate of the invention comprises:
  • the second monomer comprises an antibody specifically binding to PD-1 or a fragment thereof, preferably, a fragment comprising one heavy chain and one light chain of the anti-PD-1 antibody.
  • the Fc fragment of the first monomer comprises a Knob mutation and the antibody heavy chain of the second monomer comprises a hole mutation, and vice versa.
  • the Knob is mutated to Knob:S354C&T366W, and/or the Hole is mutated to Y349C&T366S&L368A&Y407V.
  • the IL-2 mutein fusion protein of the present invention has the form shown in Format 1 of Figure 1A.
  • the antibody against PD-1 or its antigen-binding fragment is the anti-PD-1 antibody or its antigen-binding fragment disclosed in WO2017024465A1.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises one or more CDRs (preferably three CDRs, namely HCDR1, HCDR2H and HCDR3; or LCDR1, LCDR2 and LCDR3, more preferably 6 CDRs, namely HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3), or VH and/or comprising an anti-PD-1 antibody or an antigen-binding fragment thereof disclosed in WO2017024465A1 VL, or comprising the heavy and/or light chains of said antibody.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2 and HCDR3. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2 and LCDR3. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises 3 complementarity determining regions (HCDR) from the heavy chain variable region and 3 complementarity determining regions (LCDR) from the light chain variable region .
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH). In some aspects, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a light chain variable region (VH). In some aspects, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementarity determining regions (CDRs), HCDR1, HCDR2, and HCDR3, from the heavy chain variable region. In some embodiments, the light chain variable region comprises three complementarity determining regions (CDRs), LCDR1, LCDR2 and LCDR3, from the light chain variable region.
  • CDRs complementarity determining regions
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises an antibody heavy chain.
  • the anti-PD-1 antibody heavy chain comprises a heavy chain variable region and a heavy chain constant region.
  • an anti-PD-1 antibody or antigen-binding fragment thereof of the invention comprises an antibody light chain.
  • the light chain of an anti-PD-1 antibody of the invention comprises a light chain variable region and a light chain constant region.
  • the anti-PD-1 antibody or antigen-binding fragment thereof of the invention further comprises a heavy chain and a light chain.
  • the heavy chain variable region VH is variable
  • (i) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 8 or consists of said amino acid sequence; or
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 8;
  • amino acid sequence (iii) comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 8 , more preferably amino acid conservative substitutions), the amino acid sequence consists of said amino acid sequence, preferably, said amino acid changes do not occur in the CDR region.
  • the light chain variable region VL is variable
  • (i) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 15 or consists of said amino acid sequence; or
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 15;
  • amino acid sequence (iii) comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 15 , more preferably amino acid conservative substitutions), the amino acid sequence consists of said amino acid sequence, preferably, said amino acid changes do not occur in the CDR region.
  • the three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2 and HCDR3 are selected from
  • the three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2 and LCDR3 are selected from
  • the sequence contains at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three LCDR regions .
  • HCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO:9, or HCDR1 comprises one, two or three changes (preferably Amino acid substitutions, preferably conservative substitutions) amino acid sequence.
  • HCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 10, or HCDR2 comprises one, two or three changes compared to the amino acid sequence of SEQ ID NO: 10 (preferably Amino acid substitutions, preferably conservative substitutions) amino acid sequence.
  • the HCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 11, or the HCDR3 comprises one, two or three changes compared to the amino acid sequence of SEQ ID NO: 11 (preferably Amino acid substitutions, preferably conservative substitutions) amino acid sequence.
  • LCDR1 comprises the amino acid sequence of SEQ ID NO: 16, or consists of said amino acid sequence, or LCDR1 comprises one, two or three changes (preferably Amino acid substitutions, preferably conservative substitutions) amino acid sequence.
  • the LCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 17, or the LCDR2 comprises one, two or three changes compared to the amino acid sequence of SEQ ID NO: 17 (preferably Amino acid substitutions, preferably conservative substitutions) amino acid sequence.
  • the LCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 18, or the LCDR3 comprises one, two or three changes compared to the amino acid sequence of SEQ ID NO: 18 (preferably Amino acid substitutions, preferably conservative substitutions) amino acid sequence.
  • the heavy chain constant region HC of the anti-PD-1 antibody or its antigen-binding fragment is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of IgG1, for example with L234A & L235A (LALA) Heavy chain constant region of IgGl mutated.
  • a knob-ino-hole mutation is introduced into the heavy chain constant region, such as the introduction of S354C and T366W mutations to obtain an antibody heavy chain comprising a knob mutation, and/or the introduction of Y349C&T366S&L368A&Y407V mutations to obtain a hole mutation antibody heavy chains.
  • the light chain constant region of the anti-PD-1 antibody or antigen-binding fragment thereof is a lambda or kappa light chain constant region.
  • the heavy chain constant region of the antibody or antigen-binding fragment thereof is the heavy chain constant region of the antibody or antigen-binding fragment thereof.
  • amino acid sequence comprising one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acids compared with the amino acid sequence selected from SEQ ID NO: 21
  • the amino acid sequence that is altered preferably amino acid substitution, more preferably amino acid conservative substitution
  • the heavy chain constant region of the antibody or antigen-binding fragment thereof comprises a hole mutation
  • amino acid sequence comprising one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acids compared with the amino acid sequence selected from SEQ ID NO: 13
  • the amino acid sequence that is altered preferably amino acid substitution, more preferably amino acid conservative substitution
  • the heavy chain of the antibody or antigen-binding fragment thereof comprising a hole mutation
  • amino acid sequence comprising one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acids compared with the amino acid sequence selected from SEQ ID NO: 14
  • the amino acid sequence that is altered preferably amino acid substitution, more preferably amino acid conservative substitution
  • the heavy chain of the antibody or antigen-binding fragment thereof is the heavy chain of the antibody or antigen-binding fragment thereof.
  • the light chain constant region of the antibody or antigen-binding fragment thereof is the light chain constant region of the antibody or antigen-binding fragment thereof.
  • amino acid sequence comprising one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acids compared with the amino acid sequence selected from SEQ ID NO: 19
  • the amino acid sequence that is altered preferably amino acid substitution, more preferably amino acid conservative substitution
  • the light chain of the antibody or antigen-binding fragment thereof is the light chain of the antibody or antigen-binding fragment thereof.
  • amino acid sequence comprising one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acids compared with the amino acid sequence selected from SEQ ID NO: 20
  • the altered (preferably amino acid substitution, more preferably amino acid conservative substitution) amino acid sequence is or consists of said amino acid sequence.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises:
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises:
  • HCDR1, HCDR2, and HCDR3 as shown in the following amino acid sequences: SEQ ID NO:9, 10, and 11, respectively, and LCDR1, LCDR2, and LCDR3: SEQ ID NO:16, 17, and 18, respectively, as shown in the following amino acid sequences.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises:
  • amino acid sequence set forth in SEQ ID NO: 8 comprising the amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto or VH consisting of said amino acid sequence, and comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence shown in SEQ ID NO: 15 Amino acid sequences with % or 99% identity or VL consisting of said amino acid sequences.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises
  • the anti-PD-1 antibody fragment in the immunoconjugate comprises or consists of one heavy chain and one light chain.
  • the anti-PD-1 antibody fragment comprises
  • the immunoconjugate of the present invention preferably compared to anti-PD-1 antibody, compared to IL-2 mutein or its fusion protein with Fc, and/or compared to known anti-PD-1 antibody and IL- 2
  • the immunoconjugate of the mutein has one or more or all of the following properties:
  • (1) have reduced or eliminated binding affinity to IL-2R ⁇ , particularly compared to wild-type IL-2 or a fusion protein thereof;
  • the activity is lower in T cells that do not express PD-1 (such as CD8+ or CD4+ T cells), and the activity is higher in T cells that express PD-1 (such as CD8+ or CD4+ T cells), Demonstrated high selectivity for PD-1, especially compared to known immunoconjugates comprising anti-PD-1 antibody and IL-2 mutein;
  • the present invention provides nucleic acids encoding any chain or any monomer or domain in any of the above IL-2 muteins or fusion proteins or dimeric molecules or conjugates.
  • the polynucleotide sequence encoding the mutein of the present invention can be generated by de novo solid-phase DNA synthesis or by PCR mutagenesis of an existing sequence encoding wild-type IL-2 using methods well known in the art.
  • the polynucleotides and nucleic acids of the present invention may contain a segment encoding a secretory signal peptide and be operably linked to a segment encoding a mutein of the present invention, thereby directing the secretory expression of the mutein of the present invention.
  • the invention also provides vectors comprising nucleic acids of the invention.
  • the vector is an expression vector, such as a eukaryotic expression vector.
  • Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).
  • YACs yeast artificial chromosomes
  • the expression vector of the present invention is a pYDO_017 expression vector.
  • the invention also provides host cells comprising said nucleic acid or said vector.
  • Host cells suitable for replicating and supporting expression of mutant IL-2 proteins or fusions or dimers or immunoconjugates are well known in the art. Such cells can be transfected or transduced with specific expression vectors and large numbers of vector-containing cells can be grown for seeding large-scale fermenters to obtain sufficient quantities of IL-2 mutants or fusions or dimers or Immunoconjugates are used in clinical applications.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells (eg CHO cells or 293 cells).
  • Examples of useful mammalian host cell lines are the monkey kidney CV1 line (COS-7) transformed with SV40; Rat kidney cells (BHK), mouse sertoli cells (TM4 cells, as described e.g. in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1 ), vero cells ( VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (HepG2), mouse mammary tumor cells (MMT060562 ), TRI cells (as described eg in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC5 cells and FS4 cells.
  • COS-7 monkey kidney CV1 line transformed with SV40
  • Rat kidney cells BHK
  • mouse sertoli cells TM4 cells, as described e.g. in Mather, Biol Reprod 23, 243-251 (1980)
  • monkey kidney cells
  • CHO Chinese hamster ovary
  • dhfr-CHO cells Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)
  • myeloma cell lines such as YO, NSO, P3X63, and Sp2/0.
  • the host cells are eukaryotic cells, preferably mammalian cells such as Chinese Hamster Ovary (CHO) cells, Human Embryonic Kidney (HEK) cells or lymphocytes (eg YO, NSO, Sp20 cells).
  • CHO Chinese Hamster Ovary
  • HEK Human Embryonic Kidney
  • the present invention provides a method for preparing the IL-2 mutein or fusion or dimer or conjugate of the present invention, wherein said method comprises, in a suitable IL-2 mutein or fusion or dimer or Under conditions for conjugate expression, culturing a host cell comprising nucleic acid encoding said protein or fusion or dimer or conjugate, as provided above, and optionally extracting said host cell (or host cell media) to recover the protein or fusion or dimer or conjugate.
  • the vector comprising the nucleic acid encoding the IL-2 mutein is transferred into cells for expression, then the cells (or cell culture supernatant) are collected, the IL-2 mutein is extracted, and purified, to obtain the IL-2 mutein.
  • said purification method is an affinity purification method.
  • said purification method is ion exchange purification.
  • a vector comprising a nucleic acid encoding an Fc-fused IL-2 mutein is transformed into a cell for expression, and then the cells (or cell culture supernatant) are collected to extract the Fc-fused IL-2 mutein, and purified to obtain the Fc-fused IL-2 mutein.
  • said purification method is an affinity purification method.
  • said purification method is ion exchange purification.
  • the vector comprising the nucleic acid encoding the IL-2 mutein fused with Fc, the nucleic acid encoding the heavy chain of the PD-1 antibody, and the nucleic acid encoding the light chain of the PD-1 antibody is transferred into the cell, so that they are respectively Expression and assembly into an immunoconjugate, followed by collection of cells (or cell culture supernatant), extraction of the immunoconjugate, and purification to obtain the immunoconjugate.
  • said purification method is an affinity purification method.
  • said purification method is ion exchange purification.
  • IL-2 muteins provided herein can be identified, screened for, or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art.
  • the IL-2 mutein of the present invention can be tested for its binding activity to IL-2 receptor.
  • binding to human IL-2R ⁇ or ⁇ protein or IL-2R ⁇ or IL-2R ⁇ can be determined by methods known in the art, such as ELISA, Western blot, etc., or exemplary methods disclosed in the Examples herein.
  • assays can be performed using flow cytometry in which cells transfected to express a mutein on the cell surface, such as yeast display cells, are combined with labeled (e.g., biotin-labeled) IL-2R ⁇ or ⁇ proteins or IL-2R ⁇ Or IL-2R ⁇ complex to react.
  • binding of muteins to receptors including binding kinetics (eg, KD values), can be determined in SPR assays using IL-2-Fc fusion or dimeric molecular formats.
  • signaling and/or immune activation effects that occur downstream of receptor binding can be measured.
  • assays for identifying biologically active mutant IL-2 proteins are provided.
  • Biological activity may include, for example, the ability to induce the proliferation of T and/or NK cells and/or Treg cells with IL-2 receptors, the ability to induce T and/or NK cells and/or Treg cells with IL-2 receptors
  • the ability to signal IL-2 the reduced ability to induce apoptosis in T cells, the ability to induce tumor regression and/or improve survival, and reduced in vivo toxic properties, such as reduced vascular permeability.
  • the present invention also provides mutant IL-2 proteins, Fc fusions thereof and dimeric molecules comprising the same having such biological activity in vivo and/or in vitro.
  • a suitable assay for testing the ability of IL-2 muteins of the invention (e.g., in the form of dimeric molecules) to stimulate NK cells to produce IFN- ⁇ may comprise the step of: mixing cultured NK cells with mutants of the invention IL-2 protein was incubated, and then the IFN- ⁇ concentration in the medium was measured by ELISA.
  • IL-2 signaling induces several signaling pathways and involves JAK (Janus kinase) and STAT (signal transducer and activator of transcription) signaling molecules.
  • IL-2 Interaction of IL-2 with receptor ⁇ and ⁇ subunits results in phosphorylation of the receptor as well as JAK1 and JAK3 (which bind to ⁇ and ⁇ subunits, respectively).
  • STAT5 then binds to phosphorylated receptors and phosphorylates itself on very important tyrosine residues. This results in STAT5 dissociation from the receptor, STAT5 dimerization, and translocation of STAT5 dimers to the nucleus where they promote transcription of target genes.
  • STAT5 dissociation from the receptor, STAT5 dimerization, and translocation of STAT5 dimers to the nucleus where they promote transcription of target genes.
  • the ability of a mutant IL-2 polypeptide to induce signaling through the IL-2 receptor can be assessed, eg, by measuring phosphorylation of STAT5. Details of this method have been disclosed in the Examples. For example, PBMCs can be treated with a mutant IL-2 polypeptide or fusion or dimer or immunoconjugate
  • the above-mentioned activity or level of IL-2 can be measured in cells expressing the antigen.
  • mutated IL-2 or a fusion or dimer or immunoconjugate thereof on tumor growth and survival can be assessed in a variety of animal tumor models known in the art.
  • xenografts of cancer cell lines can be implanted into immunodeficient mice and treated with mutant IL-2 polypeptides or fusions or dimers or immunoconjugates of the invention.
  • Mutant IL-2 polypeptides, fusions, dimeric molecules, and immunoconjugates of the invention can be tested for in vivo anti-tumor effects based on tumor inhibition rates (eg, calculated relative to an isotype control antibody).
  • mutant IL-2 polypeptides, fusions, dimeric molecules and immunoconjugates of the invention can be assayed based on the change in body weight of the animal (e.g., relative to pre-dose, absolute change in body weight or percent change in body weight). toxicity in the body. The in vivo toxicity can also be determined based on mortality, lifetime observations (visible symptoms of adverse effects, eg behavior, body weight, body temperature) and clinical and anatomic pathology (eg measurement of blood chemistry and/or histopathological analysis).
  • the pharmacological properties of the mutein of the present invention can be characterized by methods known in the art, for example, the expression level and the purity of the product.
  • the protein content can be determined on the cell culture fluid collected by centrifugation.
  • the assay may be performed after a one-step purification of the collected cell culture fluid, eg, after a one-step affinity chromatography purification.
  • the purity can be determined after performing a one-step affinity chromatography purification on the culture supernatant of the harvested production cells to detect the purification performance of the mutant protein.
  • the mutant protein of the present invention after being purified by this step of affinity chromatography, has a purity significantly better than that of the wild-type protein, indicating that the mutant protein of the present invention has better purification performance.
  • the purity determination method can be any conventional method known in the art, including but not limited to, SEC-HPLC method.
  • mutant IL-2 polypeptides or fusions or dimers or immunoconjugates of the invention can be characterized by methods known in the art.
  • the present invention also includes compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising mutant IL-2 polypeptides or fusions or dimers or immunoconjugates and compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising mutant IL-2 polypeptides or fusions or dimers encoding mutant IL-2 polypeptides or fusions or dimers.
  • compositions of polynucleotides of bodies or immunoconjugates may also optionally contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.
  • the pharmaceutical composition comprising the mutant IL-2 polypeptide or fusion or dimer or immunoconjugate of the present invention can be prepared by conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical compositions can be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the protein into preparations which can be used pharmaceutically. Suitable formulations are dependent on the chosen route of administration.
  • Immunoconjugates can be formulated into compositions in free acid or base, neutral or salt form.
  • a pharmaceutically acceptable salt is one that substantially retains the biological activity of the free acid or base.
  • These include acid addition salts, such as those formed with free amino groups of proteinaceous compositions, or with inorganic acids (such as for example hydrochloric acid or phosphoric acid) or with organic acids such as acetic acid, oxalic acid, tartaric acid or mandelic acid formation.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or iron; or organic bases such as isopropylamine, trimethylamine, histidine or procaine.
  • Pharmaceutically acceptable salts tend to be more soluble in aqueous and other protic solvents than the corresponding free base forms.
  • the invention also provides a combination product comprising a mutant IL-2 polypeptide or fusion or dimer or immunoconjugate of the invention, and one or more other therapeutic agents (e.g., chemotherapeutics, other antibodies, cytotoxic agents, vaccines, anti-infective agents, etc.). Combinations of the invention may be used in the methods of treatment of the invention.
  • therapeutic agents e.g., chemotherapeutics, other antibodies, cytotoxic agents, vaccines, anti-infective agents, etc.
  • the combination product is used to prevent or treat cancer.
  • the invention relates to a method of preventing or treating a disease in a subject, such as cancer, said method comprising administering to said subject an effective amount of any mutant IL-2 polypeptide or fusion described herein or Dimers or immunoconjugates.
  • Cancer can be early, middle or advanced or metastatic.
  • the cancer can be a solid tumor or a hematological tumor.
  • the cancer is a gastrointestinal tumor or melanoma, such as colon or colorectal cancer.
  • the tumor is a tumor or cancer that is resistant to a known drug, such as a known anti-PD-1 antibody, such as a refractory tumor or cancer.
  • the cancer is a cancer characterized by elevated protein levels and/or nucleic acid levels (eg, increased expression) of PD-1, PD-L1, and/or PD-L2.
  • mutant IL-2 polypeptides or fusions or dimers or immunoconjugates of the invention can be used to stimulate the immune system of a host, eg, enhance a cellular immune response.
  • "Stimulating the immune system" may include an overall increase in immune function, an increase in T cell function, an increase in B cell function, a recovery in lymphocyte function, an increase in IL-2 receptor expression, an increase in T cell responsiveness. High, elevated natural killer cell activity or lymphokine-activated killer (LAK) cell activity, any one or more of these.
  • LAK lymphokine-activated killer
  • the mutant IL-2 polypeptides or fusions or dimers or immunoconjugates of the invention can be administered by any suitable method, including parenteral Topically, intrapulmonarily and intranasally, and, if required for local treatment, intralesionally.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example by injection, eg intravenous or subcutaneous injection, depending in part on whether the administration is short-term or chronic.
  • Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.
  • the appropriate dosage of the mutant IL-2 polypeptide or fusion or dimer or immunoconjugate of the invention (when used alone or in combination with one or more other therapeutic agents) will depend on Depending on the type of disease being treated, the type of antibody, the severity and course of the disease, prophylactic or therapeutic administration, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician .
  • the antibody is suitably administered to the patient in one treatment or over a series of treatments.
  • mutant IL-2 polypeptides or fusion proteins or dimers or immunoconjugates of the invention can be administered to patients at higher doses without toxicity.
  • the present invention also provides the use of the mutant IL-2 polypeptide or fusion or dimer or immunoconjugate of the present invention in the preparation of a medicament for the aforementioned method (eg, for treatment).
  • the present invention designs a mechanism that can specifically bind to human PD-1 and block the combination of PD-1 and PD-L1 to release the immune brake mechanism. At the same time, it can also specifically bind to the IL-2 receptor on T cells or NK cells to play a role.
  • An immunoconjugate molecule ( ⁇ PD-1/IL-2m immunoconjugate) that activates and expands T or NK cells, said immunoconjugate molecule comprising an anti-PD-1 antibody and an IL-2 mutant capable of Enhance the effect of PD-1 antibody immunotherapy.
  • the molecular form of the immunoconjugate of the present invention is shown in Figure 1A, including two parts: 1) the second monomer: it is derived from an antibody that binds to PD-1, and the sequence of the antibody that binds to PD-1 is derived from WO2017024465A1; and 2 ) first monomer: IL-2 mutant (IL-2 mutein or IL-2m), which is modified as follows: According to the crystal structure 2ERJ of IL-2 and receptor complex (Fig.
  • the receptor binding interface selects mutation sites to reduce the binding of IL-2 to the receptor; optimize the B'C' loop region sequence of IL-2 (A73-R83, wild type is SEQ ID NO:40) to improve IL-2
  • the druggability of the loop region is optimized by using the human IL-15 B'C' loop region (AGDASIH, SEQ ID NO: 39) to replace the B'C' loop region of IL-2, or for IL-2 B'C
  • the last 4 amino acids of the 'loop region were deleted, resulting in a truncated IL-2B 'C' loop region (AQSKNFH, SEQ ID NO:41).
  • IL-2-Fc fusion protein and IL-2 mutant-anti-PD-1 antibody immunoconjugate used in the examples, see the sequence listing, and the IL-2 mutation information is shown in the table below.
  • the IL-2 receptor IL-2R ⁇ (Uiprot: P01589, aa22-217) was attached to the C-terminus of the sequence with avi tag (GLNDIFEAQKIEWHE, the tag peptide can be biotinylated by BirA enzyme) and 6 histidine tags (HHHHHH), constructed on the pTT5 vector (Addgene), then transfected into HEK293 cells for expression, and obtained IL-2R ⁇ by affinity purification through a nickel column (Histrap excel, GE, 17-3712-06).
  • avi tag avi tag
  • HHHHHHHH 6 histidine tags
  • the IL-2R ⁇ complex is based on the Fc heterodimerization of Knobs in holes, the sequence of IL-2R ⁇ is constructed to the N-terminal of Fc-Knob (SEQ ID NO:37), and the sequence of IL-2R ⁇ is constructed to Fc-Hole The N-terminus (SEQ ID NO:38) of , respectively, was constructed on the pcDNA3.1 vector, and then co-transformed and expressed in cells.
  • the vectors containing IL-2R ⁇ and IL-2R ⁇ were co-transfected into HEK293 cells for expression by transient transfection.
  • plasmid DNA and transfection reagent PEI (Polysciences, 23966) in a clean bench, take 3 mL of Opti-MEM medium (Gibco product number: 31985-070) and add it to a 50 ml centrifuge tube, add 30 ⁇ g of the corresponding plasmid DNA, and use 0.22 Filter the Opti-MEM medium containing the plasmid with a filter head of ⁇ m, then add 90 ⁇ g PEI (1 g/L) and let stand for 20 minutes. Gently pour the DNA/PEI mixture into 27 mL of HEK293 cells and mix well, and continue to culture for 6 days at 37°C and 8% CO 2 .
  • Opti-MEM medium Gibco product number: 31985-070
  • Nickel column affinity purification The nickel column (5ml Histrap excel, GE, 17-3712-06) used for purification was soaked in 0.1M NaOH for 2h, and then washed with 5-10 times column volume of ultrapure water to remove the lye.
  • the collected protein was concentrated by ultrafiltration and exchanged into PBS (Gibco, 70011-044), and then further separated and purified with superdex200increase (GE, 10/300GL, 10245605) to collect the elution peak of the monomer, the balance of the column and the elution buffer
  • the liquid is PBS.
  • Mabselect affinity purification The cells were centrifuged at 13000rpm for 20min, the supernatant was collected, and the supernatant was purified with a prepacked column Hitrap Mabselect Sure. The operation is as follows: equilibrate the packing column with 5 times column volume of equilibrium solution (0.2M Tris, 0.15M NaCl, pH7.2) before purification; pass the collected supernatant through the column, and then wash the packing column with 10 times column volume of equilibrium solution , to remove non-specific binding proteins; wash the filler with 5 times the column volume of elution buffer (0.1M sodium citrate, pH 3.5), and collect the eluate; add 80 ⁇ L Tris (2M Tris) to every 1ml of eluate, The concentrated tube was exchanged into PBS buffer, and the concentration and purity were determined.
  • equilibrium solution 0.2M Tris, 0.15M NaCl, pH7.2
  • Ion exchange purification The heterodimer molecules in the bispecific molecules are separated by ion exchange chromatography, and the homodimer impurities are removed.
  • the heavy chain Hole and light chain of the anti-PD-1 antibody were constructed on the pcDNA3.1 vector, and the IL-2 protein was connected to the N-terminal of the IgG1Fc Knob through a linker and constructed on the pcDNA3.1 vector.
