WO2023061005A1 - 新型抗体-细胞因子融合蛋白及其制备方法和用途 - Google Patents
新型抗体-细胞因子融合蛋白及其制备方法和用途 Download PDFInfo
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- 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]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
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- A61P37/00—Drugs for immunological or allergic disorders
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- C—CHEMISTRY; METALLURGY
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- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
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- 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
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- C07K14/55—IL-2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
Definitions
- the present application relates to antibody-cytokine fusion proteins and uses thereof. Specifically, the present application relates to a fusion protein (IL-2/IL-2 antibody fusion protein) of interleukin-2 (Interleukin-2, IL-2) and an anti-IL-2 antibody.
- IL-2 receptor ⁇ subunit IL2R ⁇ , CD25
- IL-2 receptor aggregates containing ⁇ subunits ie, IL2R ⁇ / ⁇ or IL2R ⁇ / ⁇ / ⁇
- IL2R ⁇ / ⁇ IL-2 receptor aggregates containing ⁇ subunits (ie, IL2R ⁇ / ⁇ or IL2R ⁇ / ⁇ / ⁇ ) bind, but can bind to IL-2 receptor ⁇ , ⁇ subunit dimer (IL2R ⁇ / ⁇ ) binds and activates the signal transduction pathway mediated by it.
- the present application further relates to bispecific molecules and immunoconjugates comprising the IL-2/IL-2 antibody fusion protein, nucleic acid molecules encoding the IL-2/IL-2 antibody fusion protein, and nucleic acid molecules comprising the nucleic acid molecule Expression vectors and host cells.
- the present application also provides a method for preparing the IL-2/IL-2 antibody fusion protein, a pharmaceutical composition comprising the IL-2/IL-2 antibody fusion protein, and the use of the IL-2/IL-2 antibody fusion protein in treating diseases. 2 Methods and uses of antibody fusion proteins or pharmaceutical compositions thereof.
- Interleukin-2 (Interleukin-2, IL-2) is a globular glycoprotein with a molecular weight of about 15.5kDa, which has dual functions of inducing immune suppression and immune activation, and plays a role in regulating the production of various immune cells. , survival, expansion and homeostasis.
- the mature IL-2 protein contains 133 amino acid residues, which are folded to form 4 antiparallel amphipathic ⁇ -helical structures, and then these 4 ⁇ -helices are folded to form a higher-level structure that is essential for its functional activity (Smith, Science 1988, 240:1169-1176; Bazan, Science 1992, 257:410-413).
- IL-2 exerts its immune regulatory function in vivo by binding to the cell surface IL-2 receptor (IL2R) and triggering downstream signaling cascades (Kreig, PNAS 2010, 107:11906-11911).
- IL2R cell surface IL-2 receptor
- IL2Rs are expressed on the surface of different immune cells, among which high-affinity IL2Rs are constitutively highly expressed on CD4 + Foxp3 + regulatory T cells (Treg), and additionally on activated T cells (CD4 + T cells and CD8 + T cells) Also transiently induced; intermediate-affinity IL2R, despite low expression on resting (or naive) effector T cells (Teff) such as CD8 + Teff cells, is more effective in CD8 + Teff cells and memory phenotypes that have undergone antigenic stimulation (memory phenotype, MP) is highly expressed on CD8 + T cells and natural killer (NK) cells (Fontenot, Nat Immunol 2005,6:1142–1151; Josefowicz, Annu Rev Immunol 2012,30:531–564; Malek&Bayer, Nat Rev Immunol 2004, 4:665–674).
- NK natural killer
- the signal transduction triggered by IL-2 is achieved by binding and inducing the interaction between IL2R ⁇ and ⁇ c to form a heterodimer, and then activating multiple downstream kinases.
- the ⁇ subunit is particularly important for increasing the binding affinity of IL-2 to IL2R, it has no role in signal transduction of immune cells (Kreig, PNAS 2010, 107:11906-11911).
- IL-2 is mainly produced by activated T cells, especially CD4 + helper T (Th) cells, and acts on neighboring cells expressing IL2R in an autocrine or paracrine manner to activate immune responses or maintain the normal regulatory defense mechanism of the immune system steady state.
- Th helper T
- IL-2 can stimulate the proliferation and differentiation of helper T cells (Zhu, Annual review of immunology 2010,28:445-489; Liao, Nat Immunol 2008,9:1288-1296; Liao, Nat Immunol 2011,12:551- 559), maintain the development and steady-state proliferation of Treg cells (Cheng, Immunol Rev 2011,241:63-76), induce the generation of cytotoxic T lymphocytes (cytotoxic T lymphocytes, CTL) and enhance their cytotoxicity, induce peripheral Blood lymphocytes differentiate into cytotoxic cells and lymphokine-activated killer cells (lymphokine-activated killer, LAK) (Liao, Immunity 2013,38:13-25), enhance the immunity of tumor infiltrating lymphocytes (Tumor infiltrating lymphocytes, TIL) function, promote the proliferation and differentiation of B cells, enhance the production of immunoglobulin by activated B cells, and stimulate the production, proliferation and activation of NK cells and a series of biological functions (Waldmann, Nat Rev
- IL-2 expands the above lymphocyte subsets in vivo, especially by stimulating and activating effector cells (including CD8 + T cells and NK cells) to play an anti-tumor effect.
- effector cells including CD8 + T cells and NK cells
- This characteristic of IL-2 makes IL-2 immunotherapy a One of the effective means of treating certain malignant tumors.
- Recombinant human IL-2 protein (rhIL-2) drug aldesleukin (Aldesleukin, trade name ) was approved in the 1990s for the treatment of metastatic renal cell carcinoma and malignant melanoma.
- Aldesleukin is an engineered rhIL-2, the difference between its structure and natural human IL-2 protein (hIL-2) is that aldesleukin has no glycosylation modification, lacks N-terminal alanine, and its Cysteine at position 125 was replaced by a serine residue.
- the complete response rate (complete response rate) of aldesleukin in various clinical trials for the treatment of metastatic renal cell carcinoma and malignant melanoma is low, about 7% (Atkins, J Clin Oncol 1999, 17:2105-2116; Fisher, Cancer J Sci Am 2000, 6 Suppl 1:S55-S57; Klapper, Cancer 2008, 113:293–301).
- aldesleukin Since aldesleukin has a short half-life in vivo, and CD8 + T cells and NK cells with anti-tumor activity express IL2R with medium affinity, high doses of aldesleukin are needed Interleukins can effectively activate CD8 + T cells and NK cells. However, high doses of aldesleukin can bind and activate the high-affinity IL2R expressed on endothelial cells and type-2 innate lymphoid cells (ILC2), which can lead to vascular leak syndrome (vascular leak syndrome).
- ILC2 type-2 innate lymphoid cells
- VLS Leak Syndrome, characterized by increased vascular permeability, causing extravasation of plasma proteins and intravascular fluid into the extravascular space and multiple organs, resulting in a decrease in blood pressure and a compensatory increase in heart rate, pulmonary and skin edema, and hepatocellular injury
- aldesleukin characterized by increased vascular permeability, causing extravasation of plasma proteins and intravascular fluid into the extravascular space and multiple organs, resulting in a decrease in blood pressure and a compensatory increase in heart rate, pulmonary and skin edema, and hepatocellular injury
- aldesleukin [aldesleukin] Package Insert, 2012).
- the low response rate of aldesleukin therapy is also related to the dual role of IL-2 in maintaining immune cell homeostasis. IL-2 not only promotes the activation and proliferation of effector cells, but also maintains peripheral immune tolerance by regulating Treg cells.
- Treg cells highly express high-affinity IL2R, IL-2 will preferentially bind to Treg cells, promote the proliferation and activation of peripheral Treg cells, thereby inhibiting the anti-tumor immune activity mediated by CD8 + T cells and NK cells (Fontenot, Nat Immunol 2005 , 6:1142-1151; D'Cruz, Nature Immunol 2005, 6:1152-1159; Maloy, Nature Immunol 2005, 97:189-192; Boyman, Nat Rev Immunol 2012, 12:180–190; Facciabene, Cancer Res 2012, 72:2162-2171). It can be seen that due to the dual role of IL-2 in immune regulation, the anti-tumor immune activity of effector cells activated in aldesleukin therapy will be inhibited by IL-2-dependent Treg cells, which limits its clinical efficacy.
- IL-2 variant molecules are being researched and developed.
- the key amino acid residues in the sequence that bind to each subunit of IL2R are mutated to reduce the binding to IL2R ⁇ in order to reduce the toxicity caused by high-affinity IL2R and the activation of Treg, or optimize the binding to IL2R ⁇ and/or ⁇ c to enhance Enhance anti-tumor activity on the proliferation and activation of CD8 + T cells and NK cells.
- IL-2 polyethylene glycol
- PEG polyethylene glycol
- Nektar's Bempegaldesleukin which is composed of aldesleukin coupled with an average of 6 dissociable PEG groups, which not only prolongs IL-2 It can also block the interaction between IL-2 and high-affinity IL-2R by coupling the PEG group on the lysine residue near the IL2R ⁇ binding site (Deborah H, Clin Cancer Res 2016,22:680 -690);
- THOR-707 developed by Synthorx has adopted another way to achieve PEG modification of IL-2, which utilizes engineered bacteria to incorporate unnatural amino acid residues into the IL-2 protein sequence, and then converts the non-dissociable
- the PEG moiety of ⁇ - ⁇ is conjugated to unnatural amino acid residues, thereby generating a homogeneous PEG-modified "non- ⁇ " IL-2 molecule, which Synthorx reported was able to induce peripheral naive T cells and NK cells expansion
- Alkermes' Nemvaleukin alfa is a fusion of IL-2 variants and IL2R ⁇ (CD25) extracellular domain fragments, thereby blocking the interaction of the drug candidate with IL2R ⁇ on Treg cells (Boni, Journal of Clinical Oncology 2021 , 39:15 Suppl, 2513).
- Boyman et al. formed antigen-antibody complexes between IL-2 and anti-IL-2 antibodies to block the binding of IL-2 to IL2R ⁇ , thereby selectively activating CD8 + T cells and NK cells expressing medium-affinity IL2R to enhance anti-IL2R.
- the domain used to block the binding of IL-2 and IL2R ⁇ is usually in a binding-dissociation dynamic equilibrium with the modified IL-2. Due to the high affinity between IL2R and IL-2 The modified IL-2 variants cannot completely avoid being competitively bound by high-affinity IL2R; secondly, some variants have increased immunogenicity due to the introduction of mutations in the IL-2 sequence, which induces
- the neutralizing anti-drug antibodies (anti-drug antibodies, ADA) not only limit the clinical application of IL-2 products, but also may inhibit the immune regulation function of endogenous IL-2 and cause potential safety risks.
- aldesleukin is an aglycosylated modified protein expressed by Escherichia coli, which is an amphiphilic molecule. It exists in the form of microaggregates produced by the aggregation of an average of 27 rhIL-2 molecules, and it was found in clinical trials that 60% to 75% There is ADA (Proleukin (aldesleukin) injection instructions) in the tested patients; BAY 50-479 mutates the 88th asparagine residue of hIL-2 to arginine (ie N88R), which appeared in the clinical phase 1 study ADA (Margolin, Clin Cancer Res 2007, 13:3312-3319) that recognizes this site was not recognized; THOR-707 contains unnatural amino acid sequences, which are usually recognized as foreign proteins by the body and may cause The body produces ADA (Levin, Nature 2012, 484:529-533).
- NKTR-214 Another PEG-modified IL-2 molecule, NKTR-214, although providing a kinetically slow release of IL-2, PEGylation also interferes with the binding of IL-2 to IL-2R ⁇ and ⁇ c, thereby Block IL-2 stimulation against tumor effector cells (Silva, Nature 2019, 565:186–191).
- Anti-IL2R ⁇ and ⁇ c bispecific antibodies cannot selectively distinguish between medium-affinity IL2R and high-affinity IL2R in activating the downstream signaling pathway of IL2R, so they will indiscriminately induce the activation and expansion of Treg cells and effector cells.
- the present application provides a kind of IL-2 receptor polymer (namely IL2R ⁇ / ⁇ or IL2R ⁇ / ⁇ / ⁇ ) that does not bind to IL2R ⁇ and contains ⁇ subunits, only selectively binds to IL2R ⁇ / ⁇ and can activate
- IL-2 receptor polymer namely IL2R ⁇ / ⁇ or IL2R ⁇ / ⁇ / ⁇
- the novel IL-2/IL-2 antibody fusion protein of IL2R ⁇ / ⁇ receptor-mediated signal transduction pathway circumvents the following technical problems to address unmet clinical needs:
- DLT Dose Limiting Toxicity
- Treg cell-mediated tumor immunosuppression Existing IL-2 products and/or IL-2 products under development will bind to the high-affinity IL2R on the surface of Treg cells in tumor tissue or in the tumor microenvironment to activate Treg cells and promote Its proliferation, in turn, will also promote the proliferation of other immunosuppressive cells in the tumor microenvironment, produce tumor immunosuppressive effect, and thus affect the curative effect.
- IL-2 product aldesleukin is an aglycosylated modified protein expressed by Escherichia coli, and Naturally present in the form of microaggregates, leading to the induction of ADA when the drug is administered in vivo.
- IL-2 products under research for example, IL-2 variants with amino acid mutations introduced at the IL2R ⁇ binding site, and IL-2 variants with non-natural amino acid sequences introduced at the PEG site-directed modification site, etc., when applied to the human body May trigger the body to produce ADA.
- ADA against IL-2 and/or IL-2 variants will not only limit the clinical application of IL-2 products, but also may interact with endogenous IL-2 and cause safety risks.
- Short half-life of existing IL-2 products and narrow therapeutic window For example, the serum half-life of aldesleukin, a marketed IL-2 product, is only 85 minutes, while CD8 + T cells and NK cells with anti-tumor activity express It is a medium-affinity IL2R, so high doses of aldesleukin are needed to activate the anti-tumor activity of CD8 + T cells and NK cells, but high doses of aldesleukins will preferentially activate the immunosuppressive response and induce systemic toxic side effects, so aldesleukin The therapeutic window for interleukins is extremely narrow.
- IL-2 products under development can relatively selectively bind to medium-affinity IL2R, they can still bind to high-affinity IL2R to activate immunosuppression and induce systemic toxicity, so the dosage will be limited when applied to clinical treatment , resulting in a narrow therapeutic window and poor patient compliance.
- the molecules of existing IL-2 products and/or IL-2 products under research are still structurally unstable or easy to dissociate (such as the complex of IL-2 and anti-IL-2 antibody), and it is not easy to produce and prepare them with good homogeneity.
- the problem of qualitative products are still structurally unstable or easy to dissociate (such as the complex of IL-2 and anti-IL-2 antibody), and it is not easy to produce and prepare them with good homogeneity.
- the application provides a human interleukin-2 (hIL-2) variant.
- the hIL-2 variant is wild-type hIL-2 (as shown in SEQ ID NO: 1) which can bind to the anti-IL-2 antibody or antigen-binding fragment of the present application after amino acid mutation, and the mutation has no effect on IL-2. 2 had no significant effect on the biological activity.
- the application provides an isolated anti-IL-2 antibody or an antigen-binding fragment thereof, which can compete with IL2R ⁇ for binding to the hIL-2 variant of the application and block
- the binding of the hIL-2 variant to IL2R ⁇ , the binding affinity of the anti-IL-2 antibody or its antigen-binding fragment to the hIL-2 variant of the present application is high, and dissociation is not easy to occur.
- the application provides a novel IL-2/IL-2 antibody fusion protein, which comprises:
- hIL-2 human interleukin 2
- the hIL-2/IL-2 antibody fusion protein provided by this application has the following characteristics:
- the hIL-2 is wild-type hIL-2 as shown in SEQ ID NO: 1;
- the wild-type hIL-2 shown contains at least one, or two, or more than two amino acid mutations other than cysteine, and the variant retains the ability to specifically bind to IL2R.
- said amino acid mutation is at said antigen-antibody binding interface of said hIL-2 or said variant and an anti-IL-2 antibody or antigen-binding fragment thereof.
- said amino acid mutation is within said antigen-antibody binding interface.
- the amino acid mutation is outside the antigen-antibody binding interface.
- the amino acid mutations are located within and outside of the antigen-antibody binding interface.
- the anti-IL-2 antibodies include anti-mouse IL-2 (mIL-2) antibodies or chimeric antibodies derived therefrom and/or humanized antibodies (such as S4B6 antibodies and humanized antibodies thereof ), anti-IL-2 humanized antibodies (such as NARA1 antibody and TCB2 antibody), or anti-hIL-2 antibodies.
- mIL-2 anti-mouse IL-2
- said anti-mIL-2 antibody, or a chimeric or humanized antibody derived therefrom, or an antigen-binding fragment thereof is capable of competing with IL2R ⁇ for binding to said hIL-2 variant and is capable of blocking said hIL-2 variant.
- the anti-hIL-2 antibody can specifically recognize the IL2R ⁇ binding site of the hIL-2, and can specifically bind the hIL-2.
- the antigen-antibody binding interface of the anti-IL-2 antibody or antigen-binding fragment thereof and the hIL-2 or variant thereof comprises a disulfide bond through - the introduction of at least one cysteine residue into the sequence of said anti-IL-2 antibody or antigen-binding fragment thereof at the antibody-binding interface and through said hIL-2 or variant thereof at said antigen-antibody binding interface
- the sequence is formed by introducing at least one cysteine residue.
- the introduction of at least one cysteine residue is by amino acid substitution or point mutation, insertion or addition, or any combination thereof.
- the present application relates to isolated nucleic acid molecules (also referred to as "polynucleosides”) encoding hIL-2 variants, anti-IL-2 antibodies or antigen-binding fragments thereof, and hIL-2/IL-2 antibody fusion proteins herein. acid”), as well as expression vectors comprising said nucleic acids and host cells comprising said nucleic acids or expression vectors.
- the present application also relates to methods of making hIL-2 variants, anti-IL-2 antibodies or antigen-binding fragments thereof, and hIL-2/IL-2 antibody fusion proteins herein using said host cells.
- the application relates to bispecific molecules, immunoconjugates, chimeric antigen receptors, immunoconjugates, chimeric antigen receptors, or hIL-2/IL-2 antibody fusion proteins comprising an anti-IL-2 antibody or an antigen-binding fragment thereof herein, or an hIL-2/IL-2 antibody fusion protein.
- the present application relates to a pharmaceutical composition
- a pharmaceutical composition comprising the anti-IL-2 antibody of the present application or its antigen-binding fragment, or hIL-2/IL-2 antibody fusion protein, or comprising the Anti-IL-2 antibody or antigen-binding fragment thereof, or hIL-2/IL-2 antibody fusion protein bispecific molecule, immunoconjugate, chimeric antigen receptor, engineered T cell receptor, or oncolytic virus , and a pharmaceutically acceptable carrier.
- the present application relates to a kit comprising an effective amount of the anti-IL-2 antibody of the present application or an antigen-binding fragment thereof, or hIL-2/IL-2 antibody fusion protein, or comprising the present application
- the anti-IL-2 antibody or antigen-binding fragment thereof, or the pharmaceutical composition of hIL-2/IL-2 antibody fusion protein, and/or another anti-cancer agent and/or immunomodulator, the anti-cancer agent Including but not limited to microtubule disruptors, antimetabolites, topoisomerase inhibitors, DNA intercalators, alkylating agents, hormone therapies, kinase inhibitors (e.g., tyrosine kinase inhibitors including but not limited to EGFR inhibitors HER2 inhibitors, HER3 inhibitors, IGFR inhibitors, PI3K inhibitors and Met inhibitors), receptor antagonists, activators of tumor cell apoptosis (including but not limited to IAP inhibitors, Bcl2 inhibitors, MC11 inhibitors agent
- the present application relates to a kit comprising an effective amount of the anti-IL-2 antibody of the present application or an antigen-binding fragment thereof, or hIL-2/IL-2 antibody fusion protein, or comprising the present application
- the pharmaceutical composition of the anti-IL-2 antibody or its antigen-binding fragment or hIL-2/IL-2 antibody fusion protein of the application is provided.
- the present application relates to the use of the anti-IL-2 antibody or antigen-binding fragment thereof herein, or hIL-2/IL-2 antibody fusion protein for preparing a pharmaceutical composition or preparation for treating diseases
- said Diseases include, but are not limited to, proliferative diseases or infections, immunodeficiency diseases including, but not limited to, cancer and other cell proliferation disorders.
- the present application relates to the anti-IL-2 antibody or antigen-binding fragment thereof, or hIL-2/IL-2 antibody fusion protein herein, or a pharmaceutical composition comprising it, for use in the treatment of proliferative diseases or infections, immunodeficiency diseases .
- the present application relates to a method for treating a proliferative disease or infection, an immunodeficiency disease, comprising administering the anti-IL-2 antibody or antigen-binding fragment thereof herein, or hIL-2/IL-2 to a subject in need thereof Antibody fusion protein, or a pharmaceutical composition comprising it.
- the present application relates to a method of treating a disease, condition or disorder by increasing the immune response of the host, the method comprising administering to the subject an effective amount of the anti-IL-2 antibody of the present application or an antigen-binding fragment thereof , or hIL-2/IL-2 antibody fusion protein, or a pharmaceutical composition comprising the anti-IL-2 antibody of the present application or its antigen-binding fragment, or hIL-2/IL-2 antibody fusion protein, or comprising the application of the Kits or kits of anti-IL-2 antibodies or antigen-binding fragments thereof, or hIL-2/IL-2 antibody fusion proteins to specifically activate effector cells expressing IL2R ⁇ / ⁇ receptors (for example, CD8 + T cells and NK cells) to improve disease conditions.
- IL2R ⁇ / ⁇ receptors for example, CD8 + T cells and NK cells
- the present application relates to a method of stimulating the immune system, the method comprising administering to a subject an effective amount of the anti-IL-2 antibody of the present application or an antigen-binding fragment thereof, or hIL-2/IL-2 Antibody fusion protein, pharmaceutical composition comprising the anti-IL-2 antibody of the present application or its antigen-binding fragment, or hIL-2/IL-2 antibody fusion protein, or comprising the anti-IL-2 antibody of the present application or its antigen-binding fragment , or hIL-2/IL-2 antibody fusion protein kit or kit to stimulate the immune system.
- the present application is directed to a method of treating, alleviating or preventing a disease condition in which stimulation of the host's immune system is beneficial (i.e., ameliorating the disease condition by stimulating the host's immune system), said method comprising administering to said affected
- the subject is administered a therapeutically effective amount of the anti-IL-2 antibody of the present application or its antigen-binding fragment, or hIL-2/IL-2 antibody fusion protein, or comprising the application of the anti-IL-2 antibody or its antigen-binding fragment, or A pharmaceutical composition of hIL-2/IL-2 antibody fusion protein, or a pharmaceutical kit or kit comprising the anti-IL-2 antibody of the present application or an antigen-binding fragment thereof, or hIL-2/IL-2 antibody fusion protein, to
- the disease state is improved by enhancing the subject's cellular immune response, the disease state includes insufficient host immune response or immune deficiency, and further the disease state is a proliferative disease or infection.
- proliferative diseases include cancer and immunodeficiency
- FIG. 1A Proliferation of CTLL-2 cell lines (Fig. 1A) and NK-92 cell lines (Fig. 1B) against hIL-2 variants (hIL2-DM and hIL2-TM) and controls (mIL-2 and hIL-2). response.
- Figure 2 ELISA detection of binding activity of S4B6 humanized antibody to antigen mIL2-his or hIL2-his.
- Figure 3 Fortebio detected the binding of hIL2 variant hIL2-DM to hIL2R ⁇ and hIL2R ⁇ after binding to K2G11.
- FIG. 4 Schematic diagram of the structure of a representative hIL-2/IL-2 antibody fusion protein of the present application, and a three-dimensional schematic diagram of the antigen-antibody binding interface of an example hIL-2/IL-2 antibody fusion protein.
- the C-terminus of hIL-2 or hIL-2 variant is connected to the N-terminus of hIL-2 antibody heavy chain through a linker, and the disulfide bond between hIL-2 or hIL-2 variant and hIL-2 antibody (shown by dashed line)
- Figure 4A Located at the antigen-antibody binding interface (Figure 4A); disulfide bonds at the binding interface of hIL-2 variants (K64G and N90R) in exemplary hIL-2/IL-2 antibody fusion proteins and anti-IL-2 humanized antibodies was formed by introducing a cysteine residue in each of the hIL-2 variants (K64G and N90R) and the anti-IL-2 humanized antibody (Fig. 4B).
- FIG. 5 ELISA detection of the binding activity of the hIL-2/IL-2 antibody fusion protein IC-Cy08 containing disulfide bonds and the control (hIL2-DM-Fc) to hCD25 ( Figure 5A) and hCD122 ( Figure 5B).
- FIG. 6A Proliferation of CTLL-2 cell line (Figure 6A) and NK-92 cell line ( Figure 6B) to IL-2/IL-2 antibody fusion protein IC-Cy08 containing disulfide bond and control (hIL-2) response.
- FIG. 7 Flow cytometry detection of CTLL-2 cell line in the IL-2 signaling pathway under the action of hIL-2/IL-2 antibody fusion protein IC-Cy08 containing disulfide bond and control (hIL-2) Result graph of STAT5 phosphorylation.
- FIG 8 ELISA detection of the antibody light chain-optimized hIL-2/IL-2 antibody fusion protein IC-Cy08 mutant containing disulfide bonds and the control (hIL2-DM-Fc and parental IC-Cy08) and hCD25 ( Figure 8A) and binding activity of hCD122 ( Figure 8B).
- FIG. 9 CTLL-2 cell line (Fig. 9A) and NK-92 cell line (Fig. 9B) to the IL-2/IL-2 antibody fusion protein IC-Cy08 mutant containing disulfide bond optimized through antibody light chain and Proliferative responses of controls (hIL-2 and parental IC-Cy08).
- FIG. 10 CTLL-2 cell line (Figure 10A) and NK-92 cell line ( Figure 10B) to hIL-2 amino acid sequence optimized hIL-2/IL-2 antibody fusion protein IC-Cy08 mutation containing disulfide bonds Proliferative responses of somatic as well as controls (hIL-2 and parental IC-Cy08).
- CTLL-2 cell line (Fig. 11A) and NK-92 cell line (Fig. 11B) hIL-2/IL-2 antibody fusion protein IC-Cy08-pITM after pI mutation and TM mutation of the heavy chain constant region and proliferative responses of controls (hIL-2 and parental IC-Cy08).
- FIG. 12A ELISA detection based on K2G11 disulfide bond-containing hIL-2/IL-2 antibody fusion proteins K2G11-Cy04, K2G11-Cy05 and K2G11-Cy06 and the control group (including H6K3(H)-Cy08 and H6K3(H)- Cy10) binding activity to hCD25 ( FIG. 12A ) and hCD122 ( FIG. 12B ), respectively.
- FIG. 13A CTLL-2 cell line ( Figure 13A) and NK-92 cell line ( Figure 13B) to hIL-2/IL-2 antibody fusion protein K2G11-Cy04, K2G11-Cy05 and K2G11- Proliferation responses of Cy06 and controls (including H6K3(H)-Cy08, H6K3(H)-Cy10, and antigen-antibody complexes formed by K2G11 and hIL2-DM( ⁇ 3,C125L)).
- Figure 14 Fortebio detected the binding of K2G11-Cy05 to hIL2R ⁇ , hIL2R ⁇ / ⁇ and hIL2R ⁇ / ⁇ / ⁇ .
- Figure 15 ELISA method to detect the binding activity of hIL-2/IL-2 antibody fusion protein (K2G11-Cy05) containing disulfide bond and control (NARA1leukin) to hCD25.
- Figure 16 Using the reporter gene method ( Figure 16A) and cell proliferation method (CTLL-2 cell line and NK-92 [NK-92(CD25-KO)] cell line with knockout CD25, Figure 16B) to detect the presence of disulfide Keyed hIL-2/IL-2 antibody fusion protein (K2G11-Cy05) as well as controls (NARA1leukin and wild-type hIL-2) activate IL2R ⁇ / ⁇ / ⁇ and/or hIL2R ⁇ / ⁇ receptor activity.
- K2G11-Cy05 disulfide Keyed hIL-2/IL-2 antibody fusion protein
- NARA1leukin and wild-type hIL-2 activate IL2R ⁇ / ⁇ / ⁇ and/or hIL2R ⁇ / ⁇ receptor activity.
- Figure 17 ForteBio detection of anti-IL-2 humanized antibody (K2G11-pITM) containing pI mutation and TM mutation in the heavy chain constant region and hIL-2/IL-2 antibody fusion protein constructed based on it and containing disulfide bonds (K2G11-Cy05), and the binding kinetics of the control antibody (K2G11) to Fc receptors, C1q and FcRn, respectively.
- K2G11-pITM anti-IL-2 humanized antibody
- K2G11-Cy05 disulfide bonds
- the reporter gene method detects the ADCC activity of the hIL-2/IL-2 antibody fusion protein (K2G11-Cy05) containing disulfide bonds that introduces pI mutation and TM mutation into the antibody constant region. Tuximab) was used as a positive control group.
- FIG. 19A and FIG. 19B After hIL-2/IL-2 antibody fusion proteins (K2G11-Cy04 and K2G11-Cy05) were incubated with cynomolgus monkey serum (M6, M7) or healthy human serum (H2, H3) for different times, the NK- The 92 cell proliferation method (FIG. 19A and FIG. 19B) and the reporter gene method (FIG. 19C) were used to detect changes in the biological activity of the samples, and the curves marked K2G11-Cy04 and K2G11-Cy05 in the figure represent control samples without serum treatment.
- Figure 20 Drug-time curves of anti-IL-2 antibody K2G11-pITM with heavy chain constant region engineered and anti-IL-2 antibody K2G11 without heavy chain constant region engineered in BALB/c mice.
- Figure 21 Flow cytometry detection of CD8 + T in peripheral blood samples of cynomolgus monkeys after administration of hIL-2/IL-2 antibody fusion protein K2G11-Cy05 engineered with heavy chain constant region and containing disulfide bonds , the number of NK and Treg cells and the expression of their proliferation marker Ki-67 (i.e. the proportion of Ki67 + cells). Arrows in the diagram indicate the time points of administration.
- Figure 22 Flow cytometry detection of CD4 + in the spleen of C57BL/6 mice after receiving the hIL-2/IL-2 antibody fusion protein K2G11-Cy05 engineered with a heavy chain constant region and containing disulfide bonds Changes in the number of CD25 + FoxP3 + Treg cells, CD8 + CD44 high T cells, and NK1.1 + cells (Figure 22A), changes in the number of conventional CD4 + T cells ( Figure 22C), and CD8 + CD44 high T cells and Changes in the absolute count ratio of Treg cells and the absolute count ratio of NK1.1 + cells to Treg cells ( FIG. 22B ), and changes in the body weight of mice in each administration group ( FIG. 22D ).
- Figure 23 The anti-tumor effect of hIL-2/IL-2 antibody fusion protein K2G11-Cy05 with heavy chain constant region engineered and containing disulfide bond and its control molecule NARA1leukin at different doses in mouse CT26 colon cancer model active.
- Figure 24 ELISA method to detect the binding activity of hIL-2/NARA1 antibody fusion protein containing disulfide bond and control molecule (hIL2-DM-Fc) to hCD25 ( Figure 24A) and hCD122 ( Figure 24B).
- FIG 25 ELISA method to detect the binding activity of hIL-2/TCB2 antibody fusion protein containing disulfide bond and control molecule (hIL2-DM-Fc) to hCD25 ( Figure 25A) and hCD122 ( Figure 25B).
- FIG. 26 Proliferative responses of CTLL-2 cell lines (Fig. 26A) and NK-92 cell lines (Fig. 26B) to hIL-2/NARA1 antibody fusion proteins containing disulfide bonds and a control molecule (wild-type hIL-2).
- FIG. 27 Proliferative response of CTLL-2 cell lines (Fig. 27A) and NK-92 cell lines (Fig. 27B) to hIL-2/TCB2 antibody fusion protein containing disulfide bonds and a control molecule (wild-type hIL-2).
- Interleukin-2 Interleukin-2
- IL-2 Interleukin-2
- IL2 IL2
- Human IL-2 (hIL-2) described herein refers to mature human IL-2 protein, also known as "wild-type hIL-2” or “wild-type IL-2” or “WT IL-2", which A polypeptide having the following amino acid sequence:
- Unprocessed human IL-2 additionally comprises a signal peptide of N-terminal 20 amino acids and has the sequence of SEQ ID NO: 229, and mature (or processed) IL-2 protein is a polypeptide from which the signal peptide has been removed.
- the hIL-2 variants (or IL-2 muteins) described herein are variants of wild-type hIL-2, and the variants may contain one, two, or more than two amino acids in the wild-type IL-2 amino acid sequence Amino acid mutations, which may be amino acid substitutions, deletions, insertions and additions. In some embodiments, preferred amino acid mutations are amino acid substitutions, insertions or additions, or combinations thereof. In other embodiments, preferred amino acid mutations are amino acid deletions.
- hIL-2 variants having enhanced binding affinity to the anti-IL-2 antibodies or antigen-binding fragments described herein are obtained by introducing mutations at the amino acid positions of the specific mutations described herein, said The enhanced binding affinity can be that the wild-type IL-2 that cannot cross-react with the anti-IL-2 antibody or antigen-binding fragment can cross-react with the anti-IL-2 antibody or antigen-binding fragment after amino acid mutation, or has Moderate binding affinity (eg, below 10 -8 M), or high binding affinity (eg, below 10 -9 M, even below 10 -10 M).
- Moderate binding affinity eg, below 10 -8 M
- high binding affinity eg, below 10 -9 M, even below 10 -10 M.
- mutations are introduced at specific mutated amino acid positions described herein to obtain hIL-2 variants capable of forming ultrastable complexes with anti-IL-2 antibodies or antigen-binding fragments described herein. In some embodiments, mutations are introduced at specific mutated amino acid positions described herein to obtain hIL-2 variants with modifications that eliminate glycosylation. In some embodiments, mutations are introduced at specific cysteine residue positions described herein to result in hIL-2 variants that eliminate free cysteines.
- amino acid positions in the IL-2 protein or polypeptide are the amino acid positions numbered according to SEQ ID NO:1.
- K64 it refers to the 64th lysine residue (K) in the amino acid sequence shown in SEQ ID NO: 1, or the corresponding position on other IL-2 polypeptide sequences after alignment amino acid residues.
- IL2R refers to the IL-2 receptor.
- the high-affinity IL2R is composed of three chains or subunits: IL-2R ⁇ (also known as CD25), IL-2R ⁇ (also known as CD122), and a common gamma chain, ⁇ c (also known as CD132).
- High affinity IL2Rs can be expressed as “IL2R ⁇ ” or “IL2R ⁇ / ⁇ / ⁇ ”.
- IL-2R ⁇ and ⁇ c is used interchangeably with the terms "IL-2R ⁇ ” or “IL2R ⁇ / ⁇ ” and refers to a medium affinity IL-2R that lacks an ⁇ subunit and contains ⁇ and ⁇ subunits.
- IL2R ⁇ binding sites or “IL2R ⁇ binding region” refers to the region where IL-2 protein and IL2R ⁇ protein interact and interact with each other observed through the resolved crystal structure.
- affinity refers to the intrinsic binding ability of the interaction between a molecule (eg, receptor or antigen) and its counterpart (eg, ligand or antibody), ie the strength of the sum of all non-covalent interactions.
- binding affinity is the intrinsic binding affinity used to reflect a 1:1 interaction between members of a binding pair (eg, receptor and ligand or antigen and antibody).
- the affinity of a molecule X for its partner Y can usually be expressed in terms of the equilibrium dissociation constant (K D ), which is the dissociation rate constant and the association rate constant (K diss or K off ) and K Binding (Ka or Kon) ratio. Affinity can be measured by common methods known in the art, including the methods used in this application.
- regulatory T cell refers to a specific type of CD4 + T cell that is capable of suppressing the response of other T cells (eg, effector T cells).
- Regulatory T cells are characterized by constitutively high expression of IL2R ⁇ and the transcription factor FOXP3 and play a key role in the induction and maintenance of peripheral self-tolerance to antigens, including tumor antigens.
- Regulatory T cells require IL-2 to induce activation of their suppressive functional activity against effector T cells.
- antibody is meant to include one or more proteins encoded substantially or in part by immunoglobulin genes or fragments of immunoglobulin genes. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as a myriad of immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively.
- Antibodies can be of any isotype/class (eg, IgG, IgM, IgA, IgD, and IgE) or of any subclass (eg, IgGl, IgG2, IgG3, IgG4, IgAl, IgA2).
- Typical immunoglobulin (eg, antibody) structural units include tetramers. Each tetramer is composed of the same two pairs of polypeptide chains, each pair having one "light” chain (about 25 kD) and one "heavy” chain (about 50-70 kD). Both the light and heavy chains are divided into regions of structural and functional homology. The terms “constant” and “variable” are used both structurally and functionally.
- each chain defines a variable (V) region or domain of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
- V variable region or domain of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
- Antibody as used herein is the broadest and includes various antibody structures, as long as they can exhibit the desired antigen-binding activity, said antibodies include but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies) and antibody fragments.
- Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments resulting from protease digestion.
- the term "antigen-binding fragment" of an antibody refers to a portion of an antibody comprising one, or two, or more than two CDRs or any other antigen-binding (for example, IL-2 protein) is an antibody fragment formed from an antibody fragment that does not have the structure of a complete antibody.
- Antigen-binding fragments can bind to the same antigen as intact antibodies.
- the antigen-binding fragment may contain one, or two, or more than two CDRs from a particular human antibody grafted to a framework region from one, or two, or more than two different human antibodies .
- Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragment, Fd fragment, disulfide bond-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv') , disulfide bond stabilized diabody (dsdiabody), single chain antibody molecule (scFv), scFv dimer (bivalent diabody), bivalent single chain antibody (BsFv), camelized single domain antibody (camelized single domain antibody), nanobody, domain antibody and diabody.
- a "Fab” fragment of an antibody refers to an antibody fragment that is disulfide-bonded by a light chain (including a light chain variable region and a light chain constant region) and a heavy chain variable region and CH1.
- a “Fab'” fragment refers to a Fab fragment that includes part of the hinge region.
- F(ab')2 refers to a dimer of Fab.
- the "Fc” fragment of an antibody is an antibody fragment formed by linking CH2 and CH3 of the heavy chain through a disulfide bond.
- the Fc fragment of an antibody is responsible for a variety of effector functions, such as determining the half-life of the antibody in serum, mediating immune responses, for example, antibody-dependent cell-mediated cytotoxicity (Antibody-dependent cell-mediated cytotoxicity, ADCC), Activates complement-dependent cytotoxicity (Complement Dependent Cytotoxicity, CDC) or antibody-dependent cell-mediated phagocytosis (Antibody Dependent Cellular Phagocytosis, ADCP), but does not participate in antigen binding.
- the "Fv" fragment of an antibody refers to the smallest fragment of an antibody that contains the complete antigen binding site. The Fv fragment consists of the variable region of one light chain and the variable region of one heavy chain.
- the "Fd fragment” of an antibody refers to the heavy chain portion of the Fab fragment, including VH, CH1 and part of the hinge region.
- Single-chain Fv antibody scFv
- Single-chain Fv antibody refers to an engineered antibody formed by directly linking the variable region of the light chain to the variable region of the heavy chain or through a peptide chain (Huston JS et al., Proc Natl Acad Sci USA 1988, 85:5879-5883).
- (dsFv)2 contains three peptide chains: it means that two VH groups are connected by a polypeptide linker, and are combined with two VL groups by disulfide bonds.
- a "bispecific ds diabody” contains VL1-VH2 (linked by a polypeptide linker) and VH1-VL2 (also linked by a polypeptide linker), which are combined by a disulfide bond between VH1 and VL1.
- "Bispecific dsFv” or “dsFv-dsFv” contains three polypeptide chains: VH1-VH2 fragments, of which the heavy chains of the two are connected by a polypeptide linker (such as: a long elastic linker), and are respectively connected to the VL1 and VL2 fragments combine, each pair of heavy and light chains paired by disulfide bonds has a different antigenic specificity.
- a "scFv dimer” is a bivalent diabody or a bivalent single chain antibody (BsFv) containing two VH-VL (linked by a polypeptide linker) fragments that are dimerized, where the VH of one fragment is bound to the other The VL's cooperate to form two binding sites that can target the same antigen (or antigen-binding epitope) or different antigens (or antigen-binding epitopes).
- scFv dimers are bispecific, bifunctional antibodies comprising interconnected VL1-VH2 (linked by a polypeptide linker) and VH1-VL2 (linked by a polypeptide linker), wherein VH1 and VL1 cooperates, VH2 and VL2 cooperate, and each cooperative pair has a different antigen specificity.
- Single-chain antibody Fv-Fc scFv-Fc
- “Camelized single domain antibody (Camelized single domain antibody)” "heavy chain antibody” or “HCAb (Heavy-chain-only antibodies, HCAb)” all refer to antibodies containing two VH domains without light chains (Riechmann L&Muyldermans S, J Immunol Methods 1999, 231:25-38; Muyldermans S, J Biotechnol 2001, 74:277-302; Patent Application WO94/04678; Patent Application WO94/25591; Patent U.S. Patent No. 6005079). Heavy chain antibodies were originally discovered in camelids, which include camels, dromedaries, and llamas.
- camelized antibodies (camelized antibodies) have full function of antigen binding (Hamers-Casterman C et al., Nature 1993, 363:446-448; Nguyen VK et al., Heavy-chain antibodies in Camelidae: a case of evolutionary innovation, Immunogenetics 2002, 54:39-47; Nguyen VK et al., Immunology 2003, 109:93-101).
- the variable region (VHH domain) of a heavy chain antibody is the smallest known antigen-binding unit produced by acquired immunity (Koch-Nolte F et al., FASEB J 2007, 21:3490-3498).
- a “Nanobody” is an antibody fragment consisting of a VHH domain from a heavy chain antibody and two constant regions, CH2 and CH3.
- Domain antibody refers to an antibody fragment that contains only one heavy chain variable region or one light chain variable region.
- two or more VH domains are covalently joined by a polypeptide linker and form a bivalent domain antibody.
- the two VH domains of bivalent domain antibodies can target the same or different antigens.
- “Diabodies” include small antibody fragments with two antigen-binding sites that contain a VH domain and a VL domain (such as VH-VL or VL-VH) linked on the same polypeptide chain (Holliger P etc., Proc Natl Acad Sci USA 1993,90:6444-6448; patent EP404097; patent application WO93/11161).
- the linker between the two domains is so short that the two domains on the same chain cannot pair with each other, thus forcing the two domains to pair with the complementary domains of another chain, forming two antibody binding sites.
- the two antibody binding sites can be targeted to bind the same or different antigens (or antigen binding epitopes).
- the antibody domain of the anti-IL-2 antibody or hIL-2/IL-2 antibody fusion protein of the present application includes all of the full-length antibody or its antigen-binding fragment (refer to the definition above), and optionally includes an antibody capable of interacting with IL2R ⁇ . All or part of the antibody variable region that competitively binds IL-2, and optionally includes one, or two, or more than two regions encoded by the V gene and/or the D gene and/or the J gene.
- immunoglobulin molecule refers to a protein having the structure of a naturally occurring antibody.
- immunoglobulins of the IgG class are heterotetrameric glycoproteins of approximately 150,000 Daltons composed of two light chains and two heavy chains linked by disulfide bonds. From N-terminus to C-terminus, each heavy chain has a variable region (VH, also called variable heavy domain or heavy chain variable domain), followed by 3 constant domains (CH1, CH2 and CH3, also called heavy chain constant region).
- each light chain has a variable region (VL, also called variable light domain or light chain variable domain or light chain variable region), followed by a constant light domain (also called is the light chain constant region, CL).
- VL variable light domain
- constant light domain also called is the light chain constant region, CL.
- the heavy chains of immunoglobulins can be classified as ⁇ (IgA), ⁇ (IgD), ⁇ (IgE), ⁇ (IgG) or ⁇ (IgM), some of which can be further divided into subclasses, such as ⁇ 1 (IgG1), ⁇ 2 (IgG2 ), ⁇ 3 (IgG3), ⁇ 4 (IgG4), ⁇ 1 (IgA1) and ⁇ 2 (IgA2).
- Immunoglobulins Based on the amino acid sequence of their constant domains, the light chains of immunoglobulins can be divided into what are called kappa ( ⁇ ) and lambda ( ⁇ ). Immunoglobulins generally consist of two Fab molecules and an Fc domain connected by an immunoglobulin hinge region.
- VL light chain variable region
- VH heavy chain variable region
- VL and VH refer to polypeptides comprising VL or VH , respectively.
- Endogenous V L is encoded by gene segments V (variable) and J (junction)
- endogenous V H is encoded by V, D (diversity) and J.
- Both VL or VH include hypervariable regions CDRs (complementarity determining regions) and framework regions (FRs).
- variable region or “V region” are used interchangeably to refer to a heavy chain variable region or a light chain variable region comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. V regions can be naturally occurring, recombinant or synthetic.
- antibody light chain variable regions and/or antibody heavy chain variable regions may sometimes be collectively referred to as “antibody variable regions” or “antibody chains.”
- an "antibody light chain variable region” or “antibody heavy chain variable region” and/or an “antibody variable region” and/or an “antibody chain” optionally comprises an incorporated cysteine residues in the polypeptide sequence.
- CDR complementarity determining region
- HVR hypervariable region
- the CDR is the target protein binding site of the antibody, and the CDR is structurally complementary to the epitope of the target protein, so the binding site has the specificity of binding this target protein.
- native four-chain antibodies contain six CDRs, three in the VH (HCDR1, HCDR2, and HCDR3) and three in the VL (LCDR1, LCDR2, and LCDR3).
- the rest of the VL or VH regions except for the CDRs, namely the framework regions (FRs) have less amino acid sequence variation (Kuby, Immunology, 4th edition, Chapter 4, W H Freeman & Co., 2000).
- CDRs and FRs can be determined using various definition methods well known in the art, for example, Kabat, Chothia, IMGT, and Contact (see, e.g., Kabat et al. 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.
- an antigen binding site is also described in: Ruiz et al., Nucleic Acids Res 2000, 28:219-221; Lefran MP, Nucleic Acids Res 2001, 29:207-209; Lefranc MP, The Immunologist 1999, 7 :132-136; Lefranc MP et al., Dev Comp Immunol 2003,27:55-77; MacCallum et al., J Mol Biol 1996,262:732-745; Martin et al., Proc Natl Acad Sci USA 1989,86:9268-9272; Martin et al., Methods Enzymol 1991, 203:121-153; Rees et al., In: Sternberg M J E (eds.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996).
- the application includes any one of the defined methods to determine the antibody portion of the hIL-2/IL-2 antibody fusion protein of the application or the CDR in the anti-IL-2 antibody or antigen-binding fragment thereof of the application, and Table 1 shows the use of different The amino acid residues of the antibody CDRs determined by the definition method. The exact number of amino acid residues encompassing a particular CDR varies with the sequence of the CDR. Once the amino acid sequence of the variable region of the antibody is determined, those skilled in the art can determine the CDR of the antibody by conventional methods including but not limited to the above definitions.
- Kabat et al. also defined a numbering system for variable region sequences, which can be applied to any antibody.
- One of ordinary skill in the art will be able to unambiguously apply this "Kabat numbering" system to the variable region sequence of any antibody without relying on any experimental data other than the antibody sequence itself to determine the variable region sequence.
- the numbering of specific amino acid residue positions in the variable region of the antibody portion of the anti-IL-2 antibody or hIL-2/IL-2 antibody fusion protein of the present application is determined according to the Kabat numbering system.
- Fc region refers to the C-terminal region of an immunoglobulin heavy chain, which contains at least a part of a constant region, such as an immunoglobulin except for the first constant region (CH1). Heavy chain constant region.
- the Fc region used herein includes native sequence Fc region and/or Fc region variants, and may be a part of the anti-IL-2 antibody or hIL-2/IL-2 antibody fusion protein of the present application.
- a human IgG heavy chain Fc region is generally defined as comprising a cysteine residue at position 226 or a proline residue at position 230 at its carboxy-terminus, It is according to the EU numbering system/scheme, as found in Kabat et al.) (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, VA).
- the term "monoclonal antibody” refers to an antibody obtained from a population of antibodies that is substantially homogeneous, that is, the individual antibodies comprising the population are identical except for natural mutations that may be present in minor amounts. of. Monoclonal antibodies exhibit a single binding specificity and affinity for a particular epitope.
- chimeric antibody refers to an antibody that contains sequences derived from two different antibodies (eg, US Pat. No. 4,816,567), usually derived from different species.
- chimeric antibodies comprise human and rodent antibody fragments, typically human constant regions and mouse variable regions.
- Methods for producing chimeric antibodies include conventional recombinant DNA and gene transfection techniques known to those of ordinary skill in the art (e.g. Morrison SL et al., Proc Natl Acad Sci USA 1984, 81:6851-6855; Patent US 5,202,238 and Patent US 5,204,244).
- humanized antibody or antigen-binding fragment refers to an antibody or antigen-binding fragment comprising non-human animal-derived CDRs, human-derived FR regions, and human-derived constant regions. Since the humanized antibody or antigen-binding fragment has reduced immunogenicity, it can be used as a therapeutic agent for humans.
- the non-human animal is a mammal such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.
- the humanized antibody or antigen-binding fragment consists substantially entirely of human sequences except for the CDR sequences which are non-human.
- the human-derived FR region may comprise the same amino acid sequence as the human antibody from which it was derived, or it may comprise some amino acid changes, e.g., no more than 10, 9, 8, 7, 6, 5 , 4, 3, 2 or 1 amino acid changes.
- the amino acid change may be present only in the heavy chain FR region, only in the light chain FR region, or in both chains.
- corresponding human germline sequence refers to an antibody variable region amino acid sequence or subsequence that is compared to all other known human germline immunoglobulin variable region amino acid sequences. Or the subsequence has the highest amino acid sequence identity to a reference human germline immunoglobulin variable region amino acid sequence.
- the corresponding human germline sequences may be framework regions alone, complementarity determining regions, framework regions and complementarity determining regions, variable regions, or other combinations of variable region sequences or subsequences. Sequence identity can be determined using the methods described herein, eg, by aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art.
- the corresponding human germline nucleic acid or amino acid sequence may be at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% identical to a reference human germline immunoglobulin variable region amino acid sequence , 98%, 99% or 100% sequence identity.
- the antibody part of the anti-IL-2 antibody or hIL-2/IL-2 antibody fusion protein of the present application can be selected from any of the following forms, or two, or more than two forms, including chimeric forms, non-human forms, human
- the humanized form or the fully human form as long as the form can compete with human IL2R ⁇ for binding to hIL-2 or its variants and can inhibit the binding of human IL2R ⁇ to hIL-2 or its variants, while retaining hIL-2 or its variants. Binding activity and functional activity of IL2R ⁇ / ⁇ .
- the term “specifically binds” or “binding specificity” or “specific for” or “binds” refers to a binding reaction that determines a heterogeneous population of proteins and other biological agents (e.g., In a biological sample (such as blood, serum, plasma, or tissue samples), the presence of the target molecule (or protein or antigen) means that the binding is selective for the target molecule and can discriminate those that are not desired or non-specific interactions.
- an antibody that specifically binds a target molecule (which may be an antigen) binds the target molecule with greater affinity, binding activity, more readily and/or consistently than the antibody binds to other non-target molecules. longer.
- the antibody specifically binds to the target antigen with an equilibrium dissociation constant (K D ) of ⁇ 1 ⁇ , ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, or ⁇ 0.1 nM.
- K D equilibrium dissociation constant
- the term "antigen-antibody binding interface” refers to the amino acid residues that participate in the antigen-antibody interaction (e.g., by binding, steric hindrance, stabilization/destabilization, spatial distribution) when the antibody specifically binds the antigen.
- the spatial structure region formed by the base includes the antigenic epitope and the antigen-binding region of the antibody.
- the amino acid residues of the antigen-antibody binding interface may or may not be in contiguous sequence. For example, when the interface is three-dimensional, the amino acid residues within the interface may be located separately at different positions on the linear sequence.
- epipe means a protein-determining region capable of specifically binding to an antibody.
- Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. The difference between conformational epitopes and non-conformational epitopes is that antibody binding to the former will be lost in the presence of denaturing solvents, but binding to the latter will not be lost.
- epitope-binding domain or “antigen-binding domain” or “antigen-binding region” are used interchangeably and refer to a specific interaction with a binding site on a target epitope (e.g., by A portion of a binding molecule (eg, an antibody or an epitope-binding fragment or derivative thereof) that binds, sterically hinders, stabilizes/destabilizes, spatially distributes).
- a binding molecule eg, an antibody or an epitope-binding fragment or derivative thereof
- Antigen binding region in the present application also refers to one, or both, of antibodies that retain specific interaction (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) with an IL-2 epitope. or two or more fragments.
- fused or “fused” when applied to amino acid sequences refer to the combination of two or more amino acid sequences into a non- A naturally occurring single amino acid sequence.
- a fusion amino acid sequence can be produced by genetic recombination of two encoding polynucleotide sequences and can be expressed by introducing a construct containing the recombined polynucleotide into a host cell.
- antibody variant refers to an antibody polypeptide sequence that contains at least one amino acid mutation in the variable region of a reference antibody.
- a variant may be substantially homologous or substantially identical to the unmodified antibody.
- one, two, three, four, five and/or six CDRs of the antibody portion of the anti-IL-2 antibody or hIL-2/IL-2 antibody fusion protein of the present application carry amino acid Mutations to increase the degree of humanization of the antibody or antibody portion, enhance specificity or affinity for IL-2 variants, and/or form ultrastable complex structures with IL-2 or variants thereof.
- amino acid mutations are carried out in one, two, three and/or four FRs of the antibody portion of the anti-IL-2 antibody or hIL-2/IL-2 antibody fusion protein of the application to improve the The degree of humanization of the antibody or antibody portion and/or formation of ultrastable complex structures with IL-2 or variants thereof.
- one or more than two amino acid mutations are carried out in the CDR and FR of the antibody part of the anti-IL-2 antibody or hIL-2/IL-2 antibody fusion protein of the present application to improve the antibody or antibody part.
- the degree of humanization enhance the specificity or affinity for IL-2 variants, and/or form ultra-stable complex structures with IL-2 or variants thereof.
- the amino acid mutation comprises amino acid substitution, deletion, insertion or any combination thereof.
- amino acid substitutions include conservative amino acid substitutions and non-conservative amino acid substitutions.
- Conservative amino acid substitutions involve the substitution of another amino acid in the same class (such as similar chemical properties or functions), and non-conservative substitutions involve substitution of this amino acid with another amino acid in another class.
- a category of amino acids One of ordinary skill in the art can make conservative amino acid substitutions based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
- nonpolar (hydrophobic) amino acids include alanine (Ala, A), leucine (Leu, L), isoleucine (Ile, I), valine (Val, V), Proline (Pro, P), phenylalanine (Phe, F), tryptophan (Trp, W) and methionine (Met, M);
- polar neutral amino acids including glycine (Gly, G) , serine (Ser, S), threonine (Thr, T), cysteine (Cys, C), tyrosine (Tyr, Y), asparagine (Asn, N) and glutamine (Gln , Q);
- positively charged (basic) amino acids including arginine (Arg, R), lysine (Lys, K) and histidine (His, H);
- negatively charged (Acidic) amino acids include aspartic acid (Asp, D) and glutamic acid (Glu
- Non-conservative amino acid substitutions may lead to large changes in the properties or functions of biomacromolecules.
- Substitutions that are generally expected to produce the greatest change in the properties of a polypeptide include, but are not limited to: (a) Substitution of (or being substituted by) a hydrophilic residue such as serine or threonine such as leucine, isoleucine, phenylalanine , valine or alanine and other hydrophobic residues; (b) cysteine or proline replaces (or is replaced by) any other amino acid residue; (c) such as lysyl, arginyl or group Residues with positively charged side chains such as aminoacyl are substituted for (or are substituted by) negatively charged residues such as glutamyl or aspartyl; or (d) residues with bulky side chains such as phenylalanine Substituting (or being substituted for) residues that do not have side chains such as glycine.
- the site or region for the introduction of amino acid sequence mutations can be determined in advance, the potential changes in the properties or functions of biomacromolecules brought about by non-conservative substitutions are unpredictable.
- the hIL-2 variants of the present application have obtained unexpected large changes in function by creatively carrying out non-conservative amino acid substitutions on the basis of wild-type hIL-2, for example, hIL-2 is not related to this
- the anti-IL-2 antibody binding of the application has obtained a large change in binding through non-conservative amino acid substitutions.
- the first and second are used only for the convenience of distinguishing the position of the introduction of cysteine.
- the use of these terms is not intended to confer a particular order of substitution of particular amino acid residues for cysteine residues or addition or insertion of cysteine residues unless expressly so stated.
- nucleic acid or polypeptide sequences As used herein, the terms “identical” or “identity” or “percent identity” or “percent sequence identity” are used interchangeably between two or more nucleic acid or polypeptide sequences and refer to After the sequences (or nucleic acid sequences) are aligned, and if necessary, spacers are introduced to maximize the number of identical amino acids (or nucleic acids), in the candidate sequence, the amino acid (or nucleic acid) residues that are identical to the reference sequence account for the majority of the candidate sequence The percentage of amino acid (or nucleic acid) residues. Conservative substitutions of such amino acid residues may or may not be considered identical residues.
- isolated when referring to a nucleic acid (or polynucleotide) or protein means that the nucleic acid or protein is substantially free of other cellular components with which it is associated in its native state, preferably in a homogeneous state, e.g. , an isolated nucleic acid or protein can be one that has been removed from its native or natural environment. Isolated nucleic acids or proteins can be lyophilized or in aqueous solution. Generally, its purity and homogeneity can be determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. as the main ingredient in the preparation. Proteins are basically obtained by purification.
- nucleic acid or protein yields essentially one band in an electrophoretic gel.
- recombinant proteins expressed in host cells are considered isolated, and the same applies to separation, fractionation, or fractionation by any technique known to those skilled in the art. or substantially purified native or recombinant protein.
- an "isolated antibody” refers to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds hIL-2 is substantially free of antibodies that specifically bind hIL-2).
- An isolated antibody that specifically binds hIL-2 may, however, be cross-reactive with other antigens, such as IL-2 molecules from other species, eg, mouse IL-2.
- an isolated antibody can be substantially free of other cellular material and/or chemicals.
- the recombinant polynucleotide encoding the polypeptide or protein of the present application (such as hIL-2 or its variants, anti-IL-2 antibody, hIL-2/IL-2 antibody fusion protein) contained in the vector is replaced by considered separate.
- isolated polynucleotides include recombinant polynucleotides contained in heterologous host cells or purified (partially or substantially) polynucleotides in solution.
- An isolated polynucleotide includes a polynucleotide molecule contained in cells that normally contain the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location other than its natural chromosomal location.
- An isolated polynucleotide or nucleic acid of the present application also includes such molecules produced synthetically.
- a polynucleotide or nucleic acid may be or may include a regulatory element such as a promoter, ribosomal binding site, or transcription terminator.
- engineering is considered to include any manipulation of the polypeptide or protein backbone or post-translational modification of naturally occurring or recombinant proteins or polypeptides or fragments thereof.