  • the above three plasmids were combined with 3 times High-quality PEI (for example, transfection of 3ml HEK293, requires 1ug heavy chain Hole + 1ug light chain + 1ug IL-2m-linker-Fc-Knob + 9ug PEI) to co-transfect HEK293 cells, express and prepare each immune protein used in this example Conjugate molecules.
  • the preparation of cells and the collection and purification of samples are the same as the preparation methods of the above receptors.
  • the equilibrium dissociation constant (K D ) of the immunoconjugate of the present invention binding to human IL-2 receptor (IL2R ⁇ or IL-2R ⁇ ) was measured by surface plasmon resonance (SPR). Based on the SPR principle, when a beam of polarized light is incident on the end face of the prism at a certain angle, surface plasmon waves will be generated at the interface between the prism and the gold film, causing free electrons in the metal film to resonate, that is, surface plasmon resonance. During analysis, a layer of protein is immobilized on the surface of the sensor chip first, and then the sample to be tested flows over the surface of the chip.
  • the refractive index of the gold film surface will change, eventually leading to The change of SPR angle, by detecting the change of SPR angle, information such as affinity and kinetic constant of the analyte can be obtained.
  • Biacore T200 (Cytiva) was used to measure the K D of the immunoconjugate and the IL-2 receptor. After the chip surface, the affinity and kinetic constants were obtained by detecting the association and dissociation between the chip surface protein and the studied immunoconjugate and the control molecule in the mobile phase.
  • the method includes chip preparation and affinity detection.
  • the assay process used 10 ⁇ HBS-EP+(BR-1006-69, Cytiva) diluted 10 times as the experimental buffer.
  • the amino coupling kit (BR-1006-33, Cytiva) was used to couple SA to the surface of the CM5 chip (29-1496-03, Cytiva).
  • 1M ethanolamine was injected to activate the remaining active sites to close.
  • Each cycle of affinity detection involves capture of the receptor, binding of a concentration of the molecule of interest, and regeneration of the chip.
  • the molecules after gradient dilution (molecular concentration gradient is 0-400nM) flowed over the surface of the chip from low concentration to high concentration at a flow rate of 30 ⁇ l/min, the binding time was 180s, and the dissociation time was 300s. Finally, the chip was regenerated using 5mM NaOH (BR-1003-58, Cytiva). Data results were analyzed using Biacore T200 analysis software (version 3.1), using analysis 1:1 binding or steady-state analysis models.
  • Biacore T200 (Cytiva, T200) was used to measure the affinity of the immunoconjugate to be studied or the control molecule to human PD1 (Cat. No.: PD1-H5221, ACRO Biosystem).
  • the specific method was as follows: the molecule to be studied was captured on a chip coupled with Protein A (29127555, Cytiva) surface, the affinity and kinetic constants were obtained by detecting the binding and dissociation between the chip surface molecules and the antigen in the mobile phase.
  • the assay process used 10 ⁇ HBS-EP+(BR-1006-69, Cytiva) diluted 10 times as the experimental buffer. Each cycle of affinity detection includes capture of the molecule to be studied, binding of a concentration of antigen, and regeneration of the chip.
  • the antigen after gradient dilution (when combined with the molecule to be studied, the antigen concentration gradient is 0-40nM) flows over the surface of the chip from low concentration to high concentration at a flow rate of 30 ⁇ l/min, the binding time is 180s, and the dissociation time is 180s. 600s. Finally, the chip was regenerated using 10mM Glycine-HCl, pH 1.5 (BR-1003-54, Cytiva). The data results were analyzed using Biacore T200 analysis software (version 3.1) using a 1:1 binding model.
  • Table 2 and Figure 3 are the binding constants and binding curves of the immunoconjugate or the control molecule and IL-2R ⁇ respectively
  • 3010 is the wild-type IL-2 sequence fused with Fc, see the sequence listing for the sequence, and its affinity is 1.09nM
  • 2061 (derived from US20180326010A1) is a control molecule.
  • the affinity between 2061 and IL-2R ⁇ is 1.48nM.
  • the affinity of IL-2 immunoconjugate in this study is weaker than that of 3010 and 2061.
  • Table 3 and Figure 4 are the affinity and binding curves of immunoconjugates or control molecules to IL-2R ⁇ , respectively.
  • the affinity of 3010 to IL-2R ⁇ is 4.38E-08M, and 2061 is no binding.
  • the bispecific molecule in this study has an affinity with IL-2R ⁇
  • the binding of -2R ⁇ was weaker than that of wild-type IL-2, but stronger than that of the control molecule 2061.
  • Table 4 and Figure 5 are the affinity and binding curves of immunoconjugates or control molecules to human PD1, respectively. Both the control molecules and the molecules in this study have strong affinity to human PD1.
  • IL-2 The binding of IL-2 to the IL-2 receptor on the surface of CTLL2 cells will activate the CTLL2JAK-STAT signaling pathway and trigger the reporter gene signal.
  • Overexpression of human PD-1 (hPD-1, uniprot:Q15116) on the surface of CTLL2 cell lines can further enhance the CTLL2JAK-STAT signaling pathway under the enrichment of hPD-1.
  • CTLL2-hPD-1 cell line construction
  • step ⁇ 2> Add the medium packaging system prepared in step ⁇ 2> to the replaced medium in step ⁇ 3>, and let stand in a 5% CO 2 incubator at 37°C for 4-6 hours.
  • CTS medium Gibco, A3021002
  • CTLL2 (Promega, CS2028B04) was infected with Lentvirus+hPD-1, and a stable CTLL2-hPD-1 cell line was obtained by pressure selection and sorting.
  • edge wells were plated with an equal volume of Assay Medium, and treated with starvation at 37°C with 5% CO 2 for 18-20 hours.
  • the results are shown in Figure 6.
  • the results in Figure 6 show that the activity of the ⁇ PD-1/IL-2m immunoconjugate in this study on PD-1-positive CTLL2 cells is stronger than that on PD-1-negative CTLL2 cells.
  • the activity (EC50) is 24-fold selectivity
  • 2063 is 42-fold selectivity
  • 2149 is 115-fold selectivity
  • 2219 is 500-fold selectivity
  • 2213 and 2214 are more than >10000-fold selectivity.
  • the results show that the immunoconjugate molecules of the present invention can selectively activate PD-1 positive CTLL2 cells.
  • the binding of IL-2 to the IL-2 receptor on the surface of T cells will activate the JAK-STAT signaling pathway of T lymphocytes, and the phosphorylation level of STAT5 is an important indicator for judging the activation level of this signaling pathway.
  • AlexaFluor TM 488 Antibody Labeling Kit (Thermo Fisher, A20181) was used to label PD-1 mAb (Innovent, ADI-11416) to prepare AF488-anti human PD-1 fluorescent antibody, which was used to label PBMC suspension cells cultured overnight.
  • the labeled PBMC suspension cells are plated in a 96-well U-shaped plate, and the number of cells is 5x10 5 cells/well.
  • tissue cell fixative (Solarbio, P1110) at 200ul/well, and centrifuge at room temperature for 400g/30min.
  • T lymphocytes After T lymphocytes are activated, under the action of PD-1, explore and verify the effect of immunoconjugates on the pSTAT5 signal of activated T lymphocytes.
  • AlexaFluor TM 488 Antibody Labeling Kit (Thermo Fisher, A20181) was used to label PD-1 mAb (Innovent, ADI-11416) to prepare AF488-anti human PD-1 fluorescent antibody, which labeled activated and resting T cells.
  • HEK-Blue TM IL-2cell reporter assay detects the activity of the immunoconjugate of the present invention
  • HEK293 cells In HEK293 cells, overexpressed IL2R (CD25, CD122, CD132), JAK3 and STAT5 genes were introduced to construct HEK293+hIL2R/SEAP cell line with IL2 signaling pathway (huPD-1- cells, HEK-Blue TM IL-2 Cells , Invivogen, hkb-il2), under the action of IL2, activate HEK293+hIL2R/SEAP cell reporter gene.
  • IL2R CD25, CD122, CD132
  • JAK3 and STAT5 genes were introduced to construct HEK293+hIL2R/SEAP cell line with IL2 signaling pathway (huPD-1- cells, HEK-Blue TM IL-2 Cells , Invivogen, hkb-il2), under the action of IL2, activate HEK293+hIL2R/SEAP cell reporter gene.
  • HEK293+hIL2R+hPD-1/SEAP cell line construct and package Lentvirus+hPD-1 lentivirus as above, infect HEK293+hIL2R/SEAP (Invivogen, hkb-il2) with Lentvirus+PD-1, pressurize screening and The HEK293+hIL2R+hPD-1/SEAP stable cell line (huPD-1+ cells) was sorted for the following experiments.
  • HEK-Blue TM IL-2 Cells (huPD-1-cells) is a HEK293 cell overexpressing the IL-2 receptor. It can be seen from Figure 8 and Table 4 that the activity of the immunoconjugate obtained in this study was 3.14 times weaker than that of 2061 in IL-2, and the maximum weakened to 2.21E+08 times.
  • the immunoconjugate can achieve strong IL-2 activity, in two Cells maintain high selectivity, for example, the selectivity of 2063 can reach 3.52 times, the selectivity of 2132 can reach 53.45 times, the selectivity of 2149 can reach 96.04 times, 2219 is 606.11 times, 2214 is 5119.78 times, 2213 is 1.57E +07 times.
  • hPD-1 knock-in mice were inoculated with MC38 cells (mouse colon cancer cell line, Shanghai Heyuan Biotechnology) to determine the bifunctional PD-1 of the present invention.
  • the experiment uses SPF grade female hPD-1 knock-in mice (purchased from Shanghai Nguiding Model Organisms), the certificate number is NO.20170010005748.
  • MC38 cells were routinely subcultured for subsequent in vivo experiments. The cells were collected by centrifugation, and the MC38 cells were resuspended in PBS (1 ⁇ ) to prepare a cell suspension with a cell concentration of 5 ⁇ 10 6 cells/ml. On day 0, 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal region of hPD-1 knock-in mice to establish the MC38 tumor-bearing mouse model.
  • mice Six days after tumor cell inoculation, the tumor volume of each mouse was detected, and the mice were divided into groups (8 mice in each group). The dosage and method of administration are shown in Table 5.
  • h-IgG is an isotype control antibody purchased from Equitech-Bio, lot number 161206-0656.
  • the concentrations of h-IgG, 2063 and 2132 were 2mg/ml, 1mg/ml and 1mg/ml respectively, administered once a week, 3 times in total (QWx3). Administration was performed on the 6th, 13th, and 20th day after MC38 cell inoculation, and the tumor volume and body weight of the mice were monitored twice a week, as shown in Figure 9A, and the monitoring ended after 24 days.
  • Tumor volume measurement: the maximum long axis (L) and maximum width axis (W) of the tumor were measured with a vernier caliper, and the tumor volume was calculated according to the following formula: V L*W 2 /2. Body weight was measured using an electronic balance.
  • the results of tumor inhibition rate are shown in Table 6: On the 24th day after inoculation, compared with h-IgG, 20mg/kg group, the tumor inhibition rates of 2063 and 2132 were 98% and 96%, respectively.
  • the results of detecting the body weight of the mice at the same time showed that there was no significant difference in the body weight of the mice on the 24th day after inoculation.
  • MC38 cells were routinely subcultured for subsequent in vivo experiments. The cells were collected by centrifugation, and the MC38 cells were resuspended in PBS (1 ⁇ ) to prepare a cell suspension with a cell concentration of 5 ⁇ 10 6 cells/ml. On day 0, 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal region of hPD-1 knock-in mice to establish the MC38 tumor-bearing mouse model.
  • h-IgG is an isotype control antibody purchased from Equitech-Bio, lot number 161206-0656.
  • the concentration of h-IgG, 2063 and IBI308 was 1mg/ml, administered once a week, a total of 3 times (QWx3). Administration was performed on the 12th, 19th, and 26th day after MC38 cell inoculation, and the tumor volume and body weight of the mice were monitored twice a week, as shown in Figure 10A, and the monitoring ended after 29 days.
  • the results of tumor inhibition rate are shown in Table 8: on the 29th day after inoculation, compared with h-IgG, 20mg/kg group, the tumor inhibition rates of 2063 and IBI308 were 112% and 56%, respectively.
  • the results of detecting the body weight of the mice FIG. 10B ) showed that there was no significant difference in the body weight of the mice on the 29th day after inoculation.
  • PD-1 antibody-resistant B16F10 cells (mouse melanoma cell line, ATCC CRL-6475) were used to inoculate hPD-1 knock-in mice to assay this assay.
  • B16F10 cells were routinely subcultured for subsequent in vivo experiments. The cells were collected by centrifugation, and the B16F10 cells were resuspended in PBS (1 ⁇ ) to prepare a cell suspension with a cell concentration of 2.5 ⁇ 10 6 cells/ml. On day 0, 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal region of hPD-1 knock-in mice to establish a B16F10 tumor-bearing mouse model.
  • mice in each group The tumor volume of each mouse was detected 7 days after tumor cell inoculation, and grouped (6 mice in each group), the dosage and method of administration are shown in Table 9.
  • h-IgG is an isotype control antibody purchased from Equitech-Bio, lot number 161206-0656.
  • the concentrations of h-IgG, IBI308, 2124 and 2063 were 1mg/ml, 1mg/ml, 0.6mg/ml and 1mg/ml respectively, administered once a week, 3 times in total (QWx3).
  • the drugs were administered on the 8th, 15th and 22nd day after B16F10 cell inoculation, and the tumor volume and body weight of the mice were monitored twice a week, as shown in Figure 11A-C, and the monitoring ended after 28 days.
  • TGI% tumor inhibition rate
  • Body weight was measured using an electronic balance. Mice were euthanized for tumors larger than 2000 mm. If more than half of the group died, the tumor growth curve for the entire group was not displayed at that time point.
  • the tumor inhibition rate results are shown in Table 10: On the 22nd day after inoculation, compared with the h-IgG group, the tumor inhibition rates of IBI308, IBI308+2214, and 2063 were 2%, 84%, and 99%, respectively, and 2063 was in CR The rate was significantly better than IBI308 and IBI308 combined with non-targeted IL2m-Fc molecules (2124). The results of detecting the body weight of the mice at the same time ( FIG. 11C ) showed that there was no significant difference in the body weight of the mice during the monitoring period.
  • MC38 cells mouse colon cancer cell line, Shanghai Heyuan Biotechnology
  • hPD-1 knock-in mice were used to inoculate hPD-1 knock-in mice to determine the immune response of the present invention.
  • SPF grade female hPD-1 knock-in mice purchased from Shanghai Nguiding Model Organisms
  • the certificate number is NO.20170010006762.
  • MC38 cells were routinely subcultured for subsequent in vivo experiments. The cells were collected by centrifugation, and the MC38 cells were resuspended in PBS (1 ⁇ ) to prepare a cell suspension with a cell concentration of 5 ⁇ 10 6 cells/ml. On day 0, 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal region of hPD-1 knock-in mice to establish the MC38 tumor-bearing mouse model.
  • mice Eight days after tumor cell inoculation, the tumor volume of each mouse was detected, and the mice were grouped (8 mice in each group), and the dosage and method of administration were shown in Table 11.
  • Table 11 Grouping, dosage and method of in vivo experiments
  • h-IgG is an isotype control antibody purchased from Equitech-Bio, lot number 161206-0656.
  • h-IgG, 2149, 10mg/kg, 2149, 20mg/kg and 2149, 40mg/kg were used at a concentration of 4mg/ml, 1mg/ml, 2mg/ml and 4mg/ml, administered once a week, a total of 3 Times (QWx3). They were administered on days 8, 15 and 22 after inoculation of MC38 cells, and the tumor volume and body weight of the mice were monitored twice a week, as shown in Figure 12A-B. If the tumor exceeds 2000mm 3 , the mice will be euthanized, and the mice will be monitored throughout the experiment until 61 days later.
  • TGI% 100%*(control group tumor volume-treatment group tumor volume)/(tumor volume of the control group – tumor volume of the control group before administration).
  • Body weight was measured using an electronic balance.
  • the tumor growth curve and survival curve are shown in Figures 12A and 12B.
  • the anti-tumor effects of different dose groups of 2149 molecules were dose-dependent, and in the 20mg/kg and 40mg/kg groups, the tumors of the mice completely regressed. And this advantage is also reflected in the survival curve in Figure 12B, 100% of the tumors of the two groups of mice regressed, while in the 10mg/kg group, only 2 of the 8 mice completely regressed the tumors.
  • the tumor inhibition rate results are shown in Table 12: on the 36th day after inoculation, compared with the h-IgG group, the tumor inhibition rates of 2149, 10 mg/kg, 2149, 20 mg/kg and 2149, 40 mg/kg were 84%, respectively, 103% and 103%.
  • the results of detecting the body weight of the mice FIG. 12C ) showed that there was no significant difference in the body weight of the mice on the 36th day after inoculation.
  • PD-1 antibody-resistant B16F10 cells (mouse melanoma cell line, ATCC CRL-6475) were used to inoculate hPD-1 knock-in mice to determine the bifunctional PD of the present invention.
  • SPF grade female hPD-1 knock-in mice purchased from Shanghai Nguiding Model Organisms
  • the certificate number is NO.20170010007909.
  • B16F10 cells were routinely subcultured for subsequent in vivo experiments. The cells were collected by centrifugation, and the B16F10 cells were resuspended in PBS (1 ⁇ ) to prepare a cell suspension with a cell concentration of 2.5 ⁇ 10 6 cells/ml. On day 0, 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal region of hPD-1 knock-in mice to establish a B16F10 tumor-bearing mouse model.
  • mice Six days after tumor cell inoculation, the tumor volume of each mouse was detected, and the mice were grouped (8 mice in each group). The dosage and method of administration are shown in Table 13.
  • h-IgG is an isotype control antibody purchased from Equitech-Bio, lot number 161206-0656.
  • the concentrations of h-IgG, IBI308, 20mg/kg, IBI308, 40mg/kg, 2149-20mg/kg and 2149, 40mg/kg are 4mg/ml, 2mg/ml, 4mg/ml, 2mg/ml and 4mg/kg respectively ml, administered once a week, a total of 3 times (QWx3).
  • the drugs were administered on the 8th, 15th, and 22nd day after B16F10 cell inoculation, and the tumor volume and body weight of the mice were monitored twice a week, as shown in Figure 13A-B, and the monitoring ended after 22 days.
  • TGI% tumor inhibition rate
  • Body weight was measured using an electronic balance. Mice were euthanized for tumors larger than 2000 mm.
  • mice 13A and 13B The tumor growth curves of mice are shown in Figures 13A and 13B.
  • IBI308 showed almost no drug effect, but the 20mg/kg and 40mg/kg groups of 2149 showed certain anti-tumor effects, and the high The anti-tumor effect of the dose was better than that of the low dose, and this effect was also reflected in the survival curve of the mice.
  • the tumor inhibition rate results are shown in Table 12: On the 15th day after inoculation, compared with the h-IgG, 40mg/kg group, IBI308, 20mg/kg, IBI308, 40mg/kg, 2149, 20mg/kg and 2149, 40mg/kg The tumor inhibition rates of kg were 29%, 27%, 82% and 86%, respectively. At the same time, the results of detecting the body weight of the mice ( FIG. 13D ) showed that there was no significant difference in the body weight of the mice on the 22nd day after inoculation.
  • PD-1 antibody-resistant B16F10 cells were used (Mouse melanoma cell line, ATCC CRL-6475) was inoculated with hPD-1 knock-in mice to determine the anti-tumor efficacy of the fusion protein (2149) of the bifunctional PD-1 antibody and IL-2 mutant molecule of the present invention.
  • the experiment used SPF grade female hPD-1 knock-in mice (purchased from Shanghai Nguiding Model Organisms), the certificate number is NO.20170010008942.
  • B16F10 cells were routinely subcultured for subsequent in vivo experiments. The cells were collected by centrifugation, and the B16F10 cells were resuspended in PBS (1 ⁇ ) to prepare a cell suspension with a cell concentration of 2.5 ⁇ 10 6 cells/ml. On day 0, 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal region of hPD-1 knock-in mice to establish a B16F10 tumor-bearing mouse model.
  • mice Eight days after tumor cell inoculation, the tumor volume of each mouse was detected, and the mice were divided into groups (7 mice in each group). The dosage and method of administration are shown in Table 15.
  • h-IgG is an isotype control antibody purchased from Equitech-Bio, lot number 161206-0656.
  • the concentration of h-IgG, IBI308, 40mg/kg, 2061, 10mg/kg, 2061, 20mg/kg, 2061, 40mg/kg, 2149, 10mg/kg, 2149, 20mg/kg and 2149-40mg/kg are respectively 4mg/ml, 2mg/ml, 4mg/ml, 2mg/ml and 4mg/ml, administered once a week, 3 times in total (QWx3).
  • the drugs were administered on the 8th, 15th, and 22nd day after B16F10 cell inoculation, and the tumor volume and body weight of the mice were monitored twice a week, as shown in Figure 14A, and the monitoring ended after 33 days.
  • Tumor volume measurement: the maximum long axis (L) and maximum width axis (W) of the tumor were measured with a vernier caliper, and the tumor volume was calculated according to the following formula: V L*W2/2.
  • Body weight was measured using an electronic balance. Mice were euthanized for tumors larger than 2000 mm. If more than half of the group died, the tumor growth curve for the entire group was not displayed at that time point.
  • the tumor inhibition rate results are shown in Table 16: On the 19th day after inoculation, compared with the h-IgG, 40mg/kg group, IBI308, 40mg/kg, 2061, 10mg/kg, 2149, 10mg/kg, 2149, 20mg/kg The tumor inhibition rates of kg and 2149-40mg/kg were 21%, 96%, 79%, 87% and 97%, respectively.
  • the 20mg/kg and 40mg/kg of 2061 were not calculated because of the significant weight loss and death of the mice after the first injection, so the TGI of this group was not calculated.
  • mice comparing the maximum administration/tolerance doses of 2061 and 2149 in this experiment, we can see that at a dose of 40mg/kg, 2149, 2061 molecules More mice survived at the dose of 10 mg/kg. For 2149, 3 of 7 mice had complete tumor regression, while only 1 mouse for 2061 had complete tumor regression.
  • the results of detecting the body weight of the mice showed that on the 29th day after inoculation, the body weight of the mice in each dose group of 2149 did not decrease, while the body weight of the mice in the 2061 group decreased.
  • hPD-1 knock-in mice were inoculated with MC38 cells (mouse colon cancer cell line, Shanghai Heyuan Biotechnology) to determine the bifunctional PD-1 antibody of the present invention Antitumor efficacy of fusion protein (2214) with IL-2 mutant molecule.
  • the experiment used SPF grade female hPD-1 knock-in mice (purchased from Shanghai Nguiding Model Organisms), the certificate number is NO.20170010010829.
  • MC38 cells were routinely subcultured for subsequent in vivo experiments. The cells were collected by centrifugation, and the MC38 cells were resuspended in PBS (1 ⁇ ) to prepare a cell suspension with a cell concentration of 5 ⁇ 10 6 cells/ml. On day 0, 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal region of hPD-1 knock-in mice to establish the MC38 tumor-bearing mouse model.
  • mice in each group The tumor volume of each mouse was detected 7 days after tumor cell inoculation, and grouped (7 mice in each group), the dosage and method of administration are shown in Table 17.
  • h-IgG is an isotype control antibody purchased from Equitech-Bio, lot number 161206-0656.
  • Both h-IgG and 2214 were used at a concentration of 4 mg/ml, administered once a week for a total of 3 times (QWx3).
  • Administration was performed on the 7th, 14th, and 21st day after MC38 cell inoculation, and the tumor volume and body weight of the mice were monitored twice a week, as shown in Figure 15A, and the monitoring ended after 56 days.
  • TGI% the relative tumor inhibition rate
  • the results of tumor inhibition rate are shown in Table 18: on the 28th day after inoculation, compared with h-IgG, 20mg/kg group, the tumor inhibition rate of 2214 was 104%. At the same time, the results of detecting the body weight of the mice ( FIG. 15C ) showed that there was no significant difference in the body weight of the mice on the 28th day after inoculation.
  • PD-1 antibody-resistant B16F10 cells (mouse melanoma cell line, ATCC CRL-6475) were used to inoculate hPD-1 knock-in mice to assay this assay.
  • SPF grade female hPD-1 knock-in mice purchased from Shanghai Nguiding Model Organisms
  • the certificate number is NO.20170010010829.
  • B16F10 cells were routinely subcultured for subsequent in vivo experiments. The cells were collected by centrifugation, and the B16F10 cells were resuspended in PBS (1 ⁇ ) to prepare a cell suspension with a cell concentration of 2.5 ⁇ 10 6 cells/ml. On day 0, 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal region of hPD-1 knock-in mice to establish a B16F10 tumor-bearing mouse model.
  • mice in each group The tumor volume of each mouse was detected 7 days after tumor cell inoculation, and grouped (7 mice in each group), the dosage and method of administration are shown in Table 19.
  • h-IgG is an isotype control antibody purchased from Equitech-Bio, lot number 161206-0656.
  • the concentration of h-IgG, 2214-20mg/kg and 2214, 40mg/kg was 4mg/ml, 2mg/ml and 4mg/ml respectively, administered once a week, 3 times in total (QWx3).
  • the drugs were administered on the 7th, 14th, and 21st day after B16F10 cell inoculation, and the tumor volume and body weight of the mice were monitored twice a week, as shown in Figure 16A-B, and the monitoring ended after 63 days.
  • Tumor volume measurement: the maximum long axis (L) and maximum width axis (W) of the tumor were measured with a vernier caliper, and the tumor volume was calculated according to the following formula: V L*W 2 /2.
  • Body weight was measured using an electronic balance. Mice were euthanized for tumors larger than 2000 mm.