- Engineering includes mutation of the amino acid sequence, modification of the glycosylation pattern or side chain groups of each amino acid, and combinations of these methods.
- polypeptide refers to a polymer of amino acids and their equivalents, rather than to a specific length of the product; thus, “peptide” and “protein” are included within the definition of polypeptide. Also included within the definition of polypeptide is an “antibody” as defined herein.
- a "derivative” is a polypeptide or fragment thereof that has one, or two, or more non-conservative or conservative amino acid substitutions relative to a second polypeptide (also referred to as a "variant"); or By covalently linking a second molecule, such as by linking a heterologous polypeptide, or a polypeptide or fragment thereof modified by glycosylation, acetylation, phosphorylation, or the like.
- derivatives may also include, for example, polypeptides containing analogs of one or more amino acids (e.g., unnatural amino acids, etc.), polypeptides with unsubstituted linkages, and known in the art (natural and non-natural amino acids, etc.) naturally occurring) other modifications.
- expression vector refers to a vehicle into which a polynucleotide or nucleic acid encoding a protein can be operatively inserted and the protein can be expressed.
- Vectors can be used to transform, transduce or transfect host cells, so that the genetic material elements carried by them can be expressed in the host cells.
- host cell refers to a cell into which exogenous polynucleotides or nucleic acids and/or vectors have been introduced.
- 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 the originally transformed cells are included in this application.
- subject refers to any subject, including, for example, humans, non-human primates (e.g., monkeys), mice, Pigs, cows, goats, rabbits, rats, guinea pigs, hamsters, horses, monkeys, sheep, or other non-human mammals; non-mammals, including, for example, non-mammalian vertebrates such as birds (e.g., chickens or ducks) or fish ; and non-mammalian invertebrates.
- the subjects and pharmaceutical compositions involved in the uses or methods of the present application are used to treat (prophylactically and/or therapeutically) a non-human animal.
- treating means alleviating a disease or condition, reducing the rate at which a disease or condition arises or develops, or reducing the risk of developing a disease or condition , or delay the development of symptoms associated with a disease or condition, reduce or terminate symptoms associated with a disease or condition, produce complete or partial reversal of a disease or condition, cure a disease or condition, or any of the above combination.
- 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 the disease are candidates for prophylactic regimens.
- prophylactic regimens refers to the administration of a drug prior to the onset of a sign or symptom, especially in a subject at risk.
- therapeutically effective amount refers to a dose or concentration effective to achieve prevention or amelioration of symptoms associated with a disease or disorder and/or lessening of the severity of the disease or disorder, at a required dosage and for a required period of time.
- a therapeutically effective amount of a formulation, antibody or antigen-binding fragment thereof, or composition of the present application may vary depending on a variety of factors, such as the disease state, age, sex and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual .
- a therapeutically effective amount can also be considered to outweigh any toxic or detrimental effects of the formulation, antibody or antigen-binding fragment thereof, or composition as a therapeutically beneficial effect.
- effective amount refers to the amount of the active ingredient or agent that is sufficient to provide clinical benefits to the subject (including but not limited to improving, alleviating or alleviating the disease, disorder or its related symptoms, delaying or stopping the progression of the disease).
- the term “pharmaceutically acceptable” or “pharmaceutically acceptable” refers to the carrier, vehicle, diluent, excipient and/or salt, generally chemically and/or in Physically compatible with the other ingredients in the formulation and physiologically compatible with the subject.
- the terms “comprising” or “comprising” or “containing” or “having” or “involving” are used interchangeably and mean the inclusion of stated elements, integers or steps, but not the exclusion of any other elements, integers or steps.
- the terms “comprising” or “comprising” or “comprising” or “having” or “involving” are used, unless otherwise specified, the situation consisting of the mentioned elements, integers or steps is also encompassed.
- the application provides a kind of hIL-2 variant, described hIL-2 variant obtains the anti-IL-2 antibody of the present application or The ability of its antigen-binding fragment to bind, and the mutation has no obvious effect on the biological activity of hIL-2.
- the hIL-2 variant of the present application has a binding affinity (K D ) value of ⁇ 5 ⁇ 10 -8 M, preferably ⁇ 1 ⁇ 10 -8 M, ⁇ 5 ⁇ 10 -9 M, or ⁇ 1 ⁇ 10 -9 M, more preferably ⁇ 6 ⁇ 10 -10 M.
- the hIL-2 variants of the present application have a KD value of about 5 ⁇ 10 -8 M to 5 ⁇ 10 -10 M, or for example 1 ⁇ 10 -8 M to 1 ⁇ 10 -10 M, 1.5 ⁇ 10 -8 M to 1 ⁇ 10 -10 M, 1 ⁇ 10 -8 M to 10 ⁇ 10 -10 M, 1 ⁇ 10 -9 M to 1 ⁇ 10 -10 M.
- the hIL-2 variant of the present application also includes other functional properties: (i) maintaining the binding activity and biological function of hIL-2 and hIL2R ⁇ / ⁇ ; (ii) the anti-IL-2 antibody or antigen-binding fragment thereof of the present application Competitively binding to the hIL-2 variant of the present application with IL2R ⁇ ; (iii) the anti-IL-2 antibody or antigen-binding fragment thereof of the present application can block the binding of the hIL-2 variant to IL-2R ⁇ .
- the hIL-2 variants of the present application also include other improved properties, for example, the mutation site is located within the antigen-antibody binding interface and is not exposed outside the antigen-antibody binding interface, thus significantly reducing the potential for inducing ADA (anti -drug antibody, anti-drug antibody) risk.
- ADA anti -drug antibody, anti-drug antibody
- the hIL-2 variant of the present application has more than 90% amino acid sequence identity, preferably more than 93% amino acid sequence identity, compared with wild-type hIL-2 (shown in SEQ ID NO: 1), However, the sequence identity is less than 100%.
- the hIL-2 variant of the present application comprises at least one, or two, or more than two amino acid mutations relative to wild-type hIL-2.
- the hIL-2 variant of the present application comprises at least one, or two, or more than two amino acid substitutions and/or deletions relative to wild-type hIL-2.
- the hIL-2 variants of the present application comprise at least one, or two, or more amino acid substitutions other than cysteine residues relative to wild-type hIL-2.
- said amino acid mutation is at said antigen-antibody binding interface.
- said amino acid mutation is within said antigen-antibody binding interface.
- the mutation site of the hIL-2 variant of the present application is buried within the binding interface between the variant and the anti-IL-2 antibody or antigen-binding fragment thereof, and the hIL of the present application
- the part of the -2 variant exposed outside the binding interface retains the same amino acid sequence as the wild-type hIL-2, thereby not only having no significant effect on the biological activity of hIL-2, but also significantly reducing the hIL-2 2 Immunogenicity of variants that significantly reduce the potential ADA of the hIL-2 variants after forming an antigen-antibody complex with the anti-IL-2 antibody or antigen-binding fragment thereof risk.
- the hIL-2 variant comprises one, two or more amino acid mutations at position 64, position 90 and/or position 104 relative to wild-type hIL-2 as shown in SEQ ID NO:1.
- the hIL-2 variant comprises amino acid mutations at one, two or three positions selected from K64, N90, and M104 relative to wild-type hIL-2.
- the hIL-2 variant comprises at least one mutation as follows: (1) The K mutation at the 64th amino acid residue of the wild-type hIL-2 shown is G, S, T, C, Y, N or Q; (2) corresponding to the wild The N mutation at the 90th amino acid residue of type hIL-2 is R, K or H; or (3) corresponding to the 104th amino acid residue of wild-type hIL-2 as shown in SEQ ID NO:1
- the M at is mutated to E or D.
- the hIL-2 variant comprises one, two or more of the following amino acid mutations relative to the wild-type hIL-2 shown in SEQ ID NO: 1: (a) in the sequence corresponding to There is an amino acid mutation K64G at the 64th amino acid residue of wild-type hIL-2 shown in SEQ ID NO: 1; or (b) at the amino acid residue corresponding to wild-type hIL-2 shown in SEQ ID NO: 1 There is an amino acid mutation N90R at the 90th amino acid residue; or (c) there is an amino acid mutation M104E at the 104th amino acid residue corresponding to wild-type hIL-2 as shown in SEQ ID NO: 1, preferably the amino acid
- the mutations are K64G and/or N90R, or K64G, N90R and M104E, more preferably the amino acid mutations are K64G and N90R.
- the amino acid mutation is located outside the antigen-antibody binding interface to solve possible problems in the downstream production and control of the hIL-2 variant.
- amino acid mutations are performed on the wild-type hIL-2 to eliminate the glycosylation modification, and the hIL-2
- the -2 variant has a deletion relative to the first three or first five amino acid residues of the N-terminus of wild-type hIL-2 as shown in SEQ ID NO: 1, or relative to the wild-type shown in SEQ ID NO: 1
- the third amino acid residue at the N-terminal of hIL-2 has an amino acid mutation, and the preferred amino acid mutation is to delete the first three amino acid residues at the N-terminal of wild-type hIL-2.
- amino acid mutations are performed on wild-type hIL-2 to eliminate free cysteine residue
- the hIL-2 variant has an amino acid mutation relative to the 125th amino acid residue of wild-type hIL-2 as shown in SEQ ID NO: 1, and the preferred amino acid mutation is C125A, C125L, C125S, C125Q or C125V.
- the amino acid mutations are located within and outside of the antigen-antibody binding interface.
- the hIL-2 variant is at the first 3 positions, or the first 5 positions, or the 3rd position, or the 64th position of the N-terminal relative to the wild-type hIL-2 shown in SEQ ID NO: 1.
- position, or position 90, or position 104, or position 125 has one or more amino acid mutations.
- the hIL-2 variant comprises: a deletion mutation of the first 3 amino acids at the N-terminal, selected from K64, N90 and M104 A substitution mutation at one or more positions, and a substitution mutation at position C125; preferred amino acid mutations are K64G and/or N90R, deletion of the first 3 amino acid residues at the N-terminus, and C125A, C125L, C125S, C125Q, or C125V; or K64G, N90R, M104E and the first 3 amino acid residues at the N-terminal are deleted, and any one selected from C125A, C125L, C125S, C125Q or C125V. More preferred amino acid mutations are K64G, N90R, deletion of the first 3 amino acid residues at the N-terminus, and C125L.
- the application provides an anti-IL-2 antibody or an antigen-binding fragment thereof, which can compete with IL2R ⁇ for binding to the hIL-2 variant of the application and block the hIL-2 variant of the application.
- the binding of the hIL-2 variants to IL2R ⁇ or IL-2 receptor aggregates containing ⁇ subunits (ie, IL2R ⁇ / ⁇ or IL2R ⁇ / ⁇ / ⁇ ), and the binding affinity of the hIL-2 variants of the present application Higher, less prone to dissociation.
- the binding affinity (K D ) value of the anti-IL-2 antibody or antigen-binding fragment thereof of the present application to the hIL-2 variant of the present application is ⁇ 5 ⁇ 10 -8 M, preferably ⁇ 1 ⁇ 10 -8 M, ⁇ 5 ⁇ 10 -9 M, or ⁇ 1 ⁇ 10 -9 M, more preferably ⁇ 6 ⁇ 10 -10 M.
- the KD value of the anti-IL-2 antibody or antigen-binding fragment thereof of the present application and the hIL-2 variant of the present application is about 5 ⁇ 10 -8 M to 5 ⁇ 10 -10 M, or for example 1 ⁇ 10 -8 M to 1 ⁇ 10 -10 M, 1.5 ⁇ 10 -8 M to 1 ⁇ 10 -10 M, 1 ⁇ 10 -8 M to 10 ⁇ 10 -10 M, 1 ⁇ 10 -9 M to 1 ⁇ 10 -10 M.
- the dissociation constant (K dis ) of the anti-IL-2 antibody or its antigen-binding fragment of the present application and the hIL-2 variant of the present application is ⁇ 5 ⁇ 10 -3 s -1 , 1 ⁇ 10 -3 s -1 , ⁇ 5 ⁇ 10 -4 s -1 or ⁇ 3 ⁇ 10 -4 s -1 .
- the antibody of the present application can compete with IL2R ⁇ for binding to hIL-2 or its variants.
- the anti-IL-2 antibody or antigen-binding fragment thereof of the present application can specifically recognize the IL2R ⁇ binding site in hIL-2 or its variant sequence.
- Antibodies of the present application may also optionally include F(ab)2, F(ab')2, Fab, Fab', scFv, single domain antibodies and the like.
- the antibodies of the present application can be chimeric antibodies, humanized antibodies, fully human antibodies, monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies and antibody fragments, as long as the antibodies can specifically recognize IL2R ⁇ binding site of hIL-2 or its variants, and can block the binding of hIL-2 or its variants to IL2R ⁇ .
- the anti-IL-2 antibody is a humanized antibody.
- the anti-IL-2 antibody is selected from anti-mouse IL-2 antibodies and humanized antibodies thereof.
- the anti-mouse IL-2 antibody is selected from an anti-mIL-2 antibody that specifically recognizes the binding site between mouse IL-2 (mIL-2) and mouse IL2R ⁇ (mIL2R ⁇ ), the Anti-mIL-2 antibodies are capable of cross-reacting with the hIL-2 variants of the present application.
- mIL-2 mouse IL-2
- mIL2R ⁇ mouse IL2R ⁇
- the anti-IL-2 antibodies of the present application include VH and VL.
- the antibody of the present application is derived from an anti-IL-2 antibody comprising the VH amino acid sequence shown in SEQ ID NO:5 and the VL amino acid sequence shown in SEQ ID NO:6, and can specifically bind to the and blocking the binding of the hIL-2 variants to IL2R ⁇ .
- the anti-IL-2 antibody of the present application is derived from an antibody comprising the VH amino acid sequence shown in SEQ ID NO: 5:
- the anti-IL-2 antibody of the present application is derived from an antibody comprising the VL amino acid sequence shown in SEQ ID NO: 6:
- the anti-IL-2 antibody of the present application comprises one, or two, or more than two CDRs in the VH amino acid sequence shown in SEQ ID NO: 5 or such as SEQ ID NOs: 7, 8 and The amino acid sequence shown in 9 or its variants, said variants include humanized antibodies or any other variants described in this application.
- the anti-IL-2 antibody comprises HCDR1 (shown in SEQ ID NO:7), HCDR2 (shown in SEQ ID NO:8) and HCDR3 (shown in SEQ ID NO:8) of the VH amino acid sequence shown in SEQ ID NO:5. Shown in SEQ ID NO:9).
- the anti-IL-2 antibody of the present application comprises one, or two, or more than two CDRs in the VL amino acid sequence shown in SEQ ID NO: 6 or such as SEQ ID NOs: 10, 11 and The amino acid sequence shown in 12 or a variant thereof, said variant comprising a humanized antibody or any other variant described in the application.
- the anti-IL-2 antibody comprises LCDR1 (shown in SEQ ID NO: 10), LCDR2 (shown in SEQ ID NO: 11) and LCDR3 (shown in SEQ ID NO: 11) of the VL amino acid sequence shown in SEQ ID NO: 6 Shown in SEQ ID NO:12).
- the anti-IL-2 antibody of the present application comprises: one, or two, or more than two CDRs of the VH amino acid sequence shown in SEQ ID NO: 5 or such as SEQ ID NOs: 7, 8 and The amino acid sequence shown in 9 or its variant; And one, or two, or more than two CDRs in the VL amino acid sequence shown in SEQ ID NO:6 or as shown in SEQ ID NOs:10, 11 and 12 Amino acid sequences or variants thereof, including humanized antibodies or any other variants described herein.
- the anti-IL-2 antibody comprises HCDR1 (shown in SEQ ID NO:7), HCDR2 (shown in SEQ ID NO:8) and HCDR3 (shown in SEQ ID NO:8) of the VH amino acid sequence shown in SEQ ID NO:5. shown in SEQ ID NO:9), and LCDR1 (shown in SEQ ID NO:10), LCDR2 (shown in SEQ ID NO:11) and LCDR3 (shown in SEQ ID NO:11) of the VL amino acid sequence shown in SEQ ID NO:6 12).
- the anti-IL-2 antibody comprises HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:8 and HCDR3 as shown in SEQ ID NO:9, and as shown in SEQ ID NO: LCDR1 as shown in ID NO:10, LCDR2 as shown in SEQ ID NO:11 and LCDR3 as shown in SEQ ID NO:12.
- VH and/or VL of the anti-IL-2 antibody of the present application can be used as a starting material for engineering to prepare the antibody described herein that can be used in humans.
- Antibodies can be engineered by mutating one, or two, or more amino acid residues within one or both variable domains (i.e., VH and/or VL), e.g., one, or One, or two, or two or more amino acid residues in two or more CDR regions and/or one, or two or more framework regions are mutated.
- variable regions of the anti-IL-2 antibodies of the present application are engineered by CDR grafting.
- Antibodies interact with target antigens primarily through the amino acid residues of the six CDRs of the heavy and light chains. Therefore, the amino acid sequences within the CDR regions between individual antibodies are more diverse than sequences outside the CDR regions (eg, FR). Because the amino acid sequences of the CDR regions are responsible for most of the antibody's interactions with antigens, recombinant antibodies can be expressed to mimic the properties of specific naturally occurring antibodies by constructing expression vectors containing the CDRs derived from specific naturally occurring antibodies. Sequence grafting to another FR sequence of an antibody with different properties (see e.g.
- the present application relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region and/or a light chain variable region
- the heavy chain can be
- the variable region comprises the amino acid sequences of the HCDR1 region, the HCDR2 region and the HCDR3 region described in the application
- the light chain variable region comprises the amino acid sequences of the LCDR1 region, the LCDR2 region and the LCDR3 region described in the application.
- the antibodies comprise the VH and VL CDR region sequences of the monoclonal antibodies of the present application, they may also comprise different framework region sequences.
- Such framework region sequences can be obtained from DNA public databases or published references relating to germline antibody gene sequences.
- the germline DNA sequences of the human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase) and, Kabat et al., 1991, supra; Tomlinson et al., J Mol Biol 1992, 227:776-798; and Cox et al., Eur J Immunol 1994, 24:827-836; the respective contents of which are expressly incorporated by reference Incorporated into this article.
- the germline DNA sequences of the human heavy and light chain variable region genes can be found in the IMGT database.
- the following heavy chain germline sequences found in human immunoglobulins are available through IMGT accession numbers: 4-59*02 (M29812), 1-69-2*01 (KF698734, IMGT000035 or Z12305).
- the following light chain germline sequences found in human immunoglobulins can be obtained through the IMGT accession numbers: 3-20*01 (X12686, M15038, M15039, X56593 or X93639), 3-11*01 ( X01668, L37726 or X92342), 1-39*01 (X59315 or X93627).
- Antibody amino acid sequences can be compared against compiled protein sequence databases using one of the sequence similarity searching methods known to those of skill in the art known as Gapped BLAST (Altschul et al., 1997, supra).
- the amino acid sequence numbers of the heavy chain CDR region, light chain CDR region, and heavy chain variable region and light chain variable region of the anti-IL-2 antibody of the present application are summarized in Table 2, wherein the heavy chain variable region CDR and the light chain Variable region CDRs are defined by the Kabat numbering system.
- CDR regions can also be defined based on other numbering systems/methods such as Chothia and IMGT, AbM or Contact numbering systems/methods of heavy chain/light chain variable region sequences, and CDR regions defined by different numbering systems/methods There are slight differences, but those skilled in the art can determine according to conventional methods, and the CDR regions defined by other numbering systems/methods and the CDR regions defined by Kabat used in this application are all within the protection scope of this application.
- the preferred framework sequence used in the anti-IL-2 antibody of the present application is the acceptor framework region structurally similar (or highly homologous) to the framework sequence of the parental antibody of the present application.
- the CDR1 region sequence, CDR2 region sequence and CDR3 region sequence of VH or VL can be respectively grafted into the acceptor framework region, and the acceptor framework region has the same or the same sequence as the germline immunoglobulin gene in which it is located. highest sequence.
- the present application chooses to transplant the CDR regions of the VH shown in SEQ ID NO:5 and the VL shown in SEQ ID NO:6 to the human IgG FR region respectively to obtain a humanized antibody, and the human
- the derivatized antibody can maintain antigen binding activity similar to that of the parent antibody comprising the VH amino acid sequence shown in SEQ ID NO:5 and the VL amino acid sequence shown in SEQ ID NO:6.
- VH and VL sequences (or CDR sequences) of other anti-IL-2 antibodies that bind to hIL-2 can also be “mixed and match".
- VH and VL chains or CDRs within these chains
- the VH sequence from a particular VH/VL pair is replaced by a structurally similar VH sequence.
- a VL sequence from a particular VH/VL pair is replaced by a structurally similar VL sequence.
- the antibody or antigen-binding fragment thereof of the present application comprises:
- the humanized antibody of the present application comprises at least 85%, 86%, 87%, 88% of the sequence shown in SEQ ID NO:45, SEQ ID NO:47 or SEQ ID NO:49 , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical VH amino acid sequences.
- the humanized antibody of the present application comprises at least 85%, 86%, 87%, 88% of the sequence shown in SEQ ID NO:46, SEQ ID NO:48 or SEQ ID NO:50 , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical VL amino acid sequences.
- the humanized antibody of the present application comprises at least 85%, 86%, 87%, 88% of the sequence shown in SEQ ID NO:45, SEQ ID NO:47 or SEQ ID NO:49 , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical VH amino acid sequence, and SEQ ID NO: 46,
- the sequence shown in SEQ ID NO:48 or SEQ ID NO:50 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical VL amino acid sequences.
- the humanized antibody of the present application comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% of the sequence shown in SEQ ID NO:45 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH amino acid sequence and the sequence shown in SEQ ID NO: 46 has at least 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical VL amino acid sequence; or with SEQ ID NO :
- the sequence shown in 47 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical VH amino acid sequence and the sequence shown in SEQ ID NO: 48 have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
- Preferred humanized antibodies comprise at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the sequence shown in SEQ ID NO: 45 %, 96%, 97%, 98%, 99% or 100% identical to the VH amino acid sequence and the sequence shown in SEQ ID NO: 46 with at least 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical VL amino acid sequence; or have at least 85% of the sequence shown in SEQ ID NO:49 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical VH amino acids
- the sequence and the sequence shown in SEQ ID NO: 48 or 50 have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%
- More preferred humanized antibodies comprise at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH amino acid sequence and the sequence shown in SEQ ID NO: 48 with at least 85%, 86%, 87%, 88%, 89%, 90% %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical VL amino acid sequences.
- CDR sequences may be grafted onto framework regions comprising one, or two, or more mutations compared to the germline sequences. For example, it has been found that mutating amino acid residues within the framework regions can maintain or enhance the antigen-binding ability of an antibody (see, e.g., US Pat. . In some embodiments, grafting the CDRs of the parent antibody described in the application to the acceptor framework region with one, or two, or more mutations than the germline sequence can improve the anti-IL- 2 The degree of humanization of antibodies.
- one, or two, or more than two amino acid mutations are carried out on the FR region of the VH shown in SEQ ID NO:49, and one or two mutations can be carried out on FR1, FR2, or FR3 , or two or more amino acid mutations, and further one, or two, or two or more amino acid mutations can be carried out on FR1, FR2 or FR3, respectively, so as to improve the humanization degree of the anti-IL-2 antibody of the present application.
- the 48th position of the VH amino acid sequence), and/or the asparagine residue at the 11th position of FR3 (corresponding to the 76th position of the VH amino acid sequence shown in SEQ ID NO: 49) is mutated.
- one, or two, or more than two amino acid mutations are carried out on the FR region of the VL shown in SEQ ID NO:48, and one, or two, or two or more amino acids can be selected to be mutated on FR3 Mutations to improve the degree of humanization of the anti-IL-2 antibody of the present application.
- the arginine residue at the 10th position of FR3 (corresponding to the 66th position of the VL amino acid sequence shown in SEQ ID NO:48) and/or the tyrosine residue at the 15th position of FR3 (corresponding to the 66th position of the VL amino acid sequence shown in SEQ ID NO:48)
- the 71st position of the VL amino acid sequence shown in :48) is mutated.
- amino acid mutations can also be performed on the VH and/or VL CDR regions of the humanized antibody to improve one or more properties of the target antibody (for example, increase the degree of humanization, increase expression, reduce potential risk of isomerization, improve binding properties).
- Site-directed mutagenesis or PCR-induced mutations can be performed, and the effect of the mutations on antibody binding ability or other functional properties can be evaluated by in vitro or in vivo detection methods known in the art.
- Mutations may be amino acid substitutions, additions or deletions, preferably amino acid substitutions. In particular, no more than 1, 2, 3, 4 or 5 amino acid residues within a CDR region are mutated.
- one, or two, or more than two amino acid mutations are carried out in the CDR region of the VH shown in SEQ ID NO:49, and one, or two, or More than two amino acid mutations, preferably, can be at the 1st alanine residue of HCDR1 (corresponding to the 31st position of VH shown in SEQ ID NO:49), the 4th leucine residue (corresponding to SEQ ID NO:49)
- the 34th position of VH shown in ID NO:49) and/or the 17th asparagine residue of HCDR2 carry out one, or two, or two more than one amino acid mutation.
- the present application also relates to isomerization engineering of antibodies.
- Antibody charge heterogeneity occurs because isomerization causes changes in the conformation of the antibody, thereby changing the surface charge of the antibody.
- the antibody of the present application does not contain an aspartic acid isomerization site. Aspartic acid isomerization can occur on the D-G sequence, resulting in an isoaspartic acid residue that reduces the stability of the polypeptide chain due to the introduction of a linker into the polypeptide chain (also known as isoaspartic acid effect).
- one, or two, or more than two amino acid mutations are carried out in the CDR region of the VL shown in SEQ ID NO:48, and one, or two, or more than two amino acid mutations can be further carried out at LCDR2 , preferably, an amino acid mutation can be carried out at the 7th aspartic acid residue of LCDR2 (corresponding to the 56th position of VL shown in SEQ ID NO: 48), to reduce the risk of aspartic acid isomerization.
- combinatorial amino acid mutations can also be performed on the CDR regions and framework regions of the variable regions of humanized antibodies to further improve one or more properties of the antibody of interest (e.g., to improve the degree, improved binding characteristics, reduced potential risk of isomerization, increased expression).
- two CDR regions, one FR region, and one CDR region of VL shown in SEQ ID NO:48 of the above-mentioned humanized antibody shown in SEQ ID NO:49 Multiple amino acid mutations are carried out in one FR region, preferably, the 4th leucine residue of HCDR1 of the VH shown in SEQ ID NO: 49 (corresponding to the 34th position of the VH shown in SEQ ID NO: 49
- the mutated amino acid sequence can be HCDR1 as shown in SEQ ID NO:14), the 17th asparagine residue of HCDR2 (corresponding to the 65th position of VH shown in SEQ ID NO:49, for example, the mutation
- the amino acid sequence behind can be HCDR2 as shown in SEQ ID NO:15 or 16), the asparagine residue at the 11th position of FR3 (corresponding to the 76th position of the VH amino acid sequence shown in SEQ ID NO:49, for example
- the mutated amino acid sequence can be the VH shown in SEQ
- the application provides an isolated anti-IL-2 monoclonal antibody or antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, said The heavy chain variable region comprises: (a) a VH CDR1 region comprising the sequence of the present application or an amino acid sequence with 1, 2, 3, 4 or 5 amino acid mutations; (b) a VH CDR2 region , which comprises the sequence of the present application or an amino acid sequence with 1, 2, 3, 4 or 5 amino acid mutations; (c) VH CDR3 region, which comprises the sequence of the present application or has 1, 2 amino acid mutations , an amino acid sequence with 3, 4 or 5 amino acid mutations; (d) a V L CDR1 region comprising the sequence of the present application or an amino acid sequence with 1, 2, 3, 4 or 5 amino acid mutations (e) V L CDR2 district, it comprises the sequence of the application or has the aminoacid sequence of 1, 2, 3, 4 or 5 amino acid mutations; And (f) V L CDR1 region
- the anti-IL-2 monoclonal antibody to the hIL-2 variant of the present application for example, ⁇ 5 ⁇ 10 -9 M, or ⁇ 1 ⁇ 10 -9 M , more preferably, ⁇ 6 ⁇ 10 ⁇ 10 M, mutating specific amino acids on the heavy chain CDR and light chain CDR of the anti-IL-2 antibody.
- the CDR3 (especially HCDR3) domain does not depend on the CDR1 domain and/or the CDR2 domain, it is very important for the binding specificity of the antigen and has greater diversity compared to the other two CDR domains.
- the present application has carried out one, two and/or three amino acid mutations to the HCDR3 comprising SEQ ID NO: 9, and the amino acid mutation is located at No. 1 of HCDR3 shown in SEQ ID NO: 9 Valine residue at position 4, tyrosine residue at position 4, and/or serine residue at position 6, preferably the amino acid mutation includes V1R, V1I, V1T, Y4H, Y4L, Y4W or S6A, S6R, S6N , S6D, S6Q, S6E, S6H, S6I, S6L, S6K, S6F, S6P, S6T, S6W, S6Y, S6V.
- the present application also carries out one, or two, or more than two amino acid mutations, preferably one amino acid mutation, to the HCDR2 comprising SEQ ID NO: 15, and the amino acid mutation is located in SEQ ID NO
- the 5th glutamic acid residue of HCDR2 shown in: 15, preferred amino acid mutation is E5D.
- one, two and/or three amino acid mutations on HCDR3 shown in SEQ ID NO:9 are further combined with amino acid mutations on HCDR2 shown in SEQ ID NO:15, preferred amino acids
- the mutation is one, two and/or three amino acid mutations of V1I, Y4L, and S6T on HCDR3 shown in SEQ ID NO:9 combined with the amino acid mutation E5D on HCDR2 shown in SEQ ID NO:15.