  • mice survival curve is shown in Figure 16A, both doses of 2214 can significantly prolong the survival of mice.
  • FIG. 16C the results of detecting the body weight of the mice showed that there was no significant difference in the body weight of the mice on the 22nd day after inoculation.

Abstract

提供了新型白介素2(IL-2)突变蛋白及其用途，其与野生型IL-2相比，具有改善的性质，例如，改善的IL-2受体结合性质和改善的成药性的IL-2突变蛋白。还提供包含该IL-2突变蛋白的融合蛋白、二聚体、免疫缀合物以及编码该IL-2突变蛋白、二聚体、免疫缀合物的核酸、包含该核酸的载体和宿主细胞。进一步提供制备该IL-2突变蛋白、融合蛋白、二聚体和免疫缀合物的方法、包含其的药物组合物和治疗用途。

Description

白介素2突变体以及其融合蛋白 技术领域
本发明涉及新型白介素2(IL-2)突变蛋白及其用途。具体地，本发明涉及，与野生型IL-2相比，具有改善的性质，例如，改善的IL-2受体结合性质和改善的成药性的IL-2突变蛋白。本发明还提供包含该IL-2突变蛋白的融合蛋白、二聚体、免疫缀合物，以及编码该IL-2突变蛋白、二聚体、免疫缀合物的核酸、包含该核酸的载体和宿主细胞。更具体地，本发明尤其提供一种包含IL-2突变蛋白和抗PD-1抗体的免疫缀合物。本发明进一步提供制备该IL-2突变蛋白、融合蛋白、二聚体和免疫缀合物的方法、包含其的药物组合物和治疗用途。
背景技术
白介素-2(IL-2)，也称作T细胞生长因子(TCGF)，是一种主要由活化的T细胞，尤其是CD4 +T辅助细胞产生的多功能细胞因子。在真核细胞中，人IL-2(uniprot:P60568)作为153个氨基酸的前体多肽合成，在去除N端20个氨基酸后，产生成熟的分泌性IL-2。其它物种的IL-2的序列也已经公开，参见NCBI Ref Seq No.NP032392(小鼠)、NP446288(大鼠)或NP517425(黑猩猩)。
白介素2具有4个反平行的、两亲性α螺旋，此4个α螺旋形成其功能必不可少的四级结构(Smith,Science 240,1169-76(1988)；Bazan,Science257,410-413(1992))。在大多数情况下，IL-2通过三种不同受体起作用：白介素2受体α(IL-2Rα；CD25)、白介素2受体β(IL-2Rβ；CD122)和白介素2受体γ(IL-2Rγ；CD132)。IL-2Rβ和IL-2Rγ对于IL-2的信号传导至关重要，而IL-2Rα(CD25)对于信号传导不是必需的，但可以赋予IL-2对受体的高亲和力结合(Krieg等,Proc Natl Acad Sci 107,11906-11(2010))。由IL-2Rα,β,和γ联合形成的三聚体受体(IL-2αβγ)为IL-2高亲和力受体(KD约10pM)，由β和γ组成的二聚体受体(IL-2βγ)为中间亲和力受体(KD约1nM)，单独由α亚基形成的IL-2受体为低亲和力受体。
免疫细胞表达二聚体或三聚体IL-2受体。二聚体受体在细胞毒性CD8 +T细胞和天然杀伤细胞(NK)上表达，而三聚体受体主要在激活的淋巴细胞和CD4 +CD25 +FoxP3 +抑制性调节T细胞(Treg)上表达(Byman,O.和Sprent.J.Nat.Rev.Immunol.12,180-190(2012))。由于静息状态的效应T细胞和NK细胞在细胞表面上没有CD25，故对于IL-2相对不敏感。而Treg细胞在体内一贯表达最高水平的CD25，因此，正常情况下IL-2会优先刺激Treg细胞增殖。
IL-2通过与不同细胞上IL-2受体的结合，在免疫反应中介导多重作用。一方面，IL-2具有免疫系统刺激作用，可以刺激T细胞和天然杀伤(NK)细胞增殖和分化。因此，IL-2已经被批准作为免疫治疗剂用于癌症和慢性病毒感染的治疗。而另一方面，IL-2也可以促进免疫抑制性CD4 +CD25 +调节性T细胞(即，Treg细胞)的维持(Fontenot等,Nature Immunol 6,1142-51(2005)；D’Cruz和Klein,Nature Immunol 6,1152-59(2005)；Maloy和Powrie,Nature Immunol6,1171-72(2005))，在患者中引起由激活的Treg细胞导致的免疫抑制。
此外，多年的临床实践经验发现,尽管高剂量IL-2能在癌症例如黑色素瘤和肾癌治疗中带来显著的临床药效,但是同时也会造成药物相关的严重毒副作用,包括血管渗漏综合症和低血压等心血管系统毒性。研究表明,这些毒性很有可能是由于IL-2对淋巴细胞(尤其是T细胞和NK细胞)的过度激活，刺激释放炎症因子所致。例如，这会导致血管内皮细胞收缩，增大细胞间的间隙,造成组织液外流，从而引起血管渗漏副作用。
临床上使用IL-2的另外一个限制性问题是其极短的半衰期导致给药的困难。由于IL-2分子量只有15KDa,会主要通过肾小球的过滤作用进行清除,人体半衰期只有1小时左右。为了达到足够高的人体暴露量,在临床上需要每隔8小时输注一次大剂量IL-2。然而，频繁的给药不仅给病人带来很重的负担,更重要的是大剂量输注IL-2会造成很高的峰值血药浓度 (Cmax),这很可能是造成药物毒性的另外一个关键因素。
已经采取数种办法来克服这些与IL-2免疫疗法有关的问题。例如,已经发现在体内IL-2与某些抗IL-2单克隆抗体的组合增强IL-2的治疗效果(Kamim ura e t a l.,JImmunol 177,306-14(2006)；Boyman et al.,Science 311,1924-27(2006))。还提出了一些IL-2分子的改造方案。例如，Helen R.Mott等公开了具有消除的IL-2Rα结合能力的人IL-2的突变蛋白F42A。Rodrigo Vazquez-Lombardi等人(Nature Communications,8:15373,DOI:10.1038/ncomms15373)也提出一种具有消除的IL-2Rα结合能力的三重突变人IL-2突变蛋白IL-2 3X，该蛋白在氨基酸残基位置38,43和61分别具有残基突变R38D+K43E+E61R。CN1309705A公开了导致IL-2与IL-2Rβγ结合降低的D20、N88和Q126位置的突变。这些突变蛋白在药代动力学和/或药效学性质方面仍有缺陷，并且在哺乳动物细胞中表达时也存在表达量低和/或分子稳定性较差的问题。
编程性细胞死亡蛋白1(PD-1或CD279)是CD28受体家族的一个抑制性成员,该家族还包括CD28,CTLA-4,ICOS和BTLA。PD-1是一种细胞表面受体且在激活的B细胞,T细胞,和髓样细胞上表达。PD-1的结构是一种单体1型跨膜蛋白,由免疫球蛋白可变样胞外域和含有免疫受体基于酪氨酸的抑制性基序(ITIM)和免疫受体基于酪氨酸的转换基序(ITSM)的胞质域组成。已经鉴定出PD-1的两种配体,PD-Ll和PD-L2,它们显示在结合PD-1后下调T细胞激活。PD-Ll和PD-L2均是B7同系物,结合PD-1但不结合其它CD28家族成员。PD-1的一种配体,PD-Ll在多种人癌症中是丰富的。PD-1和PD-Ll之间的相互作用导致肿瘤浸润性淋巴细胞减少,T细胞受体介导的增殖降低,和癌性细胞的免疫逃脱。
本领域已知多种结合PD-1的抗体，例如在WO2017024465A1中公开的PD-1抗体。
因此，本领域需要进一步开发具有改善性质(例如降低的与其受体的结合，改善的成药性等)的新的IL-2分子，特别是与PD-1抗体的免疫缀合物。
发明内容
本发明涉及以下实施方案：
1.一种免疫缀合物,其包含(i)结合PD-1的抗体和(ii)IL-2突变蛋白，所述突变蛋白，与野生型IL-2(优选人IL-2，更优选包含SEQ ID NO:3序列的IL-2)相比，包含突变：
(i)在IL-2与IL-2Rα结合界面上，尤其是在位置35和/或42上，具有消除或降低对IL-2Rα受体的结合亲合力的突变；
和/或
(ii)在IL-2与IL-2Rβγ结合界面上，尤其是在选自位置88、127和/或130的至少一个位置上，具有弱化对IL-2Rβγ受体的结合的突变；
以及
(iii)缩短的B’C’环区(即，连接氨基酸残基aa72和aa84的序列)，优选地，所述缩短的环区具有小于10，9,8，7，6，或5个的氨基酸长度，且优选7个氨基酸长度；优选地，所述缩短的B’C’环区导致改善的蛋白表达量和/或纯度，以及
任选地(iv)在IL-2的N末端，尤其是在位置3上，具有去除IL2N末端的O糖修饰的突变，其中氨基酸位置根据SEQ ID NO:3编号。
2.根据实施方案1的免疫缀合物,其中所述突变蛋白，相对于野生型IL-2，包含：
(i)N88D；
N88R；
N88R+S130R；
F42A+N88R+S127E；
F42A+N88R+S127E；或
K35E+N88R+S127E；
和
(ii)B’C’环区序列AGDASIH或AQSKNFH；
以及任选地(iii)T3A。
3.根据实施方案1的免疫缀合物，其中所述IL-2突变蛋白包含SEQ ID NO：4、23、25、27、29或31的氨基酸序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成。
4.根据实施方案1-3中任一项的免疫缀合物，其中所述免疫缀合物包含：
第一单体，其包含与Fc片段融合的IL-2突变蛋白；和
第二单体，其包含特异性结合PD-1的抗体或其片段，优选地，所述片段包含所述抗PD-1抗体的一条重链和一条轻链。
5.根据实施方案4的免疫缀合物，其中第一单体的Fc片段中包含Knob突变，第二单体的抗体重链中包含hole突变，或者第一单体的Fc片段中包含hole突变，第二单体的抗体重链中包含knob突变。
6.根据实施方案4或5的免疫缀合物，其中所述第一单体中的Fc片段是IgG1、IgG2、IgG3或IgG4的Fc片段，优选地包含SEQ ID NO:6、42或43的氨基酸序列或由其组成。
7.根据实施方案4-6中任一项的免疫缀合物，其中所述与Fc片段融合的IL-2突变蛋白包含SEQ ID NO:7、24、26、28、30或32的氨基酸序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成。
8.根据实施方案4-7中任一项的免疫缀合物，其中所述PD-1抗体或其抗原结合片段包含重链，所述重链包含重链可变区，所述重链可变区包含分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO:9、10和11。
9.根据实施方案4-8中任一项的免疫缀合物，其中所述PD-1抗体或其抗原结合片段包含轻链，所述轻链包含轻链可变区，其中所述轻链可变区包含分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3:SEQ ID NO:16、17和18。
10.根据实施方案4-9中任一项的免疫缀合物，其中所述抗PD-1抗体或其抗原结合片段包含：包含SEQ ID NO:8所示的氨基酸序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的重链可变区，和包含SEQ ID NO:15所示的氨基酸序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的轻链可变区。
11.根据实施方案4-9中任一项的免疫缀合物，其中所述抗PD-1抗体或其抗原结合片段包含包含SEQ ID NO:14或22所示的氨基酸序列或与其具有至85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的重链；和包含SEQ ID NO:20所示的氨基酸序列或与其具有至85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的轻链。
12.根据实施方案1-11任一项的免疫缀合物，其中所述IL-2突变蛋白与Fc通过接头连接，或所述IL-2突变蛋白与所述抗PD-1抗体通过接头连接，优选地所述接头为(GGGGS) n,其中n＝1、2、3或4，例如所述接头为SEQ ID NO:5。
13.一种分离的多核苷酸，其编码实施方案1-12中任一项的免疫缀合物中的一条或多条链，或第一单体和/或第二单体。
14.一种表达载体，其包含实施方案13的多核苷酸。
15.一种宿主细胞，其包含实施方案13的多核苷酸或实施方案14的载体，优选所述宿主细胞是酵母细胞或哺乳动物细胞，特别是HEK293细胞或CHO细胞。
16.一种用于生产实施方案1-12中任一项的免疫缀合物的方法，包括，在适于表达所述免疫缀合物的条件下，培养实施方案15的宿主细胞。
17.一种药物组合物，其包含实施方案1-12中任一项的免疫缀合物，任选地和药用辅料。
18.实施方案1-12中任一项的免疫缀合物或实施方案17的药物组合物在制备用于预防和/或治疗癌症的药物中的用途，优选地，所述癌症是实体肿瘤或血液肿瘤，例如胃肠道肿瘤或黑色素瘤，例如结肠直肠癌或结肠癌；例如，所述癌症是PD-1抗体治疗耐受性癌症。
19.实施方案18所述的用途，其中所述药物组合物还包含第二治疗剂。
20.一种预防和/或治疗受试者癌症的方法，所述方法包括，向所述受试者施用实施方案1-12中任一项的免疫缀合物或实施方案17的药物组合物，优选地，所述癌症是实体肿瘤或血液肿瘤，例如胃肠道肿瘤或黑色素瘤，例如结肠直肠癌或结肠癌；例如，所述癌症是PD-1抗体治疗耐受性癌症。
21.根据实施方案20中任一项所述的方法，其中所述突变蛋白、所述融合蛋白或所述药物组合物与第二治疗剂以组合疗法施用。
附图说明
图1A是本发明的抗PD-1与IL-2突变体的免疫缀合物的分子结构，图1B是分子2124和3010的IL-2-Fc融合蛋白的分子结构。
图2是IL-2与受体结合的晶体结构(PDB:2ERJ)。
图3是免疫缀合物和对照分子与IL-2Rβγ的结合曲线。
图4是免疫缀合物或对照分子与IL-2Rα的结合曲线。
图5是免疫缀合物或对照分子与人PD1的结合曲线。
图6是免疫缀合物或对照分子在CTLL2WT(huPD1-)和CTLL2-hPD-1(huPD1+)中的活性测定。
图7是免疫缀合物或对照分子分别在PD-1-和PD-1+的T细胞群(CD4或CD8)中的活性。
图8显示了免疫缀合物分别在HEK-Blue TM IL-2 Cells(huPD-1-细胞)和过表达的PD-1的细胞(HEK293+hIL2R+hPD-1/SEAP稳转细胞系(huPD-1+细胞))中的活性。
图9A显示了2132和2063对小鼠抗肿瘤作用的影响；图9B显示了2132和2063对小鼠体重的影响。
图10A显示了2063对小鼠MC38肿瘤的抗肿瘤作用的影响；图10B显示了2063对小鼠体重的影响。
图11A显示了2063对小鼠B16F10肿瘤的抗肿瘤作用的影响；图11B显示了2063对小鼠B16F10肿瘤的抗肿瘤作用的影响-个体肿瘤数值；图11C显示了2063对小鼠体重的影响。
图12A显示了2149对小鼠MC38肿瘤的抗肿瘤作用的影响；图12B显示了2149对小鼠MC38肿瘤的抗肿瘤作用的影响-生存曲线；图12C显示了2149对小鼠体重的影响。
图13A显示了2149对小鼠B16F10肿瘤的抗肿瘤作用的影响；图13B显示了2149对小鼠B16F10肿瘤的抗肿瘤作用的影响-个体肿瘤数值；图13C显示了2149对小鼠B16F10肿瘤的抗肿瘤作用的影响-生存曲线；图13D显示了2149对小鼠体重的影响。
图14A显示了2061和2149对小鼠B16F10肿瘤的抗肿瘤作用的影响-个体肿瘤数值；图14B显示了2061和2149对小鼠B16F10肿瘤的抗肿瘤作用的影响-生存曲线图；图14C显示了2061和2149对小鼠体重的影响。
图15A显示了2214对小鼠抗肿瘤作用的影响；图15B显示了2214对小鼠抗肿瘤作用的影响-生存曲线；图15C显示了2214对小鼠体重的影响。
图16A显示了2214对小鼠B16F10肿瘤的抗肿瘤作用的影响-生存曲线；图16B显示了2214对小鼠B16F10肿瘤的抗肿瘤作用的影响-生存曲线；图16C显示了2214对小鼠体重的影响。
发明详述：
I.定义
在下文详细描述本发明前，应理解本发明不限于本文中描述的特定方法学、方案和试剂，因为这些可以变化。还应理解本文中使用的术语仅为了描述具体实施方案，而并不意图限制本发明的范围，其仅会由所附权利要求书限制。除非另外定义，本文中使用的所有技术和科学术语与本发明所属领域中普通技术人员通常的理解具有相同的含义。
为了解释本说明书，将使用以下定义，并且只要适当，以单数形式使用的术语也可以包括复数，并且反之亦然。要理解，本文所用的术语仅是为了描述具体的实施方案，并且不意欲是限制性的。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
如本文所用，术语“和/或”意指可选项中的任一项或可选项的两项或多项。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”或“包括”某个突变或突变组合的IL-2突变蛋白时，也旨在涵盖仅具有所述突变或突变组合的IL-2突变蛋白。
在本文中，野生型“白介素-2”或“IL-2”是指作为引入本发明突变或突变组合的模板的亲本IL-2蛋白，优选天然存在的IL-2蛋白，例如来源于人、小鼠、大鼠、非人灵长类动物的天然IL-2蛋白，包括未经加工的(例如未去除信号肽)的形式和经加工的(例如去除了信号肽)的形式。包含信号肽的一个全长天然人IL-2序列显示于SEQ ID NO:1中，其成熟蛋白的序列显示于SEQ ID NO:2中。此外，该表述也包括天然存在的IL-2等位基因变体和剪接变体、同种型、同源物、和物种同源物。该表述也包括天然IL-2的变体，例如，所述变体可以与天然IL-2具有至少95％-99％或更高同一性或具有不超过1-10个或1-5个氨基酸突变(例如，保守取代)，并优选与天然IL-2蛋白具有基本相同的IL-2Rα结合亲合力和/或IL2Rβγ结合亲合力。因此，在一些实施方案中，野生型IL-2相比于天然IL-2蛋白可以包含不影响其对IL-2受体结合的氨基酸突变，例如，在125位引入了突变C125S的天然人IL-2蛋白(uniprot:P60568)属于本发明的野生型IL-2。一个包含C125S突变的野生型人IL-2蛋白的实例显示于SEQ ID NO:3中。在一些实施方案中，野生型IL-2序列可以与SEQ ID NO:1或2或3的氨基酸序列具有至少85％，95％，甚至至少96％,97％,98％,或99％或更高的氨基酸序列同一性。
在本文中，氨基酸突变可以是氨基酸取代、缺失、插入和添加。可以进行取代、缺失、插入和添加的任意组合来获得具有期望性质(例如降低的IL-2Rα结合亲合力和/或改善的成药性和/或弱化的IL-2Rβγ)的最终突变蛋白构建体。氨基酸缺失和插入包括在多肽序列的氨基和/或羧基末端的缺失和插入，也包括在多肽序列内部的缺失和插入。例如，可以在全长人IL-2位置1缺失丙氨酸残基，或在B’C’环区缺失一个或数个氨基酸以缩短该环区的长度。在一些实施方案中，优选的氨基酸突变是氨基酸取代，例如单氨基酸取代的组合或氨基酸序列区段的置换。例如，可以是将野生型IL-2的B’C’环区序列整体或部分替换为不同的序列(例如IL-15 的B’C’环)，优选地以获得长度缩短的B’C’环区序列。
在本发明中，当提及IL-2蛋白或IL-2序列区段中的氨基酸位置时，通过参考野生型人IL-2蛋白(也称作IL-2 WT)的氨基酸序列SEQ ID NO:3，予以确定。可以通过与SEQ ID NO:3进行氨基酸序列比对，鉴定在其它IL-2蛋白或多肽(包括全长序列或截短片段)上的对应氨基酸位置。因此，在本发明中，除非另有说明，否则IL-2蛋白或多肽的氨基酸位置为根据SEQ ID NO:3编号的氨基酸位置。例如，当提及“F42”时，是指SEQ ID NO:3的第42位苯丙氨酸残基F，或经比对在其它IL-2多肽序列上的对应位置的氨基酸残基。同时，为了便于理解和对比，当本发明的突变涉及某些特定区段的位点截短或缺失(例如，B’C’环区的序列，即SEQ ID NO:3的第73-83位共11个氨基酸残基)时，在已给定具体突变区域及其突变方式的情况下，该区域之外的氨基酸残基编号仍然维持不变，例如，将B’C’环区的序列，即SEQ ID NO:3的第73-83位共11个氨基酸残基，截短为7个氨基酸残基之后，编号中的80-83不再分配，而紧跟B’C’环区的下一位氨基酸残基位置编号仍然是84。为进行氨基酸位置确定而实施的序列比对，可以使用可从https://blast.ncbi.nlm.nih.gov/Blast.cgi获得的Basic Local Alignment Search Tool，采用默认参数进行。
在本文中，在提及IL-2突变蛋白时，以如下方式来描述单氨基酸取代：[原始氨基酸残基/位置/取代的氨基酸残基]。例如，位置35的赖氨酸取代为谷氨酸，可以表示为K35E。当在一个给定位置(例如K35位)可以有多种可选氨基酸取代方式(例如D,E)时，该氨基酸取代可以表示为：K35D/E。相应地，可以通过加号“+”或“-”将各单氨基酸取代连接起来，以表示在多个给定位置的组合突变。例如F42A、N88R和S127E位置的组合突变，可以表示为：F42A+N88R+S127E，或F42A-N88R-S127E。
在本文中，可以通过在比较窗内比较两条最佳比对的序列来确定“序列同一性百分比”。优选地，在参考序列(例如SEQ ID NO:3)的全长上确定序列同一性。用于比较的序列比对方法是本领域内公知的。适用于确定序列同一性百分比的算法包括例如BLAST和BLAST 2.0算法(参见Altschul等，Nuc.Acids Res.25:3389-402,1977和Altschul等J.Mol.Biol.215:403-10,1990)。可通过美国国家生物技术信息中心(National Center for Biotechnology Information)公众获取用于进行BLAST分析的软件。出于本申请的目的，同一性百分比采用可从https://blast.ncbi.nlm.nih.gov/Blast.cgi获得的Basic Local Alignment Search Tool，利用默认参数来确定。
如本文中使用的，术语“保守取代”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的生物学功能的氨基酸取代。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守取代。典型的保守型氨基酸取代是指将一种氨基酸取代为具有相似的化学性质(例如电荷或疏水性)的另一种氨基酸。功能上相似氨基酸的保守性置换表是本领域熟知的。在本发明中，保守取代残基来自以下的保守替代表X，尤其是表X中的优选保守氨基酸取代残基。
表X
Figure PCTCN2022120265-appb-000001
Figure PCTCN2022120265-appb-000002
例如，野生型IL-2蛋白可以相对于SEQ ID NO:1-3之一具有保守氨基酸取代，或仅具有保守氨基酸取代，且在一个优选实施方案中，保守取代不超过10个氨基酸残基，如1,2,3,4,5,6,7,8,9,10个残基。再例如，本发明的突变IL-2蛋白可以相对于本文中具体给出的IL-2突变蛋白序列(例如SEQ ID NO:4、23、25、27、29和31之任一)具有保守氨基酸取代，或仅具有保守氨基酸取代且在一个优选实施方案中，保守取代不超过10个氨基酸残基，如1,2,3,4,5,6,7,8,9,10个残基。
“亲和力”或“结合亲和力”可以用于反映结合对子的成员之间相互作用的内在结合能力。分子X对其结合配偶体Y的亲和力可以由平衡解离常数(K D)表示，平衡解离常数是解离速率常数和结合速率常数(分别是k dis和k on)的比值。结合亲和力可以由本领域已知的常见方法测量。用于测量亲和力的一个具体方法是本文中的SPR亲和力测定技术或BLI测定技术。
在本文中，抗原结合分子是可以特异性结合抗原的多肽分子，例如，免疫球蛋白分子、抗体或抗体片段，例如Fab片段和scFv片段。在一个实施方案中，本发明的抗原结合分子是针对免疫检查点分子作为抗原的结合分子，例如抗体，例如单克隆抗体。在一个实施方案中，免疫检查点分子为PD-1或PD-L1或PD-L2。
在本文中，抗体Fc片段是指含有至少一部分的恒定区的免疫球蛋白重链的C-端区域，并且可以包括天然序列Fc片段和变体Fc片段。天然序列Fc片段涵盖天然存在的各种免疫球蛋白Fc序列，例如各种Ig亚型以及其同种异型的Fc区(Gestur Vidarsson等,IgG subclasses and allotypes:from structure to effector functions，20October 2014,doi:10.3389/fimmu.2014.00520.)在一个实施方案中，人IgG重链Fc片段从重链的Cys226或从Pro230延伸至羧基端。在另一实施方案中，Fc-片段的C-端赖氨酸(Lys447)可以存在或可以不存在。在另一些实施方案中，Fc片段是包含突变的变体Fc片段，例如包含L234A-L235A突变。除非本文中另外指出，Fc 片段中的氨基酸残基的编号根据EU编号系统，也称为EU索引，如Kabat，E.A.等，Sequences of Proteins of Immunological Interest，第5版，Public Health Service,National Institutes of Health，Bethesda，MD(1991)，NIH Publication 91-3242中所述。在一些实施方案中，抗体Fc片段可以在N端带有IgG1铰链序列或部分IgG1铰链序列，例如根据EU编号，E216到T225的序列或D221到T225的序列。在所述铰链序列中可以含有突变。
IL-2蛋白属于具有四个α螺旋束(A,B,C,D)结构的短链I型细胞因子家族成员。在本文中，术语“B’C’Loop”或“B’C’环区”或“B’C’环序列”可互换使用，是指IL-2蛋白的B螺旋和C螺旋之间的连接序列。通过IL-2的晶体结构分析(例如PDB:2ERJ)，可以确定一个IL-2蛋白的B’C’环序列。为了本发明的目的，根据SEQ ID NO:3的编号，该B’C’环序列是指IL-2多肽中连接位置72的残基和位置84的残基的序列。在SEQ ID NO:1、2和3的野生型IL-2蛋白中，该连接序列包括A73-R83共11个氨基酸。相应地，在本文中，术语“缩短的环区”或“缩短的B’C’环区”是指相对于野生型IL-2蛋白，突变蛋白具有长度减小的B’C’环序列，即，根据SEQ ID NO:3的编号，氨基酸残基aa72和aa84之间的连接序列长度缩短。“缩短的环区”可以通过环序列的替换或截短来实现。所述替换或截短可以发生在B’C’环序列的任何区域或部分。例如，替换或截短可是环区A73-R83序列的替换(例如替换为IL-15B’C环区)或自该序列C端的一个或多个氨基酸残基截短。再例如，替换或截短可以是环区Q74-R83序列的替换或自该序列C端的一个或多个氨基酸残基截短。在进行所述替换或截短后，如果需要，可以进一步在环区序列中引入单氨基酸替代，例如用于消除糖基化的氨基酸替代，和/或回复突变，以进一步改善突变蛋白的性能，例如成药性能。因此，在本文中，经突变后的缩短B’C’环区可以通过在引入突变后连接位置72的残基和位置84的残基之间的序列来描述。