- the present application carries out one, or two, or more than two amino acid mutations to LCDR1 shown in SEQ ID NO:10 and/or LCDR2 shown in SEQ ID NO:17, and the amino acid mutation Including the 5th aspartic acid residue or the 7th glycine residue in LCDR1 shown in SEQ ID NO:10, and/or the 1st serine residue or the 3rd serine residue in LCDR2 shown in SEQ ID NO:17 Threonine, the amino acid mutation includes D5N, D5H, D5I, D5L, D5F, D5Y and/or G7A, G7R, G7N, G7Q, G7I, G7L, G7S, G7T on the LCDR1 shown in SEQ ID NO:10 , G7D, and/or S1R, S1Q, S1T, S1W, S1Y or T3G on LCDR2 shown in SEQ ID NO:17, preferred amino acid mutations include D5N, D
- any one, or two, or more than two mutations of the heavy chain CDR region and/or any one, or two, or more than two mutations of the light chain CDR region can be selected.
- it can maintain the VH amino acid sequence shown in SEQ ID NO:63 and the VL amino acid sequence shown in SEQ ID NO:64 The binding affinity of the anti-IL-2 humanized antibody of the present application to the hIL-2 variant of the present application.
- the anti-IL-2 human The binding affinity of the derivatized antibody to the hIL-2 variant is ⁇ 10 -9 M, even ⁇ 6 ⁇ 10 -10 M.
- one of the mutations in the light chain CDR region may be selected or combined with any one, two, three or four mutations in the heavy chain CDR region, wherein the amino acid mutations in the light chain CDR region include D28Y (aspartic acid at position 28 of the VL amino acid sequence shown in SEQ ID NO: 64, corresponding to LCDR1 position 5 shown in SEQ ID NO: 10), the amino acid mutation in the heavy chain CDR region includes V95I ( Located at the first valine residue of HCDR3 shown in SEQ ID NO: 9, corresponding to the 95th position of the VH amino acid sequence shown in SEQ ID NO: 63), E53D (located in HCDR2 shown in SEQ ID NO: 15 The 5th glutamic acid residue, corresponding to the 53rd position of the VH amino acid sequence shown in SEQ ID NO:63), S100T (located at the 6th serine residue of HCDR3 shown in SEQ ID NO:9, corresponding to The 100th position of the VH amino acid sequence shown
- two amino acid mutations in the light chain CDR region are selected or combined with any one, two, three or four amino acid mutations in the heavy chain CDR region, and the amino acid mutation in the light chain CDR region is Including D28Y and G30S (located at the 30th glycine in the VL amino acid sequence shown in SEQ ID NO: 64, corresponding to the 7th position of LCDR1 shown in SEQ ID NO: 10) or S50Q (located in the VL shown in SEQ ID NO: 64
- the 50th serine in the amino acid sequence corresponds to the first position of LCDR2 shown in SEQ ID NO: 17)
- the one amino acid mutation in the heavy chain CDR region includes V95I, E53D, Y98L or S100T
- the two amino acid mutations in the heavy chain CDR region The mutation includes V95I and any one selected from E53D, S100T or Y98L
- the three amino acid mutations in the heavy chain CDR region include V95I and any two selected from E53D, S
- three of the light chain CDR region mutations are selected or combined with any one, two, three or four mutations of the heavy chain CDR region, and the light chain CDR region mutations include D28Y , G30S and S50Q, one amino acid mutation in the heavy chain CDR region includes V95I, E53D, Y98L or S100T, and the two mutations in the heavy chain CDR region include V95I and any one selected from E53D, S100T or Y98L, the The three mutations in the heavy chain CDR region include V95I and any two selected from E53D, S100T or Y98L, and the four amino acid mutations in the heavy chain CDR region include V95I, E53D, S100T and Y98L.
- the combination of the amino acid mutation in the heavy chain CDR region and the amino acid mutation in the light chain CDR region includes the combination of four amino acid mutations in the heavy chain CDR region and the D28Y amino acid mutation in the light chain CDR region, or the combination of the amino acid mutation D28Y in the heavy chain CDR region.
- a combination of three or four amino acid mutations and two amino acid mutations D28Y and S50Q or G30S in the light chain CDR region the three amino acid mutations in the heavy chain CDR region are V95I and any two selected from E53D, S100T or Y98L, Or a combination of two, three or four amino acid mutations in the heavy chain CDR region and three amino acid mutations D28Y, G30S and S50Q in the light chain CDR region, the two amino acid mutations in the heavy chain CDR region are V95I and Y98L, the heavy chain CDR region
- the three amino acid mutations in the chain CDR region are V95I and any two selected from E53D, S100T or Y98L, specifically including the amino acid mutations V95I, S100T, Y98L and E53D in the heavy chain CDR region and the amino acid mutation D28Y in the light chain CDR region, and the heavy chain CDR region Region amino acid mutation V95I, E53D and Y98L and light chain CDR
- the mutation combinations can significantly improve the anti-IL-2 humanized antibody comprising the VH amino acid sequence shown in SEQ ID NO:63 and the VL amino acid sequence shown in SEQ ID NO:64 of the present application and the hIL of the present application.
- -2 variant has a binding affinity up to about 5 ⁇ 10 ⁇ 10 M, and significantly reduces the dissociation constant (K dis ) of the humanized antibody to the hIL-2 variant to about 10 ⁇ 3 s ⁇ 1 or lower, even ⁇ 3 ⁇ 10 -4 s -1 , or the equilibrium dissociation constant (K D ) of said humanization and said hIL-2 variant is reduced by more than 2 orders of magnitude, even 3 more than an order of magnitude.
- the antibody of the present application comprises a heavy chain variable region comprising a CDR1 sequence, a CDR2 sequence and a CDR3 sequence and/or a light chain variable region comprising a CDR1 sequence, a CDR2 sequence and a CDR3 sequence, wherein:
- the heavy chain variable region CDR1 sequence comprises the sequences listed in Table 2 and/or conservative modifications thereof; and/or
- the heavy chain variable region CDR2 sequence comprises the sequences listed in Table 2 and/or conservative modifications thereof; and/or
- the heavy chain variable region CDR3 sequence comprises the sequences listed in Table 2 and conservative modifications thereof;
- light chain variable region CDR1 sequence and/or CDR2 sequence and/or CDR3 sequence comprises the sequence listed in Table 2 and/or its conservative modification
- the antibody competes with IL2R ⁇ for binding to the hIL-2 variant of the present application and blocks the binding of the hIL-2 variant to IL2R ⁇ .
- the antibody or antigen-binding fragment thereof of the present application comprises: HCDR1 as shown in SEQ ID NO: 7, SEQ ID NO: 13 or SEQ ID NO: 14; such as SEQ ID NO: 8, SEQ ID NO HCDR2 shown in: 15, SEQ ID NO: 16 or SEQ ID NO: 19; as SEQ ID NO: 9, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, HCDR3 set forth in SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:29 or SEQ ID NO:34; and
- SEQ ID NO: 10 SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ LCDR1 shown in ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43 or SEQ ID NO:44; such as SEQ ID NO:11, SEQ ID NO: 17.
- LCDR2 as shown in SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:32 or SEQ ID NO:33
- LCDR3 as shown in SEQ ID NO:12 or SEQ ID NO:38.
- the antibody or antigen-binding fragment thereof of the present application comprises: HCDR1 as shown in SEQ ID NO: 7 or 14, HCDR2 as shown in SEQ ID NO: 8, 15, 16 or 19, as shown in SEQ ID NO: 8, 15, 16 or 19 HCDR3 identified by ID NO: 9, 20, 21, 22, 23, 24 or 25; and
- LCDR1 as shown in SEQ ID NO: 10, 26 or 27, LCDR2 as shown in SEQ ID NO: 11, 17, 18 or 28, LCDR3 as shown in SEQ ID NO: 12.
- the antibody or antigen-binding fragment thereof of the present application comprises: 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 86 or 112 have at least 85% (e.g., at least 88%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical heavy chain variable region VH; , 79, 80, 81, 82, 83, 84, 85, 87, 88, 89, 90, 91, 92, 93, 104, 105, 106, 107, 108, 109, 110 or 111 have at least 85% (eg, at least 88%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity of the light chain variable region VL.
- the anti-IL-2 humanized antibody of the present application also has one or more of the following functional properties, for example, the binding affinity to the hIL-2 variant of the present application can reach about 5 ⁇ 10 -10 M, more importantly , the dissociation constant between the humanized antibody and the hIL-2 variant is reduced to about 3 ⁇ 10 -4 1/s, making the two relatively less prone to dissociation, significantly reducing the The probability of exposing the IL2R ⁇ binding site of hIL-2; the degree of humanization of the antibody of this application is further improved, and the mutated amino acid residues on the hIL-2 variant of this application are all buried in the antigen-antibody binding interface state, thus greatly reducing the risk of inducing ADA.
- the binding affinity to the hIL-2 variant of the present application can reach about 5 ⁇ 10 -10 M, more importantly , the dissociation constant between the humanized antibody and the hIL-2 variant is reduced to about 3 ⁇ 10 -4 1/s, making the two relatively less prone to dissociation, significantly
- the present application also provides a hIL-2/IL-2 antibody fusion protein, the fusion protein is between hIL-2 or the hIL-2 variant of the present application and the antigen of the anti-IL-2 antibody or antigen-binding fragment -
- the design of the antibody binding interface introduces a disulfide bond to form an ultra-stable covalent binding structure between the two to ensure that the ⁇ subunit receptor binding site of hIL-2 or its variants is always buried in the constructed
- the interior of the fusion protein molecule will not be exposed due to the dissociation of the antigen-antibody complex, and will not be competitively bound by the high-affinity IL2R because the affinity of the antigen-antibody is not strong enough.
- the hIL-2/IL-2 antibody fusion protein of the present application which comprises:
- hIL-2 wild-type human interleukin 2
- the hIL-2/IL-2 antibody fusion protein of the present application does not bind to the ⁇ subunit of IL2R, or the ⁇ / ⁇ dimer, or the ⁇ / ⁇ / ⁇ trimer, but retains the IL2R ⁇ and ⁇ c receptor subunits Binding and functional activity of IL2R ⁇ / ⁇ .
- the hIL-2/IL-2 antibody fusion protein of the present application has the following functional properties:
- the hIL-2/IL-2 antibody fusion protein of the present application does not bind to the high-affinity IL2R on the surface of Treg cells, effectively avoiding the tumor immunosuppressive effect mediated by Treg cells.
- the hIL-2/IL-2 antibody fusion protein of the present application does not interact with normal tissues (for example, vascular endothelium, pulmonary capillary endothelium, etc.) or ILC2 cells (Type-2innate lymphoid cells, type II congenital lymphoid cells) ), thereby significantly improving the toxic side effects (eg, VLS, pulmonary edema, hypotension, etc.) induced by existing IL-2 products in vivo.
- normal tissues for example, vascular endothelium, pulmonary capillary endothelium, etc.
- ILC2 cells Type-2innate lymphoid cells, type II congenital lymphoid cells
- the hIL-2/IL-2 antibody fusion protein of the present application has a stable molecular structure, and there will be no dissociation of the hIL-2/hIL-2 antibody complex in the prior art, thus effectively avoiding hIL-2 Dissociation of the 2/hIL-2 antibody complex exposes the IL2R ⁇ binding site of hIL-2, thereby avoiding the potential narrow therapeutic window caused by high-affinity IL-2R-mediated toxicity and Treg activation .
- the hIL-2/IL-2 antibody fusion protein of the present application can not only specifically bind to effector T (Teff) cells (such as memory CD8 + T cells) and/or NK cells, and promote Teff cells and/or NK Cell proliferation, can also induce IL-2 signaling in Teff cells and/or NK cells, activate their tumor killing activity, but will not activate IL-2 signaling in Treg cells and its cell activity, and will not affect conventional CD4 + T cells (conventional CD4 + T cells, Tconv) have adverse effects.
- the hIL-2/IL-2 antibody fusion protein of the present application has low immunogenicity, and all mutation sequences are embedded in the antigen-antibody binding interface, which significantly reduces the potential risk of ADA.
- the hIL-2/IL-2 antibody fusion protein of the present application has good stability, is easy to produce and prepare, and has a high expression level, which is conducive to reducing production costs.
- the hIL-2/IL-2 antibody fusion protein of the present application has a long half-life, has effective and long-lasting immune stimulation and anti-tumor activity in vivo, and does not activate or induce the body's tumor immunosuppression.
- the antigen-antibody binding interface of the hIL-2 or the hIL-2 variant of the present application and the anti-IL-2 antibody or its antigen-binding fragment comprises a disulfide bond, and the disulfide bond is formed by binding to the antigen-antibody At least one cysteine residue introduced in the sequence of the anti-IL-2 antibody or antigen-binding fragment thereof of the interface is combined with at least one cysteine residue introduced in the sequence of hIL-2 or a variant thereof of the antigen-antibody binding interface formed by cysteine residues.
- the anti-IL-2 antibody or antigen-binding fragment thereof can compete with IL2R ⁇ for binding to the hIL-2 or its variant, and can block the interaction of the hIL-2 or its variant with IL2R ⁇ or a protein containing an ⁇ subunit Binding of IL-2 receptor aggregates (ie, IL2R ⁇ / ⁇ or IL2R ⁇ / ⁇ / ⁇ ).
- the anti-IL-2 antibody specifically binds to the IL2R ⁇ binding site of the hIL-2 or variant thereof.
- the binding affinity (K D ) of the anti-IL-2 antibody or antigen-binding fragment thereof to the hIL-2 variant is ⁇ 5 ⁇ 10 -8 M, preferably, ⁇ 1 ⁇ 10 -8 M , ⁇ 5 ⁇ 10 -9 M, or ⁇ 1 ⁇ 10 -9 M, more preferably, ⁇ 6 ⁇ 10 -10 M.
- the dissociation constant (K dis ) of the anti-IL-2 antibody or antigen-binding fragment thereof with the hIL-2 variant is ⁇ 5 ⁇ 10 -3 s -1 , 1 ⁇ 10 -3 s -1 1. ⁇ 5 ⁇ 10 -4 s -1 or ⁇ 3 ⁇ 10 -4 s -1 .
- the antigen-antibody binding interface of the hIL-2 or the hIL-2 variant of the present application and the anti-IL-2 antibody or its antigen-binding fragment comprises the binding interface of hIL-2 or its variant and IL2R ⁇ , or the The antigen-antibody binding interface partially overlaps with the binding interface of hIL-2 or the hIL-2 variant of the present application with IL2R ⁇ .
- the antigen-antibody binding interface between the hIL-2 or its variant and the anti-IL-2 antibody or its antigen-binding fragment is located at the AB loop of the hIL-2 or its variant, B helix, C helix, BC loop and/or CD loop, preferably located in the AB loop, B helix, C helix and CD loop of the hIL-2 or its variants; the IL2R ⁇ of the hIL-2 or its variants
- the binding interface is located on the AB loop and B helix of hIL-2 or its variants.
- the binding interface between the hIL-2 or its variant and the anti-IL-2 antibody or its antigen-binding fragment is located at position 30-45 of the hIL-2 or its variant or its Adjacent amino acid residues, amino acid residues 57-77 or adjacent amino acid residues, and/or amino acid residues 90-111 or adjacent amino acid residues.
- the binding interface of the hIL-2 or variant thereof to the anti-IL-2 antibody or antigen-binding fragment thereof is located in the variable region of the anti-IL-2 antibody or antigen-binding fragment thereof.
- the hIL-2 variant in the hIL-2/IL-2 antibody fusion protein of the present application comprises the aforementioned hIL-2 variant of the present application, which is relative to the wild type shown in SEQ ID NO:1 hIL-2 comprises one or more of the following amino acid mutations: (a) having an amino acid mutation K64G at amino acid residue 64 corresponding to wild-type hIL-2 as shown in SEQ ID NO: 1; or (b) There is an amino acid mutation N90R at the 90th amino acid residue corresponding to wild-type hIL-2 shown in SEQ ID NO: 1; or (c) corresponding to wild-type hIL-2 shown in SEQ ID NO: 1
- the 104th amino acid residue of 2 has an amino acid mutation M104E, preferably the amino acid mutation is K64G and/or N90R, or K64G, N90R and M104E, more preferably the amino acid mutation is K64G and N90R.
- the hIL-2 variant comprises the amino acid sequence shown in SEQ
- the anti-IL-2 antibody or antigen-binding fragment thereof in the hIL-2/IL-2 antibody fusion protein of the present application comprises the aforementioned anti-IL-2 antibody or antigen-binding fragment thereof of the present application and other specific An anti-IL-2 humanized antibody or an antigen-binding fragment thereof that specifically recognizes the IL2R ⁇ binding site on hIL-2, and other preferred anti-IL-2 humanized antibodies are selected from NARA1 as described in PCT published patent application WO2016005950 and as described in PCT TCB2 described in published patent application WO 2018217058.
- the anti-IL-2 antibody or antigen-binding fragment thereof in the hIL-2/IL-2 antibody fusion protein of the present application comprises the HCDR1 amino acid sequence shown in SEQ ID NO: 7, the amino acid sequence shown in SEQ ID NO: 8 HCDR2 amino acid sequence shown, HCDR3 amino acid sequence shown in SEQ ID NO: 9, and LCDR1 amino acid sequence shown in SEQ ID NO: 10, LCDR2 amino acid sequence shown in SEQ ID NO: 11, shown in SEQ ID NO: 12 LCDR3 amino acid sequence, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences.
- the sequence is
- the above-mentioned antibody or antigen-binding fragment thereof comprises: HCDR1 as shown in SEQ ID NO: 7, SEQ ID NO: 13 or SEQ ID NO: 14; such as SEQ ID NO: 8, SEQ ID NO: 15.
- SEQ ID NO: 10 SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ LCDR1 shown in ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43 or SEQ ID NO:44; such as SEQ ID NO:11, SEQ ID NO: 17.
- LCDR2 as shown in SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:32 or SEQ ID NO:33
- LCDR3 as shown in SEQ ID NO:12 or SEQ ID NO:38.
- the above-mentioned antibody or antigen-binding fragment thereof comprises: HCDR1 as shown in SEQ ID NO: 7 or 14, HCDR2 as shown in SEQ ID NO: 8, 15, 16 or 19, as shown in SEQ ID NO : HCDR3 shown in 9, 20, 21, 22, 23, 24, 25, 29 or 34; and
- LCDR1 as shown in SEQ ID NO: 10, 26, 27, 30, 31, 35, 36 or 37
- LCDR2 as shown in SEQ ID NO: 11, 17, 18, 28, 32 or 33
- LCDR3 as shown in 12 or 38.
- the anti-IL-2 antibody or antigen-binding fragment thereof in the hIL-2/IL-2 antibody fusion protein of the present application comprises HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3, which comprise:
- the anti-IL-2 antibody or antigen-binding fragment thereof in the hIL-2/IL-2 antibody fusion protein of the present application comprises HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3, which comprise:
- the anti-IL-2 antibody or antigen-binding fragment thereof in the hIL-2/IL-2 antibody fusion protein of the present application comprises HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3, which comprise:
- the anti-IL-2 antibody or antigen-binding fragment thereof in the hIL-2/IL-2 antibody fusion protein of the present application further comprises the heavy chain variable region having the HCDR1, HCDR2 and HCDR3 and the heavy chain variable region having the LCDR1 , LCDR2 and LCDR3 light chain variable regions.
- the antibody or antigen-binding fragment thereof of the present application comprises: 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 86 or 112 have at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical heavy chain variable region VH; , 79, 80, 81, 82, 83, 84, 85, 87, 88, 89, 90, 91, 92, 93, 104, 105, 106, 107, 108, 109, 110 or 111 have at least 80% (eg, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity of the light chain variable region VL.
- the anti-IL-2 antibody or antigen-binding fragment thereof in the hIL-2/IL-2 antibody fusion protein of the present application comprises the heavy chain variable region having the HCDR1, HCDR2 and HCDR3 and the The light chain variable region of LCDR1, LCDR2 and LCDR3, which comprises at least 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences, preferably is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences.
- the anti-IL-2 antibody or antigen-binding fragment thereof in the hIL-2/IL-2 antibody fusion protein of the present application comprises the heavy chain variable region having the HCDR1, HCDR2 and HCDR3 and the The light chain variable region of LCDR1, LCDR2 and LCDR3, which comprises at least 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences; or Comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences.
- the anti-IL-2 antibody or antigen-binding fragment thereof in the hIL-2/IL-2 antibody fusion protein of the present application comprises the heavy chain variable region with the HCDR1, HCDR2 and HCDR3 and the The light chain variable regions of LCDR1, LCDR2, and LCDR3 comprise at least 85%, 86%, 87%, 88%, 89% of the VH shown in SEQ ID NO:63 and the VL shown in SEQ ID NO:64, respectively. %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences.
- the anti-IL-2 antibody or antigen-binding fragment thereof in the hIL-2/IL-2 antibody fusion protein of the present application comprises the heavy chain variable region with the HCDR1, HCDR2 and HCDR3 and the The light chain variable regions of LCDR1, LCDR2 and LCDR3 comprise at least 85% of the VH shown in any one of SEQ ID NO: 63, 65-75 and the VL shown in any one of SEQ ID NO: 76-79, respectively. %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences .
- the anti-IL-2 antibody or antigen-binding fragment thereof in the hIL-2/IL-2 antibody fusion protein of the present application comprises the heavy chain variable region with the HCDR1, HCDR2 and HCDR3 and the The light chain variable regions of LCDR1, LCDR2 and LCDR3 comprise at least 85%, 86%, 87%, 88%, 89% of the VH shown in SEQ ID NO:71 and the VL shown in SEQ ID NO:76, respectively. %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences.
- the anti-IL-2 antibody or antigen-binding fragment thereof in the hIL-2/IL-2 antibody fusion protein of the present application comprises the heavy chain variable region with the HCDR1, HCDR2 and HCDR3 and the The light chain variable regions of LCDR1, LCDR2 and LCDR3 comprise at least 85%, 86%, 87%, 88%, 89% of the VH shown in SEQ ID NO:72 and the VL shown in SEQ ID NO:77, respectively. %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences.
- the anti-IL-2 antibody or antigen-binding fragment thereof in the hIL-2/IL-2 antibody fusion protein of the present application comprises the heavy chain variable region with the HCDR1, HCDR2 and HCDR3 and the The light chain variable regions of LCDR1, LCDR2, and LCDR3 comprise at least 85%, 86%, 87%, or 85%, 86%, 87%, or 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences.
- the anti-IL-2 antibody or antigen-binding fragment thereof in the hIL-2/IL-2 antibody fusion protein of the present application comprises the heavy chain variable region with the HCDR1, HCDR2 and HCDR3 and the The light chain variable regions of LCDR1, LCDR2, and LCDR3 comprise at least 85%, 86%, 87%, 85%, 86%, 87%, and 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences.
- the antigen-antibody binding interface of the hIL-2 variant in the hIL-2/IL-2 antibody fusion protein of the present application and the anti-IL-2 antibody or antigen-binding fragment thereof comprises a disulfide bond, said The antigen-antibody binding interface overlaps with the binding interface of the hIL-2 variants to IL2R ⁇ .
- the antigen-antibody binding interface between the hIL-2 variant and the anti-IL-2 antibody or its antigen-binding fragment is located at the AB loop, B helix, C
- the helix and CD loop are preferably located at amino acid residues 41-45 or adjacent to them, amino acid residues 57-68 or adjacent to them, and amino acid residues 90-107 or adjacent to said hIL-2 variants. amino acid residues.
- the antigen-antibody binding interface of the hIL-2 variant in the hIL-2/IL-2 antibody fusion protein of the present application and the anti-IL-2 antibody or antigen-binding fragment thereof comprises a disulfide bond, said The disulfide bond is through at least one cysteine residue introduced in the sequence of the anti-IL-2 antibody or antigen-binding fragment thereof at the antigen-antibody binding interface and through the hIL-2 at the antigen-antibody binding interface. 2 formed by the introduction of at least one cysteine residue in the sequence of the variant.
- “Introduction” or “introduced” as used in the present application refers to the parent polypeptide (for example, the wild-type hIL-2 or variant thereof, anti-IL-2 antibody or antigen-binding fragment thereof described in the present application) Corresponding original or natural amino acid residues are mutated, including substitutions, insertions or additions.
- an amino acid residue at a specific position in a parental polypeptide eg, wild-type hIL-2 or a variant thereof, an anti-IL-2 antibody or an antigen-binding fragment thereof as described herein
- its corresponding introduced cysteine residue is a cysteine residue that is different from the original amino acid residue but located at the same position on the parent polypeptide.
- the introduced cysteine residue is a cysteine residue that replaces the original residue at the same position.
- the introduced cysteine residue is an insertion or addition of a cysteine residue or a polypeptide fragment comprising a cysteine residue at the same position or adjacent to it.
- the antigen-antibody binding interface of the hIL-2 variant in the hIL-2/IL-2 antibody fusion protein and the anti-IL-2 antibody or antigen-binding fragment thereof comprises two or two Cysteine residues introduced above to form the disulfide bonds.
- the hIL-2 variant is at the 42nd, 60th, 61st, 64th, 65th position relative to the wild-type hIL-2 shown in SEQ ID NO: 1 , 102nd and 104th amino acid residues have one, or two, or more than two amino acid mutations to introduce the first cysteine residue.
- the anti-IL-2 antibody is selected from the anti-IL-2 humanized antibody of the present application or an antigen-binding fragment thereof comprising the heavy chain variable region and/or the HCDR1, HCDR2 and HCDR3
- the light chain variable region having said LCDR1, LCDR2 and LCDR3 comprises at least 85%, 86%, 87% of the VH shown in SEQ ID NO:63 and/or the VL shown in SEQ ID NO:64, respectively , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence;
- the VH shown in any one of ID NOs: 63, 65-75 and/or the VL shown in any one of SEQ ID NOs: 76-79 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence, and said antibody or
- the hIL-2 variant may be manipulated in order to mutate an amino acid residue at a specific site in the hIL-2 variant and/or the anti-IL-2 antibody or antigen-binding fragment thereof to a cysteine residue and/or the coding sequence of an anti-IL-2 antibody or antigen-binding fragment thereof, for example by substituting codons encoding native residues with codons encoding cysteine residues, or at positions encoding native residues or A codon encoding a cysteine residue was inserted or added at an adjacent position.
- the hIL-2 variant at the antigen-antibody binding interface of the hIL-2/IL-2 antibody fusion protein of the present application introduces the first cysteine residue by way of amino acid mutation, where
- the anti-IL-2 humanized antibody or antigen-binding fragment thereof at the antigen-antibody binding interface introduces a second cysteine residue by way of amino acid mutation, and the preferred amino acid mutation is amino acid substitution, insertion or addition, for example, non- A cysteine residue may be substituted for a cysteine residue, or a cysteine residue may be inserted or added at or adjacent to a non-cysteine residue or contain a cysteine residue
- the position of substitution or the position of insertion or addition can be determined such that the non-cysteine residue is substituted into a cysteine residue or a cysteine residue is inserted or added or contains cysteine
- the hIL-2 variant of the antigen-antibody binding interface of the present application introduces the first cysteine residue by way of amino acid mutation, where
- disulfide bond-forming cysteine residues are in close enough proximity, and/or that the substitution or insertion or addition does not significantly disturb the hIL-2 variant, or anti-IL -2 antibody or its antigen-binding fragment, or the tertiary structure of the hIL-2/IL-2 antibody fusion protein itself, and the formed disulfide bond should be located at the antigen-antibody binding interface.
- Suitable methods known in the art including but not limited to distance mapping by photodetection, computer modeling, NMR spectroscopy or X-ray crystallography, to determine the distance between two amino acid residues to be mutated. distance and angle.
- the target protein for example, the antigen-antibody complex formed by IL-2 and anti-IL-2 antibody
- its protein crystal structure can be obtained from public databases (such as PDB database), or use such as X X-ray crystallography; computer software can also be used to determine distances and angles between amino acid residues based on protein crystal structure data.
- the site capable of correct pairing is located at the antigen-antibody binding interface between the hIL-2 variant and the anti-IL-2 humanized antibody or antigen-binding fragment thereof, and the paired amino acid residues Close enough, for example, the space between paired amino acid residues is less than 10 angstroms, preferably less than 5 angstroms.
- the mIL-2/S4B6 antigen-antibody complex crystal structure (as shown in PDB ID: 4YUE ) in mIL-2 and the distance of each amino acid residue of S4B6, to select suitable for introducing the first and second cysteine respectively in the hIL-2 variant and the anti-IL-2 humanized antibody or antigen fragment thereof position the amino acid residues and allow them to pair correctly.
- the pairing site for introducing a cysteine residue or the pairing amino acid residue to be mutated is determined, those skilled in the art can easily convert the codon to be mutated by methods known in the art (for example, PCR mutagenesis) Mutation to cysteine codon.
- the formation of said disulfide bonds can be determined by suitable methods known in the art. For example, it can be judged by means of mass spectrometry.
- the disulfide bond can stabilize the hIL-2/IL-2 antibody fusion protein, and this stabilization effect is reflected in various aspects.
- the disulfide bond can make the expression of the hIL-2/IL-2 antibody fusion protein have a higher proportion of monomer components, and/or have good thermal stability and serum stability, and/or construct
- a specific biological activity for example, to be able to better block the binding of the hIL-2 variant to the ⁇ subunit of IL2R, or the ⁇ / ⁇ dimer, or the ⁇ / ⁇ / ⁇ trimer, and to retain binding and functional activity to IL2R ⁇ / ⁇
- stable fusion protein in order to have a specific biological activity (for example, to be able to better block the binding of the hIL-2 variant to the ⁇ subunit of IL2R, or the ⁇ / ⁇ dimer, or the ⁇ / ⁇ / ⁇ trimer, and to retain binding and functional activity to IL2R ⁇ / ⁇ ) stable fusion protein, and/or expressed and assembled into a
- the hIL-2 variant in said hIL-2/IL-2 antibody fusion protein forms a disulfide at the antigen-antibody binding interface of an anti-IL-2 humanized antibody or antigen-binding fragment thereof.