在本文中，“IL-2Rα结合界面”突变是指，发生在IL-2与IL-2Rα(即，CD25)相互作用的氨基酸位点上的突变。可以通过分析IL-2与其受体复合物的晶体结构(例如PDB:1Z92)，确定这些相互作用位点。在一些实施方案中，所述突变尤其是指在IL-2的氨基酸残基35-72区域的突变，特别是在如下氨基酸位点上的突变：35；37；38；41；42；43；45；61；62；68；72。优选地，与引入所述突变前的对应蛋白相比，包含所述突变的IL-2蛋白具有降低或消除的IL-2Rα结合。
在本文中，“IL-2βγ结合界面”突变是指，发生在IL-2与IL-2Rβγ(即，CD122和CD132)相互作用的氨基酸位点上的突变。可以通过IL-2与其受体复合物的晶体结构分析(例如PDB:2ERJ)，确定这些相互作用氨基酸位点。在一些实施方案中，所述突变尤其是指在IL-2的氨基酸残基12-20、氨基酸残基84-95、和氨基酸残基126-130区域的突变，特别是在如下氨基酸位点上的突变：12,15,16,19,20,84,87,88,91,92,95,126,127,130。优选地，与引入所述突变前的对应蛋白相比，包含所述突变的IL-2蛋白具有减弱的IL-2Rβγ结合。
在本文中，对IL-2Rβγ受体结合而言，“弱化”IL-2蛋白分子是指，在IL-2Rβγ结合界面引入突变，其中相对于引入此突变前的对应IL-2蛋白，所述突变导致降低的对IL-2Rβγ受体的结合亲合力。进一步优选地，相对于对应蛋白，所述弱化分子具有降低的T细胞(例如CD8+T细胞或CD4+T细胞)和/或NK细胞激活活性。例如通过检测所述弱化分子和对应蛋白激活T细胞pSTAT5信号的EC50值比值，下降倍数可达到例如5倍以上，例如10倍以上，或50倍以上，或100倍以上，或甚至1000倍以上。例如，相对于对应蛋白，弱化分子对T细胞的激活活性可以下降10-50倍，或50-100倍，或100-1000倍，或更高倍数。因此，在本发明中，在一些实施方案中，本发明的弱化分子具有“弱化”的对IL-2Rβγ受体的结合亲合力和“弱化”的T细胞激活活性。
“抗原结合片段”指与完整抗体不同的分子，其包含完整抗体的一部分且结合完整抗体所结合的抗原。抗体片段的例子包括但不限于Fv，Fab，Fab’，Fab’-SH，F(ab’) 2；dAb(domain antibody)；线性抗体；单链抗体(例如scFv)；单结构域抗体例如VHH；双价抗体或其片段；或骆驼科抗 体。
术语“抗原”是指引发免疫应答的分子。这种免疫应答可能涉及抗体产生或特异性免疫细胞的活化，或两者兼有。技术人员将理解，任何大分子，包括基本上所有的蛋白质或肽，都可以用作抗原。此外，抗原可以衍生自重组或基因组DNA。在一些实施方案中，本发明所述的抗原是肿瘤相关抗原，即与肿瘤的发生、发展或进展有关联的抗原，例如PD-1、PD-L1或PD-L2。
“互补决定区”或“CDR区”或“CDR”是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变结构域内的CDR被称作HCDR1、HCDR2和HCDR3，而位于抗体轻链可变结构域内的CDR被称作LCDR1、LCDR2和LCDR3。在一个给定的轻链可变区或重链可变区氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派系统的任一种或其组合确定，所述指派系统包括例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883，Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))，基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest,第4版,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(在万维网上imgt.cines.fr/上)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。
例如，根据不同的CDR确定方案，每一个CDR的残基如下所述。
Figure PCTCN2022120265-appb-000003
CDR也可以基于与参考CDR序列(例如本发明示例性CDR之任一)具有相同的Kabat编号位置而确定。
除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”涵盖以上述任一种方式确定的CDR序列。
除非另有说明，否则在本发明中，当提及抗体可变区中的残基位置(包括重链可变区残基和轻链可变区残基)时，是指根据Kabat编号系统(Kabat等人，Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.(1991))的编号位置。
在一个实施方案中，本发明抗体的重链可变区和轻链可变区CDR是依据North编号方案定义的CDR序列。
如本文所用的术语“接头”是指使得能够直接连接融合蛋白的不同部分的任何分子。在融合蛋白不同部分之间建立共价连接的接头的实例包括肽接头和非蛋白质聚合物，包括但不限于聚乙二醇(PEG)、聚丙二醇、聚氧化烯或聚乙二醇、聚丙二醇的共聚物。根据本发明的术语“肽接头”是指氨基酸的序列，其中所述序列将融合蛋白的第一部分的氨基酸序列连接至融合蛋白的第二部分。例如，肽接头可以将融合蛋白的IL-2部分与Fc结构域或其片段连接。例如，肽接头也可以将抗体与IL-2连接，诸如将抗体重链的C末端与IL-2连接。优选地，所述肽接头具有这样的长度，其足以连接两个实体，其方式使得它们维持它们相对于彼此的构象，使得不妨碍期望的活性。肽接头可以主要包括或可以不主要包括以下氨基酸残基：Gly、Ser、Ala或Thr。有用的接头包括甘氨酸-丝氨酸聚合物，包括例如(GS) n、(GSGGS) n、(GGGGS) n、(GGGS) n和(GGGGS) nG，其中n是至少1(且优选2、3、4、5、6、7、8、9、10)的整数。有用的接头还包括甘氨酸-丙氨酸聚合物、丙氨酸-丝氨酸聚合物和其他柔性接头。优选地，本发明的接头是(GGGGS) n，其中n＝1、2、3、4或5，优选地为2。优选地，本发明的接头是SEQ ID NO:5。
“IgG形式的抗体”是指抗体的重链恒定区所属的IgG形式。所有同一型的抗体的重链恒定区都是相同的，不同型的抗体之间的重链恒定区不同。例如，IgG4形式的抗体是指其重链恒定区来自IgG4，或者IgG1形式的抗体是指其重链恒定区来自IgG1。
“人源化”抗体是指包含来自非人CDR的氨基酸残基和来自人FR的氨基酸残基的抗体。在一些实施方案中，人源化抗体将包含基本上所有的至少一个、通常两个可变结构域，其中所有或基本上所有的CDR(例如，CDR)对应于非人抗体的那些，并且所有或基本上所有的FR对应于人抗体的那些。人源化抗体任选可以包含至少一部分的来源于人抗体的抗体恒定区。抗体(例如非人抗体)的“人源化形式”是指已经进行了人源化的抗体。
“人抗体”或“全人抗体”或“全人源抗体”可以互换使用，其指具有这样的氨基酸序列的抗体，所述氨基酸序列对应于下述抗体的氨基酸序列，所述抗体由人或人细胞生成或来源于非人来源，其利用人抗体库或其它人抗体编码序列。人抗体的这种定义明确排除包含非人抗原结合残基的人源化抗体。
本发明的免疫缀合物中的抗体部分可以是人源化抗体、人抗体或嵌合抗体。
本文使用的术语“融合物”是指通过连接两个或多个最初分开的蛋白/基因/化合物形成的融合物。如果构成融合物的实体是蛋白质，则被称为融合蛋白。融合蛋白涵盖在本申请的融合物的范围内。例如，IL-2与Fc二聚体连接可以构成IL-2融合蛋白。构成融合物的两个实体分子之间的连接可以通过或不通过接头实现。
如本文中使用的术语“免疫缀合物”指包括至少一个IL-2分子和至少一个抗体或抗体片段的多肽分子。如本文中描述的，IL-2分子可以通过多种相互作用及以多种构造连接至抗体。例如，IL-2与Fc的融合蛋白与抗体分子的包含重链和轻链的片段可以通过二聚化构成免疫缀合物。优选地，本发明的免疫缀合物具有图1A所示的结构，或IL-2部分与PD-1抗体部分互换的图1A所示的结构。
如本文中使用的,术语“第一”和“第二”就Fc结构域或单体等而言为了在有超过一个每类模 块时便于区分而使用。除非明确如此陈述,这些术语的使用不意图赋予免疫缀合物的特定次序或取向。
本文所述的术语“治疗剂”涵盖在预防或治疗肿瘤，例如癌症中有效的任何物质，包括化疗剂、细胞因子、血管生成抑制剂、细胞毒性剂、其它抗体、小分子药物或免疫调节剂(例如免疫抑制剂)。
术语“有效量”指本发明的抗体或片段或组合物或组合的这样的量或剂量，其以单一或多次剂量施用患者后，在需要治疗或预防的患者中产生预期效果。“有效量”可以涵盖“治疗有效量”或“预防有效量”。
“治疗有效量”指以需要的剂量并持续需要的时间段，有效实现所需治疗结果的量。治疗有效量也是这样的一个量，其中抗体或抗体片段或组合物或组合的任何有毒或有害作用不及治疗有益作用。相对于未治疗的对象，“治疗有效量”优选地抑制可度量参数(例如肿瘤体积)至少约40％、甚至更优选地至少约50％、55％、60％、65％、70％、75％、80％、85％、90％甚至100％。“预防有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防结果的量。通常，由于预防性剂量在对象中在疾病较早阶段之前或在疾病较早阶段使用，故预防有效量将小于治疗有效量。
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可交换地使用且是指其中引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“转化的细胞”，其包括初级转化的细胞和来源于其的后代，而不考虑传代的数目。后代在核酸内容上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括在最初转化的细胞中筛选或选择的具有相同功能或生物学活性的突变体后代。
本文所使用的术语“标记”是指被直接或间接缀合或融合至试剂(诸如多核苷酸探针或抗体)并且促进其所缀合或融合的试剂的检测的化合物或组合物。标记本身可以是可检测的(例如，放射性同位素标记或荧光标记)或在酶促标记的情况下可以催化可检测的底物化合物或组合物的化学改变。术语旨在涵盖通过将可检测物质偶联(即，物理连接)至探针或抗体来直接标记探针或抗体以及通过与直接标记的另一种试剂反应来间接标记探针或抗体。
“个体”或“受试者”包括哺乳动物。哺乳动物包括但不限于，家养动物(例如，牛，羊，猫，狗和马)，灵长类动物(例如，人和非人灵长类动物如猴)，兔，以及啮齿类动物(例如，小鼠和大鼠)。在一些实施方案中，个体或受试者是人。
术语“抗肿瘤作用”指可以通过多种手段展示的生物学效果，包括但不限于例如，肿瘤体积减少、肿瘤细胞数目减少、肿瘤细胞增殖减少或肿瘤细胞存活减少。在一些实施方案中，抗肿瘤作用还涉及在不减少受试者体重的情况下的抗肿瘤作用。
术语“肿瘤”和“癌症”在本文中互换地使用，涵盖实体瘤和血液肿瘤。
术语“癌症”指向或描述哺乳动物中特征通常为细胞生长不受调节的生理疾患在某些实施方案中，适合于通过本发明的抗体来治疗的癌症包括实体肿瘤或血液肿瘤等，包括癌症的转移性形式。术语“肿瘤”指所有赘生性(neoplastic)细胞生长和增殖，无论是恶性的还是良性的，及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”、“癌性”和“肿瘤”在本文中提到时并不互相排斥。
术语“药用辅料”指与活性物质一起施用的稀释剂、佐剂(例如弗氏佐剂(完全和不完全的))、赋形剂、载体或稳定剂等。
术语“药物组合物”指这样的组合物，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用所述组合物的受试者具有不可接受的毒性的另外的成分。
术语“药物组合或组合产品”是指非固定组合产品或固定组合产品，包括但不限于药盒、药物组合物。术语“非固定组合”意指活性成分(例如，(i)本发明的突变蛋白或融合物、以及 (ii)其他治疗剂)以分开的实体被同时、无特定时间限制或以相同或不同的时间间隔、依次地施用于患者，其中这类施用在患者体内提供预防或治疗有效水平的两种或更多种活性剂。术语“固定组合”意指两种或更多种活性剂以单个实体的形式被同时施用于患者。优选对两种或更多种活性剂的剂量和/或时间间隔进行选择，从而使各部分的联合使用能够在治疗疾病或病症时产生大于单独使用任何一种成分所能达到的效果。各成分可以各自呈单独的制剂形式，其制剂形式可以相同也可以不同。
术语“组合疗法”是指施用两种或更多种治疗剂或治疗方式(例如放射疗法或手术)以治疗本文所述疾病。这种施用包括以基本上同时的方式共同施用这些治疗剂，例如以具有固定比例的活性成分的单一胶囊。或者，这种施用包括对于各个活性成分在多种或在分开的容器(例如片剂、胶囊、粉末和液体)中的共同施用。粉末和/或液体可以在施用前重构或稀释至所需剂量。此外，这种施用还包括以大致相同的时间或在不同的时间以顺序的方式使用每种类型的治疗剂。在任一情况下，治疗方案将提供药物组合在治疗本文所述的病症或病状中的有益作用。
用于本文时，“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转已存在的症状、病症、病况或疾病的进展或严重性。
用于本文时，“预防”包括对疾病或病症或特定疾病或病症的症状的发生或发展的抑制。在一些实施方式中，具有癌症家族病史的受试者是预防性方案的候选。通常，在癌症的背景中，术语“预防”是指在癌症的病征或症状发生前，特别是在具有癌症风险的受试者中发生前的药物施用。
术语“载体”当在本文中使用时是指能够增殖与其相连的另一个核酸的核酸分子。该术语包括作为自我复制核酸结构的载体以及结合到已经引入其的宿主细胞的基因组中的载体。一些载体能够指导与其可操作相连的核酸的表达。这样的载体在本文中被称为“表达载体”。
“受试者/患者/个体样品”指从患者或受试者得到的细胞或流体的集合。组织或细胞样品的来源可以是实体组织，像来自新鲜的、冷冻的和/或保存的器官或组织样品或活检样品或穿刺样品；血液或任何血液组分；体液，诸如脑脊液、羊膜液(羊水)、腹膜液(腹水)、或间隙液；来自受试者的妊娠或发育任何时间的细胞。组织样品可能包含在自然界中天然不与组织混杂的化合物，诸如防腐剂、抗凝剂、缓冲剂、固定剂、营养物、抗生素、等等。
II.本发明的IL-2突变蛋白
本发明IL-2突变蛋白的有利生物学性质
本发明人经长期研究发现，可以如下实施如下分子突变和改造或组合所述突变和改造中的一项或多项，来同时改善IL-2的药效、降低IL-2的毒副作用、和实现良好的生产性能：
(i)在IL-2与IL-2Rβγ受体的结合界面引入特定的残基突变，弱化与IL-2Rβγ受体的结合，一定程度地下调IL-2的活性。通过包含弱化IL-2Rβγ受体结合的此类突变，本发明的IL-2突变蛋白可以在激活淋巴细胞杀伤肿瘤的同时,避免由淋巴细胞过度激活导致的大量炎症因子释放,和由此带来的药物相关毒性。此外，该弱化突变还可以通过减低本发明IL-2突变蛋白与广泛存在于淋巴细胞上的IL-2受体的结合亲和力，减少由IL-2受体介导的IL-2突变蛋白清除，延迟IL-2突变蛋白的作用时效；
(ii)将本发明的突变IL-2蛋白构建成IL-2-Fc二聚体。该二聚体的形成可以增加本发明IL-2突变蛋白的分子量，极大地减小肾清除,并通过FcRn介导的体内循环回收,进一步延长IL2-Fc融合蛋白的半衰期。从而，克服由IL-2的短半衰期和高频大剂量施用带来的高峰值血 药浓度问题；
(iii)对IL-2的B’C’loop结构进行改造，例如分别使用IL-15分子的loop进行替换或者将IL-2分子B’C’loop进行截短。该B’C’环突变可以显著地增强本发明IL-2突变蛋白中B’C’loop结构的稳定性,显著地提升IL-2突变蛋白和由其构建的IL-2-Fc二聚体分子的生产性能，例如显著提升的表达量和纯度；
(iv)保持与野生型IL-2基本上相当的IL-2Rα结合活性；或可以进一步组合如下突变：(v)在IL-2与IL-2Rα受体的结合界面的一个或多个特定的突变，以改变IL-2突变蛋白对IL-2Rα的结合性能。而且，本发明人发现，在本发明的突变蛋白中，在保持上述优良性质的同时，还可以根据需要，调节IL-2突变蛋白与IL-2Rα的结合活性，以满足IL-2在抗肿瘤或自身免疫疾病治疗等多方面不同的成药需求，从而，进一步赋予本发明突变蛋白优良的药效性质。
由此，通过对序列的改造，本发明的IL2-Fc系列分子，一方面弱化与其受体IL2Rβ/γ的结合亲和力，实现了更优秀的药代动力学实验结果和药效结果，另一方面在成药性例如蛋白表达量和纯度方面得到了显著的提升。
因此，本发明提供了具有改善的成药性质和改善的IL-2受体结合性质的IL-2突变蛋白。包含本发明IL-2突变蛋白的IL-2-Fc分子可以有效地避免对淋巴细胞的强激动造成的炎症因子的过度释放,具有更加平稳长效的药代特性。因此，可以用较低的单次给药剂量来达到足够高的人体药物暴露量,避免Cmax过高导致的药物相关毒性。此外，更有意义的是,虽然本发明的长效IL-2-Fc分子相对于天然IL-2具有弱化的淋巴细胞免疫刺激活性,但是因为药代动力学性质的显著改善,本发明分子的体内有效药物浓度持续的时间更长,可以发挥较长时间的对淋巴细胞的持续刺激,达到与天然IL-2分子相当甚至更好的药效动力学效果,在动物中实现更好的抗肿瘤药效和耐受性。
此外，还可以将本发明的具有有利生物学性质的突变IL-2蛋白与抗原结合分子(例如抗体或其片段)形成免疫缀合物，在激活并扩增T细胞或NK细胞的同时，增强抗原结合分子的免疫或免疫治疗效果。
改善的成药性质
在一些实施方案中，本发明IL-2突变蛋白具有改善的成药性质，例如，当在哺乳动物细胞例如HEK293或CHO细胞中表达时，尤其是以Fc融合蛋白表达时，具有选自以下的一项或多项性质：(i)优于野生型IL-2蛋白的表达量；和(ii)易于纯化至更高的蛋白纯度。
在本发明的一些实施方案中，本发明IL-2突变蛋白与野生IL-2相比表现出表达水平的增加。在本发明的一些实施方案中，增加的表达发生在哺乳动物细胞表达系统中。表达水平可通过允许定量或半定量分析细胞培养上清液(优选一步亲和层析纯化后的上清液)中的重组IL-2蛋白量的任何合适方法来测定。例如，可以通过蛋白质印迹或ELISA，评估样品中的重组IL-2蛋白量。在一些实施方案中，本发明IL-2突变蛋白，与野生型IL-2相比，在哺乳动物细胞中的表达量增加至少1.1倍，或至少1.5倍，或至少2倍、3倍或4倍以上，或至少5、6、7、8或9倍，或甚至10倍以上，例如大约10，15、20，25,30和35倍。
在一些实施方案中，如通过测定蛋白A亲和层析后纯化蛋白的纯度所显示的，本发明IL-2突变蛋白-Fc融合物，相对于野生型IL-2蛋白融合物，表现出更高的纯度。在一些实施方案中，蛋白纯度通过SEC-HPLC技术检测。在一些优选的实施方案中，在一步蛋白A亲和层析纯化后，本发明IL-2突变蛋白-Fc融合物的纯度可以达到70％，或80％，或90％以上，优选地92％,93％,94％,95％,98％或99％以上。
在一些实施方案中，如通过测定蛋白A亲和层析后纯化蛋白的纯度所显示的，本发明IL-2-Fc二聚体蛋白，相对于由野生型IL-2蛋白形成的对应IL-2-Fc二聚体蛋白，表现出更高的纯度。在一些实施方案中，蛋白纯度通过SEC-HPLC技术检测。在一些优选的实施方案中，在一步蛋白A亲和层析纯化后，本发明IL-2-Fc二聚体蛋白的纯度可以达到70％，或80％，或90％以上，优选地92％,93％,94％,95％,98％或99％以上。
弱化的IL-2βγ受体结合性
通过在IL-2Rβγ结合界面引入突变，在一些实施方案中，本发明的IL-2突变蛋白，相对于引入所述突变前的对应蛋白，具有弱化的IL-2βγ结合亲和力。
在一些实施方案中，相对于引入IL-2Rβγ结合界面突变进行弱化前，本发明的IL-2突变蛋白对IL-2Rβ和/或IL-2Rβγ受体的结合亲和力降低。在一些实施方案中，相对于进行弱化前，本发明的IL-2突变蛋白对IL-2Rβ受体的结合亲和力降低，例如降低1-20倍或更高，包括在一些实施方案中，去除对IL-2Rβ受体的结合。在一些实施方案中，相对于进行弱化前，本发明的IL-2突变蛋白对IL-2Rβγ受体的结合亲和力降低，例如降低1-100倍或更高。在一些实施方案中，本发明的IL-2突变蛋白不与IL-2Rβ受体的结合，但仍能够与IL-2Rβγ受体结合，优选地其中所述与IL-2Rβγ受体的结合，相比于进行弱化前，可以例如降低1-100倍，如大约20-80倍。可以通过SPR亲和测定技术，测定本发明IL-2突变蛋白，例如与Fc片段融合的本发明IL-2突变蛋白或其二聚体分子，与受体IL-2Rβ或IL-2Rβγ受体的平衡解离常数(K D)来确定结合亲和力。
通过在IL-2Rβγ结合界面引入突变，在一些实施方案中，本发明的IL-2突变蛋白，相对于引入所述突变前的对应蛋白，具有弱化的IL-2活性，例如选自以下至少一项的IL-2活性：
-相比于弱化前，降低对T细胞(例如CD4+和CD8+T细胞，例如CD4+/CD8+CD25-T细胞，CD4+CD25+T细胞)的激活；
-相比于弱化前，降低对NK细胞的激活；
-相比于弱化前，降低由IL-2刺激的T细胞/NK细胞炎症因子释放。
在一个实施方案中，相对于弱化前，本发明IL-2突变蛋白导致减少的由IL-2介导的淋巴细胞(例如T细胞和/或NK细胞)激活和/或增殖。在一个实施方案中，淋巴细胞是CD4+和CD8+T细胞，例如CD25 -T细胞。在一个实施方案中，在STAT5磷酸化测定试验中，通过检测IL-2突变蛋白在淋巴细胞例如T细胞或NK细胞中对STAT5磷酸化信号的激活，来鉴定IL-2突变蛋白激活CD4+和CD8+T细胞的能力。例如，如本申请实施例中所述，可以通过流式细胞术分析细胞中的STAT5磷酸化，确定半最大有效浓度(EC50)。例如通过检测所述本发明的IL-2弱化分子和对应蛋白激活T细胞STAT5磷酸化信号的EC50值的比值，本发明的IL-2突变分子具有“弱化”的T细胞激活活性。根据该比值，本发明IL-2突变分子的T细胞激活活性下降可达到例如5倍以上，例如10倍以上，或50倍以上，或100倍以上，或甚至1000倍以上。 例如，相对于对应蛋白，本发明IL-2突变分子对T细胞的激活活性可以下降10-50倍，或50-100倍，或100-1000倍，或更高倍数。在一些优选实施方案中，本发明IL-2突变蛋白相对于野生型IL-2，具有减少的由细胞表面IL-2受体介导的IL-2清除，表现出增加的体内半衰期。
在一些优选实施方案中，本发明IL-2突变蛋白相对于野生型IL-2具有减小的由IL-2与其受体介导的体内毒性。
保持或改变(优选地弱化的)的IL-2Rα受体结合
IL-2蛋白通过与IL-2受体相互作用来引发信号传导和发挥功能。野生型IL-2对不同IL-2受体显示出不同的亲合力。在静息效应细胞(包括CD8 +T细胞和NK细胞)上表达与野生型IL-2具有较低亲合力的IL-2β和γ受体。在调节性T细胞(Treg)细胞和激活的效应细胞上表达与野生型IL-2具有高亲合力的IL-2Rα。由于高亲合力的原因，野生型IL-2会优先结合细胞表面的IL-2Rα，再招募IL-2Rβγ，通过IL-2Rβγ释放下游p-STAT5信号，刺激Treg细胞和激活的效应细胞。不受理论的束缚，改变IL-2对IL-2Rα受体的亲合力，将改变IL-2优先激活CD25 +细胞的偏向性，改变IL-2介导的Treg细胞的免疫下调作用。
在一些实施方案中，本发明的IL-2突变蛋白，相对于野生型IL-2，具有保持或改变的IL-2Rα受体结合能力。
在一些实施方案中，本发明的IL-2突变蛋白，相对于野生型IL-2，保持对IL-2Rα受体的结合。在本文中，表述“保持对IL-2Rα受体的结合”是指IL-2突变蛋白相对于野生型IL-2蛋白与IL-2Rα受体具有相当的结合活性。优选地，“相当的结合活性”为，当采用相同的测量方式测定时，IL-2突变蛋白与野生型IL-2蛋白的结合活性数值(例如结合亲和力K D)之间的比值在1:20至20:1之间，优选地，1:10至10:1之间。优选地，IL-2突变蛋白相对于野生型IL-2，不具有IL-2Rα结合界面突变。
在一些实施方案中，本发明的IL-2突变蛋白是弱化IL-2突变分子，且保持对IL-2Rα受体的结合。在再一些实施方案中，本发明的弱化IL-2突变蛋白，相对于野生型IL-2，不具有IL-2Rα结合界面突变。优选地，所述弱化IL-2突变分子具有改善的Treg选择性和/或改善的NK细胞(例如，CD3 -CD56 +NK细胞)选择性。在一个实施方案中，在STAT5磷酸化测定试验中，通过检测IL-2突变蛋白在不同淋巴细胞例如Treg细胞、NK细胞、CD4+和CD8+效应T细胞中对STAT5磷酸化信号的激活，来鉴定IL-2突变蛋白对淋巴细胞的选择性。在一个实施方案中，在STAT5磷酸化测定试验中，IL-2突变蛋白的选择性可以通过选择性激活特定的(一种或多种)淋巴细胞而不实质性激活其它淋巴细胞的IL-2突变蛋白剂量窗口来反映。例如，在一些实施方案中，本发明的弱化IL-2突变蛋白可以表现出改善的Treg选择性和/或改善的NK细胞(CD3 -CD56 +NK细胞)选择性，所述选择性相对于效应T细胞，例如CD25 -/low CD4+和/或CD8+效应T淋巴细胞而言。在再一些实施方案中，改善的选择性可以反映为IL-2突变蛋白的低药物相关毒性。
在另一些实施方案中，本发明的IL-2突变蛋白，相对于野生型IL-2，引入IL-2Rα结合界面突变，所述突变导致IL-2突变蛋白具有降低或消除的IL-2Rα受体结合。
在再一些实施方案中，本发明的该IL-2突变蛋白，相对于野生型IL-2，降低IL-2优先激活CD25 +细胞的偏向性。在再一些实施方案中，本发明的该IL-2突变蛋白，相对于野生型IL-2， 降低IL-2介导的Treg细胞的免疫下调作用。
在另一些实施方案中，本发明的IL-2突变蛋白具有免疫下调作用。在再一些实施方案中，本发明的IL-2突变蛋白可以用于自身免疫疾病的治疗。
因此，在一些实施方案中，本发明的IL-2突变蛋白具有选自例如以下的一项或多项的改善性质：
-与野生型IL-2相比，保持或改变(例如降低或增加，优选地降低的)对IL-2R受体(IL-2Rαβγ、IL-2Rα和/或IL2Rαβγ)的结合亲合力；
-与野生型IL-2相比，保持或改变(例如降低或增加)对CD25+细胞(例如CD8+T细胞和Treg细胞)的激活；
-与野生型IL-2相比，保持或改变(例如去除或降低，或增加)IL-2优先激活CD25+细胞(例如Treg细胞)的偏向性；
-与野生型IL-2相比，保持或改变(例如降低或增加)由IL-2诱导的经Treg细胞引起的免疫反应下调作用。
在一些实施方案中，相对于野生型IL-2(例如SEQ ID NO:1或SEQ ID NO:3中所示IL-2 WT)，本发明IL-2突变蛋白对IL-2Rα受体的结合亲和力降低至少5倍、至少10倍、或至少25倍，尤其是至少30倍、50倍或100倍以上。在优选的实施方案中，本发明的突变蛋白不结合IL-2受体α。可以通过SPR亲和测定技术，测定本发明IL-2突变蛋白，例如与Fc片段融合的本发明IL-2突变蛋白或其二聚体分子，与受体IL-2Rα受体的平衡解离常数(K D)来确定结合亲和力。
在一个实施方案中，相对于野生型IL-2，本发明IL-2突变蛋白导致减少的由IL-2介导的CD25 +细胞激活和/或增殖。在一个实施方案中，CD25 +细胞是CD25 +CD8 +T细胞。在另一实施方案中，CD25 +细胞是Treg细胞。在一个实施方案中，在STAT5磷酸化测定试验中，通过检测IL-2突变蛋白在CD25 +细胞中对STAT5磷酸化信号的激活，来鉴定IL-2突变蛋白激活CD25 +细胞的能力。例如，如本申请实施例中所述，可以通过流式细胞术分析细胞中的STAT5磷酸化，确定半最大有效浓度(EC50)。