- the pairing of the first cysteine residue and the second cysteine residue of the bond is selected from the following one, or two, or a combination of more than two paired amino acid mutations:
- the first cysteine residue is the amino acid mutation E60C that the hIL-2 variant has relative to the 60th amino acid residue of wild-type hIL-2 as shown in SEQ ID NO:1
- the sequence shown in ID NO: 10, 26 or 27) is obtained by introducing an amino acid mutation G7C or S7C at position 7 of LCDR1; and/or
- the first cysteine residue is the amino acid mutation E61C that the hIL-2 variant has relative to the 61st amino acid residue of the wild-type hIL-2 shown in SEQ ID NO: 1
- the amino acid mutation G7C or S7C is introduced into the 7th position of LCDR1, or
- the first cysteine residue is the amino acid mutation M104C that the hIL-2 variant has relative to the 104th amino acid residue of wild-type hIL-2 as shown in SEQ ID NO:1
- the first cysteine residue is the amino acid mutation T102C that the hIL-2 variant has relative to the 102nd amino acid residue of wild-type hIL-2 as shown in SEQ ID NO:1
- the second cysteine residue is obtained by introducing an amino acid mutation S67C at position 67 of the light chain variable region of the anti-IL-2 humanized antibody or antigen-binding fragment thereof, or
- the second cysteine residue is obtained by introducing an amino acid mutation G68C at position 68 of the light chain variable region of the anti-IL-2 humanized antibody or its antigen-binding fragment; and/or
- the first cysteine residue is the amino acid mutation K64C that the hIL-2 variant has relative to the 64th amino acid residue of wild-type hIL-2 as shown in SEQ ID NO:1 ,and
- the second cysteine residue is obtained by introducing an amino acid mutation Y4C at position 4 of LCDR3 with the amino acid sequence LQHYSTPYT (SEQ ID NO: 12) of the anti-IL-2 humanized antibody or its antigen-binding fragment. get.
- the anti-IL-2 humanized antibody or its antigen-binding fragment in the hIL-2/IL-2 antibody fusion protein forms the first half of the disulfide bond with the antigen-antibody binding interface of the hIL-2 variant
- the pairing of a cystine residue and a second cysteine residue is selected from the following one, or two, or a combination of more than two paired amino acid mutations:
- the first cysteine residue is the amino acid mutation M104C that the hIL-2 variant has relative to the 104th amino acid residue of wild-type hIL-2 as shown in SEQ ID NO:1
- the second cysteine residue is obtained by introducing an amino acid mutation T3C at the third position of LCDR2 of the anti-IL-2 humanized antibody or its antigen-binding fragment, and the anti-IL-2 human
- the first cysteine residue is the amino acid mutation T102C that the hIL-2 variant has relative to the 102nd amino acid residue of wild-type hIL-2 as shown in SEQ ID NO:1
- the second cysteine residue is obtained by introducing an amino acid mutation S67C at position 67 of the light chain variable region of the anti-IL-2 humanized antibody or its antigen-binding fragment
- the anti-IL-2 -2 Humanized antibody or binding fragment thereof comprises 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% of the VL shown in SEQ ID NO:77 %, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences, or
- the second cysteine residue is obtained by introducing an amino acid mutation G68C at position 68 of the light chain variable region of the anti-IL-2 humanized antibody or its antigen-binding fragment, and the anti-IL-2 2
- the humanized antibody or its binding fragment comprises 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% of the VL shown in SEQ ID NO:77 , 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences; and/or
- the first cysteine residue is the amino acid mutation F42C that the hIL-2 variant has relative to the 42nd amino acid residue of wild-type hIL-2 as shown in SEQ ID NO:1 , or amino acid mutation K64C at amino acid residue 64, or amino acid mutation P65C at amino acid residue 65
- the anti-IL-2 humanized antibody or antigen-binding fragment thereof in the hIL-2/IL-2 antibody fusion protein forms the second disulfide bond with the antigen-antibody binding interface of the hIL-2 variant.
- the pairing of a cysteine residue and a second cysteine residue is selected from the following one, or two, or a combination of more than two paired amino acid mutations:
- the first cysteine residue is the amino acid mutation M104C that the hIL-2 variant has relative to the 104th amino acid residue of wild-type hIL-2 as shown in SEQ ID NO:1
- the first cysteine residue is the amino acid mutation T102C that the hIL-2 variant has relative to the 102nd amino acid residue of wild-type hIL-2 as shown in SEQ ID NO:1
- the second cysteine residue is obtained by introducing an amino acid mutation S67C at position 67 of the light chain variable region of the anti-IL-2 humanized antibody or its antigen-binding fragment
- the anti-IL-2 -2 Humanized antibody or binding fragment thereof comprises 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% of the VL shown in SEQ ID NO:77 %, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences, or
- the second cysteine residue is obtained by introducing an amino acid mutation G68C at position 68 of the light chain variable region of the anti-IL-2 humanized antibody or its antigen-binding fragment, and the anti-IL-2 2
- the humanized antibody or binding fragment thereof comprises 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% of the VL shown in SEQ ID NO:77 , 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences; and/or
- the first cysteine residue is the amino acid mutation P65C that the hIL-2 variant has relative to the 65th amino acid residue of wild-type hIL-2 as shown in SEQ ID NO:1
- the first cysteine residue is the amino acid mutation F42C that the hIL-2 variant has relative to the 42nd amino acid residue of wild-type hIL-2 as shown in SEQ ID NO:1
- the use of the above-mentioned paired amino acid mutations can not only ensure the formation of the antigen-antibody binding interface of the anti-IL-2 humanized antibody or its antigen-binding fragment in the hIL-2/IL-2 antibody fusion protein and the hIL-2 variant.
- the designed disulfide bond connection ensures that the ⁇ receptor binding sites of the hIL-2 variants are all buried inside the IL-2/IL-2 antibody fusion protein molecule (ie, the antigen-antibody binding interface), and the fusion
- antigen-antibody dissociation will not occur to expose the ⁇ subunit receptor binding site of the hIL-2 variant, and it cannot be competitively bound to the hIL-2 variant by the high-affinity IL2R in vivo, thereby Significantly reduce systemic toxic side effects and Treg cell-mediated immunosuppressive effects caused by activation of high-affinity IL2R.
- the hIL-2/IL-2 antibody fusion protein is different from the antigen-antibody complex formed by IL-2 and anti-IL-2 antibody.
- the antigen-antibody complex formed by IL-2 and anti-IL-2 antibody is prone to dissociation and is easily competed by high-affinity IL2R for binding to IL-2, for example, it can stimulate Treg with high expression of CD25 in vivo and in vitro cell proliferation and promotes immunosuppressive responses.
- the hIL-2/IL-2 antibody fusion protein comprises HCDR1, HCDR2, HCDR3 of the heavy chain variable region and LCDR1, LCDR2, LCDR3 of the light chain variable region, and the HCDR3, LCDR1 , LCDR2 and/or LCDR3 comprise one, or two, or more than two cysteine residue mutations, HCDR1, HCDR2, HCDR3 of the heavy chain variable region and LCDR1, LCDR2, LCDR3 of the light chain variable region include:
- the hIL-2/IL-2 antibody fusion protein comprises HCDR1, HCDR2, HCDR3 of the heavy chain variable region and LCDR1, LCDR2, LCDR3 of the light chain variable region comprising:
- the hIL-2/IL-2 antibody fusion protein comprises the heavy chain variable region with the HCDR1, HCDR2 and HCDR3, and/or the light chain with the LCDR1, LCDR2 and LCDR3 can be Variable region, wherein said HCDR3, LCDR1, LCDR2 and/or LCDR3 comprise one, or two, or more than two cysteine residue mutations, and said heavy chain variable region comprises a sequence corresponding to SEQ ID NO:63 respectively , 86, 74 or 112 have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% or 100% identical amino acid sequence, and/or the light chain variable region comprises an amino acid sequence corresponding to SEQ ID NO: 64, 77, 80, 81, 82, 83, 84, 85, 104, 105, 106, respectively , 107, 108, 109, 110 or 111 have at least 85%
- the heavy chain variable region comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
- light chain variable region comprising SEQ ID NO: 64, 87, 88, 89 , 90, 91, 107, 108 or 109 have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99% or 100% identical amino acid sequences.
- the heavy chain variable region and the light chain variable region comprise: the heavy chain variable region shown in SEQ ID NO: 86 and SEQ ID NO: 64, 87, 88, 89, 90 or 91
- the light chain variable regions shown have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% %, 99% or 100% identical amino acid sequence; or have at least 85% with the heavy chain variable region shown in SEQ ID NO:74 and the light chain variable region shown in SEQ ID NO:107, 108 or 109, respectively , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences.
- the hIL-2/IL-2 antibody fusion protein of the present application when applied in vivo, it can not only significantly promote the proliferation of Teff cells and/or NK cells, but also induce IL-2 in Teff cells and/or NK cells.
- the hIL-2/IL-2 antibody fusion protein of this application does not combine with the ⁇ subunit of IL2R, or ⁇ / ⁇ dimer, or ⁇ / ⁇ / ⁇ trimer Therefore, it not only blocks its binding to the high-affinity IL2R on the surface of Treg cells, does not activate Treg cells to induce tumor immunosuppression, but also does not bind to cells or tissues that express CD25 or express high-affinity IL2R, thereby Significantly reduced the toxic and side effects of existing IL-2 products in vivo (for example, VLS, pulmonary edema, hypotension, etc.); in addition, the administration of the hIL-2/IL-2 antibody fusion protein of the present application did not find any effect on conventional CD4 + T Cells (Tconv) produce adverse effects.
- Tconv T Cells
- the present application also introduces a disulfide bond at the binding interface of the antigen-antibody complex formed by wild-type hIL-2 and other anti-IL-2 humanized antibodies that specifically recognize the IL2R ⁇ site of hIL-2 , the antigen-antibody complex is selected from hIL-2:NARA1 antigen-antibody complex and hIL-2:TCB2 antigen-antibody complex.
- mutating pairs of amino acid residues whose spatial distance between amino acids in the antigen-antibody binding interface of the complex is less than 10 angstroms (preferably less than 5 angstroms) to pairs of cysteine residues to form disulfide bonds, It is also possible to more stably embed the hIL2R ⁇ binding site of IL-2 on the antigen-antibody binding interface, further reduce or eliminate the activity of the antigen-antibody complex to competitively bind to IL2R ⁇ or high-affinity IL2R, and maintain Binding activity of hIL-2 to IL2R ⁇ / ⁇ .
- the binding interface of the hIL-2:NARA1 antigen-antibody complex comprises the binding interface of hIL-2 and IL2R ⁇ , and the binding interface of the hIL-2:NARA1 antigen-antibody complex is located at the The AB loop, B helix and BC loop of hIL-2 are described.
- the binding interface of the hIL-2:NARA1 antigen-antibody complex is located at the 30-43 or adjacent amino acid residues and the 71-77 or adjacent amino acid residues of the hIL-2. amino acid residues.
- the binding interface of the hIL-2:NARA1 antigen-antibody complex comprises two or more cysteine residues to form the disulfide bond.
- said hIL-2 is at position 34, position 37, position 41, position 42, position 68, position There is one, or two, or more than two amino acid mutations at the 73rd, 75th, 77th and 111th amino acid residues to introduce the first cysteine residue.
- the amino acid mutation includes the 1st position in HCDR1 of NARA1 or its antigen-binding fragment, and/or the 2nd, 6th and/or 8th position in HCDR2, and/or the 3rd and/or 8th position in HCDR3 position, and/or at position 9 of LCDR1, and/or at position 3, position 4 and/or amino acid residue at position 6 of LCDR3.
- the pairing of the first cysteine residue and the second cysteine residue forming a disulfide bond at the binding interface of the hIL-2:NARA1 antigen-antibody complex is selected from the following one: , or two, or a combination of more than two paired amino acid mutations:
- the first cysteine residue is the amino acid mutation P34C at the 34th amino acid residue of the hIL-2
- the second cysteine residue is the NARA1 antibody or Its antigen-binding fragment is obtained by introducing an amino acid mutation A8C at the 8th position of its HCDR3, or the second cysteine residue is the anti-NARA1 antibody or its antigen-binding fragment obtained by introducing the amino acid mutation A8C at the 3rd or 4th position of LCDR3 obtained by introducing amino acid mutation S3C or N4C respectively;
- the first cysteine residue is the amino acid mutation T37C at amino acid residue 37 of the hIL-2
- the second cysteine residue is the NARA1 antibody or Its antigen-binding fragment is obtained by introducing an amino acid mutation D6C at position 6 of its LCDR3;
- the first cysteine residue is the amino acid mutation T41C at amino acid residue 41 of the hIL-2
- the second cysteine residue is the NARA1 antibody or Its antigen-binding fragment is obtained by introducing an amino acid mutation I2C at position 2 of its HCDR2;
- the first cysteine residue is the amino acid mutation F42C at amino acid residue 42 of the hIL-2
- the second cysteine residue is the NARA1 antibody or Its antigen-binding fragment is obtained by introducing an amino acid mutation G8C at position 8 of its HCDR2;
- the first cysteine residue is the amino acid mutation E68C at the 68th amino acid residue of the hIL-2
- the second cysteine residue is the NARA1 or its
- the antigen-binding fragment is obtained by introducing an amino acid mutation N1C at position 1 of HCDR1;
- the first cysteine residue is the amino acid mutation A73C at the 73rd amino acid residue of the hIL-2, and the second cysteine residue is the NARA1 or its
- the antigen-binding fragment is obtained by introducing an amino acid mutation G3C at position 3 of HCDR3; and/or
- the first cysteine residue is the amino acid mutation S75C or N77C respectively at the 75th or 77th amino acid residue of the hIL-2
- the second cysteine residue is The base is obtained by introducing an amino acid mutation D9C at the 9th position of LCDR1 of the NARA1 or its antigen-binding fragment;
- the first cysteine residue is the amino acid mutation T111C at amino acid residue 111 of the hIL-2
- the second cysteine residue is the NARA1 antibody or Its antigen-binding fragment is obtained by introducing an amino acid mutation S6C at position 6 of its HCDR2.
- the pairing of the first cysteine residue and the second cysteine residue forming a disulfide bond at the binding interface of the hIL-2:NARA1 antigen-antibody complex is selected from one or both of the following A combination of one, or two or more paired amino acid mutations:
- the first cysteine residue is the amino acid mutation T37C at amino acid residue 37 of the hIL-2
- the second cysteine residue is the NARA1 antibody or Its antigen-binding fragment is obtained by introducing an amino acid mutation D6C at position 6 of its LCDR3, or
- the first cysteine residue is the amino acid mutation E68C at amino acid residue 68 of the hIL-2
- the second cysteine residue is the NARA1 antibody or Its antigen-binding fragment is obtained by introducing an amino acid mutation N1C at position 1 of its HCDR1;
- the first cysteine residue is the amino acid mutation S75C or N77C at the 75th or 77th amino acid residue of the hIL-2, and the second cysteine residue
- the NARA1 antibody or its antigen-binding fragment is obtained by introducing an amino acid mutation D9C at position 9 of its LCDR1; and/or
- the first cysteine residue is the amino acid mutation T111C at amino acid residue 111 of the hIL-2
- the second cysteine residue is the NARA1 antibody or Its antigen-binding fragment is obtained by introducing an amino acid mutation S6C at position 6 of its HCDR2.
- the hIL-2/NARA1 antibody fusion protein comprises HCDR1, HCDR2, HCDR3 of the heavy chain variable region and LCDR1, LCDR2, LCDR3 of the light chain variable region, and the HCDR1, HCDR2, HCDR3 , LCDR1, and/or LCDR3 comprise one, or two, or more than two cysteine residue substitutions, the HCDR1, HCDR2, HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, LCDR3 includes: (1) HCDR1 with amino acid sequence as shown in SEQ ID NO:142 or 148, (2) HCDR2 with amino acid sequence as shown in SEQ ID NO:143, 149, 150 or 151, (3) amino acid sequence as shown in SEQ ID NO:143, 149, 150 or 151 HCDR3 shown in ID NO:144, 152 or 153, (4) LCDR1 shown in amino acid sequence as SEQ ID NO:145 or 154, (5) LCDR2 shown in SEQ ID NO:146 amino acid sequence, (6)
- HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 of the light chain variable region include:
- Amino acid sequence such as HCDR1 shown in SEQ ID NO:142, amino acid sequence such as HCDR2 shown in SEQ ID NO:149, amino acid sequence such as HCDR3 shown in SEQ ID NO:144, amino acid sequence such as SEQ ID NO:145
- the LCDR1 shown the amino acid sequence is LCDR2 shown in SEQ ID NO:146
- the amino acid sequence is LCDR3 shown in SEQ ID NO:147;
- Amino acid sequence such as HCDR1 shown in SEQ ID NO:142, amino acid sequence such as HCDR2 shown in SEQ ID NO:143, amino acid sequence such as HCDR3 shown in SEQ ID NO:152 or 153, amino acid sequence such as SEQ ID NO LCDR1 shown in :145, the amino acid sequence is LCDR2 shown in SEQ ID NO:146, and the LCDR3 shown in amino acid sequence is SEQ ID NO:147;
- HCDR1 shown in SEQ ID NO:142 amino acid sequence such as HCDR2 shown in SEQ ID NO:143
- amino acid sequence such as HCDR3 shown in SEQ ID NO:144 amino acid sequence such as SEQ ID NO:145
- the LCDR1 shown amino acid sequence is LCDR2 shown in SEQ ID NO:146
- the amino acid sequence is LCDR3 shown in SEQ ID NO:155, 156 or 157; and/or
- Amino acid sequence such as HCDR1 shown in SEQ ID NO:148, amino acid sequence such as HCDR2 shown in SEQ ID NO:143, amino acid sequence such as HCDR3 shown in SEQ ID NO:144, amino acid sequence such as SEQ ID NO:145
- the LCDR1 shown the amino acid sequence is LCDR2 shown in SEQ ID NO:146
- the amino acid sequence is LCDR3 shown in SEQ ID NO:147;
- Amino acid sequence such as HCDR1 shown in SEQ ID NO:142, amino acid sequence such as HCDR2 shown in SEQ ID NO:150 or 151, amino acid sequence such as HCDR3 shown in SEQ ID NO:144, amino acid sequence such as SEQ ID NO LCDR1 shown in: 145, the amino acid sequence is LCDR2 shown in SEQ ID NO: 146, and the LCDR3 shown in amino acid sequence is SEQ ID NO: 147.
- the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 of the light chain variable region include:
- Amino acid sequence such as HCDR1 shown in SEQ ID NO:142, amino acid sequence such as HCDR2 shown in SEQ ID NO:149, amino acid sequence such as HCDR3 shown in SEQ ID NO:144, amino acid sequence such as SEQ ID NO:145
- the LCDR1 shown the amino acid sequence is LCDR2 shown in SEQ ID NO:146
- the amino acid sequence is LCDR3 shown in SEQ ID NO:147;
- Amino acid sequence such as HCDR1 shown in SEQ ID NO:148, amino acid sequence such as HCDR2 shown in SEQ ID NO:143, amino acid sequence such as HCDR3 shown in SEQ ID NO:144, amino acid sequence such as SEQ ID NO:145 As shown in LCDR1, the amino acid sequence is LCDR2 shown in SEQ ID NO:146, and the amino acid sequence is LCDR3 shown in SEQ ID NO:147.
- the hIL-2/NARA1 antibody fusion protein comprises the heavy chain variable region with the HCDR1, HCDR2 and HCDR3 and/or the light chain variable region with the LCDR1, LCDR2 and LCDR3,
- the HCDR1, HCDR2, HCDR3, LCDR1, and/or LCDR3 comprise one, or two, or more than two cysteine residue mutations
- the heavy chain variable region comprises the same sequence as SEQ ID NO:140,
- the sequence shown at 158, 159, 160, 161, 162 or 163 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
- the light chain variable region comprises at least 85% of the sequence shown in SEQ ID NO: 141, 164, 165, 166 or 167, respectively %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%
- (1) have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences; or
- the hIL-2/NARA1 antibody fusion protein comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and light chain variable region include:
- (1) have at least 85%, 86%, 87%, 88%, 89% with the heavy chain variable region shown in SEQ ID NO: 158 or 161 and the light chain variable region shown in SEQ ID NO: 141, respectively , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences; or
- the hIL-2/NARA1 antibody fusion protein described in this application has no binding activity to IL2R ⁇ , but maintains its binding activity to IL2R ⁇ / ⁇ .
- the binding interface of the hIL-2: TCB2 antigen-antibody complex comprises the binding interface of hIL-2 and IL2R ⁇ , and the binding interface of the hIL-2: TCB2 antigen-antibody complex is located at the The AB loop, B helix and CD loop of hIL-2 are described.
- the binding interface of the hIL-2: TCB2 antigen-antibody complex is located at the 34-45 or adjacent amino acid residues, 62-76 or adjacent amino acid residues of the hIL-2. The amino acid residue at position 111 or its adjacent amino acid residues.
- the binding interface of the hIL-2:TCB2 antigen-antibody complex comprises two or more cysteine residues to form the disulfide bond.
- said hIL-2 is at position 34, position 36, position 37, position 38, position 42, position 38 relative to wild type hIL-2 shown in SEQ ID NO: 1.
- the amino acid mutation includes position 6 of HCDR2 of TCB2 or its antigen-binding fragment, and/or position 3, position 5, position 6, and/or position 7 of HCDR3, and/or position 4 of LCDR1 , located at the 4th, 5th, and/or 6th amino acid residue of LCDR3.
- the pairing of the first cysteine residue and the second cysteine residue forming a disulfide bond at the binding interface of the hIL-2:TCB2 antigen-antibody complex is selected from the following one , or two, or a combination of more than two paired amino acid mutations:
- the first cysteine residue is the amino acid mutation P34C at the 34th amino acid residue of the hIL-2
- the second cysteine residue is the TCB2 or its
- the antigen-binding fragment is obtained by introducing the amino acid mutation T4C at the 4th position of its LCDR1, or the second cysteine is the TCB2 or its antigen-binding fragment by introducing at the 4th or 5th position of its LCDR3 respectively obtained by the amino acid mutation D4C or N5C;
- the first cysteine residue is the amino acid mutation L36C at the 36th amino acid residue of the hIL-2, and the second cysteine residue is the TCB2 or its
- the antigen-binding fragment is obtained by introducing an amino acid mutation N5C at position 5 of its LCDR3;
- the first cysteine residue is the amino acid mutation T37C at the 37th amino acid residue of the hIL-2
- the second cysteine residue is the TCB2 or its
- the antigen-binding fragment is obtained by introducing an amino acid mutation N5C or L6C at position 5 or position 6 of LCDR3, respectively;
- the first cysteine residue is the amino acid mutation R38C at the 38th amino acid residue of the hIL-2
- the second cysteine residue is the TCB2 or its
- the antigen-binding fragment is obtained by introducing an amino acid mutation F7C at position 7 of HCDR3;
- the first cysteine residue is the amino acid mutation F42C at the 42nd amino acid residue of the hIL-2
- the second cysteine residue is the TCB2 or its
- the antigen-binding fragment is obtained by introducing an amino acid mutation R5C at position 5 of HCDR3;
- the first cysteine residue is the amino acid mutation E68C at the 68th amino acid residue of the hIL-2
- the second cysteine residue is the TCB2 or its
- the antigen-binding fragment is obtained by introducing an amino acid mutation A3C or G6C at position 3 or position 6 of HCDR3, respectively;
- the first cysteine residue is the amino acid mutation D109C at the 109th amino acid residue of the hIL-2
- the second cysteine residue is the TCB2 or its
- the antigen-binding fragment is obtained by introducing an amino acid mutation D6C at position 6 of HCDR2.
- the pairing of the first cysteine residue and the second cysteine residue forming a disulfide bond at the binding interface of the hIL-2: TCB2 antigen-antibody complex is selected from one or both of the following A combination of one, or two or more paired amino acid mutations:
- the first cysteine residue is the amino acid mutation P34C at the 34th amino acid residue of the hIL-2, and the second cysteine is the TCB2 or its antigen-binding
- the fragment is obtained by introducing an amino acid mutation D4C or N5C at position 4 or position 5 of LCDR3, respectively; and/or
- the first cysteine residue is the amino acid mutation L36C at the 36th amino acid residue of the hIL-2, and the second cysteine residue is the TCB2 or its
- the antigen-binding fragment is obtained by introducing an amino acid mutation N5C at position 5 of its LCDR3;
- the first cysteine residue is the amino acid mutation T37C at the 37th amino acid residue of the hIL-2
- the second cysteine residue is the TCB2 or its
- the antigen-binding fragment is obtained by respectively introducing the amino acid mutation L6C at position 6 of its LCDR3; and/or
- the first cysteine residue is the amino acid mutation F42C at the 42nd amino acid residue of the hIL-2
- the second cysteine residue is the TCB2 or its
- the antigen-binding fragment is obtained by introducing an amino acid mutation R5C at position 5 of HCDR3;
- the first cysteine residue is the amino acid mutation E68C at the 68th amino acid residue of the hIL-2
- the second cysteine residue is the TCB2 or its
- the antigen-binding fragments are obtained by respectively introducing the amino acid mutation A3C at the third position of HCDR3.
- the hIL-2/TCB2 antibody fusion protein comprises HCDR1, HCDR2, HCDR3 of the heavy chain variable region and LCDR1, LCDR2, LCDR3 of the light chain variable region, and the HCDR2, HCDR3, LCDR1 , and/or LCDR3 comprises one, or two, or more than two cysteine residue substitutions
- the HCDR1, HCDR2, HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, LCDR3 of the light chain variable region include : (1) amino acid sequence such as HCDR1 shown in SEQ ID NO:170, (2) amino acid sequence such as HCDR2 shown in SEQ ID NO:171 or 176, (3) amino acid sequence such as SEQ ID NO:172,177,178 HCDR3 shown in , 179 or 180, (4) amino acid sequence such as LCDR1 shown in SEQ ID NO:173 or 181, (5) amino acid sequence such as LCDR2 shown in SEQ ID NO:174, (6) amino acid sequence such as SEQ ID NO:
- the hIL-2/TCB2 antibody fusion protein comprises HCDR1, HCDR2, HCDR3 of the heavy chain variable region and LCDR1, LCDR2, LCDR3 of the light chain variable region, including:
- Amino acid sequence such as HCDR1 shown in SEQ ID NO:170, amino acid sequence such as HCDR2 shown in SEQ ID NO:171, amino acid sequence such as HCDR3 shown in SEQ ID NO:177, 178, 179 or 180, amino acid sequence LCDR1 as shown in SEQ ID NO:173, LCDR2 as shown in SEQ ID NO:174 in amino acid sequence, LCDR3 as shown in SEQ ID NO:175 in amino acid sequence; And/or
- amino acid sequence such as HCDR1 shown in SEQ ID NO:170, amino acid sequence such as HCDR2 shown in SEQ ID NO:171, amino acid sequence such as HCDR3 shown in SEQ ID NO:172, amino acid sequence such as SEQ ID NO:181
- the LCDR1 shown the amino acid sequence is LCDR2 shown in SEQ ID NO:174
- the amino acid sequence is LCDR3 shown in SEQ ID NO:175;
- Amino acid sequence such as HCDR1 shown in SEQ ID NO:170, amino acid sequence such as HCDR2 shown in SEQ ID NO:171, amino acid sequence such as HCDR3 shown in SEQ ID NO:172, amino acid sequence such as SEQ ID NO:173 As shown in LCDR1, the amino acid sequence is LCDR2 shown in SEQ ID NO:174, and the amino acid sequence is LCDR3 shown in SEQ ID NO:182, 183 or 184.
- the hIL-2/TCB2 antibody fusion protein comprises HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and LCDR1, LCDR2, and LCDR3 of the light chain variable region, which include:
- Amino acid sequence such as HCDR1 shown in SEQ ID NO:170, amino acid sequence such as HCDR2 shown in SEQ ID NO:171, amino acid sequence such as HCDR3 shown in SEQ ID NO:177 or 178, amino acid sequence such as SEQ ID NO LCDR1 shown in :173, the amino acid sequence is LCDR2 shown in SEQ ID NO:174, and the LCDR3 shown in amino acid sequence is SEQ ID NO:175;
- Amino acid sequence such as HCDR1 shown in SEQ ID NO:170, amino acid sequence such as HCDR2 shown in SEQ ID NO:171, amino acid sequence such as HCDR3 shown in SEQ ID NO:172, amino acid sequence such as SEQ ID NO:173 As shown in LCDR1, the amino acid sequence is LCDR2 shown in SEQ ID NO:174, and the amino acid sequence is LCDR3 shown in SEQ ID NO:182, 183 or 184.
- the hIL-2/TCB2 antibody fusion protein comprises a heavy chain variable region with the HCDR1, HCDR2 and HCDR3 and/or a light chain variable region with the LCDR1, LCDR2 and LCDR3, so
- the HCDR2, HCDR3, LCDR1, and/or LCDR3 comprise one, or two, or more than two cysteine residue mutations, and the heavy chain variable region comprises the same sequence as SEQ ID NO: 168, 185, 186, respectively.
- 187, 188 or 189 have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% or 100% identical amino acid sequence, and/or the light chain variable region comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences.
- the hIL-2/TCB2 antibody fusion protein comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region comprising:
- (1) have at least 85%, 86%, 87% with the heavy chain variable region shown in SEQ ID NO: 185, 186, 187, 188 or 189 and the light chain variable region shown in SEQ ID NO: 169, respectively , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences; or
- the hIL-2/TCB2 antibody fusion protein comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region include:
- (1) have at least 85%, 86%, 87%, 88%, 89% with the heavy chain variable region shown in SEQ ID NO: 185 or 186 and the light chain variable region shown in SEQ ID NO: 169, respectively , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences;
- the hIL-2/TCB2 antibody fusion protein described in this application does not have binding activity to IL2R ⁇ , but maintains or enhances its binding activity to IL2R ⁇ / ⁇ .
- the heavy chain variable region and its CDR region and the light chain variable region and its CDR region (defined by the Kabat numbering system) of the antibody or antigen-binding fragment portion thereof of the exemplary hIL-2/IL-2 antibody fusion proteins of the application number, as shown in Table 3.
- the IL2R ⁇ binding site of hIL-2 or its variants is always embedded inside the molecule described in the application (i.e. antigen-antibody binding interface), and retain the IL2R ⁇ / ⁇ binding site of IL-2 or its variants outside the molecule, thereby maintaining the binding affinity with IL2R ⁇ / ⁇ and specifically activating the biological activity of IL2R ⁇ / ⁇ (including Promote the proliferation of Teff cells and/or NK cells, activate the tumor killing activity of Teff cells and/or NK cells), and eliminate the combination with high-affinity IL2R and its derived toxicity (including expression on normal tissues and/or ILC2 cells VLS, pulmonary edema, hypotension, etc.
- the molecular structure described in the present application is stabilized, and there will be no dissociation similar to the hIL-2/IL-2 antibody complex, and the high-affinity IL2R cannot be competitively bound to hIL-2 or its variant body, thereby significantly reducing the risk of potential clinical application of the hIL-2/IL-2 antibody complex; thirdly, the constructed molecule has low immunogenicity, which greatly reduces the risk of ADA; fourthly, the constructed The molecule is easy to produce and prepare, and the obtained product has good homogeneity, which is not only beneficial to quality control, but also has high stability and is not easy to aggregate, which significantly reduces potential adverse effects caused by product quality.
- the hIL-2/IL-2 antibody fusion protein of the present application also includes the hIL-2 or its variant fused to or operably linked to the hIL-2 provided by the present application through a linker sequence.
- This application is concerned with the position of hIL-2 or its variants linked to the anti-IL-2 antibody or its binding fragment, the type and length of the linker, and whether the linker will affect the binding activity of hIL-2 or its variants to IL2R ⁇ / ⁇
- the linker selected should also have low immunogenicity. Flexible peptides can be chosen as linkers in this context.
- the linker sequence includes G4S or GAF, and one, two, or more than two copies of the linker can be used. In other embodiments, 2, 3, 4 or 5 copies of the G4S or GAF linker or any other linker sequence known in the art suitable for the fusion proteins disclosed herein may be used herein.
- Preferred linkers include (G4S)4, (G4S)3, (GAF)2, which can ensure the stability of the internal structure of the IL-2/IL-2 antibody fusion protein constructed, and the more preferred linker is (G4S) 4.
- linker refers to a molecule (including but not limited to unmodified or modified amino acids or amino acid sequences) that connects two compounds, such as two polypeptides.
- a linker may consist of a single, or two, or more than two linking molecules, or may include a linking molecule and at least one spacer molecule intended to separate the linking molecule and the compound at a specific distance.
- operably linked refers to the linking of amino acid sequences, peptides or proteins with different functional properties, such as linking hIL-2 or its variants with anti-IL-2 via a linker sequence as described herein. Linkage of antibodies or antigen-binding fragments thereof.
- the present application links the hIL-2 variant to the heavy chain variable region or light chain variable region of the anti-IL-2 humanized antibody of the present application through a linker.
- the linker is connected to the C-terminus of the hIL-2 variant and the N-terminus of the heavy chain of the anti-IL-2 antibody, which can ensure less impact on the binding of the hIL-2 variant to IL2R ⁇ / ⁇ .
- the present application also connects hIL-2 to other anti-IL-2 humanized antibody heavy chain variable regions or light chain variable regions that specifically recognize the IL2R ⁇ binding site of hIL-2 through a linker .
- the linker is connected to the C-terminus of hIL-2 and the N-terminus of the variable region of the light chain of the NARA1 antibody, which can ensure less impact on the binding of hIL-2 and IL2R ⁇ / ⁇ .
- more than 4 copies of the linker can be selected to be connected to the C-terminal of hIL-2 and the N-terminal of the heavy chain variable region of the NARA1 antibody, which will also not significantly affect the binding activity of hIL-2 and IL2R ⁇ / ⁇ .
- the linker is connected to the C-terminus of hIL-2 and the N-terminus of the variable region of the light chain of the TCB2 antibody, which can ensure less impact on the binding of hIL-2 and IL2R ⁇ / ⁇ .
- more than 4 copies of the linker can be selected to be connected to the C-terminal of hIL-2 and the N-terminal of the heavy chain variable region of the TCB2 antibody, and it will not affect the binding activity of hIL-2 and IL2R ⁇ / ⁇ . Significantly affected.
- the linker selected in this application is not only beneficial to the structural stability of the hIL-2/IL-2 antibody fusion protein molecule of this application, production preparation and homogeneity products, but also has low immunogenicity.
- the present application carried out further optimization on the basis of the above-mentioned hIL-2/IL-2 antibody fusion protein, including the optimization of the anti-IL-2 antibody or its antigen-binding fragment in the fusion protein molecule and the optimization of hIL-2 variants.
- the present application uses the first aspartic acid or the 27th gluten in the light chain variable region of the anti-IL-2 antibody (including anti-mIL-2 antibody and each humanized antibody derived therefrom)
- Amino acid amide located in the light chain CDR1 has undergone amino acid mutations, the amino acid mutations include D1E, Q27D, Q27E, Q27S, Q27G, Q27K or Q27R, and the preferred amino acid mutations include D1E, Q27D, Q27E, Q27S or Q27G, which can significantly improve The expression level of the hIL-2/IL-2 antibody fusion protein of the present application.
- the hIL-2/IL-2 antibody fusion protein comprises the following HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3: the amino acid sequence is HCDR1 as shown in SEQ ID NO: 14, and the amino acid sequence is as follows HCDR2 shown in SEQ ID NO:15, the amino acid sequence is HCDR3 shown in SEQ ID NO:29, the amino acid sequence is LCDR1 shown in any one of SEQ ID NOs:39-44, the amino acid sequence is shown in SEQ ID NO:17 The LCDR2, and the LCDR3 of amino acid sequence as shown in SEQ ID NO:12.
- the hIL-2/IL-2 antibody fusion protein comprises the following HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3: the amino acid sequence is HCDR1 as shown in SEQ ID NO: 14, and the amino acid sequence is as follows HCDR2 shown in SEQ ID NO:15, the amino acid sequence is HCDR3 shown in SEQ ID NO:29, the amino acid sequence is LCDR1 shown in any one of SEQ ID NOs:39-42, the amino acid sequence is shown in SEQ ID NO:17 The LCDR2, and the LCDR3 of amino acid sequence as shown in SEQ ID NO:12.
- the hIL-2/IL-2 antibody fusion protein comprises the following heavy chain variable region and/or light chain variable region, and the heavy chain variable region comprises the same sequence as SEQ ID NO:86
- the sequence shown has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
- the light chain variable region comprises at least 85%, 86%, 87%, 88%, 89%, 90% of the sequence shown in any one of SEQ ID NOs:87-93, respectively %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences.
- the hIL-2/IL-2 antibody fusion protein comprises the following heavy chain variable region and/or light chain variable region, and the heavy chain variable region comprises the same sequence as SEQ ID NO:86
- the sequence shown has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% Or 100% identical amino acid sequence
- the light chain variable region comprises at least 85%, 86%, 87%, 88%, 89%, 90% of the sequence shown in any one of SEQ ID NOs:87-91 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences.
- the present application in order to facilitate the production and preparation of the hIL-2/IL-2 antibody fusion protein of the present application, increase the expression yield, facilitate quality control and generate homogeneous products, and reduce potential ADA risks, the present application
- the hIL-2 variant molecules were further optimized, including:
- the glycosylation sites present in the hIL-2 variants can be mutated (eg, deleted) or left unchanged.
- the term "glycosylation site” in this application refers to an amino acid residue whose side chain can be connected to a carbohydrate moiety (such as an oligosaccharide structure) when it is used in a polypeptide sequence, and it is convenient to mutate this amino acid residue. to achieve the elimination of natural glycosylation modifications.
- the hIL-2/IL-2 antibody fusion protein of the present application at least one native glycosylation site is absent or present in said hIL-2 variant.
- the partial glycosylation site of the hIL-2 variant is deleted, including deleting the N-terminal of the hIL-2 variant The first 3 or 5 amino acid residues, or mutating the third threonine residue, preferably deleting the first 3 amino acid residues at the N-terminus (for example, as shown in SEQ ID NO: 134); and/or
- the first polypeptide and the second polypeptide of the hIL-2/IL-2 antibody fusion protein of the present application are provided.
- the hIL-2/IL-2 antibody fusion protein of the present application comprises a first polypeptide and a second polypeptide, and the first polypeptide comprises hIL-2 or a variant thereof with a cysteine residue mutation operatively Linked to one variable region derived from said anti-IL-2 antibody or antigen-binding fragment thereof, the second polypeptide comprises another variable region of said anti-IL-2 antibody or antigen-binding fragment thereof.
- the first polypeptide comprises a variable region of hIL-2 or a variant thereof having a cysteine residue mutation, a linker, and the anti-IL-2 antibody or an antigen-binding fragment thereof, optionally in One variable region of the anti-IL-2 antibody or antigen-binding fragment thereof in the first polypeptide and/or another variable region of the anti-IL-2 antibody or antigen-binding fragment thereof in the second polypeptide are halved Cystine residues are mutated so that correctly paired and stable disulfide bonds can be formed with hIL-2 or variants thereof having mutated cysteine residues, and said disulfide bonds are located in said hIL-2 or A variant thereof binds to the antigen-antibody binding interface of said anti-IL-2 antibody or antigen-binding fragment thereof.
- the hIL-2/IL-2 antibody fusion protein comprises a first polypeptide comprising a hIL-2 variant with a cysteine residue mutation and a second polypeptide , a linker and the heavy chain variable region of the anti-IL-2 antibody or an antigen-binding fragment thereof, and the second polypeptide comprises the light chain variable region of the anti-IL-2 antibody or an antigen-binding fragment thereof.
- the first polypeptide comprises at least 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
- the second polypeptide comprises the same amino acid sequence as SEQ ID NO : 64, 77, 80-85, 87-93, or any one of the sequences shown in 104-111 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences.
- the hIL-2/IL-2 antibody fusion protein comprises a first polypeptide and a second polypeptide comprising:
- (1) have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences; or
- the hIL-2/IL-2 antibody fusion protein comprises a first polypeptide and a second polypeptide comprising:
- (1) have at least 85%, 86%, and 87% with the first polypeptide shown in any one of SEQ ID NO:101, 102, 103, and 133-138 and the second polypeptide shown in SEQ ID NO:64, respectively , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences; or
- the hIL-2/IL-2 antibody fusion protein comprises a first polypeptide and a second polypeptide comprising:
- (1) have at least 85%, 86%, 87%, 88%, 89% with the first polypeptide shown in SEQ ID NO:113 and the second polypeptide shown in SEQ ID NO:104, 105 or 106, respectively , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences; or
- (7) have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences.
- the hIL-2/IL-2 antibody fusion protein comprises a first polypeptide and a second polypeptide comprising:
- (1) have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences; or
- the hIL-2/IL-2 antibody fusion protein comprises a first polypeptide comprising hIL-2 with a cysteine residue mutation, a linker and a second polypeptide. and the light chain variable region of the anti-IL-2 antibody or an antigen-binding fragment thereof, and the second polypeptide comprises the heavy chain variable region of the anti-IL-2 antibody or an antigen-binding fragment thereof.
- the first polypeptide comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% of the sequence shown in any one of SEQ ID NO:206-226. %, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
- said second polypeptide comprises the same amino acid sequence as SEQ ID NO: 140, 158-163, 168, Or any one of the sequences shown in 185-189 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% or 100% identical amino acid sequences.
- the hIL-2/IL-2 antibody fusion protein comprises a first polypeptide and a second polypeptide comprising:
- (1) have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences; or
- (7) have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences.
- the hIL-2/IL-2 antibody fusion protein comprises a first polypeptide and a second polypeptide comprising:
- (1) have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences; or
- the hIL-2/IL-2 antibody fusion protein comprises a first polypeptide and a second polypeptide comprising:
- the hIL-2/IL-2 antibody fusion protein comprises a first polypeptide and a second polypeptide comprising:
- (1) have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences; or
- the anti-IL-2 antibody of the present application or its antigen-binding fragment and/or the hIL-2/IL-2 antibody fusion protein of the present application can also comprise a constant region, which includes an antibody heavy chain constant region and a light chain constant region .
- the heavy chain constant region of the present application includes native and mutein forms of the Fc region of the human IgG heavy chain constant region, and also includes truncated forms of polypeptides containing a hinge region that promotes dimer formation.
- the Fc region comprises antibody CH2 and CH3 domains. Fusion proteins (and oligomers formed thereby) comprising an Fc portion offer the advantages of easy affinity chromatography purification by protein A or protein G, as well as increased serum half-life.
- Preferred Fc regions are derived from human IgG, including IgGl, IgG2, IgG3 and IgG4.
- the positions of specific amino acid residues of the Fc region are determined according to the EU numbering system.
- ADCC antibody-dependent cellular cytotoxicity
- ADCP antibody-dependent cellular phagocytosis
- CDC complement dependence Cytotoxicity
- ADCC and ADCP are mediated through the binding of Fc to Fc receptors (FcRs) on the surface of immune cells
- FcRs Fc receptors
- the immune cells include monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, NK cells and T cells.
- CDC is mediated through the binding of Fc to proteins of the complement system such as CIq (Ward et al., Ther Immunol 1995, 2:77-94).
- the anti-IL-2 antibody of the present application and/or the hIL-2/IL-2 antibody fusion protein of the present application comprise an engineered IgG Fc region to reduce its mediated effector function.
- Exemplary Fc molecules with reduced effector function include Fc molecules with the following amino acid substitutions:
- V234A/G237A (IgG2)
- a preferred engineered Fc region is a human IgG1 Fc with L234F/L235E/P331S (EU numbering system) amino acid substitutions capable of reducing binding of the Fc region to one or more Fc ⁇ Rs and C1q (Oganesyan V et al., Acta Crystallogr D Biol Crystallogr 2008,64:700-704; US Patent No.: US5624821, US6194551).
- the Fc ⁇ Rs protein family includes Fc ⁇ RI (also known as CD64), including isoforms Fc ⁇ RIa, Fc ⁇ RIb, and Fc ⁇ RIc; Fc ⁇ RII (also known as CD32), including isoforms Fc ⁇ RIIa, Fc ⁇ RIIb, and Fc ⁇ RIIc; and Fc ⁇ RIII (also known as CD16), including Isoforms FcyRIIIa and FcyRIIIb (Jefferis et al., Immunol Lett 2002, 82:57-65).
- FcyRI, FcyRIIa, FcyRIIc and FcyRIIIa can induce ADCC, endocytosis, phagocytosis and/or cytokine release.
- Binding properties include, but are not limited to, binding specificity, binding affinity constant (K D ), dissociation and association rates (K dis and K a , respectively), and binding affinity, any one or more of which can be determined by those skilled in the art.
- K D binding affinity constant
- K dis and K a dissociation and association rates
- binding affinity any one or more of which can be determined by those skilled in the art.
- the introduction of L234F/L235E/P331S into the heavy chain constant region of the anti-IL-2 antibody of the present application and/or the hIL-2/IL-2 antibody fusion protein of the present application has the effect of reducing one or more Fc ⁇ R
- the Fc ⁇ R may be Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIb, Fc ⁇ RIIIa (158F) and Fc ⁇ RIIIa (158V).
- the introduction of the L234F/L235E/P331S mutation in the heavy chain constant region can make the anti-IL-2 antibody and/or hIL-2/IL-2 antibody fusion protein and Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIb, Fc ⁇ RIIIa (158F) And/or Fc ⁇ RIIIa (158V) does not substantially bind.
- the introduction of L234F/L235E/P331S mutations in the heavy chain constant region renders the anti-IL-2 antibody and/or hIL-2/IL-2 antibody fusion protein substantially non-binding to C1q.
- the introduction of L234F/L235E/P331S mutations in the heavy chain constant region does not affect the binding affinity of the anti-IL-2 antibody and/or hIL-2/IL-2 antibody fusion protein to FcRn.
- a preferred engineered Fc region is a human IgG1 Fc with L234F/L235E/P331S (EU numbering system) amino acid substitutions.
- introducing L234F/L235E/P331S into the heavy chain constant region can significantly reduce the ADCC activity of the anti-IL-2 antibody and/or hIL-2/IL-2 antibody fusion protein of the present application.
- ADCC activity of the anti-IL-2 antibody and/or hIL-2/IL-2 antibody fusion protein of the present application is significantly weakened or eliminated compared with antibodies without amino acid mutations in the Fc region.
- the anti-IL-2 antibody and/or hIL-2/IL-2 antibody fusion protein of the present application shows that its ADCC activity is weakened to undetectable, and the weakening or elimination of ADCC activity may be caused by the antibody of the present application and/or Or the affinity of the fusion protein to Fc ⁇ R is significantly reduced.
- the anti-IL-2 antibody of the present application and/or the hIL-2/IL-2 antibody fusion protein of the present application comprise an engineered IgG1 constant region, by reducing the antibody and/or fusion protein isoelectric point (pI) to prolong its half-life in vivo.
- Changes in the isoelectric point of an antibody and/or fusion protein can affect its tissue distribution and pharmacokinetic (PK) properties; generally, a decrease in pI results in reduced nonspecific tissue absorption and metabolism/degradation and increased in vivo half-life ( Boswell CA et al., Bioconjug Chem 2010, 21:2153-2163; Li et al., MAbs 2014, 6:1255-1264).
- mutating amino acids at specific sites in the heavy chain constant region of the anti-IL-2 antibody and/or hIL-2/IL-2 antibody fusion protein of the present application for example, using an acidic amino acid (such as aspartic acid) Acid or glutamic acid) to replace basic amino acids (such as lysine or arginine) or neutral amino acids (such as asparagine or glutamine), can reduce the pI of the antibody and/or antibody fusion protein.
- an acidic amino acid such as aspartic acid
- Basic amino acids such as lysine or arginine
- neutral amino acids such as asparagine or glutamine
- one approach to minimize the risk of immunogenicity due to such mutations is to take advantage of amino acid sequence differences between natural isoforms of the human protein to make substitutions.
- human IgG subclasses ie IgG1, IgG2, IgG3 and IgG4 Isotype differences between provide mutations with low risk of immunogenicity.
- the mutation can be performed by comparing the amino acid sequences of the constant regions of IgG1, IgG2, IgG3 and IgG4, and mutating the non-acidic or basic amino acid residues in the IgG1 constant region to the corresponding positions in other IgG subtypes.
- the mutation can be selected from one or more of N203D, K274Q and Q419E, so as to reduce the pI value of IgG1 protein.
- the anti-IL-2 antibody and/or hIL-2/IL-2 antibody fusion protein of the present application comprises a heavy chain constant region with mutations comprising N203D/K274Q/Q419E (EU numbering system) , compared to the antibody and/or fusion protein without the mutation in the heavy chain constant region, the pI value of the anti-IL-2 antibody and/or hIL-2/IL-2 antibody fusion protein of the present application is reduced by about 1-2 unit. The pI value can be obtained by theoretical calculation or experimental measurement.
- the anti-IL-2 antibody and/or hIL-2/IL-2 antibody fusion protein of the present application having a reduced pI value exhibits significantly improved pharmacokinetic characteristics, including relative to untreated
- the mutated antibody and/or fusion protein, the anti-IL-2 antibody and/or hIL-2/IL-2 antibody fusion protein of the present application has obtained at least 2-3 times increased elimination half-life (t 1/2 ), and and/or more than 30% increased area under the drug-time curve (AUC).
- the present application provides a nucleic acid encoding a hIL-2 variant, an anti-IL-2 antibody or an antigen-binding fragment thereof, or a hIL-2/IL-2 antibody fusion protein.
- the present application also includes polynucleotide variants encoding the amino acid sequences described herein.
- the nucleotide sequence corresponding to the amino acid sequence described in this application, used as a probe or primer for nucleic acid isolation, or provided by a database can be obtained by back-translation of the amino acid sequence.
- a polymerase chain reaction (PCR) procedure can be used to isolate and amplify hIL-2 variants, muteins, and anti-IL-2 antibodies or antigen-binding fragments thereof, hIL-2/IL-2 antibody fusion proteins of the present application.
- Oligonucleotides defining the desired ends of the combination of DNA fragments serve as 5' and 3' primers.
- the oligonucleotides may additionally contain recognition sites for restriction endonucleases to facilitate the combined insertion of the amplified DNA fragments into the expression vector.
- PCR technology is found in Saiki et al., Science 1988,239:487-491; Recombinant DNA Methodology, edited by Wu et al., Academic Press, Inc., San Diego (1989), pp. 189-196; PCR Protocols: A Guide to Methods and Applications, edited by Innis et al., Academic Press, Inc. (1990).
- Nucleic acid molecules of the present application include single- and double-stranded forms of DNA and RNA, and corresponding complementary sequences, including isolated nucleic acid molecules, preferably derived from at least one isolation in substantially pure form and in amounts or concentrations that can be determined by standard biochemical methods. Identification, manipulation and recovery of DNA or RNA of its component nucleotide sequences (as described for example in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) method). Preferably, such sequences include open reading frames provided and/or constructed as internal non-translated sequences or intron-interrupted open reading frames not normally present in eukaryotic genes. Sequences of non-translated DNA may be present 5' or 3' to the open reading frame, where said sequences do not interfere with the manipulation or expression of the coding region.
- hIL-2 variants, anti-IL-2 antibodies or antigen-binding fragments thereof, and hIL-2/IL-2 antibody fusion proteins of the present application are prepared by using PCR mutagenesis or other methods known to those of ordinary skill in the art. Technique Site-specific mutagenesis of nucleotides in DNA encoding hIL-2 variants, anti-IL-2 antibodies or antigen-binding fragments thereof, or hIL-2/IL-2 antibody fusion proteins to generate coding variants DNA, thereafter expressing the recombinant DNA in cell culture as outlined herein.
- hIL-2 variants, anti-IL-2 antibodies or antigen-binding fragments thereof, and hIL-2/IL-2 antibody fusion proteins can also be prepared by in vitro synthesis using established techniques.
- the hIL-2 variants, anti-IL-2 antibodies or antigen-binding fragments thereof, or hIL-2/IL-2 antibody fusion proteins of the present application are composed of extremely Numerous nucleic acids are encoded, and nucleic acids of each portion are within the scope of this application and can be made using standard techniques. Therefore, having identified a specific amino acid sequence, those skilled in the art can obtain the hIL-2 variants, anti-IL-2 antibodies or antigen-binding fragments thereof, or hIL-2/IL-2 antibody fusion proteins of the present application without changing the specific amino acid sequence. Many different nucleic acids can be prepared by simply modifying their respective coding sequences by one, or two, or more than two codons by means of the amino acid sequence.
- the present application also provides expression vectors of nucleic acids encoding hIL-2 variants, anti-IL-2 antibodies or antigen-binding fragments thereof, or hIL-2/IL-2 antibody fusion proteins herein.
- the nucleic acid encoding the hIL-2 variant, anti-IL-2 antibody or antigen-binding fragment thereof, or hIL-2/IL-2 antibody fusion protein of the present application can be constructed in a suitable vector to be introduced into host cells for target protein expression.
- Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
- the nucleic acid encoding the target protein in the vector is operably linked to the promoter.
- operably linked refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence, and The control sequences thus control the transcription and/or translation of other nucleic acid sequences.
- a nucleic acid expression control sequence e.g., a promoter, a signal sequence, or an array of transcriptional regulator binding sites
- Suitable vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal virus etc.
- artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal virus etc.
- Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillo
- a vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
- the vector may also contain an origin of replication.
- a vector may also include components to facilitate its entry into cells, including, but not limited to, viral particles, liposomes, or protein coats.
- the present application also provides a host cell comprising a nucleic acid or an expression vector encoding the hIL-2 variant, anti-IL-2 antibody or antigen-binding fragment thereof, or hIL-2/IL-2 antibody fusion protein of the present application .
- the cells can be eukaryotic cells, for example, mammalian host cells, including but not limited to, SV40 transformed monkey kidney cell line CV1 (COS-7, ATCC, CRL-1651), human embryonic kidney cell line (293 or suspension culture 293 cell subclones of Graham et al., J Gen Virol 1977,36:59-74), young hamster kidney cells (BHK-21, ATCC, CCL-10), Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al.
- mammalian host cells including but not limited to, SV40 transformed monkey kidney cell line CV1 (COS-7, ATCC, CRL-1651), human embryonic kidney cell line (293 or suspension culture 293 cell subclones of Graham et al., J Gen Virol 1977,36:59-74), young hamster kidney cells (BHK-21, ATCC, CCL-10), Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al.
- the present application provides methods for preparing hIL-2 variants, anti-IL-2 antibodies or antigen-binding fragments thereof, and hIL-2/IL-2 antibody fusion proteins of the present application using the host cells.
- the method comprises transfecting a nucleic acid or an expression vector encoding the hIL-2 variant of the present application, an anti-IL-2 antibody or an antigen-binding fragment thereof, or a hIL-2/IL-2 antibody fusion protein into a host cell, and The host cells are cultured in culture for a period of time to express the hIL-2 variants, anti-IL-2 antibodies or antigen-binding fragments thereof, or hIL-2/IL-2 antibody fusion proteins of the present application.
- commercially available media can be used as the media.
- the expressed hIL-2 variant, anti-IL-2 antibody or antigen-binding fragment thereof, or hIL-2/IL-2 antibody fusion protein is secreted into the culture medium in which the host cell is grown.
- Antibodies are recovered from the culture medium using standard protein purification methods, such as centrifugation or ultrafiltration to remove impurities, or purification of the resultant by affinity chromatography; other purification techniques such as anion or cation exchange chromatography, hydrophobic interaction chromatography and hydroxyapatite chromatography.
- the present application also provides a bispecific antibody, which comprises the anti-IL-2 antibody or antigen fragment thereof of the present application, or hIL-2/IL-2 antibody fusion protein, and one of the two arms of the bispecific antibody is One arm comprises the anti-IL-2 antibody or its antigenic fragment of the present application, or hIL-2/IL-2 antibody fusion protein, while the other arm comprises antigens (preferably cancer-associated antigens or immune checkpoints) other than hIL-2 Antibodies specific to protein antigens, or antibodies or antigen-binding fragments thereof that specifically bind to antigens associated with immune effector cells.
- the antigen associated with cancer can be selected from HER2, EGFR, VEGF, VEGFR, CD19, CD20, CD30, CD33, CD52, BCMA, SLAMF7, MUC1, MUC16, EphB2, E-selectin, EpCam, CEA, PSMA, PSA , ERBb3, c-MET, ILT3, ILT7, 5T4, GPC-3, DLL3, etc.
- the immune checkpoint protein antigen can be selected from PD-1, PD-L1, PD-L2, TIM3, CTLA-4, LAG- 3. CD47, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD40, CD40L, OX40, CD160, 2B4 or TGFR.
- bispecific molecules of the present application can have many different forms.
- a bispecific molecule retains the traditional antibody format, except instead of having two binding arms of the same specificity, it has two binding arms of different specificities, each of which can be formed by a peptide Two single-chain antibody fragments (scFv) connected by a chain to construct a bispecific molecule, the so-called Bs(scFv)2 structure.
- Bispecific molecules can also comprise two different F(ab) fragments linked by a peptidyl linker.
- the application provides an immunoconjugate comprising the anti-IL-2 antibody or antigen-binding fragment thereof herein, or hIL-2/IL-2 antibody fusion protein.
- the anti-IL-2 antibody or antigen-binding fragment thereof of the present application, or hIL-2/IL-2 antibody fusion protein can be conjugated with a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC).
- a therapeutic agent such as an antibody-drug conjugate (ADC).
- Suitable therapeutic agents include cytotoxins, alkylating agents, DNA minor groove binders, DNA intercalators, DNA crosslinkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerization Enzyme I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics and antimitotics.
- the linker coupled with the antibody and the therapeutic agent can choose a cleavable linker (Cleavable linker), such as a peptidyl linker, a disulfide bond or a hydrazone linker, or a non-cleavable linker (Non-cleavable linker).
- a cleavable linker such as a peptidyl linker, a disulfide bond or a hydrazone linker
- Non-cleavable linker Non-cleavable linker
- the present application also provides chimeric antigen receptors, engineered T cell receptors, or lysed T cell receptors comprising the anti-IL-2 antibodies or antigen-binding fragments thereof, or hIL-2/IL-2 antibody fusion proteins herein. tumor virus.
- the application provides a pharmaceutical composition
- a pharmaceutical composition comprising the anti-IL-2 antibody or antigen-binding fragment thereof of the application, or hIL-2/IL-2 antibody fusion protein (or immunoconjugate, or bispecific molecule, or chimeric antigen receptor, or engineered T cell receptor, or oncolytic virus), and a pharmaceutically acceptable carrier.
- the pharmaceutical composition may contain any kind of excipients.
- Excipients that can be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coating agents, disintegrants, lubricants , sweeteners, preservatives, isotonic agents or combinations thereof. Selection and use of suitable excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th ed. (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.