在一个实施方案中，相对于野生型IL-2，本发明IL-2突变蛋白去除或降低IL-2对CD25 +细胞优先激活的偏向性。在一个实施方案中，CD25 +细胞是CD25 +CD8 +T细胞。在另一实施方案中，CD25 +细胞是Treg细胞。在一个实施方案中，在STAT5磷酸化测定试验中，通过检测IL-2突变蛋白分别在CD25 -细胞中和在CD25 +细胞中激活STAT5磷酸化信号的EC50值，来鉴定IL-2突变蛋白激活CD25 -细胞的能力。例如，通过计算在CD25 -和CD25 +T细胞上激活STAT5磷酸化信号的EC50值的比值，确定IL-2突变蛋白对CD25 +细胞的激活偏向性。优选地，相对于野生型蛋白，突变蛋白对CD25 +的偏向性降低了至少10倍，优选至少100倍，150倍，200倍，300倍或更高。
在一些实施方案中，本发明的IL-2突变蛋白具有PCT/CN2021/081840所示的突变蛋白的性质，所述专利以其全文引入本发明。
本发明的突变蛋白
一方面，本发明提供一种IL-2突变蛋白，其中所述突变蛋白，与野生型IL-2(优选人IL-2，更优选包含SEQ ID NO:3序列的IL-2)相比，包含突变：
(i)在IL-2与IL-2Rα结合界面上，尤其是在位置35和/或42上，具有消除或降低对IL-2Rα受体的结合亲合力的突变；
和/或
(ii)在IL-2与IL-2Rβγ结合界面上，尤其是在选自位置88、127和/或130的至少一个位置上，具有弱化/降低的对IL-2Rβγ受体的结合的突变；
以及
(iii)缩短的B’C’环区(即，连接氨基酸残基aa72和aa84的序列)，优选地，所述缩短的环区具有小于10，9,8，7，6，或5个的氨基酸长度，且优选7个氨基酸长度；优选地，所述缩短的B’C’环区导致改善的蛋白表达量和/或纯度，
其中氨基酸位置根据SEQ ID NO:3编号。
在一个实施方案中，所述突变蛋白包含突变(i)和(iii)或包含突变(ii)和(iii)或包含(i)、(ii)和(iiii)。
IL-2Rβγ结合界面突变
适用于本发明突变蛋白的IL-2Rβγ结合界面突变，可以是任何能够与本发明其他突变组合并导致弱化或降低的IL-2Rβγ结合亲和力和/或弱化的淋巴细胞(例如T细胞/NK细胞)激活活性的突变。
这样的突变的例子包括，但不限于：在IL-2与IL-2Rβγ结合界面上，尤其是在选自位置88、127和130的至少一个位置上，导致弱化或减低的IL-2Rβγ受体结合的突变。
在一些实施方案中，IL-2Rβγ结合界面突变包含选自以下的突变中的一个或多个或以下突变的组合：
N88D,N88R,S127E,S130R,N88R+S130R，N88R+S127E。
在另一些实施方案中，包含本发明的IL-2Rβγ结合界面突变的本发明IL-2突变蛋白具有弱化或降低的IL-2Rβγ结合，例如采用SPR亲和力测定，弱化或降低的IL-2Rβγ结合亲和力。
B’C’环区突变
在一个方面，本发明的IL-2突变蛋白，相对于野生型IL-2，包含B’C’环区突变，优选地所述突变导致稳定性增加的B’C’环区；更优选地，所述突变导致本发明IL-2突变蛋白具有改善的成药性，例如，增加的表达量和/或纯度。
在一些实施方案中，所引入的突变导致，与野生型IL-2(优选人IL-2，更优选包含SEQ ID NO:3序列的IL-2)相比，突变蛋白包含缩短的B’C’环区(即，氨基酸残基aa72和aa84之间的连接序列长度缩短)。
优选地，所述缩短的环区具有小于10，9,8，7，6个或5个氨基酸长度，且优选7个氨基酸长度，其中氨基酸残基根据SEQ ID NO:3编号。
在本文中，适用于本发明的B’C’环区突变包括B’C’环区的截短和替换。在一个实施方案中，所述突变包括B’C’环区氨基酸残基aa73至aa83的截短(例如截短B’C’环区的1、2、3、或4个氨基酸)或替换，例如截短为A(Q/G)SKN(F/I)H，优选地为AQSKNFH，或替换为SGDASIH。在另一实施方案中，所述突变包括B’C’环区氨基酸残基aa74至aa83的截短或替换，例如截短为(Q/G)SKN(F/I)H，或替换为GDASIH或AGDASIH。
在一些实施方案中，本发明的IL-2突变蛋白包含B’C’环嵌合突变。相对于野生型IL-2，所述突变蛋白包含对连接aa72至aa84的序列的全部或部分替代，例如替代为来自其它四螺旋短链细胞因子家族成员的短B’C’环序列。可以通过晶体结构的superpose，从其它四螺旋短链细胞因子IL家族成员，例如IL-15，IL-4，IL-21，或来自非人物种(如小鼠)的IL家族成员，鉴定适用于替代野生型IL-2的短B’C’环。在一个实施方案中，用于替代的序列为来自白介素IL-15(尤其是人IL-15)的B’C’环序列。在一个实施方案中，所述替代包括B’C’环区氨基酸残基aa73至aa83的替代。在另一实施方案中，所述替代包括B’C’环区氨基酸残基aa74至aa83的替代。优选地，经替代后，本发明IL-2突变蛋白具有选自以下的B’C’环序列(即，连接aa72至aa84的序列)：SGDASIH或AGDASIH，优选地AGDASIH。
在一些实施方案中，本发明IL-2突变蛋白包含B’C’环截短突变。相对于野生型IL-2，所述突变蛋白包含对连接aa72至aa84的序列的截短。在一个实施方案中，所述截短包括B’C’环区氨基酸残基aa73至aa83的截短。在另一实施方案中，所述截短包括B’C’环区氨基酸残基aa74至aa83的截短。例如，可以自C端截短1、2、3或4个氨基酸。优选地，经截短后，本发明IL-2突变蛋白的B’C’环区具有序列A(Q/G)SKN(F/I)H，优选地为AQSKNFH。优选地，经截短后，本发明IL-2突变蛋白具有选自以下的B’C’环序列(即，连接aa72至aa84的序列)：
B’C’loop序列
AQSKNFH
AGSKNFH
AQSANFH
AQSANIH
在一个优选的实施方案中，本发明IL-2突变蛋白包含选自以下的B’C’环区序列(即，连接aa72至aa84的序列)：AQSKNFH或AGDASIH。
IL-2Rα结合界面突变
在一个方面，本发明的IL-2突变蛋白，相对于野生型IL-2，在IL-2Rα结合界面，优选地在位置35和/或42上，包含一个或多个突变。优选地，所述突变消除或降低对IL-2Rα受体的结合亲合力。
在一些优选的实施方案中，本发明的IL-2Rα结合界面突变包含突变K35E和/或F42A。
在另一些实施方案中，包含本发明的IL-2Rα结合界面突变的本发明IL-2突变蛋白具有改变的IL-2Rα结合，例如采用SPR亲和力测定，改变的(优选降低或消除的)IL-2Rα结合。
其它突变
除了上述“IL-2Rβγ结合界面突变”、“B’C’环区突变”和“IL-2Rα结合界面突变”，本发明的 IL-2突变蛋白还可以在其它区域或位置上具有1个或多个突变，只要其保留本发明IL-2突变蛋白的上述一个或多个有益性质即可。例如，本发明IL-2突变蛋白还可以包含在位置125的取代，例如C125S，C125A,C125T,或C125V，以提供额外的优点，例如改善的表达或同质性或稳定性(参见例如，美国专利号4,518,584)。再例如，本发明IL-2突变蛋白还可以包含在位置3的取代，例如T3A，以去除IL2N末端的O糖修饰。本领域技术人员知晓如何确定可以并入本发明IL-2突变蛋白中的额外突变。
优选的示例性突变组合
在一些优选实施方案中，本发明IL-2突变蛋白具有弱化的IL-2Rβγ结合，且具有选自以下一项或全部两项的改善性质：(i)降低的(或消除的)IL-2Rα结合；和(ii)改善的表达水平和纯度。在一些实施方案中，所述IL-2突变蛋白相对于野生型IL-2蛋白保持IL-2Rα受体结合。
在一些实施方案中，本发明提供IL-2突变蛋白，其中所述突变蛋白，相对于野生型IL-2，包含：
(i)N88D；
N88R；
N88R+S130R；
F42A+N88R+S127E；或
K35E+N88R+S127E；
和
(ii)B’C’环区序列AGDASIH或AQSKNFH；
以及任选地(iii)T3A。
在一些实施方案中，本发明提供IL-2突变蛋白，其中所述突变蛋白，相对于野生型IL-2，包含：
(i)N88D；
N88R；
N88R+S130R；
F42A+N88R+S127E；或
K35E+N88R+S127E；
和
(ii)B’C’环区序列AGDASIH或AQSKNFH；
以及(iii)T3A。
在一些实施方案中，本发明的IL-2突变蛋白
(i)包含与SEQ ID NO:4的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:4的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与SEQ ID NO:4的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变，
优选地，其中所述突变蛋白包含N88D取代和B’C’环区序列AGDASIH，以及任选地T3A。在一些实施方案中，本发明的IL-2突变蛋白
(i)包含与SEQ ID NO:23的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:23的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与SEQ ID NO:23的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变，
优选地，其中所述突变蛋白包含N88R取代和B’C’环区序列AGDASIH，以及任选地T3A。在一些实施方案中，本发明的IL-2突变蛋白
(i)包含与SEQ ID NO:25的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:25的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与SEQ ID NO:25的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变，
优选地，其中所述突变蛋白包含N88R+S130R取代和B’C’环区序列AGDASIH，以及任选地T3A。
在一些实施方案中，本发明的IL-2突变蛋白
(i)包含与SEQ ID NO:27的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:27的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与SEQ ID NO:27的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变，
优选地，其中所述突变蛋白包含F42A+N88R+S127E取代和B’C’环区序列AQSKNFH， 以及任选地T3A。
在一些实施方案中，本发明的IL-2突变蛋白
(i)包含与SEQ ID NO:29的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:29的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与SEQ ID NO:29的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变，
优选地，其中所述突变蛋白包含F42A+N88R+S127E取代和B’C’环区序列AGDASIH，以及任选地T3A。
在一些实施方案中，本发明的IL-2突变蛋白
(i)包含与SEQ ID NO:31的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:31的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与SEQ ID NO:31的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变，
优选地，其中所述突变蛋白包含K35E+N88R+S127E取代和B’C’环区序列AQSKNFH，以及任选地T3A。
关于适用于本发明的突变以及突变组合，也可以参见本申请人的共同待决申请PCT/CN2021/081840。该申请整体并入本文作为参考。
IL-2突变蛋白与野生型蛋白之间的序列差异性可以用序列同一性表述，也可以用两者之间差异氨基酸的数量来表达。在一个实施方案中，IL-2突变蛋白与野生型蛋白之间具有至少85％，86％,87％,88％,89％同一性，优选90％以上同一性，优选95％，但优选不超过97％，更优选不超过96％同一性。在另一实施方案中，除了本发明的上述突变外，IL-2突变蛋白与野生型蛋白之间还可以具有不超过15个，例如1-10个，或1-5个突变，例如，0,1,2，3,4个突变。在一个实施方案中，所述其余突变可以是保守取代。
III.融合蛋白和IL-2-Fc二聚体蛋白
在一个方面，本发明还提供包含本发明IL-2突变蛋白的融合蛋白。在一个优选的实施方案中，本发明IL-突变蛋白与可以赋予改善的药代动力学性质的另一多肽融合，例如清蛋白，更优选抗体Fc片段。
在一个实施方案中，本发明提供IL-2突变蛋白融合蛋白，其包含与抗体Fc片段融合的本发明IL-2突变蛋白。
用于本发明的Fc片段可以包含减小或去除效应子功能的突变。在一个优选的实施方案中， Fc片段具有减少的由Fc区介导的效应子功能，例如减少的或消除的ADCC和/或ADCP和/或CDC效应子功能。例如，在一些特别的实施方案中，用于本发明的Fc片段具有降低与Fcγ受体结合的L234A/L235A突变或L234A/L235E/G237A。
在再一优选的实施方案中，Fc片段可以具有导致增加的血清半衰期的突变，例如改善Fc片段与FcRn结合的突变。
在一些实施方案中，与IL-2突变蛋白融合的Fc片段为人的IgG Fc，例如，人IgG1Fc,人IgG2Fc,或人IgG4Fc。在一个实施方案中，Fc片段包含氨基酸序列SEQ ID NO:6或12或42或43或与其具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性或由其组成。
在一些实施方案中，IL-2突变蛋白直接或通过接头与Fc融合。在一些实施方案中，可以选择接头以提高Fc融合蛋白对于CD25 -T细胞的激活作用。在一个实施方案中，接头是(GGGGS) n或GSGS，更优选为(GGGGS) 2。
在再一方面，本发明也提供包含与Fc片段融合的本发明IL-2突变蛋白的二聚体分子。这样的二聚体分子将分子量提高到60-80KDa,极大地减小了肾清除,而且通过FcRn介导的体内循环回收,能进一步的延长IL2-Fc融合蛋白的半衰期。
在一些实施方案中，本发明提供IL-2-Fc二聚体蛋白，其是同二聚体，其中第一单体和第二单体分别由自N端到C端包含：i)IL-2突变蛋白；ii)接头；和iii)Fc片段。
在另一些实施方案中，本发明提供IL-2-Fc二聚体蛋白，其是异二聚体，包含：
a)第一单体，其中所述第一单体自N端到C端包含：i)IL-2突变蛋白；ii)接头；和iii)第一Fc片段；和
b)第二单体，其中所述第二单体包含第二Fc片段。
在一些实施方案中，第一Fc片段和第二Fc片段分别包含促进第一单体与第二单体形成异二聚体的第一和第二异二聚化突变。在一些优选实施方案中，第一和第二异二聚化突变包含Knob:Hole突变组合，例如突变组合T366W/S354C:Y349C/T366S/L368A/Y407V。
在一些优选实施方案中，第一Fc片段上的第一异二聚体突变包含Knob突变，第二Fc片段的第二异二聚体突变包含Hole突变；或者，第一Fc片段上的第一异二聚体突变包含Hole突变，第二Fc片段的第二异二聚体突变包含Knob突变。
如本领域技术人员理解的，适用于本发明融合蛋白和二聚体分子的Fc片段可以是任何抗体Fc片段。在一个实施方案中，本发明的Fc片段是效应物功能沉默的。
在一个实施方案中，在一个或多个选自以下的特性上修饰Fc片段：Fc区的效应子功能和Fc区的补体激活功能。在一个实施方案中，所述效应子功能或补体激活功能相对于相同同种型的野生型Fc区已经被降低或消除。在一个实施方案中，通过选自以下的方法来降低或消除效应子功能：降低Fc区的糖基化、使用天然具有降低或消除的效应子功能的Fc同种型、和Fc区修饰。
在一个实施方案中，通过降低Fc区的糖基化来降低或消除效应子功能。在一个实施方案中，通过选自以下的方法来降低Fc区的糖基化：在不允许野生型糖基化的环境中生产本发明的融合蛋白或二聚体分子；除去Fc区上已经存在的碳水化合物基团；和修饰Fc区使得不发生野生型糖基化。在一个实施方案中，通过修饰使得不发生野生型糖基化的方法来降低Fc区的糖基化，如在Fc区的位置297包含突变，使得该位置的野生型天冬酰胺残基被另一个干扰 该位置糖基化的氨基酸替代，例如N297A突变。
在一个实施方案中，通过至少一个Fc区修饰来降低或消除效应子功能。在一个实施方案中，至少一个Fc区修饰选自：选自以下位置的、损害与一个或多个Fc受体的结合的Fc区点突变：238、239、248、249、252、254、265、268、269、270、272、278、289、292、293、294、295、296、297、298、301、303、322、324、327、329、333、335、338、340、373、376、382、388、389、414、416、419、434、435、437、438和439；选自以下位置的损害C1q结合的Fc区点突变：270、322、329和321；以及CH1结构域的位置132的点突变。在一个实施方案中，通过Fc区的点突变L234A&L235A(即LALA突变)降低或消除效应子功能。在一个实施方案中，所述修饰是选自以下位置的损害C1q结合的Fc区点突变：270、322、329和321。在另一个实施方案中，所述修饰是消除一些Fc区。
如本领域技术人员理解的，为了促进本发明的异二聚体形成，本发明二聚体分子的Fc片段可以包含利于第一单体与第二单体二聚化的突变。优选地，基于Knob-in-Hole技术，在第一单体和第二单体中引入相应的Knob突变和Hole突变。
因此，在一个实施方案中，本发明的二聚体分子包含:
i)任选具有突变P329G、L234A和L235A或具有突变L234A和L235A的、人IgG1亚类的同二聚Fc-区，或
ii)任选具有突变P329G、S228P和L235E的、人IgG4亚类的同二聚Fc-区，或
iii)异二聚Fc-区，其中
a)一个Fc-区多肽包含突变T366W，而另一个Fc-区多肽包含突变T366S、L368A和Y407V，或
b)一个Fc-区多肽包含突变T366W和Y349C，而另一个Fc-区多肽包含突变T366S、L368A、Y407V和S354C，或
c)一个Fc-区多肽包含突变T366W和S354C，而另一个Fc-区多肽包含突变T366S、L368A、Y407V和Y349C，
或
iv)人IgG4亚类的异二聚Fc-区，其中两个Fc-区多肽都包含突变P329G、L234A和L235A，且
a)一个Fc-区多肽包含突变T366W，而另一个Fc-区多肽包含突变T366S、L368A和Y407V，或
b)一个Fc-区多肽包含突变T366W和Y349C，而另一个Fc-区多肽包含突变T366S、L368A、Y407V和S354C，或
c)一个Fc-区多肽包含突变T366W和S354C，而另一个Fc-区多肽包含突变T366S、L368A、Y407V和Y349C，
或
v)人IgG4亚类的异二聚Fc-区，其中两个Fc-区多肽都包含突变P329G、S228P和L235E，且
a)一个Fc-区多肽包含突变T366W，而另一个Fc-区多肽包含突变T366S、L368A和Y407V，或
b)一个Fc-区多肽包含突变T366W和Y349C，而另一个Fc-区多肽包含突变T366S、L368A、Y407V和S354C，或
c)一个Fc-区多肽包含突变T366W和S354C，而另一个Fc-区多肽包含突变T366S、L368A、Y407V和Y349C。
在一些实施方案中,Fc区还包含其他利于异二聚体纯化的突变。例如，可以将H435R突变(Eric J.Smith,，Scientific Reports|5:17943|DOI:10.1038/srep17943)引入异二聚体的Fc区之一中(例如，带Hole突变的Fc区)，以利于使用蛋白A纯化异二聚体。在另一些实施方案中，对于包含铰链区的异二聚体单体，也可以在铰链区中引入突变，例如C220S，以利于异二聚体的形成。
适用于本发明的融合蛋白的Fc区也可用于本发明的免疫缀合物中的Fc部分。
在一些实施方案中，与Fc区融合的IL-2突变蛋白
(i)包含与SEQ ID NO:7、24、26、28、30或32的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:7、24、26、28、30或32的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与SEQ ID NO:7、24、26、28、30或32的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变，
其中所述IL-2突变蛋白包含本文所述的突变。
在一些实施方案中，本发明的与Fc融合的IL-2突变蛋白
(i)包含与SEQ ID NO:7的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:7的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与SEQ ID NO:7的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变，
优选地，其中所述突变蛋白包含N88D取代和B’C’环区序列AGDASIH，以及任选地T3A。
在一些实施方案中，本发明的与Fc融合的IL-2突变蛋白
(i)包含与SEQ ID NO:24的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:24的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与SEQ ID NO:24的氨基酸序列相比具有1个或多个(优选不超过10个，更优 选不超过5、4、3、2、1个)的氨基酸改变，
优选地，其中所述突变蛋白包含N88R取代和B’C’环区序列AGDASIH，以及任选地T3A。
在一些实施方案中，本发明的与Fc融合的IL-2突变蛋白
(i)包含与SEQ ID NO:26的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:26的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与SEQ ID NO:26的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变，
优选地，其中所述突变蛋白包含N88R+S130R取代和B’C’环区序列AGDASIH，以及任选地T3A。
在一些实施方案中，本发明的与Fc融合的IL-2突变蛋白
(i)包含与SEQ ID NO:28的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:28的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与SEQ ID NO:28的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变，
优选地，其中所述突变蛋白包含F42A+N88R+S127E取代和B’C’环区序列AQSKNFH，以及任选地T3A。
在一些实施方案中，本发明的与Fc融合的IL-2突变蛋白
(i)包含与SEQ ID NO:30的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:30的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与SEQ ID NO:30的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变，
优选地，其中所述突变蛋白包含F42A+N88R+S127E取代和B’C’环区序列AGDASIH，以及任选地T3A。
在一些实施方案中，本发明的与Fc融合的IL-2突变蛋白
(i)包含与SEQ ID NO:32的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:32的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与SEQ ID NO:32的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变，
优选地，其中所述突变蛋白包含K35E+N88R+S127E取代和B’C’环区序列AQSKNFH，以及任选地T3A。
如本领域技术人员明了的，适用于本发明融合蛋白和二聚体分子中连接IL-2突变蛋白和Fc片段的接头可以是本领域已知的任何接头。在一些实施方案中，接头可以包含IgG1铰链，或可以包含选自以下的接头序列：(GS)n,(GSGGS)n,(GGGGS)n,and(GGGS)n，其中n是至少1的整数。优选地，接头包含(G 4S) 2，即GGGGSGGGGS(SEQ ID NO:5)。
IV.免疫缀合物
本发明还提供免疫缀合物，其包含本发明的IL2突变蛋白或IL-2突变蛋白融合蛋白(例如与Fc片段融合的融合蛋白)和抗原结合分子。优选地，抗原结合分子是免疫球蛋白分子，特别是IgG分子，或抗体或抗体片段，特别是Fab分子和scFv分子或半抗体(包含一条重链和一条轻链，或由一条重链和一条轻链组成)。
在一些实施方案中，所述抗原结合分子特异性结合肿瘤细胞上或肿瘤环境中呈现的抗原，特别优选的是PD-1、PD-L1和/或PD-L2。由此，本发明免疫缀合物在施用于受试者体内后可以靶向肿瘤细胞或肿瘤环境，从而提供进一步的治疗益处，例如以更低的剂量进行治疗的可行性和由此带来的低副作用；增强的免疫治疗效应或抗肿瘤效应等。
在本发明的免疫缀合物中，本发明IL-2突变蛋白可以直接或通过接头与另一分子或抗原结合分子连接，且在一些实施方案中，在两者之间包含蛋白水解切割位点。在本发明的免疫缀合物中，本发明的IL-2突变蛋白或其融合蛋白还可以通过二聚化与另一分子或抗原结合分子连接。
在一些实施方案中，所述抗体是针对肿瘤相关抗原的抗体，例如PD-1、PD-L1或PD-L2。
适用于与IL-2突变蛋白连接的抗体可以是完整抗体，或其抗原结合片段。在一些实施方案中，本发明的抗体是IgG1形式的抗体或IgG2形式的抗体或IgG3形式的抗体或IgG4形式的抗体，优选的，为IgG1形式的抗体。在一些实施方案中，本发明的抗体是单克隆抗体。在一些实施方案中，本发明的抗体是人源化的。在一些实施方案中，本发明的抗体是人抗体。在一些实施方案中，本发明的抗体是嵌合抗体。在一个实施方案中，本发明的抗体的抗原结合片段选自以下的抗体片段：Fab、Fab’、Fab’-SH、Fv、单链抗体(例如scFv)、(Fab’)2、单结构域抗体例如VHH、dAb(domain antibody)或线性抗体或半抗体。
在本发明的一个实施方案中，本发明的免疫缀合物包含IL-2突变蛋白或其融合蛋白与抗PD-1抗体或其抗原结合片段。
在本发明的一个实施方案中，本发明的免疫缀合物包含：
第一单体，其包含与Fc片段融合的IL-2突变蛋白；和
第二单体，其包含特异性结合PD-1的抗体或其片段，优选地，包含所述抗PD-1抗体的一条重链和一条轻链的片段。
在一些实施方案中，第一单体的Fc片段中包含Knob突变，第二单体的抗体重链中包含hole突变，反之亦然。在一些实施方案中，Knob突变为Knob：S354C&T366W，和/或Hole突变为Y349C&T366S&L368A&Y407V。
在一个具体实施方案中，本发明的IL-2突变蛋白融合蛋白具有如图1A的Format 1所示的形式。
在本发明的一个实施方案中，所述针对PD-1的抗体或其抗原结合片段为WO2017024465A1中公开的抗PD-1抗体或其抗原结合片段。在一个实施方案中，所述抗PD-1抗体或其抗原结合片段包含WO2017024465A1中公开的抗PD-1抗体或其抗原结合片段的一个或多个CDR(优选3个CDR，即HCDR1、HCDR2H和HCDR3；或LCDR1、LCDR2和LCDR3，更优选6个CDR，即HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3)，或包含WO2017024465A1中公开的抗PD-1抗体或其抗原结合片段的VH和/或VL，或包含所述抗体的重链和/或轻链。
在一些实施方案中，所述抗PD-1抗体或其抗原结合片段包含3个来自重链可变区的互补决定区(HCDR)，HCDR1、HCDR2和HCDR3。在一些实施方案中，所述抗PD-1抗体或其抗原结合片段包含3个来自轻链可变区的互补决定区(LCDR)，LCDR1、LCDR2和LCDR3。在一些实施方案中，所述抗PD-1抗体或其抗原结合片段包含3个来自重链可变区的互补决定区(HCDR)和3个来自轻链可变区的互补决定区(LCDR)。
在一些方面中，所述抗PD-1抗体或其抗原结合片段包含重链可变区(VH)。在一些方面中，所述抗PD-1抗体或其抗原结合片段包含轻链可变区(VH)。在一些方面中，所述抗PD-1抗体或其抗原结合片段包含重链可变区(VH)和轻链可变区(VL)。在一些实施方案中，所述重链可变区包含3个来自重链可变区的互补决定区(CDR)，HCDR1、HCDR2和HCDR3。在一些实施方案中，所述轻链可变区包含3个来自轻链可变区的互补决定区(CDR)，LCDR1、LCDR2和LCDR3。
在一些实施方案中，所述抗PD-1抗体或其抗原结合片段包含抗体重链。在一些实施方案中，所述抗PD-1抗体重链包含重链可变区和重链恒定区。在一些实施方案中，本发明抗PD-1抗体或其抗原结合片段包含抗体轻链。在一些实施方案中，本发明的抗PD-1抗体轻链包含轻链可变区和轻链恒定区。在一些实施方案中，本发明抗PD-1抗体或其抗原结合片段还包含重链和轻链。
在一些实施方案中，所述重链可变区VH
(i)包含与SEQ ID NO:8的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:8的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与SEQ ID NO:8的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列由所述氨基酸序列组成，优选地，所述氨基酸改变不发生在CDR区中。
在一些实施方案中，所述轻链可变区VL
(i)包含与SEQ ID NO:15的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:15的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与SEQ ID NO:15的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列由所述氨基酸序列组成，优选地，所述氨基酸改变不发生在CDR区中。
在一些实施方案中，所述3个来自重链可变区的互补决定区(HCDR)，HCDR1、HCDR2和HCDR3选自
(i)如SEQ ID NO:8所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，或
(ii)相对于(i)中的序列，在所述三个HCDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列。
在一些实施方案中，所述3个来自轻链可变区的互补决定区(LCDR)，LCDR1、LCDR2和LCDR3选自
(i)如SEQ ID NO:15所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3，或
(ii)相对于(i)中的序列，在所述三个LCDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列。
在一些实施方案中，HCDR1包含SEQ ID NO:9的氨基酸序列，或由所述氨基酸序列组成，或者HCDR1包含与SEQ ID NO:9的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，HCDR2包含SEQ ID NO:10的氨基酸序列，或由所述氨基酸序列组成，或者HCDR2包含与SEQ ID NO:10的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，HCDR3包含SEQ ID NO:11的氨基酸序列，或由所述氨基酸序列组成，或者HCDR3包含与SEQ ID NO:11的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，LCDR1包含SEQ ID NO:16的氨基酸序列，或由所述氨基酸序列组成，或者LCDR1包含与SEQ ID NO:16的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，LCDR2包含SEQ ID NO:17的氨基酸序列，或由所述氨基酸序列组成，或者LCDR2包含与SEQ ID NO:17的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，LCDR3包含SEQ ID NO:18的氨基酸序列，或由所述氨基酸序列组成，或者LCDR3包含与SEQ ID NO:18的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，所述抗PD-1抗体或其抗原结合片段重链恒定区HC为IgG1、IgG2、IgG3或IgG4的重链恒定区，优选的IgG1的重链恒定区，例如带有L234A&L235A(LALA)突变的IgG1的重链恒定区。在一些实施方案中，向重链恒定区中引入knob-ino-hole的突变，例如引入S354C和T366W的突变，获得包含knob突变的抗体重链，和/或引入Y349C&T366S&L368A&Y407V的突变，获得包含hole突变的抗体重链。在一些实施方案中，所述抗PD-1抗体或其抗原结合片段轻链恒定区为lambda或Kappa轻链恒定区。
在一些实施方案中，所述抗体或其抗原结合片段的重链恒定区
(i)包含与选自SEQ ID NO:21的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；
(ii)包含选自SEQ ID NO:21的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与选自SEQ ID NO:21的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由所述氨基酸序列组成。
在一些实施方案中，所述抗体或其抗原结合片段的包含hole突变的重链恒定区
(i)包含与选自SEQ ID NO:13的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；
(ii)包含选自SEQ ID NO:13的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与选自SEQ ID NO:13的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由所述氨基酸序列组成。
在一些实施方案中，所述抗体或其抗原结合片段的包含hole突变的重链
(i)包含与选自SEQ ID NO:14的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；
(ii)包含选自SEQ ID NO:14的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与选自SEQ ID NO:14的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由所述氨基酸序列组成。
在一些实施方案中，所述抗体或其抗原结合片段的的重链
(i)包含与选自SEQ ID NO:22的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；
(ii)包含选自SEQ ID NO:22的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与选自SEQ ID NO:22的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由所述氨基酸序列组成。
在一些实施方案中，所述抗体或其抗原结合片段的轻链恒定区
(i)包含与选自SEQ ID NO:19的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；
(ii)包含选自SEQ ID NO:19的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与选自SEQ ID NO:19的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由所述氨基酸序列组成。
在一些实施方案中，所述抗体或其抗原结合片段的轻链
(i)包含与选自SEQ ID NO:20的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；
(ii)包含选自SEQ ID NO:20的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与选自SEQ ID NO:20的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由所述氨基酸序列组成。
在本发明的一些具体实施方案中，所述抗PD-1抗体或其抗原结合片段包含：
如SEQ ID NO:8所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:15所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3。
在本发明的一些具体实施方案中，所述抗PD-1抗体或其抗原结合片段包含：
分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO:9、10和11，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3:SEQ ID NO:16、17和18。
在本发明的一些具体实施方案中，所述抗PD-1抗体或其抗原结合片段包含：
包含SEQ ID NO:8所示的氨基酸序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO:15所示的氨基酸序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的VL。
在本发明的一些具体的实施方案中，所述抗PD-1抗体或其抗原结合片段包含
包含SEQ ID NO:14或22所示的氨基酸序列或与其具有至85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的重链；和包含SEQ ID NO:20所示的氨基酸序列或与其具有至85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的轻链。
在一些优选的实施方案中，所述免疫缀合物中的抗PD-1抗体片段包含一条重链和一条轻链或由其组成。
在本发明的一些具体的实施方案中，所述抗PD-1抗体片段包含
包含SEQ ID NO:14所示的氨基酸序列或与其具有至85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的包含hole突变的重链；
和包含SEQ ID NO:20所示的氨基酸序列或与其具有至85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的轻链。
本发明的免疫缀合物，优选地相比抗PD-1抗体、相比IL-2突变蛋白或其与Fc的融合蛋白，和/或相比已知的包含抗PD-1抗体和IL-2突变蛋白的免疫缀合物，具有如下一个或多个或全部性质：
(1)具有降低的或消除的与IL-2Rα结合的亲和力，特别是相比野生型IL-2或其融合蛋白；
(2)具有降低的与IL-2Rβγ结合的亲和力，特别是相比IL-2突变蛋白或其与Fc的融合蛋白，和/或相比已知的包含抗PD-1抗体和IL-2突变蛋白的免疫缀合物；
(3)能够选择性激活表达PD-1的细胞，即对PD-1具有高度选择性；
(4)在不表达PD-1的T细胞(例如CD8+或CD4+T细胞)中的活性较低，而在表达PD-1的T细胞(例如CD8+或CD4+T细胞)中活性较高，表明其对PD-1具有高选择性，特别是相比已知的包含抗PD-1抗体和IL-2突变蛋白的免疫缀合物；
(5)在表达(例如过表达)IL-2受体的细胞中IL-2的活性更弱，特别是相比已知的包含抗PD-1抗体和IL-2突变蛋白的免疫缀合物；
(6)在表达IL-2受体和PD-1的细胞中，相比表达IL-2受体但是不表达PD-1的细胞中，具有更强的IL-2活性，表明对PD-1阳性细胞的选择性；
(7)优选地相比抗PD-1抗体、相比IL-2突变蛋白或其与Fc的融合蛋白，或相比抗PD-1抗体与IL-2突变蛋白或其融合蛋白的联合，和/或相比已知的包含抗PD-1抗体和IL-2突变蛋白的免疫缀合物，具有更强的抗肿瘤作用，和/或更低的毒性(例如不影响或较低影响被治疗对象的体重)。
V.多核苷酸、载体和宿主
本发明提供编码以上任何IL-2突变蛋白或融合蛋白或二聚体分子或缀合物中的任一条链或任何单体或结构域的核酸。可以采用本领域熟知的方法，通过从头固相DNA合成或通过PCR诱变编码野生型IL-2的现有序列，产生编码本发明突变蛋白的多核苷酸序列。此外，本发明的多核苷酸和核酸可以包含编码分泌信号肽的区段，并与编码本发明突变蛋白的区段可操作连接，从而可以指导本发明突变蛋白的分泌性表达。
本发明也提供包含本发明核酸的载体。在一个实施方案中，载体是表达载体，例如真核表达载体。载体包括但不限于病毒、质粒、粘粒、λ噬菌体或酵母人工染色体(YAC)。在优选的实施方案中，本发明的表达载体是pYDO_017表达载体。
本发明也提供包含所述核酸或所述载体的宿主细胞。适用于复制并支持突变IL-2蛋白或融合物或二聚体或免疫缀合物表达的宿主细胞是本领域中公知的。可以用特定的表达载体转染或转导这类细胞，并且可以生长大量的含载体细胞以用于接种大规模发酵罐，从而获得充足量的IL-2突变体或融合物或二聚体或免疫缀合物用于临床应用。在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞(例如CHO细胞或293细胞)。可用的哺乳动物宿主细胞系的例子是由SV40转化的猴肾CV1系(COS-7)；人胚胎肾系(293或293T细胞，如例如记载于Graham等,JGenVirol36,59(1977))、幼仑鼠肾细胞(BHK)、小鼠塞托利(sertoli)细胞(TM4细胞，如例如记载于Mather,BiolReprod23,243-251(1980))、猴肾细胞(CV1)、非洲绿猴肾细胞(VERO-76)、人宫颈癌细胞(HELA)、犬肾细胞(MDCK)，buffalo大鼠肝细胞(BRL3A)、人肺细胞(W138)、人肝细胞(HepG2)、小鼠乳房肿瘤细胞(MMT060562)、TRI细胞(如例如记载于Mather等,AnnalsN.Y.AcadSci383,44-68(1982))、MRC5细胞和FS4细胞。其它可用的哺乳动物宿主细胞系包括中国仓鼠卵巢(CHO)细胞，包括dhfr-CHO细胞(Urlaub等,ProcNatlAcadSciUSA77,4216(1980))；和骨髓瘤细胞系如YO、NS0、P3X63和Sp2/0。在一个实施方案中，宿主细胞是真核生物细胞，优选为哺乳动物细胞如中国仓鼠卵巢(CHO)细胞、人胚胎肾(HEK)细胞或淋巴细胞(例如Y0、NS0、Sp20细胞)。
VI.制备方法
再一方面，本发明提供制备本发明IL-2突变蛋白或融合物或二聚体或缀合物的方法，其中所述方法包括，在适合IL-2突变蛋白或融合物或二聚体或缀合物表达的条件下，培养包含编码所述蛋白或融合物或二聚体或缀合物的核酸的宿主细胞，如上文所提供的，和任选地从所述宿主细胞(或宿主细胞培养基)回收所述蛋白或融合物或二聚体或缀合物。
在一个实施方案中，将包含编码IL-2突变蛋白的核酸的载体转入细胞中使其表达，随后收集细胞(或细胞培养上清)，提取所述IL-2突变蛋白，并进行纯化，以获得所述IL-2突变蛋白。在一个具体的实施方案中，所述纯化方法是亲和纯化方法。在另一个具体的实施方案中，所述纯化方法是离子交换纯化。
在一个实施方案中，将包含编码与Fc融合的IL-2突变蛋白的核酸的载体转入细胞中使其表达，随后收集细胞(或细胞培养上清)，提取所述Fc融合的IL-2突变蛋白，并进行纯化，以获得所述Fc融合的IL-2突变蛋白。在一个具体的实施方案中，所述纯化方法是亲和纯化方法。在另一个具体的实施方案中，所述纯化方法是离子交换纯化。
在一个实施方案中，将包含编码与Fc融合的IL-2突变蛋白的核酸、编码PD-1抗体重链的核酸和编码PD-1抗体轻链的核酸的载体转入细胞中，使其分别表达并组装为免疫缀合物，随后收集细胞(或细胞培养上清)，提取所述免疫缀合物，并进行纯化，以获得所述免疫缀合物。在一个具体的实施方案中，所述纯化方法是亲和纯化方法。在另一个具体的实施方案中，所述纯化方法是离子交换纯化。
VII.测定法
可以通过本领域中已知的多种测定法对本文中提供IL-2突变蛋白进行鉴定，筛选，或表征其物理/化学特性和/或生物学活性。
一方面，可以对本发明的IL-2突变蛋白，测试其与IL-2受体的结合活性。例如，可以通过本领域已知的方法，诸如ELISA，Western印迹等，或本文实施例公开的例示性方法，来测定与人IL-2Rα或β蛋白或IL-2Rβγ或IL-2Rαβγ的结合。例如，可以使用流式细胞术进行测定，其中使经转染在细胞表面上表达突变蛋白的细胞例如酵母展示细胞，与标记的(例如生物素标记的)IL-2Rα或β蛋白或IL-2Rβγ或IL-2Rαβγ复合物进行反应。备选地，突变蛋白与受体的结合，包括结合动力学(例如K D值)，可以使用IL-2-Fc融合物或二聚体分子形式，在SPR测定法中测定。
再一方面，可以通过测定在受体结合下游发生的信号传导和/或免疫激活效应。来间接测量IL-2突变蛋白结合IL-2受体的能力。
因此，在一些实施方案中，提供了用于鉴定具有生物学活性的突变IL-2蛋白的测定法。生物学活性可以包括，例如诱导具有IL-2受体的T和/或NK细胞和/或Treg细胞增殖的能力、诱导具有IL-2受体的T和/或NK细胞和/或Treg细胞中IL-2信号传导的能力、降低的诱导T细胞中细胞凋亡的能力、诱导肿瘤消退和/或改善存活的能力、和降低的体内毒性性质，例如降低的血管通透性。本发明也提供体内和/或体外具有这类生物学活性的突变IL-2蛋白、其Fc融合物和包含其的二聚体分子。
本领域中公知多种方法可以用于测定IL-2的生物学活性。例如，用于测试本发明IL-2突变蛋白(例如以二聚体分子形式)刺激NK细胞生成IFN-γ的能力的合适测定法，可以包括如下步骤：将培养的NK细胞与本发明的突变IL-2蛋白温育，并随后通过ELISA测量培养基中的IFN-γ浓度。IL-2信号传导诱导数个信号传导途径，并且牵涉JAK(Janus激酶)和STAT(信号转导物和转录的激活剂)信号传导分子。
IL-2与受体β和γ亚基的相互作用导致受体以及JAK1和JAK3(分别与β和γ亚基结合)的磷酸化。然后，STAT5与磷酸化受体结合，并自身在非常重要的酪氨酸残基上磷酸化。这导致STAT5从受体解离、STAT5二聚化以及STAT5二聚体移位至细胞核，在该处它们促进靶基因的转录。由此，可以例如通过测量STAT5的磷酸化，评估突变体IL-2多肽经由IL-2受体诱导信号传导的能力。此方法的详情已经披露在实施例中。例如，可以将PBMC用本发明的突变体IL-2多肽或融合物或二聚体或免疫缀合物处理，并通过流式细胞术测定磷酸化STAT5的水平。
在本发明的与针对抗原的抗体的免疫缀合物的情况下，可以通过在表达抗原的细胞中测定IL-2的上述活性或水平。
再有，可以在多种本领域中已知的动物肿瘤模型中评估突变的IL-2或其融合物或二聚体或免疫缀合物对肿瘤生长和存活的影响。例如，可以将癌症细胞系的异种移植物植入免疫缺陷型小鼠，并用本发明的突变体IL-2多肽或融合物或二聚体或免疫缀合物处理。可以基于肿瘤抑制率(例如，相对于同种型对照抗体计算)，检测本发明的突变体IL-2多肽、融合物、二聚体分子和免疫缀合物的体内抗肿瘤效应。此外，可以基于动物的体重变化(例如，相对于给药前，绝对体重的变化或体重变化百分比)来来测定本发明的突变体IL-2多肽、融合物、二聚体分子和免疫缀合物在体内的毒性。也可以基于死亡率、生命期观察(不良作用的可见症状，例如行为、体重、体温)以及临床和解剖病理学(例如测量血液化学值和/或组织病理学分析)来测定所述体内毒性。
再一方面，可以通过本领域已知的方法，表征本发明突变蛋白的成药性能，例如，表达量和产品的纯度。对于表达量的测定，当突变蛋白自培养细胞中分泌表达到培养上清液中时，可以对离心收集的细胞培养液进行蛋白含量的测定。或者，可以在对收集的细胞培养液进行一个步骤的纯化后，例如在一步亲和层析纯化后，进行测定。对于产品纯度的测定，可以在对收获的生产细胞的培养上清液实施一步亲和层析纯化后，进行纯度确定，以检测突变蛋白的纯化性能。优选地，本发明突变蛋白，按照此一步亲和层析纯化后，具有显著优于野生型蛋白的纯度，表明本发明突变蛋白具有更好的纯化性能。纯度确定方法可以是本领域已知的任何常规方法，包括但不限于，SEC-HPLC法。
再一方面，可以通过本领域已知的方法，表征本发明突变体IL-2多肽或融合物或二聚体或免疫缀合物的药代动力学性质，例如半衰期。
VIII.药物组合物和药物制剂
本发明还包括包含突变体IL-2多肽或融合物或二聚体或免疫缀合物的组合物(包括药物组合物或药物制剂)和包含编码突变体IL-2多肽或融合物或二聚体或免疫缀合物的多核苷酸的组合物。这些组合物还可以任选地包含合适的药用辅料，如本领域中已知的药用载体、药用赋形剂，包括缓冲剂。
可以通过常规的混合,溶解,乳化,包囊,包载或冻干过程来制备包含本发明的突变体IL-2多肽或融合物或二聚体或免疫缀合物的药学组合物。可以以常规方式配制药学组合物,其使用一种或多种有助于将蛋白质加工成可药学使用的制剂的生理学可接受载体,稀释剂,赋形剂或辅助剂。合适的配制剂依赖于选择的施用路径。
可以将免疫缀合物以游离酸或碱,中性或盐形式配制成组合物。药学可接受盐是基本保留游离酸或碱的生物学活性的盐。这些包括酸加成盐(acid addition salt),例如与蛋白质性组合物的游离氨基基团形成的那些,或与无机酸(如例如氢氯酸或磷酸)或与有机酸如乙酸,草酸,酒石酸或扁桃酸形成的。与游离羧基基团形成的盐还可以自无机碱如例如氢氧化钠,钾,铵,钙或铁；或有机碱如异丙胺,三甲胺,组氨酸或普鲁卡因(procaine)衍生。药用盐倾向于比相应的游离碱形式更可溶于水性溶剂和其它质子溶剂中。
IX.组合产品
在一方面，本发明还提供了组合产品，其包含本发明的突变体IL-2多肽或融合物或二聚体或免疫缀合物，以及一种或多种其它治疗剂(例如化疗剂、其他抗体、细胞毒性剂、疫苗、抗感染活性剂等)。本发明的组合产品可用于本发明的治疗方法中。
在一些实施方案中，所述组合产品用于预防或治疗癌症。
X.治疗方法和用途
在一方面中，本发明涉及预防或治疗受试者疾病，如癌症的方法，所述方法包括向所述受试者施用有效量的本文所述的任何突变体IL-2多肽或融合物或二聚体或免疫缀合物。癌症可以处于早期、中期或晚期或是转移性癌。在一些实施方案中，癌症可以是实体肿瘤或血液肿瘤。在一些实施方案中，所述癌症是胃肠道肿瘤或黑素瘤，例如结肠癌或结直肠癌。在一些实施方案中，所述肿瘤是对已知药物，例如已知抗PD-1抗体具有耐受性的肿瘤或癌症，例如难治性肿瘤或癌症。
在一些实施方案中，所述癌症是表征为具有升高的PD-1、PD-L1和/或PD-L2的蛋白质水平和/或核酸水平(例如表达升高)的癌症。在一些实施方案中，本发明的突变体IL-2多肽或融合物或二聚体或免疫缀合物能够用于刺激宿主的免疫系统，例如增强细胞的免疫应答。。依照任何上述实施方案的“刺激免疫系统”可包括免疫功能整体升高,T细胞功能升高,B细胞功能升高,淋巴细胞功能恢复,IL-2受体表达升高,T细胞响应性升高,天然杀伤细胞活性或淋巴因子激活的杀伤(LAK)细胞活性升高,等等中任一项或多项。
本发明的突变体IL-2多肽或融合物或二聚体或免疫缀合物(以及包含其的药物组合物，其任选地另外的治疗剂)可以通过任何合适的方法给药，包括肠胃外给药，肺内给药和鼻内给药，并且，如果局部治疗需要，病灶内给药。肠胃外输注包括肌内、静脉内、动脉内、腹膜内或皮下给药。在一定程度上根据用药是短期或长期性而定，可通过任何适合途径，例如通过注射，例如静脉内或皮下注射用药。本文中涵盖各种用药时程，包括，但不限于，单次给药或在多个时间点多次给药、推注给药及脉冲输注。
为了预防或治疗疾病，本发明的突变体IL-2多肽或融合物或二聚体或免疫缀合物的合适剂量(当单独或与一种或多种其他的治疗剂组合使用时)将取决于待治疗疾病的类型、抗体的类型、疾病的严重性和进程、以预防目的施用还是以治疗目的施用、以前的治疗、患者的临床病史和对所述抗体的应答，和主治医师的判断力。所述抗体以一次治疗或经过一系列治疗合适地施用于患者。在一些实施方案中，本发明的突变体IL-2多肽或融合蛋白或二聚体或免疫缀合物可以以较高的剂量施用于患者而不会产生毒性。
再一方面，本发明也提供本发明突变体IL-2多肽或融合物或二聚体或免疫缀合物在制备用于前述方法(例如用于治疗)的药物中的用途。
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成限制本发明的保护范围。
本发明的这些以及其它方面和实施方案在附图(附图简述紧随其后)和以下的发明详述中得到描述并且示例于以下实施例中。上文以及整个本申请中所论述的任何或所有特征可以在本发明的各种实施方案中组合。以下实施例进一步说明本发明，然而，应理解实施例以说明 而非限定的方式来描述，并且本领域技术人员可以进行多种修改。
实施例1.抗-hPD-1与IL-2突变体的免疫缀合物设计