- the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion).
- the active ingredient can be coated in a material to protect it from acids and other natural conditions that might inactivate it.
- parenteral administration refers to parenteral and topical modes of administration including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intrathecal, intraorbital, intracardiac, dermal Intra-, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.
- antibodies of the present application may be administered by non-parenteral routes (eg, topical, epidermal, or mucosal administration routes), eg, intranasal, oral, vaginal, rectal, sublingual, or topical administration.
- compositions can be in the form of sterile aqueous solutions or dispersions. They can also form microemulsions, liposomes, or other ordered structural assemblies suitable to high drug concentrations.
- compositions with a pharmaceutically acceptable carrier will contain from about 0.01% to about 99% active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30%.
- Dosage regimens are adjusted to provide the optimum desired response (eg, a therapeutic response). For example, a single dose may be administered, divided doses may be administered or the dose may be proportionally reduced or increased as indicated by the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
- Dosage unit form refers to physically discrete units suitable as unit dosages for the treatment of subjects; each unit dosage contains a predetermined quantity of active ingredient calculated by calculating The active ingredient is administered together with the required pharmaceutical carrier to produce the desired therapeutic effect.
- the anti-IL-2 antibody of the present application, or hIL-2/IL-2 antibody fusion protein can also be administered as a sustained-release preparation, which can reduce the frequency of administration.
- Anti-IL-2 antibody, or hIL-2/IL-2 antibody fusion protein, or the composition administration dosage range that comprises anti-IL-2 antibody or hIL-2/IL-2 antibody fusion protein can be about 0.0001mg/ kg to 100 mg/kg body weight, usually 0.001 mg/kg to 50 mg/kg body weight.
- the anti-IL-2 antibody or antigen-binding fragment thereof of the present application or hIL-2/IL-2 antibody fusion protein, or bispecific molecule, or immunoconjugate, or chimeric antigen receptor, or engineered T cell receptor , or a "therapeutically effective dose” of an oncolytic virus is preferably capable of reducing the severity of disease symptoms, increasing the frequency and duration of disease symptoms without progression, or preventing physical damage or disability caused by disease affliction.
- a "therapeutically effective dose" for a tumor-bearing subject preferably inhibits tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, still more preferably At least about 80%.
- a therapeutically effective amount of a therapeutic antibody can reduce the size of a tumor, or improve symptoms in a subject, usually a human or other mammal.
- “Therapeutically effective dose” can also be determined differently according to various factors, including but not limited to preparation method, administration method, age, body, body weight, patient's sex or pathological condition, diet, administration, etc. Time, dosing interval, route of administration, excretion rate, and response sensitivity.
- the pharmaceutical composition can be selected from controlled-release formulations, including implants, transdermal patches, and microencapsulated delivery systems.
- Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See, e.g., Sustained and Controlled Release Drug Delivery Systems, ed. J.R. Robinson, Marcel Dekker, Inc., New York, 1978.
- the therapeutic pharmaceutical composition can be delivered by a medical device selected from the group consisting of: (1) needle-free hypodermic injection devices (e.g., U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 and 4,596,556); (2) Miniature Infusion Pumps (U.S. Patent No. 4,487,603); (3) Transdermal Devices (U.S. Patent No. 4,486,194); (4) Infusion Devices (U.S. Patent Nos. 4,447,233 and 4,447,224); and (5) Osmotic Devices (U.S. Patent Nos. Patent Nos. Patent Nos. 4,439,196 and 4,475,196); the disclosures of which are incorporated herein by reference.
- needle-free hypodermic injection devices e.g., U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,
- the anti-IL-2 antibodies or antigen-binding fragments thereof of the present application can be formulated to ensure biodistribution in vivo.
- the therapeutic antibody or antibody fusion protein of the present application crosses the blood-brain barrier, it can be formulated into liposomes, which can additionally contain targeting moieties to enhance selection to specific cells or organs sex delivery. See, e.g., U.S. Patent Nos.
- the present application provides a kit comprising the pharmaceutical composition of the anti-IL-2 antibody or antigen-binding fragment or hIL-2/IL-2 antibody fusion protein of the present application, another anticancer agent and/or or immunomodulatory agents, said anticancer agents including but not limited to microtubule disruptors, antimetabolites, topoisomerase inhibitors, DNA intercalators, alkylating agents, hormone therapies, kinase inhibitors (e.g., tyrosine Acid kinase inhibitors include but not limited to EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, IGFR inhibitors and Met inhibitors), receptor antagonists, activators of tumor cell apoptosis (including but not limited to IAP inhibitors, Bcl2 inhibitors, MC11 inhibitors, TRAIL inhibitors, CHK inhibitors), or any anti-angiogenic drugs; the immunomodulators include but are not limited to PD-1, PD-L1, PD-L2, TIM3, CTLA- 4. L
- the present application relates to a kit comprising an effective amount of the anti-IL-2 antibody of the present application or an antigen-binding fragment thereof, or hIL-2/IL-2 antibody fusion protein, or comprising the present application
- the pharmaceutical composition of the anti-IL-2 antibody or its antigen-binding fragment or hIL-2/IL-2 antibody fusion protein of the application is provided.
- the present application relates to the use of the anti-IL-2 antibody or antigen-binding fragment thereof herein, or the hIL-2/IL-2 antibody fusion protein for preparing a pharmaceutical composition or preparation for treating diseases, the diseases including Without limitation, proliferative diseases or infections, immunodeficiency diseases including but not limited to cancer and other cell proliferative disorders.
- the present application relates to the anti-IL-2 antibody or antigen-binding fragment thereof, or hIL-2/IL-2 antibody fusion protein herein, or a pharmaceutical composition comprising it, for use in the treatment of proliferative diseases or infections, immunodeficiency diseases .
- the cancer includes, but is not limited to, melanoma, lung cancer, colorectal cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, kidney cancer, liver cancer, brain cancer, esophageal cancer, gallbladder cancer, pancreatic cancer , stomach cancer, thyroid cancer, bladder cancer and lymphoma.
- the other cell proliferative disorders include, but are not limited to, hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Seza Sezary Syndrome, Waldenström's macroglobulinemia ( macroglobulinemia), histiocytosis, and any location in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (such as adrenal, parathyroid, pituitary, testis, ovary, thymus, thyroid), eye, Cell proliferative disorders other than neoplasia in the head and neck, nervous system, lymphatic system, pelvis, skin, soft tissue, spleen, and genitourinary system.
- hypergammaglobulinemia lymphoproliferative disorders
- paraproteinemias purpura
- sarcoidosis Seza Sezary Syndrome
- Waldenström's macroglobulinemia macroglobulinemia
- the immunodeficiency disease includes, but is not limited to, HIV positive, immunosuppressive disease, chronic infection.
- the present application relates to a method of treating a disease, condition or disorder by increasing the immune response of the host, the method comprising administering to the subject an effective amount of the anti-IL-2 antibody of the present application or an antigen-binding fragment thereof , or hIL-2/IL-2 antibody fusion protein, or comprising the anti-IL-2 antibody of the present application or its antigen-binding fragment, or a pharmaceutical composition of hIL-2/IL-2 antibody fusion protein, or comprising the application Kits or kits of anti-IL-2 antibodies or antigen-binding fragments thereof, or hIL-2/IL-2 antibody fusion proteins to specifically activate IL2R ⁇ / ⁇ -expressing effector cells (e.g., CD8 + T cells and NK cells ), thereby improving the condition of the disease.
- IL2R ⁇ / ⁇ -expressing effector cells e.g., CD8 + T cells and NK cells
- the present application relates to a method of stimulating the immune system, the method comprising administering to a subject an effective amount of the anti-IL-2 antibody of the present application or an antigen-binding fragment thereof, or hIL-2/IL-2 Antibody fusion protein, pharmaceutical composition comprising the anti-IL-2 antibody of the present application or its antigen-binding fragment, or hIL-2/IL-2 antibody fusion protein, or comprising the anti-IL-2 antibody of the application or its antigen-binding fragment , or a kit or kit of hIL-2/IL-2 antibody fusion protein to stimulate the immune system, which includes a general increase in immune function, an increase in T cell function, an increase in B cell function, lymphocyte Function recovery, increased expression of IL2R, increased T cell responsiveness, increased natural killer cell activity or lymphokine-activated killer cell activity, increased ratio of CD8+T cells to CD4+T cells in the subject, etc.
- the present application relates to a method of treating, alleviating or preventing a disease state in which the stimulation of the immune system of the host is beneficial, the method comprising administering to the subject a therapeutically effective amount of the anti-IL- 2 Antibodies or antigen-binding fragments thereof, or hIL-2/IL-2 antibody fusion proteins, or pharmaceutical combinations comprising the anti-IL-2 antibodies or antigen-binding fragments thereof of the application, or hIL-2/IL-2 antibody fusion proteins Drugs, or kits or kits comprising the anti-IL-2 antibody or antigen-binding fragment thereof of the present application, or hIL-2/IL-2 antibody fusion protein, to improve the disease by enhancing the cellular immune response of the subject
- said disease conditions include insufficient host immune response or immunodeficiency
- further said disease conditions are proliferative diseases or infection, immunodeficiency diseases.
- proliferative diseases include cancer and other cell proliferation disorders.
- the cancer is selected from squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal carcinoma , hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, liver tumor, breast cancer, colon cancer, Rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading melanoma, Lentigo maligna melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymph
- the method of treating, alleviating or preventing a disease condition in which stimulation of a host's immune system is beneficial further comprises administering an immune checkpoint inhibitor, an antigen-specific immunotherapeutic, a chemotherapeutic agent, an epigenetic modification agents, cytokines, growth factors, inhibitors, antibodies targeting tumor antigens, tumor vaccines, and/or other cancer therapies.
- the immune checkpoints include but are not limited to PD-1, PD-L1, PD-L2, TIM3, CTLA-4, LAG-3, CD47, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR.
- the antigen-specific immunotherapeutic agent comprises chimeric antigen receptor T cells (CAR-T cells), chimeric antigen receptor NK cells (CAR-NK cells), and/or tumor infiltrating lymphocytes cells (TIL).
- the other cancer therapy includes, but is not limited to, surgery, chemotherapy, cytotoxic agents, photodynamic therapy, immunomodulation, or radiation therapy.
- the anti-IL-2 antibody of the present application or its antigen-binding fragment, or hIL-2/IL-2 antibody fusion protein or comprise the anti-IL-2 antibody of the present application or its antigen-binding fragment, or hIL
- the pharmaceutical composition of -2/IL-2 antibody fusion protein can be combined with other therapeutic agents (for example, immune checkpoint inhibitors, antigen-specific immunotherapeutics, chemotherapeutic agents, epigenetic modifiers, cytokines, growth factors, Inhibitors, antibodies targeting tumor antigens, and/or tumor vaccines) in combination therapy and may be administered simultaneously, sequentially, or in reverse order.
- hIL-2 104th methionine residue (hIL-2/M104) side chain is slightly Long, so it may also hinder the binding of antibodies.
- hIL-2 variant containing two point mutations of K64G and N90R (the variant has his tag, named hIL2-DM-his) was constructed and expressed, and three mutations containing K64G, N90R and M104E were respectively constructed and expressed.
- a point-mutated hIL-2 variant (the variant has a his tag, named hIL2-TM-his), and wild-type mouse IL-2 (with a his tag, called mIL2-his) was used as a control group.
- each hIL-2 variant The method of construction, expression and purification of each hIL-2 variant is to obtain the gene sequence encoding wild-type hIL-2 through gene synthesis, and use PCR to perform site-directed mutations of K64G, N90R and/or M104E on hIL-2, and will contain different
- the site-directed mutation of the hIL-2 target fragment was inserted into the pcDNA3.1 expression vector, and transiently transfected into ExpiCHO-S cells (Thermo, A29127) for serum-free expression culture, and nickel column affinity chromatography was performed using the AKTA Pure system purification.
- the binding kinetics of the above three IL-2 antigens and the S4B6 chimeric antibody (chS4B6, composed of the variable region of the S4B6 antibody and the constant region of human IgG1) were detected by a molecular interaction instrument (ForteBio, model R8).
- the method is as follows: Dilute the antibody chS4B6 to be detected to 5 ⁇ g/mL with 1 ⁇ PBS solution, and add 200 ⁇ L per well into a 96-well black plate; then dilute the antigen to be detected to 100 nM with 1 ⁇ PBS solution, and make a 1:2 gradient After dilution, add 200 ⁇ L per well into the same 96-well black plate.
- Antibody Antigen K off (1/s) K on (1/Ms) K D (M) Full R 2 chS4B6:hIL2-DM-his 1.060E-02 2.691E05 3.939E-08 0.9968 chS4B6:hIL2-TM-his 4.761E-03 2.104E05 2.263E-08 0.9955 chS4B6:mIL2-his 1.823E-04 1.829E05 9.964E-10 0.9995
- CTLL-2 cell line purchased from ATCC
- NK-92 cell line purchased from ATCC
- the detection method was as follows: Centrifuge at 1000rpm to collect CTLL in logarithmic growth phase -2 cells or NK-92 cells, then resuspend the cells in complete medium without hIL-2 added, and inoculate 1 ⁇ 104 cells per well into a 96-well cell culture white plate.
- the hIL-2 variants of the samples to be tested (including hIL2-DM-his and hIL2-TM-his), and the control samples mIL2-his and hIL-2 (nearshore protein) were mixed with complete medium without hIL-2 Dilute to 500nM (for CTLL-2 cells) or 50nM (for NK-92 cells), and make a 1:5 gradient dilution; add the diluted sample to be tested at 20 ⁇ L per well into a 96-well cell culture white plate containing cells and pat to mix.
- the key site is the amino acid of the binding site between the IL-2 antigen and the IL-2 antibody
- the present application has obtained the antibody S4B6 that can bind to the mIL-2 antibody Binding hIL-2 variants that retain the biological activity of hIL-2.
- S4B6 was humanized so that it could be applied to the human body.
- CDR transplantation method to humanize the S4B6 antibody, specifically: according to the amino acid sequence of S4B6, perform CDR transplantation according to the template recommended by MOE software analysis, that is, the light chain variable region and heavy chain variable region of S4B6 and human immunoglobulin
- the gene database was compared by Blast, and the human germline (germline) IGVH and IGVK with the highest homology to the S4B6 antibody were selected as the acceptor framework for humanized transformation.
- the CDRs of the heavy chain variable region or light chain variable region of the S4B6 antibody were grafted into the selected human germline framework region to obtain the amino acid sequences of four humanized hS4B6 antibodies shown in Table 7.
- Gene synthesis encodes the sequences of each humanized antibody shown in Table 7, inserts each coding sequence into the pcDNA3.1 expression vector, transiently transfects ExpiCHO-S cells for serum-free expression culture, and uses the AKTA pure system for Protein A affinity After chromatographic purification to obtain the humanized antibodies shown in Table 7, ELISA and ForteBio kinetics were used to detect the binding activity and binding affinity of the hS4B6 humanized antibody to the antigen mIL2-his or hIL2-his, respectively.
- the ELISA detection method is as follows: Dilute goat anti-human IgG (Jackson Immunoresearch) to 1 ⁇ g/mL with 1 ⁇ PBS buffer, add 100 ⁇ L/well to the ELISA plate, and incubate overnight at 4°C. After washing the plate with PBS, a blocking solution (PBST containing 1% BSA) was added to each well, and incubated at room temperature for 1 hour. After washing the plate with PBST, the samples to be tested (including hS4B6 humanized antibody and control chimeric antibody chS4B6) at a final concentration of 13.3 nM were added to each well, and incubated at room temperature for 2 hours.
- PBST containing 1% BSA
- TMB chromogenic solution (Thermo) was added to each well, and incubated at room temperature in the dark for 3-10 minutes; after adding stop solution (2M HCl) to each well, the OD was read in a multifunctional microplate reader (Thermo, Varioskan) ( 450-570) value.
- Example 1 The method described in 1.2 of Example 1 was used to detect the binding kinetics of the hS4B6 humanized antibody and the control chimeric antibody chS4B6 to the antigen mIL2-his. The results are shown in Table 9. In the anti-IL-2 antibody after preliminary humanization, both H6K3 and H6K7 can bind to mIL-2 with high affinity, while H1K1 and H3K3 can bind to mIL-2 with moderate affinity. , indicating that all hS4B6 antibodies after preliminary humanization can specifically bind to mIL-2.
- Antibody Antigen K off (1/s) K on (1/Ms) K D (M) Full R 2 chS4B6:mIL2-his 1.823E-04 1.829E05 9.964E-10 0.9995 H1K1:mIL2-his 7.186E-04 2.769E04 2.595E-08 0.9993 H3K3:mIL2-his 2.646E-03 2.005E05 1.320E-08 0.8133 H6K3:mIL2-his 1.799E-04 1.792E05 1.004E-09 0.9993 H6K7:mIL2-his 1.212E-03 8.638E05 1.403E-09 0.9984
- the binding kinetics of the humanized antibody H6K3 to the antigen hIL-2 variant was detected using the method described in 1.2 of Example 1. The results are shown in Table 10.
- the humanized antibody H6K3 has good binding kinetics characteristics with the two hIL-2 variants, and there is no significant difference in the binding affinity between H6K3 and hIL2-DM-his and hIL2-TM-his.
- the humanized antibody H6K3 does not substantially bind to hIL-2, indicating that the antigen hIL-2 variants acquire the ability to bind to an anti-IL-2 antibody or an antigen-binding fragment thereof. ability.
- Antibody Antigen K off (1/s) K on (1/Ms) K D (M) Full R 2 H6K3:hIL2-DM-his 1.005E-02 2.220E05 4.529E-08 0.9976 H6K3:hIL2-TM-his 5.688E-03 2.372E04 2.398E-08 0.9963
- the relatively low-frequency amino acids in the light and heavy chain CDR sequences of the humanized antibody H6K3 were analyzed by the online website abYsis (http://abYsis.org/abysis/index.html) and MOE software Residues and murine amino acid residues in the FR region were analyzed, and in-depth humanization research was carried out.
- the deep humanization mutation sites are shown in Table 11.
- the deeply humanized antibodies H6K3-hu04, H6K3-hu06, H6K3-hu07, H6K3-hu08, H6K3-hu09, H6K3-hu10 and H6K3-hu12 can all bind to hIL2-DM-his, And it has good binding kinetic characteristics, indicating that these deeply humanized antibodies can specifically bind hIL-2 variant hIL2-DM.
- Antibody Antigen K off (1/s) K on (1/Ms) K D (M) Binding response value (nm) H6K3-hu01:hIL2-DM-his / / / not combined H6K3-hu02:hIL2-DM-his / / / not combined H6K3-hu03:hIL2-DM-his / / / not combined H6K3-hu04:hIL2-DM-his 1.486E-02 4.298E05 3.457E-08 0.1429 H6K3-hu05:hIL2-DM-his 1.525E-02 3.874E05 3.937E-08 0.1387 H6K3-hu06:hIL2-DM-his 1.917E-02 3.647E05 5.255E-08 0.1345 H6K3-hu07:hIL2-DM-his 1.811E-02 4.001E05 4.528E-08 0.1296 H6K3-hu08:hIL2-DM-his 2.068
- the mutation combinations of H6K3-hu04 (mutation site L34M), H6K3-hu07 (mutation site N65G) and H6K3-hu08 (mutation site N76D) were further selected as deeply humanized antibody molecules
- the heavy chain variable region, H6K3-hu09 (the mutation site is D56S) and H6K3-hu12 (the mutation site is Y71F) are combined into the light chain variable region, and the humanized antibody containing these mutation combinations is numbered hS4B6- H6K3(H), this humanized antibody can also significantly reduce the potential risk of aspartic acid isomerization during the production process.
- Example 13 After constructing, expressing and purifying the hS4B6-H6K3(H) antibody sample according to the method described in 2.1 in Example 2, use the method described in 1.2 in Example 1 to detect hS4B6-H6K3(H) and antigens hIL2-DM-his, hIL2- Binding kinetics of TM-his, or control mIL2-his.
- the test results are shown in Table 13.
- the hS4B6-H6K3(H) antibody not only has a higher degree of humanization, but also maintains the biological characteristics of highly specific binding to hIL-2 variants.
- Antibody Antigen K off (1/s) K on (1/Ms) K D (M) Full R 2 hS4B6-H6K3(H):mIL2-his 2.359E-04 9.277E05 2.543E-10 0.9998 hS4B6-H6K3(H):hIL2-DM-his 3.843E-03 2.421E04 1.587E-08 0.9971
- the heavy chain of antibody hS4B6-H6K3(H) was simulated and calculated by MOE software based on the resolved crystal structure of the S4B6:mIL2 antibody-antigen complex CDR regions and sites for site-directed mutagenesis in the light chain CDR region.
- hS4B6-H6K3 (H) light chain CDR region mutation D28Y, or D28Y/S50Q, or D28Y/G30S, or D28Y/G30S/S50Q, or its combination with the heavy chain CDR region mutation the resulting affinity All mature antibodies have significantly improved binding affinity to hIL-2 variants, and the binding affinity (equilibrium dissociation constant K D ) of hS4B6-H6K3(H) affinity matured antibody to hIL-2 variants can reach 10 -9 M, even 10 -10 M.
- the binding of hIL2-DM to IL2R ⁇ and IL2R ⁇ after forming a complex with antibody K2G11 was detected by a molecular interaction instrument (ForteBio, model Qke).
- the specific method is as follows: Dilute the antibody K2G11 to be detected to 5 ⁇ g with 1 ⁇ PBS solution /mL, and added 200 ⁇ L per well into the 96-well black plate, then diluted the antigen hIL2-DM to 100 nM with 1 ⁇ PBS solution, and added 200 ⁇ L per well into the above-mentioned 96-well black plate.
- the detection results are shown in FIG. 3 , indicating that the antibody K2G11 can block the binding of hIL2-DM to hIL2R ⁇ ( FIG. 3A ), but does not affect its binding to hIL2R ⁇ ( FIG. 3B ).
- the S4B6/mIL-2 antigen was calculated by MOE software -The distance between each amino acid residue in the crystal structure of the antibody complex, and select some amino acid residues within the range of 10 Angstroms to mutate into cysteine, so that the hIL-2 variant is compatible with the hS4B6-H6K3(H) antibody
- the combination is further stabilized through the formation of disulfide bonds.
- Table 15 shows the amino acid mutation sites at the disulfide bond formation sites between the hIL-2 variants and the hS4B6-H6K3(H) antibody. The structure of the construct containing disulfide bonds is shown in Figure 4.
- IL means IL-2
- L means antibody light chain
- H means antibody heavy chain
- the number after IL or L/H means the amino acid position corresponding to the mutation point
- IC-Cy08 Compared with the control hIL2-DM-Fc, IC-Cy08 hardly binds to hCD25, while IC-Cy07 only has a slightly reduced hCD25 binding activity; the binding activity to hCD122 is as follows: As shown in Figure 5B, compared with the control group hIL2-DM-Fc, the binding activity of IC-Cy08 and IC-Cy07 to hCD122 has been enhanced to a certain extent. This result is consistent with the literature report, that is, mIL-2 The C-helix of the protein will be allosteric to enhance the binding to mCD122. In addition, the binding activity of IC-Cy08 to hCD122 was significantly higher than that of IC-Cy07 and hIL2-DM-Fc.
- both IC-Cy08 and IC-Cy07 have enhanced binding activity to hCD122, and IC-Cy08 has almost completely lost the ability to bind to hCD25, which further indicates that the hIL-2 variant ⁇ in IC-Cy08
- the receptor binding site is completely buried inside the antibody fusion protein molecule and is not easy to dissociate, so it cannot bind to hCD25.
- the biological activities of IC-Cy07 and IC-Cy08 were detected by CTLL-2 cell line and NK-92 cell line proliferation experiments, and the control group was wild-type hIL-2.
- the activity of IC-Cy08 to promote the proliferation of CTLL-2 cells with high CD25 expression was significantly lower than that of hIL-2 in the control group ( Figure 6A and Table 16), while promoting the proliferation of NK cells with low CD25 expression
- the proliferative activity of -92 cells was equivalent to that of the control group hIL-2 (Fig.
- the combination of IL-2 and IL2R can induce the activation of intracellular JAK kinases, and then phosphorylate and activate signal transduction and activator of transcription 5 (STAT5), and finally regulate the processes involved in cells (such as proliferation, survival, etc.) and apoptosis) expression of multiple genes.
- STAT5 signal transduction and activator of transcription 5
- the activation of the IL-2/IL-2 antibody fusion protein IC-Cy08 containing a disulfide bond on the IL-2 signaling pathway was evaluated by detecting the phosphorylation of STAT5 in the CTLL2 cell line expressing high-affinity IL2R.
- the specific method was as follows: centrifugation Collect CTLL-2 cells, then resuspend the cells with complete medium and place them in a 37°C/5% CO 2 incubator for static starvation culture for 4 hours. Collect the cells by centrifugation and resuspend the cells with complete medium again, spread 5 ⁇ 105 cells per well into a 96-well cell culture U-shaped plate; use the complete medium to serially dilute the sample to be tested at a ratio of 1:5 (IC -Cy08 and wild-type hIL-2 of the control group), and added to the above-mentioned cell culture plate at a volume of 50 ⁇ L per well, and then placed the cell culture plate in a 37°C/5% CO 2 incubator for static culture for 15-30 minutes .
- CTLL-2 cells showed a significantly reduced pSTAT5 level under the action of IC-Cy08, which further indicated that the complex containing disulfide bonds was constructed
- the ⁇ -receptor binding site of the hIL-2 variant in the body IC-Cy08 molecule was stably bound to the antibody part and not exposed to the molecular surface, thus significantly reducing the binding to the high-affinity IL2R on the surface of CTLL-2 cells, thereby down-regulating Transduction of the intracellular IL-2 signaling pathway.
- mutants IC-Cy08-QS, IC-Cy08-QG, IC-Cy08-QK, and IC-Cy08-QR exhibited somewhat weakened hCD122-binding activity relative to the parent IC-Cy08, and had similar hIL2 binding activity to the control group.
- - DM-Fc equivalent hCD122 binding activity the other three mutants IC-Cy08-DE, IC-Cy08-QD and IC-Cy08-QE have equivalent hCD122 binding activity to the parent IC-Cy08, and have higher hIL2-DM-Fc increased hCD122 binding activity (Fig. 8B).
- the biological activity of the IC-Cy08 mutant was detected by CTLL-2 cell line and NK-92 cell line proliferation experiments, and the control group was wild-type hIL-2.
- the test results are shown in Figure 9, and the seven IC-Cy08 mutants all retained the proliferation-promoting effect of the parental IC-Cy08 on CTLL-2 cells ( Figure 9A and Table 19) and NK-92 cells ( Figure 9B and Table 19) activity, and compared with the control group hIL-2, the seven IC-Cy08 mutants and the parental IC-Cy08 all had significantly reduced CTLL-2 cell proliferation-promoting activity (Fig. 9A and Table 19) and comparable NK- 92 cell proliferation activity (Fig. 9B and Table 19).
- CTLL-2 and NK-92 cell proliferation assays to detect the biological activity of the IL-2/IL-2 antibody fusion protein IC-Cy08 mutant containing disulfide bonds optimized by the light chain of the antibody
- sample CTLL-2 cell proliferation activity (EC50,nM) NK-92 cell proliferation activity (EC50,nM) hIL-2 0.1469 0.01572 IC-Cy08 3.352 0.02635 IC-Cy08-DE 3.36 0.02332 IC-Cy08-QD 3.621 0.02743 IC-Cy08-QE 3.847 0.02612 IC-Cy08-QS 4.64 0.03701 IC-Cy08-QG 3.991 0.0301 IC-Cy08-QK 3.518 0.02691 IC-Cy08-QR 3.061 0.0247
- IC-Cy08-QS, IC-Cy08-QG, IC-Cy08-QK and IC-Cy08-QR exhibited attenuated hCD122-binding activity relative to parental IC-Cy08, the proliferation of CTLL-2 cells and NK cells The activity is equivalent to that of IC-Cy08, that is, it does not reduce the proliferation activity of CTLL-2 cells and NK cells. This shows that the 27th Gln of the antibody light chain in the IC-Cy08 molecule will not significantly affect the binding activity and functional activity of hIL2 and hCD122.
- mutant number hIL-2 N-terminal first three amino acids APT Amino acid residue 125 of hIL-2 IC-Cy08( ⁇ 3,WT) Delete APT C IC-Cy08( ⁇ 3,C125A) Delete APT A IC-Cy08( ⁇ 3,C125L) Delete APT L IC-Cy08( ⁇ 3,C125S) Delete APT S IC-Cy08( ⁇ 3,C125Q) Delete APT Q IC-Cy08( ⁇ 3,C125V) Delete APT V
- the IC-Cy08 mutants in Table 21 were expressed and purified according to the method described in 2.1 of Example 2, and their proliferative activities on CTLL-2 and NK-92 cells were detected according to the method described in 1.3 of Example 1.
- the experimental results are shown in Figure 10.
- the proliferative activity of each IC-Cy08 mutant on CTLL-2 cells ( Figure 10A and Table 22) and NK-92 cells ( Figure 10B and Table 22) was comparable to that of the parental IC-Cy08, and was comparable to that of the parent IC-Cy08.
- it had significantly decreased proliferation activity of CTLL-2 cells ( FIG. 10A and Table 22 ) and slightly decreased proliferation activity of NK-92 cells ( FIG. 10B and Table 22 ). This shows that by deleting the first three amino acids at the N-terminal of hIL-2 and mutating the 125th cysteine, the potential risk in the production and preparation of the molecule can be solved without significantly affecting the activity.