本发明设计了一个可以特异性结合人PD-1阻断PD-1与PD-L1的结合从而解除免疫刹车机制，同时也可以特异性结合T细胞或NK细胞上IL-2受体进而起到激活并扩增T或NK细胞的免疫缀合物分子(αPD-1/IL-2m免疫缀合物)，所述免疫缀合物分子包含抗-PD-1抗体和IL-2突变体，能够增强PD-1抗体免疫治疗的效果。
本发明的免疫缀合物分子形式如图1A所示，包括两部分：1)第二单体：其来自结合PD-1的抗体，所述结合PD-1的抗体序列来源于WO2017024465A1；和2)第一单体：IL-2突变体(IL-2突变蛋白或IL-2m)，所述突变体经过如下改造：根据IL-2与受体复合物的晶体结构2ERJ(图2)，在受体的结合界面选择突变位点，降低IL-2与受体的结合；优化IL-2的B’C’环区序列(A73-R83，野生型为SEQ ID NO:40)改善IL-2的成药性，所述环区优化为应用人IL-15 B’C’环区(AGDASIH，SEQ ID NO:39)替换IL-2的B’C’环区，或对IL-2 B’C’环区的最后4个氨基酸删除，获得截短的IL-2B’C’环区(AQSKNFH，SEQ ID NO:41)。
实施例中应用的对照分子、IL-2-Fc融合蛋白和IL-2突变体-抗PD-1抗体免疫缀合物中的序列信息参见序列表，且IL-2突变信息见下表。
表1IL-2突变体-抗PD-1抗体免疫缀合物和对照分子
Figure PCTCN2022120265-appb-000004
实施例2.IL-2受体和免疫缀合物制备
IL-2受体的表达和纯化
载体构建
将IL-2受体IL-2Rα(Uiprot:P01589,aa22-217)在序列的C末端接上avi标签(GLNDIFEAQKIEWHE，该标签肽可被BirA酶催化发生生物素化)和6个组氨酸标签(HHHHHH)，构建到pTT5载体(Addgene)上，然后转染HEK293细胞表达，通过镍柱(Histrap excel,GE,17-3712-06)亲和纯化得到IL-2Rα。
IL-2Rβγ复合物是基于Knobs in holes的Fc异源二聚，IL-2Rβ的序列被构建到Fc-Knob的N端(SEQ ID NO:37)，IL-2Rγ的序列被构建到Fc-Hole的N端(SEQ ID NO:38)，分别构建到pcDNA3.1载体上，然后在细胞中共转表达。使用瞬时转染的方法将含有IL-2Rβ的载体和IL-2Rγ的载体共转入HEK293细胞中进行表达。首先在超净工作台中准备质粒DNA和转染试剂PEI(Polysciences，23966)，取3mL Opti-MEM培养基(Gibco货号：31985-070)加入50ml离心管中，加入30μg对应质粒的DNA，利用0.22μm的滤头过滤含有质粒的Opti-MEM培养基，随后加入90μg PEI(1g/L)，静置20min。将DNA/PEI混合物轻柔倒入27 mL HEK293细胞并混匀，在37℃，8％CO 2的条件下继续培养6天。获得细胞上清用于纯化得到IL-2Rβγ复合物。镍柱亲和纯化：将纯化使用的镍柱(5ml Histrap excel,GE,17-3712-06)用0.1M NaOH浸泡2h，然后用5-10倍柱体积的超纯水冲洗，去除碱液。纯化前用5倍柱体积的结合缓冲液(20mM Tris pH 7.4,300mM NaCl)平衡纯化柱；将细胞上清通过平衡后的柱子；用10倍柱体积的冲洗缓冲液(20mM Tris 7.4,300mM NaCl,10mM imidazole)通过柱子，去除非特异性结合的杂蛋白；然后用3-5倍柱体积洗脱液(20mM Tris 7.4,300mM NaCl,100mM imidazole)将目标蛋白洗脱下来。将收集的蛋白超滤浓缩交换到PBS(Gibco，70011-044)中，然后用superdex200increase(GE,10/300GL,10245605)进一步分离纯化，收集单体的洗脱峰，柱子的平衡和洗脱缓冲液为PBS。
Mabselect亲和纯化：细胞以13000rpm离心20min，收集上清，用预装柱Hitrap Mabselect Sure纯化上清液。操作如下：纯化前用5倍柱体积的平衡液(0.2M Tris,0.15M NaCl,pH7.2)平衡填料柱；将收集的上清通过柱子，再用10倍柱体积的平衡液清洗填料柱，去除非特异性结合蛋白；用5倍柱体积的洗脱缓冲液(0.1M sodium citrate，pH 3.5)冲洗填料，收集洗脱液；每1ml洗脱液加入80μL Tris(2M Tris)，使用超滤浓缩管交换到PBS缓冲液中，并测定浓度及纯度。
离子交换纯化：通过离子交接层析分离出双特异分子中的异源二聚体分子，去除同源二聚体杂质。
免疫缀合物的制备
抗PD-1抗体的重链Hole和轻链分别构建到pcDNA3.1载体上，IL-2蛋白通过接头连接到IgG1Fc Knob的N末端并构建到pcDNA3.1载体上，将以上三质粒与3倍质量的PEI(如转染3ml HEK293，需要1ug重链Hole+1ug轻链+1ug IL-2m-接头-Fc-Knob+9ug PEI)共转染HEK293细胞，表达制备本实施例中用的各个免疫缀合物分子。
细胞的准备和样品收集纯化与上文受体的制备方法相同。
实施例3.IL-2与受体的亲和力测定
采用表面等离子共振法(SPR)测定本发明免疫缀合物结合人IL-2受体(IL2Rα或IL-2Rβγ)的平衡解离常数(K D)。基于SPR原理，当一束偏振光以一定的角度入射到棱镜端面，在棱镜与金膜的界面将产生表面等离子波，引起金属膜内自由电子产生共振，即表面等离子共振。分析时，先在传感芯片表面固定一层蛋白，然后将待测样品流过芯片表面，若样品中有能够与芯片表面的蛋白相互作用的分子，会引起金膜表面折射率变化，最终导致SPR角变化，通过检测SPR角度变化，获得被分析物的亲和力、动力学常数等信息。
本实施例通过Biacore T200(Cytiva)测定免疫缀合物与IL-2受体的K D，具体方法如下：将含有生物素标签的IL2Rα及IL2Rβγ蛋白分别捕获到偶联有SA(链霉亲和素)芯片表面后，通过检测芯片表面蛋白与流动相中的所研究的免疫缀合物和对照分子之间的结合与解离获得亲和力及动力学常数。
该方法包括芯片制备和亲和力检测。测定过程使用10倍稀释后的10×HBS-EP+(BR-1006-69,Cytiva)作为实验缓冲液。芯片制备过程使用氨基偶联试剂盒(BR-1006-33,Cytiva)，将SA偶联在CM5芯片(29-1496-03,Cytiva)表面，偶联后注入1M乙醇胺，对剩余的活化位点进行封闭。亲和力检测每个循环包括捕获受体、结合一种浓度待研究分子及芯片再生。将梯度稀释后的分子(分子浓度梯度为0-400nM)，以30μl/min的流速流由低浓度到高浓度的顺序流过芯片表面，结合时间180s，解离时间300s。最后使用5mM NaOH(BR-1003-58,Cytiva)对芯片进行再生。数据结果使用Biacore T200分析软件(版本号3.1)，使用分析1:1结合或稳态分析模型进行分析。
Biacore T200(Cytiva，T200)测定待研究的免疫缀合物或对照分子与人PD1(货号： PD1-H5221,ACRO Biosystem)的亲和力，具体方法如下：将待研究分子捕获到偶联有Protein A芯片(29127555,Cytiva)表面后，通过检测芯片表面分子与流动相中的抗原之间的结合与解离获得亲和力及动力学常数。测定过程使用10倍稀释后的10×HBS-EP+(BR-1006-69,Cytiva)作为实验缓冲液。亲和力检测每个循环包括捕获待研究分子、结合一种浓度抗原及芯片再生。将梯度稀释后的抗原(与待研究分子结合时，抗原浓度梯度为0-40nM)，以30μl/min的流速流由低浓度到高浓度的顺序流过芯片表面，结合时间180s，解离时间600s。最后使用10mM Glycine-HCl,pH 1.5(BR-1003-54,Cytiva)对芯片进行再生。数据结果使用Biacore T200分析软件(版本号3.1)，使用1:1结合模型进行分析。
表2和图3分别为免疫缀合物或对照分子与IL-2Rβγ的结合常数和结合曲线，3010为与Fc融合的野生型IL-2序列，序列参见序列表，其亲和力为1.09nM，2061(来源于US20180326010A1)是对照分子，2061与IL-2Rβγ的亲和力为1.48nM，本研究IL-2免疫缀合物的亲和力都要弱于3010和2061。
表3和图4分别为免疫缀合物或对照分子与IL-2Rα的亲和力和结合曲线，3010与IL-2Rα的亲和力为4.38E-08M，2061为不结合，本研究的双特异分子与IL-2Rα的结合要弱于野生型的IL-2，但强于对照分子2061。
表4和图5分别为免疫缀合物或对照分子与人PD1的亲和力和结合曲线，对照分子和本研究的分子都对人PD1有很强的亲和力。
表2：免疫缀合物或对照分子与IL-2Rβγ的结合常数
Figure PCTCN2022120265-appb-000005
表3.免疫缀合物或对照分子与IL-2Rα的亲和力
样品(分子编号) KD(M)
3010 4.38E-08
2061 N.B
2063 3.14E-07
2132 3.84E-07
2149 4.45E-07
2213 Weak binding
2214 Weak binding
2219 5.56E-07
表4.免疫缀合物或对照分子与人PD1的亲和力
样品(分子编 ka(1/Ms) kd(1/s) KD(M)
号)      
2061 4.601E+5 1.755E-4 3.815E-10
2063 3.923E+5 6.189E-5 1.578E-10
2132 4.303E+5 9.091E-5 2.113E-10
2149 4.015E+5 9.708E-5 2.418E-10
2213 4.002E+5 1.052E-4 2.627E-10
2214 4.276E+5 8.897E-5 2.081E-10
2219 4.260E+5 9.967E-5 2.340E-10
实施例4.免疫缀合物的体外活性测定
一、在CTLL2(huPD1-)和CTLL2-hPD-1(huPD1+)中针对PD-1的活性检测
IL-2与CTLL2细胞表面IL-2受体结合，会激活CTLL2JAK-STAT信号通路,引发报告基因信号。在CTLL2细胞系表面过表达人PD-1(hPD-1,uniprot:Q15116)，在hPD-1富集作用下，可以进一步增强CTLL2JAK-STAT信号通路。
CTLL2-hPD-1细胞系构建：
构建和包装Lentvirus+hPD-1慢病毒:
1、6x10^6 293T细胞铺T75培养瓶,以融合率达到75-80％为宜。
2、按如下表所列混合均匀包装体系，室温静置15min。
Figure PCTCN2022120265-appb-000006
3、预先弃去培养皿瓶培养基，加入6ml 10％FBS(PEAK)DMEM(ATCC)新鲜培养基。
4、将第<2>步制备的中包装体系加入第<3>步中的更换后的培养基中，37℃ 5％CO 2培养箱静置4-6h。
5、37℃ 5％CO 2培养箱静置4-6h后，更换2％减血清DMEM培养基培养，于48h和72h分别收集病毒。
6、病毒浓缩：将收集的病毒离心，并用0.45μm滤膜过滤。按照各组分体积比：病毒上清：50％PEG8000：5M NaCl＝87：10：3，混合均匀，4℃浓缩过夜。4℃，3000g，离心20min，1ml CTS培养基(Gibco，A3021002)重悬病毒，4℃溶解，-80℃保存。
用Lentvirus+hPD-1感染CTLL2(Promega,CS2028B04)，通过加压筛选和分选获得CTLL2-hPD-1稳转细胞系。
实验方法：
1、配置Assay Medium:1％MEM NEAA(Gibco,11140-050)，10％FBS(PEAK,PS-FB1)和89％IMDM(Gibco,12440-053)。
2、用Assay Medium洗涤CTLL2或CTLL2-hPD-1细胞2次。
3、用含0.4ng/ml rhIL2(R&D,202-IL)Assay Medium调整CTLL2或CTLL2-hPD-1细胞 密度,铺中间96孔白色细胞培养板(NUNC)，每孔50000个细胞。
4、边缘孔用等体积Assay Medium铺板，5％CO 2 37℃饥饿处理18-20h。
5、分别将稀释待测免疫缀合物分子加到细胞板，5％CO 2 37℃孵育6h。
6、取出培养板室温平衡15-20min,每孔添加等体积Luciferease assay system试剂(Bio-Glo)，室温孵育5-15min,酶标仪(Molecular Devices)读数。
结果见图6。图6结果显示，本研究的αPD-1/IL-2m免疫缀合物在PD-1阳性的CTLL2细胞上活性要强于PD-1阴性的CTLL2上的活性，其中2132在两种细胞之间的活性(EC50)有24倍的选择性，2063的选择性为42倍，2149的选择性为115倍，2219的选择性为500倍，2213和2214的选择性超过>10000倍。结果表明，本发明的免疫缀合物分子可以选择性激活PD-1阳性的CTLL2细胞。
二、αPD-1/IL2m免疫缀合物分子在PBMC细胞中pSTAT5信号检测
IL-2与T细胞表面IL-2受体结合，会激活T淋巴细胞JAK-STAT信号通路，其中STAT5磷酸化水平是评判该信号通路激活水平的重要指标。
实验方法：
1.PBMC细胞复苏
(1)、取液氮冻存PBMC细胞(妙通生物，货号PB100C)，37℃快速摇动融化。
(2)、将细胞缓慢加入10ml CTS培养基(Gibco)培养基需提前37℃预热，并加入100ul DNA酶(STRMCELL,货号07900)。
(3)、离心300g/8min，去除上清。
(4)、用10ml CTS重悬，转移至T75培养瓶，37℃ 5％培养箱稳定过夜。
2.pSTAT5实验
(1)、用AlexaFluor TM488Antibody Labeling Kit(Thermo Fisher,A20181)标记PD-1mAb(Innovent,ADI-11416)，制备AF488-anti human PD-1荧光抗体，荧光抗体标记过夜培养的PBMC悬浮细胞。
(2)、标记的PBMC悬浮细胞铺96孔U型板，细胞数量5x10 5cells/well。
(3)、分别将不同稀释浓度的待测免疫缀合物加入96孔型板，待检测样品与细胞37℃孵育30min。
(4)、离心400g/5min，去除上清。
(5)、按照200ul/孔，加入4％组织细胞固定液(Solarbio,P1110)，常温离心400g/30min。
(6)、按照200ul/孔，加破膜液，4℃静置30min，离心400g/10min。
(7)、按照200ul/孔，加入perm/wash Buffer(BD)，共细胞洗涤两次。
(8)、配置抗体染液，AF647-pSTAT5抗体量为3ul/100ul perm/wash Buffer/well，其余染色抗体为1ul/100ul perm/wash Buffer/well；室温孵育1.5h，perm/wash Buffer洗涤2次。
Figure PCTCN2022120265-appb-000007
Figure PCTCN2022120265-appb-000008
(9)、150ul perm/wash Buffer/well重悬，流式检测。
三、免疫缀合物分子在活化的PBMC细胞中pSTAT5信号检测
T淋巴细胞被激活活化后，在PD-1作用下，探究和验证，免疫缀合物对活化T淋巴细胞pSTAT5信号的影响。
1.PBMC细胞复苏
(1)、取液氮冻存PBMC细胞，37℃快速摇动融化。
(2)、将细胞缓慢加入10ml CTS培养基(37℃预热，并含有100ul DNA酶)。
(3)、离心300g/8min，去除上清。
(4)、用10ml CTS重悬，转移至T75培养瓶，37℃ 5％培养箱稳定过夜。
2.T淋巴细胞活化和静息
(1)、取过夜培养PBMC中悬浮细胞，细胞计数，加入等细胞数量CD3/CD28Beads，活化刺激48h。
(2)、去除Beads和培养基洗涤活化细胞。
(3)、活化细胞转至T75培养瓶，37℃ 5％静息48h。
3.pSTAT5实验
(1)、用AlexaFluor TM488Antibody Labeling Kit(Thermo Fisher,A20181)标记PD-1mAb(Innovent,ADI-11416)，制备AF488-anti human PD-1荧光抗体，荧光抗体标记活化和静息的T细胞。
(2)、细胞铺96孔U型板，细胞数量5x10 5cells/well。
(3)、分别将不同稀释检测分子加入96孔型板，待检测分子与细胞37 o孵育30min。
(4)、400g/5min离心，去除上清。
(5)、按照200ul/孔，加入4％组织细胞固定液，常温离心400g/30min。
(6)、按照200ul/孔，加破膜液，4℃静置30min，离心400g/10min。
(7)、按照200ul/孔，加入perm/wash Buffer，共细胞洗涤两次。
(8)、配置抗体染液,pSTAT5抗体(BD)量为3ul/100ul perm/wash Buffer/well，其余染色抗体为1ul/100ul perm/wash Buffer/well；室温孵育1.5h，perm/wash Buffer洗涤2次。
(9)、150ul perm/wash Buffer/well重悬，流式检测。
图7的结果显示，在PD-1阴性(PD-1-)的T细胞(CD4+PD1-T或CD8+PD1-T)上，本研究的分子活性都要比对照分子2061弱；在PD-1阳性(PD-1+)的T细胞(CD4+PD1+T或CD8+PD1+T)上，2063的活性要优于对照分子2061，说明2063对PD-1的选择性要大于2061,本研究的另外几个分子在PD-1+的T细胞上活性也是弱于2061,说明本研究的分子因高活性IL-2带来的毒性要小于2061，在体内能够耐受的剂量也要高于2061。
四、HEK-Blue TM IL-2cell reporter assay检测本发明的免疫缀合物活性
在HEK293细胞中，引入过表达IL2R(CD25,CD122,CD132)、JAK3和STAT5基因，构建拥有IL2信号通路的HEK293+hIL2R/SEAP细胞系(huPD-1-细胞，HEK-Blue TM IL-2 Cells，Invivogen,hkb-il2)，在IL2作用下，激活HEK293+hIL2R/SEAP cell reporter报告基因。
实验材料
Figure PCTCN2022120265-appb-000009
Figure PCTCN2022120265-appb-000010
HEK293+hIL2R+hPD-1/SEAP细胞系构建：如上构建和包装Lentvirus+hPD-1慢病毒，用Lentvirus+PD-1感染HEK293+h IL2R/SEAP(Invivogen,hkb-il2)，加压筛选和分选HEK293+hIL2R+hPD-1/SEAP稳转细胞系(huPD-1+细胞)，用于下述实验。
实验方法：
1.细胞消化，调整细胞密度，铺中间60孔，每孔细胞数量50000。
2.将如图所示稀释的免疫缀合物和对照分子分别加入相应细胞孔板，37℃培养20-24h。
4.取20ul细胞培养上清加入180ul QUANTI-Blue，室温15min，测定OD630。
HEK-Blue TM IL-2 Cells(huPD-1-细胞)是一个过表达了IL-2受体的HEK293细胞。从图8和表4可以看出，本研究获得的免疫缀合物在IL-2的活性比2061弱了最小3.14倍，最大的弱化到2.21E+08倍。
在过表达的PD-1的细胞(HEK293+hIL2R+hPD-1/SEAP稳转细胞系(huPD-1+细胞))上，免疫缀合物可以达到很强的IL-2活性，在两种细胞保持很高的选择性，如2063的选择性可以达到3.52倍，2132的选择性可以达到53.45倍，2149的选择性可以达到96.04倍，2219为606.11倍，2214为5119.78倍，2213为1.57E+07倍。
表4.本研究免疫缀合物在PD-1-细胞和PD-1+细胞的选择活性
  2061 2063 2132 2149 2213 2214 2219
Fold # 1 3.52 53.45 96.04 1.57E+7 5119.78 606.11
#:Fold＝分子在PD-1-reporter assay EC50/PD-1+reporter assay EC50
实施例5.免疫缀合物的体内药效实验
为了证明αPD-1/IL2m免疫缀合物在体内的药效，采用MC38细胞(小鼠结肠癌细胞系，上海和元生物)接种hPD-1敲入小鼠测定本发明的双功能PD-1抗体和IL-2突变分子免疫缀合物(2063，2132)的抗肿瘤药效。实验使用SPF等级的雌性hPD-1敲入小鼠小鼠(购自上海南方模式生物)，合格证编号为NO.20170010005748。
将MC38细胞进行常规传代培养用于后续体内实验。离心收集细胞，以PBS(1×)重悬MC38细胞，制备成细胞浓度为5×10 6个/ml细胞悬液。在第0天取0.2ml细胞悬液皮下接种至hPD-1敲入小鼠右侧腹部区域中来建立MC38荷瘤小鼠模型。
肿瘤细胞接种6天后检测各只小鼠瘤体积，进行分组(每组8只小鼠)，给药剂量和方式如表5所示。
表5：体内实验的分组、给药剂量和方式
组别 给药剂量 给药次数 给药方式
h-IgG* 20mg/kg QW x3 腹腔注射
2063 10mg/kg QW x3 腹腔注射
2132 10mg/kg QW x3 腹腔注射
*：h-IgG为同种型对照抗体，购自Equitech-Bio，批号161206-0656。
h-IgG，2063和2132的使用浓度分别为2mg/ml，1mg/ml和1mg/ml，每周给药一次，共3次(QWx3)。分别在MC38细胞接种后第6、13、20天给药，每周2次监测小鼠瘤体积与体重，如图9A所示，监测至24天后结束。
接种后第24天计算相对肿瘤抑制率(TGI％)，计算公式如下：TGI％＝100％*(对照组肿瘤体积–治疗组肿瘤体积)/(对照组肿瘤体积–对照组给药前肿瘤体积)。肿瘤体积测定：采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L*W 2/2。采用电子天平测定体重。
肿瘤抑制率结果如表6所示：在接种后第24天,与h-IgG,20mg/kg组对比，2063和2132的肿瘤抑制率分别为98％和96％。同时对小鼠体重进行检测的结果(图9B)显示，在接种后第24天,小鼠体重无显著差异。
表6、抗肿瘤药效统计
组别 肿瘤体积(mm 3) 肿瘤抑制率(％)
h-IgG 2329.61 N/A
2063 112.47 98
2132 170.91 96
为了进一步证明αPD-1/IL2m免疫缀合物体内药效优于亲本的抗PD-1单抗(Sintilimab,又称IBI308)，采用MC38细胞(小鼠结肠癌细胞系，上海和元生物)接种hPD-1敲入小鼠测定本发明的免疫缀合物(2063)的抗肿瘤药效。实验使用SPF等级的雌性hPD-1敲入小鼠小鼠(购自上海南方模式生物)，合格证编号为NO.20170010004237。
将MC38细胞进行常规传代培养用于后续体内实验。离心收集细胞，以PBS(1×)重悬MC38细胞，制备成细胞浓度为5×10 6个/ml细胞悬液。在第0天取0.2ml细胞悬液皮下接种至hPD-1敲入小鼠右侧腹部区域中来建立MC38荷瘤小鼠模型。
肿瘤细胞接种12天后检测各只小鼠瘤体积，进行分组(每组8只小鼠)，给药剂量和方式如表7所示。
表7：体内实验的分组、给药剂量和方式
组别 给药剂量 给药次数 给药方式
h-IgG* 10mg/kg QW x3 腹腔注射
2063 10mg/kg QW x3 腹腔注射
IBI308 10mg/kg QW x3 腹腔注射
*：h-IgG为同种型对照抗体，购自Equitech-Bio，批号161206-0656。
h-IgG，2063和IBI308的使用浓度均为1mg/ml，每周给药一次，共3次(QWx3)。分别在MC38细胞接种后第12、19、26天给药，每周2次监测小鼠瘤体积与体重，如图10A所 示，监测至29天后结束。接种后第29天计算相对肿瘤抑制率(TGI％)，计算公式如下：TGI％＝100％*(对照组肿瘤体积–治疗组肿瘤体积)/(对照组肿瘤体积–对照组给药前肿瘤体积)。肿瘤体积测定：采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L*W 2/2。采用电子天平测定体重。
肿瘤抑制率结果如表8所示：在接种后第29天,与h-IgG,20mg/kg组对比，2063和IBI308的肿瘤抑制率分别为112％和56％。同时对小鼠体重进行检测的结果(图10B)显示，在接种后第29天,小鼠体重无显著差异。
表8、抗肿瘤药效统计
组别 肿瘤体积(mm 3) 肿瘤抑制率(％)
h-IgG 2041.49 N/A
2063 66.19 112
IBI308 1057.37 55
为了进一步证明αPD-1/IL2m免疫缀合物的体内药效，采用PD-1抗体耐药的B16F10细胞(小鼠黑色素瘤细胞系，ATCC CRL-6475)接种hPD-1敲入小鼠测定本发明的αPD-1/IL2m免疫缀合物(2063)，亲本抗PD-1单抗(IBI308)，以及PD-1单抗和IL2m-Fc融合蛋白(2124,IL-2序列与2063相同，序列见序列表)联合给药的抗肿瘤药效。实验使用SPF等级的雌性hPD-1敲入小鼠小鼠(购自上海南方模式生物)，合格证编号为NO.20170010004768。
将B16F10细胞进行常规传代培养用于后续体内实验。离心收集细胞，以PBS(1×)重悬B16F10细胞，制备成细胞浓度为2.5×10 6个/ml细胞悬液。在第0天取0.2ml细胞悬液皮下接种至hPD-1敲入小鼠右侧腹部区域中来建立B16F10荷瘤小鼠模型。
肿瘤细胞接种7天后检测各只小鼠瘤体积，进行分组(每组6只小鼠)，给药剂量和方式如表9所示。
表9：体内实验的分组、给药剂量和方式
组别 给药剂量 给药次数 给药方式
h-IgG* 10mg/kg QW x3 腹腔注射
IBI308 10mg/kg QW x3 腹腔注射
IBI308+2124 10mg/kg+6mg/kg QW x3 腹腔注射
2063 10mg/kg QW x3 腹腔注射
*：h-IgG为同种型对照抗体，购自Equitech-Bio，批号161206-0656。
h-IgG，IBI308,2124和2063的使用浓度分别为1mg/ml,1mg/ml,0.6mg/ml和1mg/ml，每周给药一次，共3次(QWx3)。分别在B16F10细胞接种后第8、15天和22天给药，每周2次监测小鼠瘤体积与体重，如图11A-C所示，监测至28天后结束。
因为B16F10细胞容易转移诱导小鼠死亡，因此按照接种后第22天计算相对肿瘤抑制率(TGI％)，计算公式如下：TGI％＝100％*(对照组肿瘤体积–治疗组肿瘤体积)/(对照组肿瘤体积–对照组给药前肿瘤体积)。肿瘤体积测定：采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L*W 2/2。采用电子天平测定体重。肿瘤超过2000mm 3会对该小鼠进行安乐死处理。若组内超过半数死亡，整个组的肿瘤生长曲线在该时间点不显 示。
肿瘤抑制率结果如表10所示：在接种后第22天,与h-IgG组对比，IBI308,IBI308+2214,2063的肿瘤抑制率分别为2％,84％和99％，且2063在CR率明显优于IBI308以及IBI308联合无靶向的IL2m-Fc分子(2124)。同时对小鼠体重进行检测的结果(图11C)显示，在监测期间内,小鼠体重无显著差异。
表10.抗肿瘤药效统计
Figure PCTCN2022120265-appb-000011
*完全缓解率：肿瘤完全消退，肿瘤体积为0.
为了进一步证明本发明的αPD-1/IL2m免疫缀合物2149的体内药效，采用MC38细胞(小鼠结肠癌细胞系，上海和元生物)接种hPD-1敲入小鼠测定本发明的免疫缀合物(2149)的抗肿瘤药效。实验使用SPF等级的雌性hPD-1敲入小鼠小鼠(购自上海南方模式生物)，合格证编号为NO.20170010006762。
将MC38细胞进行常规传代培养用于后续体内实验。离心收集细胞，以PBS(1×)重悬MC38细胞，制备成细胞浓度为5×10 6个/ml细胞悬液。在第0天取0.2ml细胞悬液皮下接种至hPD-1敲入小鼠右侧腹部区域中来建立MC38荷瘤小鼠模型。
肿瘤细胞接种8天后检测各只小鼠瘤体积，进行分组(每组8只小鼠)，给药剂量和方式如表11所示。
表11：体内实验的分组、给药剂量和方式
组别 给药剂量 给药次数 给药方式
h-IgG* 40mg/kg QW x3 腹腔注射
2149,10mg/kg 10mg/kg QW x3 腹腔注射
2149,20mg/kg 20mg/kg QW x3 腹腔注射
2149,40mg/kg 40mg/kg QW x3 腹腔注射
*：h-IgG为同种型对照抗体，购自Equitech-Bio，批号161206-0656。
h-IgG，2149,10mg/kg，2149，20mg/kg和2149，40mg/kg的使用浓度分别为4mg/ml，1mg/ml,2mg/ml和4mg/ml，每周给药一次，共3次(QWx3)。分别在MC38细胞接种后第8、15和22天给药，每周2次监测小鼠瘤体积与体重，如图12A-B所示。肿瘤超过2000mm 3会对该小鼠进行安乐死处理，整个实验部分小鼠一直监测至61天后结束。因为部分组别小鼠肿瘤体积超过2000mm 3被安乐死，因此按照接种后第36天计算相对肿瘤抑制率(TGI％)，计算公式如下：TGI％＝100％*(对照组肿瘤体积–治疗组肿瘤体积)/(对照组肿瘤体积–对照组给药前肿瘤体积)。肿瘤体积测定：采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L*W 2/2。采用电子天平测定体重。
肿瘤生长曲线和生存曲线如图12A和12B所示，2149分子不同剂量组抗肿瘤作用呈现剂 量依赖性，并且在20mg/kg以及40mg/kg组别，小鼠肿瘤完全消退。并且这一优势在图12B的生存曲线中也有体现，该两组小鼠100％肿瘤消退，而10mg/kg组别中，8只小鼠仅有2只肿瘤完全消退。肿瘤抑制率结果如表12所示：在接种后第36天,与h-IgG组对比，2149,10mg/kg,2149,20mg/kg和2149,40mg/kg的肿瘤抑制率分别为84％，103％和103％。同时对小鼠体重进行检测的结果(图12C)显示，在接种后第36天,小鼠体重无显著差异。
表12.抗肿瘤药效统计
Figure PCTCN2022120265-appb-000012
*完全缓解率：肿瘤完全消退，肿瘤体积为0.
为了证明免疫缀合物2149的体内药效，采用PD-1抗体耐药的B16F10细胞(小鼠黑色素瘤细胞系，ATCC CRL-6475)接种hPD-1敲入小鼠测定本发明的双功能PD-1抗体和IL-2突变分子融合蛋白(2149)的抗肿瘤药效。实验使用SPF等级的雌性hPD-1敲入小鼠小鼠(购自上海南方模式生物)，合格证编号为NO.20170010007909。
将B16F10细胞进行常规传代培养用于后续体内实验。离心收集细胞，以PBS(1×)重悬B16F10细胞，制备成细胞浓度为2.5×10 6个/ml细胞悬液。在第0天取0.2ml细胞悬液皮下接种至hPD-1敲入小鼠右侧腹部区域中来建立B16F10荷瘤小鼠模型。
肿瘤细胞接种6天后检测各只小鼠瘤体积，进行分组(每组8只小鼠)，给药剂量和方式如表13所示。
表13：体内实验的分组、给药剂量和方式
组别 给药剂量 给药次数 给药方式
h-IgG* 40mg/kg QW x3 腹腔注射
IBI308,20mg/kg 20mg/kg QW x3 腹腔注射
IBI308,40mg/kg 40mg/kg QW x3 腹腔注射
2149,20mg/kg 20mg/kg QW x3 腹腔注射
2149,40mg/kg 40mg/kg QW x3 腹腔注射
*：h-IgG为同种型对照抗体，购自Equitech-Bio，批号161206-0656。
h-IgG，IBI308,20mg/kg,IBI308,40mg/kg,2149-20mg/kg和2149,40mg/kg的使用浓度分别为4mg/ml，2mg/ml,4mg/ml，2mg/ml和4mg/ml，每周给药一次，共3次(QWx3)。分别在B16F10细胞接种后第8、15、22天给药，每周2次监测小鼠瘤体积与体重，如图13A-B所示，监测至22天后结束。因为B16F10细胞容易转移诱导小鼠死亡，因此按照接种后第15天计算相对肿瘤抑制率(TGI％)，计算公式如下：TGI％＝100％*(对照组肿瘤体积–治疗组肿瘤体积)/(对照组肿瘤体积–对照组给药前肿瘤体积)。肿瘤体积测定：采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L*W 2/2。采用电子天平测 定体重。肿瘤超过2000mm 3会对该小鼠进行安乐死处理。
小鼠肿瘤生长曲线如图13A和13B所示，在PD1耐药模型中，IBI308几乎未显示出药效，但是2149的20mg/kg和40mg/kg组别展现出一定的抗肿瘤作用，且高剂量抗肿瘤作用优于低剂量，该作用同时在小鼠生存曲线中得到一定体现，高剂量组别在实验终点仍有2只小鼠肿瘤完全消退(图13C和表14)。
肿瘤抑制率结果如表12所示：在接种后第15天,与h-IgG,40mg/kg组对比，IBI308,20mg/kg,IBI308,40mg/kg,2149,20mg/kg和2149,40mg/kg的肿瘤抑制率分别为29％,27％,82％和86％。同时对小鼠体重进行检测的结果(图13D)显示，在接种后第22天,小鼠体重无显著差异。
表14.抗肿瘤药效统计
Figure PCTCN2022120265-appb-000013
为了证明αPD-1/IL2m免疫缀合物2149的体内药效优于对照药物PD-1-IL2v(分子编号2061,序列来源US20180326010A1，也参见序列表)，采用PD-1抗体耐药的B16F10细胞(小鼠黑色素瘤细胞系，ATCC CRL-6475)接种hPD-1敲入小鼠测定本发明的双功能PD-1抗体和IL-2突变分子融合蛋白(2149)的抗肿瘤药效。实验使用SPF等级的雌性hPD-1敲入小鼠小鼠(购自上海南方模式生物)，合格证编号为NO.20170010008942。
将B16F10细胞进行常规传代培养用于后续体内实验。离心收集细胞，以PBS(1×)重悬B16F10细胞，制备成细胞浓度为2.5×10 6个/ml细胞悬液。在第0天取0.2ml细胞悬液皮下接种至hPD-1敲入小鼠右侧腹部区域中来建立B16F10荷瘤小鼠模型。
肿瘤细胞接种8天后检测各只小鼠瘤体积，进行分组(每组7只小鼠)，给药剂量和方式如表15所示。
表15.体内实验的分组、给药剂量和方式
组别 给药剂量 给药次数 给药方式
h-IgG* 40mg/kg QW x3 腹腔注射
IBI308 40mg/kg QW x3 腹腔注射
2061,10mg/kg 10mg/kg QW x3 腹腔注射
2061,20mg/kg 20mg/kg QW x3 腹腔注射
2061,40mg/kg 40mg/kg QW x3 腹腔注射
2149,10mg/kg 10mg/kg QW x3 腹腔注射
2149,40mg/kg 20mg/kg QW x3 腹腔注射
2149,40mg/kg 40mg/kg QW x3 腹腔注射
*：h-IgG为同种型对照抗体，购自Equitech-Bio，批号161206-0656。
h-IgG，IBI308,40mg/kg,2061,10mg/kg,2061,20mg/kg,2061,40mg/kg,2149,10mg/kg,2149,20mg/kg和2149-40mg/kg的使用浓度分别为4mg/ml，2mg/ml,4mg/ml，2mg/ml和4mg/ml，每周给药一次，共3次(QWx3)。分别在B16F10细胞接种后第8、15、22天给药，每周2次监测小鼠瘤体积与体重，如图14A所示，监测至33天后结束。由于B16F10细胞容易转移造成小鼠死亡，因此按照接种后第19天计算相对肿瘤抑制率(TGI％)，计算公式如下：TGI％＝100％*(对照组肿瘤体积–治疗组肿瘤体积)/(对照组肿瘤体积–对照组给药前肿瘤体积)。肿瘤体积测定：采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L*W2/2。采用电子天平测定体重。肿瘤超过2000mm 3会对该小鼠进行安乐死处理。若组内超过半数死亡，整个组的肿瘤生长曲线在该时间点不显示。
肿瘤抑制率结果如表16所示：在接种后第19天,与h-IgG,40mg/kg组对比，IBI308,40mg/kg,2061,10mg/kg,2149,10mg/kg,2149,20mg/kg和2149-40mg/kg的肿瘤抑制率分别为21％,96％,79％,87％和97％。2061的20mg/kg和40mg/kg因为第一针给药后出现体重大幅下降且小鼠出现死亡，因此该组别的TGI并未计算。
并且我们对小鼠生存做了统计分析(图14B)，图示可见，对比2061和2149在本实验中最大给药/耐受剂量，我们可以看出，2149在40mg/kg剂量下、2061分子10mg/kg剂量下小鼠生存的更多，对于2149，7只小鼠中有3只肿瘤完全消退，而2061仅有1只小鼠肿瘤完全消退。同时对小鼠体重进行检测的结果(图14C)显示，在接种后第29天,2149各给药剂量组别小鼠体重均未出现下降，而2061组别的小鼠出现体重下降，在低剂量(10mg/kg)情况下，小鼠体重平均下降超过5％，中剂量(20mg/kg)和高剂量(40mg/kg)出现小鼠死亡，每组小鼠总共有7只，死亡6只，详细情况见表12。而2149相对安全，无论是低剂量，中剂量还是高剂量，体重下降均不明显，且仅在低剂量和中剂量死亡1只小鼠，而在高剂量小鼠未出现小鼠死亡，且有比2061更高的完全肿瘤缓解率(表16)。因此2149比2061药效更优，且更为安全，具有更高的治疗窗口。
表16.抗肿瘤药效统计
Figure PCTCN2022120265-appb-000014
为了验证αPD-1/IL2m免疫缀合物2214体内药效，采用MC38细胞(小鼠结肠癌细胞系，上海和元生物)接种hPD-1敲入小鼠测定本发明的双功能PD-1抗体和IL-2突变分子融合蛋白(2214)的抗肿瘤药效。实验使用SPF等级的雌性hPD-1敲入小鼠小鼠(购自上海南方模式生物)，合格证编号为NO.20170010010829。
将MC38细胞进行常规传代培养用于后续体内实验。离心收集细胞，以PBS(1×)重悬MC38 细胞，制备成细胞浓度为5×10 6个/ml细胞悬液。在第0天取0.2ml细胞悬液皮下接种至hPD-1敲入小鼠右侧腹部区域中来建立MC38荷瘤小鼠模型。
肿瘤细胞接种7天后检测各只小鼠瘤体积，进行分组(每组7只小鼠)，给药剂量和方式如表17所示。
表17.体内实验的分组、给药剂量和方式
组别 给药剂量 给药次数 给药方式
h-IgG* 40mg/kg QW x3 腹腔注射
2214 20mg/kg QW x3 腹腔注射
2214 40mg/kg QW x3 腹腔注射
*：h-IgG为同种型对照抗体，购自Equitech-Bio，批号161206-0656。
h-IgG和2214的使用浓度均为4mg/ml，每周给药一次，共3次(QWx3)。分别在MC38细胞接种后第7、14、21天给药，每周2次监测小鼠瘤体积与体重，如图15A所示，监测至56天后结束。因对照组肿瘤体积超过2000mm 3，因此我们接种后第28天计算相对肿瘤抑制率(TGI％)，计算公式如下：TGI％＝100％*(对照组肿瘤体积–治疗组肿瘤体积)/(对照组肿瘤体积–对照组给药前肿瘤体积)。肿瘤体积测定：采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L*W 2/2。采用电子天平测定体重。
肿瘤抑制率结果如表18所示：在接种后第28天,与h-IgG,20mg/kg组对比，2214的肿瘤抑制率为104％。同时对小鼠体重进行检测的结果(图15C)显示，在接种后第28天,小鼠体重无显著差异。
表18.抗肿瘤药效统计
Figure PCTCN2022120265-appb-000015
为了证明αPD-1/IL2m免疫缀合物2214的体内药效，采用PD-1抗体耐药的B16F10细胞(小鼠黑色素瘤细胞系，ATCC CRL-6475)接种hPD-1敲入小鼠测定本发明的双功能PD-1抗体和IL-2突变分子融合蛋白(2214)的抗肿瘤药效。实验使用SPF等级的雌性hPD-1敲入小鼠小鼠(购自上海南方模式生物)，合格证编号为NO.20170010010829。
将B16F10细胞进行常规传代培养用于后续体内实验。离心收集细胞，以PBS(1×)重悬B16F10细胞，制备成细胞浓度为2.5×10 6个/ml细胞悬液。在第0天取0.2ml细胞悬液皮下接种至hPD-1敲入小鼠右侧腹部区域中来建立B16F10荷瘤小鼠模型。
肿瘤细胞接种7天后检测各只小鼠瘤体积，进行分组(每组7只小鼠)，给药剂量和方式如表19所示。
表19：体内实验的分组、给药剂量和方式
组别 给药剂量 给药次数 给药方式
h-IgG* 40mg/kg QW x3 腹腔注射
2214,20mg/kg 20mg/kg QW x3 腹腔注射
2214,40mg/kg 40mg/kg QW x3 腹腔注射
*：h-IgG为同种型对照抗体，购自Equitech-Bio，批号161206-0656。
h-IgG，2214-20mg/kg和2214,40mg/kg的使用浓度分别为4mg/ml，2mg/ml和4mg/ml，每周给药一次，共3次(QWx3)。分别在B16F10细胞接种后第7、14、21天给药，每周2次监测小鼠瘤体积与体重，如图16A-B所示，监测至63天后结束。肿瘤体积测定：采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L*W 2/2。采用电子天平测定体重。肿瘤超过2000mm 3会对该小鼠进行安乐死处理。
小鼠生存曲线如图16A所示，2214的两个剂量均能显著延长小鼠的生存期。同时对小鼠体重进行检测的结果(图16C)显示，在接种后第22天,小鼠体重无显著差异。
序列表
Figure PCTCN2022120265-appb-000016
Figure PCTCN2022120265-appb-000017
Figure PCTCN2022120265-appb-000018
Figure PCTCN2022120265-appb-000019
Figure PCTCN2022120265-appb-000020
Figure PCTCN2022120265-appb-000021
Figure PCTCN2022120265-appb-000022
Figure PCTCN2022120265-appb-000023
Figure PCTCN2022120265-appb-000024
Figure PCTCN2022120265-appb-000025

Claims (21)

  1. 一种免疫缀合物,其包含(i)结合PD-1的抗体和(ii)IL-2突变蛋白，所述突变蛋白，与野生型IL-2(优选人IL-2，更优选包含SEQ ID NO:3序列的IL-2)相比，包含突变：
    (i)在IL-2与IL-2Rα结合界面上，尤其是在位置35和/或42上，具有消除或降低对IL-2Rα受体的结合亲合力的突变；
    和/或
    (ii)在IL-2与IL-2Rβγ结合界面上，尤其是在选自位置88、127和/或130的至少一个位置上，具有弱化对IL-2Rβγ受体的结合的突变；
    以及
    (iii)缩短的B’C’环区(即，连接氨基酸残基aa72和aa84的序列)，优选地，所述缩短的环区具有小于10，9,8，7，6，或5个的氨基酸长度，且优选7个氨基酸长度；优选地，所述缩短的B’C’环区导致改善的蛋白表达量和/或纯度，
    其中氨基酸位置根据SEQ ID NO:3编号。
  2. 根据权利要求1的免疫缀合物,其中所述突变蛋白，相对于野生型IL-2，包含：
    (i)N88R+S130R；
    N88D；
    N88R；
    F42A+N88R+S127E；或
    K35E+N88R+S127E；和
    (ii)B’C’环区序列AGDASIH或AQSKNFH；
    以及任选地(iii)T3A。
  3. 根据权利要求1的免疫缀合物，其中所述IL-2突变蛋白包含SEQ ID NO：4、23、25、27、29或31的氨基酸序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成。
  4. 根据权利要求1-3中任一项的免疫缀合物，其中所述免疫缀合物包含：
    第一单体，其包含与Fc片段融合的IL-2突变蛋白；和
    第二单体，其包含特异性结合PD-1的抗体或其片段，优选地，所述片段包含所述抗PD-1抗体的一条重链和一条轻链。
  5. 根据权利要求4的免疫缀合物，其中第一单体的Fc片段中包含Knob突变，第二单体的抗体重链中包含hole突变，或者第一单体的Fc片段中包含hole突变，第二单体的抗体重链中包含knob突变。
  6. 根据权利要求4或5的免疫缀合物，其中所述第一单体中的Fc片段是IgG1、IgG2、IgG3或IgG4的Fc片段，优选地包含SEQ ID NO:6、42或43的氨基酸序列或由其组成。
  7. 根据权利要求4-6中任一项的免疫缀合物，其中所述与Fc片段融合的IL-2突变蛋白包含SEQ ID NO:7、24、26、28、30或32的氨基酸序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成。
  8. 根据权利要求4-7中任一项的免疫缀合物，其中所述PD-1抗体或其抗原结合片段包含重链，所述重链包含重链可变区，所述重链可变区包含分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO:9、10和11。
  9. 根据权利要求4-8中任一项的免疫缀合物，其中所述PD-1抗体或其抗原结合 片段包含轻链，所述轻链包含轻链可变区，其中所述轻链可变区包含分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3:SEQ ID NO:16、17和18。
  10. 根据权利要求4-9中任一项的免疫缀合物，其中所述抗PD-1抗体或其抗原结合片段包含：
    包含SEQ ID NO:8所示的氨基酸序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的重链可变区，和
    包含SEQ ID NO:15所示的氨基酸序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的轻链可变区。
  11. 根据权利要求4-9中任一项的免疫缀合物，其中所述抗PD-1抗体或其抗原结合片段包含
    包含SEQ ID NO:14或22所示的氨基酸序列或与其具有至85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的重链；和
    包含SEQ ID NO:20所示的氨基酸序列或与其具有至85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的轻链。
  12. 根据权利要求1-11任一项的免疫缀合物，其中所述IL-2突变蛋白与Fc通过接头连接，或所述IL-2突变蛋白与所述抗PD-1抗体通过接头连接，优选地所述接头选自为(GGGGS) n,其中n＝1、2、3或4，例如所述接头为SEQ ID NO:5。
  13. 一种分离的多核苷酸，其编码权利要求1-12中任一项的免疫缀合物中的一条或多条链，或第一单体和/或第二单体。
  14. 一种表达载体，其包含权利要求13的多核苷酸。
  15. 一种宿主细胞，其包含权利要求13的多核苷酸或权利要求14的载体，优选所述宿主细胞是酵母细胞或哺乳动物细胞，特别是HEK293细胞或CHO细胞。
  16. 一种用于生产权利要求1-12中任一项的免疫缀合物的方法，包括，在适于表达所述免疫缀合物的条件下，培养权利要求15的宿主细胞。
  17. 一种药物组合物，其包含权利要求1-12中任一项的免疫缀合物，任选地和药用辅料。
  18. 权利要求1-12中任一项的免疫缀合物或权利要求17的药物组合物在制备用于预防和/或治疗癌症的药物中的用途，优选地，所述癌症是实体肿瘤或血液肿瘤，例如胃肠道肿瘤或黑色素瘤，例如结肠直肠癌或结肠癌；例如，所述癌症是PD-1抗体治疗耐受性癌症。
  19. 权利要求18所述的用途，其中所述药物组合物还包含第二治疗剂。
  20. 一种预防和/或治疗受试者癌症的方法，所述方法包括，向所述受试者施用权利要求1-12中任一项的免疫缀合物或权利要求17的药物组合物，优选地，所述癌症是实体肿瘤或血液肿瘤，例如胃肠道肿瘤或黑色素瘤，例如结肠直肠癌或结肠癌；例如，所述癌症是PD-1抗体治疗耐受性癌症。
  21. 根据权利要求20中任一项所述的方法，其中所述突变蛋白、所述融合蛋白或所述药物组合物与第二治疗剂以组合疗法施用。
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