- CTLL-2 and NK-92 cell proliferation assays detect the biological activity of hIL-2/IL-2 antibody fusion protein IC-Cy08 mutants containing disulfide bonds optimized by hIL-2 amino acid sequence
- sample CTLL-2 cell proliferation activity (EC50,nM) NK-92 cell proliferation activity (EC50,nM) hIL-2 0.1956 0.008233 IC-Cy08 2.332 0.01564 IC-Cy08 ( ⁇ 3, WT) 2.140 0.02636 IC-Cy08 ( ⁇ 3, C125A) 2.270 0.02469 IC-Cy08 ( ⁇ 3, C125L) 3.158 0.02812 IC-Cy08 ( ⁇ 3, C125S) 3.260 0.02886 IC-Cy08 ( ⁇ 3, C125Q) 4.425 0.03354 IC-Cy08 ( ⁇ 3, C125V) 2.407 0.02878
- the antibody part of the molecule was restructured.
- the chain constant region was engineered.
- N203D/K274Q/Q419E (EU numbering) mutation (defined as "pI mutation") was introduced into the constant region of the heavy chain hIgG1 of the antibody part of IC-Cy08.
- the L234F/L235E/P331S (EU numbering) mutation (defined as "TM mutation") was introduced into the Fc region. ”) to reduce the binding of the Fc region to Fc ⁇ Rs and C1q, thereby eliminating Fc-mediated effector functions.
- the IL-2/IL-2 antibody fusion protein IC-Cy08-pITM comprising the heavy chain constant region of the above-mentioned antibody part after pI mutation and TM mutation was expressed and purified, and according to the method in Example 1,
- the method described in 1.3 detects its proliferative activity on CTLL-2 and NK-92 cells respectively.
- the experimental results are shown in Figure 11, IC-Cy08-pITM has almost the same proliferation-promoting activity of CTLL-2 ( Figure 11A and Table 23) and NK-92 ( Figure 11B and Table 23) cells as the parental IC-Cy08.
- Table 23 CTLL-2 and NK-92 cell proliferation detection on the biological activity of hIL-2/IL-2 antibody fusion protein IC-Cy08-pITM after pI mutation and TM mutation of the heavy chain constant region of the antibody
- Example 6 hIL-2/IL-2 antibody fusion protein constructed from hS4B6-H6K3(H) affinity matured antibody and detection of its biological activity
- the antigen-antibody fusion protein of hIL2-DM ( ⁇ 3, C125L) and K2G11 was constructed according to the method described in Example 4, in order to Candidate molecules with more stable structures are obtained.
- (G4S) 4 was selected as the linker for the construction of the fusion protein, and connected to the N-terminus of the K2G11 antibody heavy chain through the C-terminus of hIL2-DM ( ⁇ 3, C125L).
- the amino acid mutation sites of the disulfide bond formation site between hIL2-DM ( ⁇ 3, C125L) and the K2G11 antibody are shown in Table 24 to further evaluate the possibility of correct disulfide bond formation.
- the structure of the hIL-2/IL-2 antibody fusion protein containing a disulfide bond is shown in Figure 4.
- IL means IL-2
- L means antibody light chain
- H means antibody heavy chain
- the number after IL or L/H means the amino acid position corresponding to the mutation point
- Table 24 The constructs in Table 24 were constructed, expressed and purified according to the method described in 2.1 of Example 2, and analyzed by SEC-HPLC to select hIL-2/IL-2 antibody fusion proteins K2G11-Cy04 and K2G11 capable of forming stable monomers -Cy05 and K2G11-Cy06 for further research, and hIL-2/IL-2 antibody fusion protein IC-Cy08(H6K3(H)-Cy08) and H6K3( H)-Cy10 served as a control.
- K2G11-Cy04, K2G11-Cy05, K2G11-Cy06 and H6K3(H)-Cy10 were almost completely incompatible with hCD25 H6K3(H)-Cy08 showed a small hCD25 binding activity at high concentrations; the binding activity to hCD122 was shown in Figure 12B, and each sample showed no significant difference in binding activity.
- K2G11-Cy04, K2G11-Cy05, K2G11-Cy06, and H6K3(H)-Cy10 were able to completely inhibit the binding of hIL-2 variants to hCD25 even at high concentrations compared with H6K3(H)-Cy08. It is also further indicated that the ⁇ subunit receptor binding site of IL-2 in them is more stably embedded in the antibody fusion protein molecule and will not be competitively bound by hCD25.
- Example 13 the biological activity of the hIL2/IL2 antibody fusion protein based on the K2G11 antibody and hIL2-DM ( ⁇ 3, 125L) was detected by CTLL-2 cell line and NK-92 cell line proliferation experiments. The detection results are shown in Figure 13.
- the hIL-2 variant hIL2-DM ( ⁇ 3, C125L), and the pre-incubated K2G11 antibody and hIL2-DM ( ⁇ 3, 125L), K2G11-Cy04, K2G11-Cy05, K2G11-Cy06 and H6K3(H)-Cy10 exhibited in promoting the proliferation activity (EC50 value) of CTLL-2 cells with high expression of CD25 significantly lower than that of H6K3(H)-Cy08 (Fig.
- H)-Cy10 has no cell proliferation activity compared to the control hIL-2, hIL-2 variant hIL2-DM ( ⁇ 3, C125L) and K2G11:hIL2-DM ( ⁇ 3, 125L) antigen-antibody complex Significant difference (Fig. 13B, Table 25), the results indicate that the ⁇ subunit receptor binding site of IL-2 in K2G11-Cy04, K2G11-Cy05, K2G11-Cy06 and H6K3(H)-Cy10 molecules is due to the disulfide bond The formation is more stably embedded in the molecule, and will not be competitively bound by the high-affinity IL2R.
- Table 25 CTLL-2 and NK-92 cell proliferation assay detection of biological activity based on K2G11 antibody containing disulfide bond IL-2/IL-2 antibody fusion protein
- Example 7 ForteBio detects the binding of hIL-2/IL-2 antibody fusion protein constructed based on K2G11 to hIL2R ⁇ , hIL2R ⁇ / ⁇ and hIL2R ⁇ / ⁇ / ⁇
- the hIL-2/IL-2 antibody fusion protein constructed based on K2G11 and hIL2-DM ( ⁇ 3, 125L) and IL2R ⁇ , IL2R ⁇ / ⁇ and IL2R ⁇ / ⁇ / ⁇ were detected by a molecular interaction instrument (ForteBio, model Qke).
- Binding detection the specific method is as follows: Dilute Biotin-CD25 (Acro) to 5 ⁇ g/mL with 1 ⁇ PBS solution, and add 200 ⁇ L per well into a 96-well black plate; then dilute hIL2R ⁇ (Acro) with 1 ⁇ PBS solution to 5 ⁇ g/mL, and add 200 ⁇ L per well into the above-mentioned 96-well black plate; then dilute the sample hIL2-DM ( ⁇ 3, C125L) and K2G11 with 1 ⁇ PBS, 5 ⁇ g/mL hIL2R ⁇ (Acro) and 5 ⁇ g/mL hIL2R ⁇ (Acro) -Cy05 to 100 nM, and added to the above 96-well black plate at 200 ⁇ L per well.
- Example 8 Comparison of the binding of hIL-2/IL-2 antibody fusion protein K2G11-Cy05 and NARA1leukin to hCD25 by ELISA
- the test results are shown in Figure 15. With the increase of the antibody concentration, NARA1leukin gradually showed the binding to hCD25, while the hIL-2/IL-2 antibody fusion protein K2G11-Cy05 of the present application did not show any binding to hCD25 within the detection range.
- the binding of hCD25 indicates that the hIL-2/IL-2 antibody fusion protein constructed by the disulfide bond covalent connection method used in this application, the binding between the hIL-2 variants inside the molecule and the anti-IL-2 antibody It is more stable and can prevent the competitive combination of hCD25 and the IL-2 component in the fusion protein to a greater extent.
- Example 9 Using reporter gene method and cell proliferation method to detect the biological activity of the hIL-2/IL-2 antibody fusion protein of the present application
- the hIL-2/IL-2 antibody fusion protein K2G11-Cy05 prepared in Example 6 was used to detect the activation of IL-2 downstream signaling pathway in high-expression CD25 cell lines by reporter gene method, and NARA1leukin protein was used as a control molecule.
- the specific method is as follows: IL-2 Bioassay cells (Promega) were collected by centrifugation, resuspended to 8 ⁇ 10 5 cells/mL with culture medium, then added to a 96-well white plate, and incubated overnight at 37°C/5% CO 2 .
- K2G11-Cy05 and NARA1leukin were serially diluted with culture medium, and wild-type hIL-2 was used as a positive control; the diluted samples were added to the above-mentioned 96-well white plate and incubated at 37°C/5% CO 2 for 6 hours.
- the 96-well plate was placed at room temperature to equilibrate for 30 minutes, then the detection reagent One-Lite Luciferase agent (Nanjing Nuoweizan) was added, and the chemiluminescence value was read with a multifunctional microplate reader.
- NARA1leukin showed a certain activity of activating the downstream signaling pathway of IL-2 at high concentrations, while the hIL-2/IL-2 antibody fusion protein of the application K2G11-Cy05 did not show obvious activity of activating signaling pathways within the detection range.
- the biological activities of hIL2/IL2 antibody fusion protein K2G11-Cy05 and NARA1leukin were detected by proliferation experiments of CTLL-2 cell line and CD25 knockout NK-92 (CD25-KO) cell line.
- K2G11-Cy05 showed a significant reduction in the pro-proliferation activity of CTLL-2 cells with high expression of CD25, and the reduction was significantly greater than that of NARA1leukin; while In CD25-knockout NK-92 (CD25-KO) cells, K2G11-Cy05 and NARA1leukin showed no significant difference in pro-cell proliferation activity relative to hIL-2.
- Example 10 Detection of the thermal stability of the anti-IL-2 antibody comprising pI mutation and TM mutation in the heavy chain constant region of the antibody and the hIL-2/IL-2 antibody fusion protein constructed based on this antibody
- Humanized antibody K2G11; hIL-2/IL-2 antibody fusion protein is prepared according to the method described in Example 6.
- the heavy chain constant region of the antibody part contains pI mutation and TM mutation K2G11-Cy04, K2G11-Cy05 and K2G11-Cy06.
- the specific detection method is as follows: Dilute each sample to 1 mg/mL with 1 ⁇ PBS and centrifuge at 4°C for 15 minutes, then use a capillary detection tube to absorb the supernatant of the sample and place it on the detection platform, and perform 2 repeated tube detection for each sample . Set up the detection program, set the temperature range as 25 to 95°C; run the program and record the data. After the program finishes running, analyze the data with analysis software.
- the results of testing the stability of the hIL-2/IL-2 antibody fusion protein are shown in Table 28.
- the heavy chain constant region of the antibody part contains pI mutation and TM mutation hIL-2/IL-2 antibody fusion protein K2G11-Cy04, K2G11 -The T m1 value of Cy05 and K2G11-Cy06 is about 65°C, which is similar to that of the anti-IL-2 humanized antibody K2G11-pITM, both of which are the denaturation temperature of the CH2 domain of the heavy chain constant region of the antibody; reflecting the three hIL-
- the T m2 values of the binding stability of the Fab region of the antibody part of the 2/IL-2 antibody fusion protein sample to the hIL-2 variant (hIL2-DM( ⁇ 3, C125L)) were all above 80°C, and the T turbility of the entire molecule
- the value of K2G11-pITM also showed a significant increase, indicating that the cysteine residue substitutions introduced in the anti
- Table 28 The thermal stability of hIL-2/IL-2 antibody fusion protein comprising pI mutation and TM mutation in the heavy chain constant region of the antibody
- Example 11 Anti-IL-2 humanized antibody containing pI mutation and TM mutation in the heavy chain constant region and detection of binding kinetics of hIL-2/IL-2 antibody fusion protein constructed based on it to Fc receptor and FcRn
- Example 1 The method described in 1.2 of Example 1 was used to detect the anti-IL-2 humanized antibody K2G11-pITM containing the pI mutation and TM mutation in the heavy chain constant region and the hIL-2/IL-2 antibody fusion protein K2G11-pITM constructed based on it. Binding kinetics of Cy05 and control antibody K2G11 to Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIb, Fc ⁇ RIIIa-158F, Fc ⁇ RIIIa-158V, FcRn, C1q and FcRn.
- Example 12 Detection of ADCC activity of hIL-2/hIL-2 antibody fusion protein by reporter gene method
- this application uses Jurkat cells (Promega) expressing Fc ⁇ RIIIa-158V and NFAT response elements to regulate the expression of firefly luciferase as effector cells.
- NK-92 cells expressing IL2R were used as target cells, and the ADCC activity of K2G11-Cy05 containing a TM mutation in the heavy chain constant region of the antibody prepared in Example 6 was detected, and the anti-EGFR antibodies cetuximab and A431 Cells were the positive control group.
- NK-92 cells and A431 cells were collected by centrifugation, resuspended with ADCC detection buffer (RPMI-1640 containing 0.5% FBS), and added to 96-well white plate at 1.5 ⁇ 10 4 /well.
- the sample to be tested was diluted in gradient with ADCC detection buffer, and added to the above-mentioned 96-well plate at 25 ⁇ L/well.
- the effector cells Jurkat cells
- 1.5 ⁇ 10 5 /well was added to the above-mentioned 96-well plate, and then placed in a 37°C/5% CO 2 incubator for overnight incubation.
- the 96-well plate was placed at room temperature to equilibrate, and then the detection reagent One-Lite Luciferase agent (Nanjing Nuoweizan) was added, and the chemiluminescence value was read using a multifunctional microplate reader.
- One-Lite Luciferase agent Najing Nuoweizan
- test results are shown in Figure 18.
- K2G11-Cy05 did not significantly stimulate the expression of luciferase in the effector cells even at the highest concentration, indicating that in the antibody part of the heavy chain
- the introduction of TM mutations into the constant region can significantly reduce the affinity between the hIL-2/IL-2 antibody fusion protein and the Fc receptor, thereby significantly reducing the ADCC activity mediated by the hIL-2/IL-2 antibody fusion protein Fc.
- Example 13 Stability of hIL-2/IL-2 antibody fusion protein in cynomolgus monkey and healthy human serum respectively
- NK-92 cells detected the changes in the biological activities of K2G11-Cy04 and K2G11-Cy05 after being incubated with cynomolgus monkey and human serum respectively
- the specific method is as follows: the hIL-2/IL-2 antibody fusion proteins K2G11-Cy04 and K2G11-Cy05 were diluted respectively in two cynomolgus monkeys (one female and one male, M6 and M7) and two healthy people (H2 and H3).
- the final concentration was 100 ⁇ g/mL, mixed and distributed into centrifuge tubes, and prepared as samples M6-Cy04, M7-Cy04, M6-Cy05 and M7-Cy05 treated with cynomolgus monkey serum, and samples treated with healthy human Serum-treated samples H2-Cy04, H3-Cy04, H2-Cy05, and H3-Cy05 were placed in a 37°C incubator for static incubation, and the time point at which the incubation started was defined as day 0 (0 hour).
- Samples were taken at designated time points and stored at -80°C for testing; samples treated with cynomolgus monkey serum were sampled at the 3rd day (72 hours), the 7th day (168 hours) and the 14th day (336 hours) hours); the sampling time points of samples treated with healthy human serum were the 7th day (168 hours), the 14th day (336 hours) and the 21st day (504 hours).
- NK-92 cells to detect the biological activity of each sample according to the method described in Example 1, and set the hIL-2/IL-2 antibody fusion protein samples K2G11-Cy04 and K2G11 without serum treatment -Cy05, and hIL-2 as a control group.
- Example 14 Detection of disulfide bonds in the hIL-2/IL-2 antibody fusion protein of the present application
- the hIL-2 variants and anti-IL-2 human in the hIL-2/IL-2 antibody fusion proteins K2G11-Cy04 and K2G11-Cy05 prepared in Example 6 were detected by liquid chromatography-mass spectrometry (LC-MS). Whether the correct paired disulfide bond is formed between the derivatized antibody K2G11.
- the specific method is as follows.
- Disulfide bond detection in K2G11-Cy04 The sample was denatured with guanidine hydrochloride, alkylated with N-ethylmaleimide (NEM), desalted after changing the medium, and digested with Trypsin (Promega, V5113). TFA (Thermo, 28904) was added to stop the reaction after DTT reduction of a part of the digested sample, and TFA was directly added to the other part of the digested sample to terminate the reaction, and the final sample was obtained for LC-MS detection. The data were analyzed by UNIFI software to obtain the measured disulfide bond connection data.
- Disulfide bond detection in K2G11-Cy05 The sample was denatured with guanidine hydrochloride, alkylated with N-ethylmaleimide (NEM), and digested with Trypsin after desalting in the medium. The samples digested with Trypsin were equally divided into Two copies were added, one was supplemented with Chymotrypsin (Promega, V106A) to continue digestion, one was supplemented with GluC (Promega, V165A) to continue digestion, after all digestion was completed, TFA was directly added to stop, and the final sample was obtained for LC-MS detection. The data were analyzed by UNIFI software to obtain the measured disulfide bond connection data.
- Example 15 Pharmacokinetic detection of anti-IL-2 antibody with pI mutation and TM mutation in the heavy chain constant region
- mice Blood was collected from the administered mice at 0.25, 1, 3, 8, 24, 48, 96, 168, 240, 336, 408 and 504 hours after the end of the administration; the supernatant plasma was collected after centrifugation at 4°C and stored at -80 °C, after all the blood samples are collected, they will be tested uniformly.
- the drug concentration in mouse plasma was detected by ELISA method, and the specific steps were as follows: Dilute goat anti-human IgG Fc fragment antibody (Jackson Immunoresearch) to 1 ⁇ g/mL with PBS, coat 96-well ELISA plate with 50 ⁇ L/well, and store at 4°C. Let stand overnight.
- 1% BSA was added to each well for blocking, and the plate was allowed to stand at room temperature for 1 hour.
- Add the diluted test sample and standard sample to the above ELISA plate, 50 ⁇ L per well, and incubate at room temperature for 2 hours.
- PBST PBS, pH7.4; 0.1% Tween-20
- HRP-labeled goat anti-human IgG F(ab)2 fragment secondary antibody Jackson Immunoresearch
- TMB chromogenic solution After washing the plate with PBST, add TMB chromogenic solution to each well, incubate at room temperature in the dark for 3-10 minutes, and then add 2M HCl to terminate the reaction.
- the OD450 and OD570 absorbance values of each well were read by a multifunctional microplate reader (Thermo Varioskan).
- the OD(450/570) value of the sample to be tested is within the quantitative range of the standard curve, then substitute and calculate the drug concentration in the plasma sample; if it is not within the quantitative range, adjust the dilution ratio of the plasma sample according to the reading value and perform the ELISA again detection. Substitute the OD (450-570) value of the sample to be tested into the standard curve, calculate the calculated concentration according to the dilution ratio, and make a drug-time curve according to the sampling time.
- the pharmacokinetic analysis was performed using the PKSolver 2.0 plug-in in Microsoft EXCEL software, and the calculation of pharmacokinetic parameters was obtained using a non-compartmental model for intravenous administration.
- the drug-time curve is shown in Figure 20.
- the anti-IL-2 antibody K2G11-pITM has significantly improved pharmacokinetic characteristics compared with the K2G11 antibody that has not introduced the pI mutation in the heavy chain constant region, and the dosage is 1 mg/kg
- the elimination half-life (t 1/2 ) and area under the drug-time curve (AUC) of the K2G11-pITM group were about 2.5 times and 1.3 times that of the K2G11 group at the same dose, respectively; the half-life and The AUCs were about 3.3 times and 1.4 times that of the K2G11 group at the same dose, respectively.
- Example 16 In vivo drug efficacy evaluation of hIL-2/hIL-2 antibody fusion protein
- the number of CD8 + T cells, NK cells and Treg cells and the proliferation marker Ki-67 were measured in the peripheral blood of cynomolgus monkeys receiving the hIL-2/hIL-2 antibody fusion protein K2G11-Cy05 prepared in Example 6 as follows
- Flow cytometry detection and analysis method add 5 ⁇ L Human TruStain FcX (Biolegend) to each flow tube, then add 100 ⁇ L whole blood containing anticoagulant to each flow tube, and incubate at room temperature for 10 minutes. Take surface staining flow antibody BV510 anti-human CD3 (BD), FITC anti-human CD4 (Biolegend), APC anti-human CD25 (Biolegend), AF700anti-human CD8 (Biolegend) and PE-Cy7anti-human CD16 (Biolegend) After mixing, add to flow tube, mix well and incubate on ice for 60min.
- BD flow antibody
- FITC anti-human CD4 Biolegend
- APC anti-human CD25 Biolegend
- AF700anti-human CD8 Biolegend
- PE-Cy7anti-human CD16 Biolegend
- Each tube was resuspended with True-Nuclear 1 ⁇ Perm buffer, PE anti-human Foxp3 (Biolegend) and eFluor 450 anti-human Ki-67 (Thermo) flow antibodies were added, and incubated at room temperature for 40 minutes in the dark.
- Cells in each tube were washed with FACS buffer, resuspended in FACS buffer, and then placed in a flow cytometer (Agilent, NovoCyte 3005) for detection.
- K2G11-Cy05 can significantly activate and increase the proliferation of CD8 + T cells (Figure 21A) and NK cells (Figure 21B) in the peripheral blood of cynomolgus monkeys, while the number of Treg cells is only at medium and high doses There was a slight transient increase under drug administration, but the expression of Ki-67 remained basically unchanged ( FIG. 21C ), which may be caused by the feedback regulation of endogenous IL-2. It can be seen that K2G11-Cy05 can selectively act on CD8 + T cells and NK cells expressing IL2R ⁇ / ⁇ in cynomolgus monkeys and promote their proliferation without significantly activating Treg cells through IL2R ⁇ / ⁇ / ⁇ . During the experiment, the body weight of the tested animals did not change significantly, and no severe adverse reactions in clinical symptoms were observed.
- Memory CD8 + T cells, NK cells, Treg cells, conventional CD4 + The number of T cells, and the ratio of memory CD8 + T cells to Treg cells and the ratio of NK cells to Treg cells were calculated, and the weight changes of each experimental mouse were counted.
- the specific method is as follows.
- Test grouping and administration method Divide C57BL/6 mice into 8 groups (5 mice in each group), specifically a negative control group: PBS; a positive control group: hIL-2 (75 ⁇ g/kg) ; Three groups of experimental control groups: hIL-2-DM ( ⁇ 3, C125L) and anti-IL-2 antibody K2G11-pITM at a molar ratio of 2:1 formed hIL-2-DM ( ⁇ 3, C125L): K2G11-pITM immunocomplex [the dose of hIL-2-DM ( ⁇ 3, C125L) is 1.5 ⁇ g/kg, 5 ⁇ g/kg and 15 ⁇ g/kg]; and three experimental groups: K2G11-Cy05 (10 ⁇ g/kg, 30 ⁇ g/kg and 100 ⁇ g/kg kg).
- PBS group and hIL-2 group were administered via tail vein once a day, a total of 4 administrations; experimental control group hIL-2-DM( ⁇ 3,C125L):K2G11-pITM immune complex and experimental group K2G11-Cy05 every two Administered once a day through the tail vein, a total of 2 administrations.
- the mice in each group were weighed every other day.
- the mice in all groups were euthanized on the second day after the last administration, and spleen cells were extracted for flow cytometry analysis.
- Flow cytometry detection and analysis method After the mouse spleen was weighed, it was washed with pre-cooled PBS and placed on a 200-mesh (75 ⁇ m) cell mesh (15-1070-ZX, Biologix) for grinding to prepare spleen single cells Suspension. After collecting cells by centrifugation, add 1 ⁇ RBC buffer (Biolegend) to each tube, shake gently and let stand at room temperature for 5 minutes, then add pre-cooled PBS to terminate the reaction, collect cells by centrifugation at 4°C and wash once with pre-cooled PBS, and resuspend the cells In pre-cooled PBS, and added to a 96-well U-shaped deep-well plate, each sample added a duplicate well.
- 1 ⁇ RBC buffer Biolegend
- the experimental data was statistically analyzed by GraphPad Prism 9 software.
- the data difference between the experimental groups was subjected to one-way analysis of variance (ANOVA) and then the difference between each experimental group and the solvent control group was tested by Dunnett. If the p value was less than 0.05, then Indicates a statistically significant difference between the two.
- hIL-2-DM( ⁇ 3,C125L):K2G11-pITM immune complex administration group compared with hIL-2 administration group, the immune complex only showed a certain degree of selectivity at high dose (15 ⁇ g/kg) NK cells were activated (p ⁇ 0.05), while the absolute counts of memory CD8 + T cells and Treg cells had no significant difference; the absolute counts of memory CD8 + T cells, NK cells and Treg cells in mice in the other two dose groups were significantly different from hIL There was no statistical difference compared with the -2 administration group.
- hIL-2-DM( ⁇ 3,C125L) K2G11-pITM immune complex dosage groups were compared with K2G11-Cy05 dosage groups, although the dosage of hIL-2 was equivalent, but the latter was more effective in inducing memory CD8 + The effect of T cell and NK cell proliferation is significantly better than the former. There was no significant change in the number of conventional CD4 + T cells in the mice of each group ( Figure 22C), and the mice in each group did not die during the experiment, and no adverse reactions in clinical symptoms were observed, and the body weight did not change. Significantly ( FIG. 22D ), thus showing that K2G11-Cy05 has good safety in vivo.
- 5 ⁇ 10 5 CT26 colon cancer cells were subcutaneously injected into Balb/C mice, and when the average tumor volume grew to about 100 mm 3 was recorded as the first day, and the tumor-bearing mice were divided into groups, including the control group PBS and NARA1leukin (0.3mg/kg), and the test group K2G11-Cy05 (0.03mg/kg, 0.1mg/kg and 0.3mg/kg), a total of 5 groups, 8 mice in each group.
- groups including the control group PBS and NARA1leukin (0.3mg/kg), and the test group K2G11-Cy05 (0.03mg/kg, 0.1mg/kg and 0.3mg/kg), a total of 5 groups, 8 mice in each group.
- Each dose group of K2G11-Cy05 has a dose-dependent inhibitory effect on tumor growth in tumor-bearing mice, and the tumor volume of mice in the K2G11-Cy05 dose 0.3 mg/kg group is significantly smaller than that of the PBS control Group.
- K2G11-Cy05 had a better inhibitory effect on tumor growth than NARA1leukin. This shows that the hIL-2/hIL-2 antibody fusion protein of the present application can selectively activate and promote the proliferation of memory CD8 + T cells and NK cells in vivo, thereby exerting a good anti-tumor effect.
- Example 17 Construction of a stable hIL-2/hIL-2 antibody fusion protein by linking other hIL-2 antibodies with hIL-2 through a disulfide bond
- hIL-2/NARA1 antibody fusion protein and hIL-2/TCB2 antibody fusion protein were respectively constructed.
- hIL-2 was connected to NARA1 or TCB2 light chain through four tandem G4S sequences The N-terminus can ensure the stability of the internal structure of the constructed fusion protein, and the fusion protein has little effect on the combination of hIL-2 and hIL2R ⁇ .
- IL means IL-2
- L means antibody light chain
- H means antibody heavy chain
- the number after IL or L/H means the amino acid position corresponding to the mutation point
- serial number amino acid residue position hIL2 sequence TCB2 sequence TCB2 introduces cysteine mutation point position TCB2-Cy01 IL:68-H:97 E68 A97 HCDR3 TCB2-Cy02 IL:42-H:99 F42 R99 HCDR3 TCB2-Cy03 IL:68-H:100 E68 G100 HCDR3 TCB2-Cy04 IL:38-H:100A R38 F100A HCDR3 TCB2-Cy05 IL:109-H:54 D109 D54 HCDR2 TCB2-Cy06 IL:34-L:27 P34 T27 LCDR1 TCB2-Cy07 IL:34-L:92 P34 D92 LCDR3 TCB2-Cy08 IL:34-L:93 P34 N93 LCDR3 TCB2-Cy09 IL:36-L:93 L36 N93 LCDR3 TCB2-Cy10 IL:37-L:93 T37 N93 LCDR
- IL means IL-2
- L means antibody light chain
- H means antibody heavy chain
- the number after IL or L/H means the amino acid position corresponding to the mutation point
- Table 33 and Table 34 were constructed, expressed and purified according to the method described in 2.1 of Example 2, and analyzed by SEC-HPLC to select the hIL-2/IL-2 antibody fusion protein NARA1- Cy01, NARA1-Cy04, NARA1-Cy05, NARA1-Cy08, and NARA1-Cy09; or TCB2-Cy01, TCB2-Cy02, TCB2-Cy07, TCB2-Cy08, TCB2-Cy09, and TCB2-Cy11 for further study.
- the hIL-2/IL-2 antibody fusion protein constructed based on NARA1 or TCB2 antibody and hCD25 and hCD122 were respectively detected. Binding activity, and set hIL2-DM-Fc as the control group.
- the hIL-2/IL-2 antibody fusion protein constructed based on NARA1 or TCB2 antibody showed significantly reduced hCD25 binding activity compared with the control group ( Figure 24A and Figure 25A), The binding activity to hCD122 even showed a certain enhancement ( Figure 24B and Figure 25B), indicating that the hIL-2/IL-2 antibody fusion protein constructed based on NARA1 or TCB2 antibody can maintain or enhance the binding of hIL-2 to hCD122 activity, and can inhibit the binding of hIL-2 and hCD25 to a certain extent, further indicating that the introduction of disulfide bonds into the antigen-antibody binding site of hIL-2/NARA1 or hIL-2/TCB2 antigen-antibody complexes can make hIL
- the ⁇ -receptor binding site of -2 is buried inside the antibody fusion protein molecule and is not easily competitively bound by hCD25 due to dissociation.
- Example 1 the biological activity of the hIL-2/IL-2 antibody fusion protein constructed based on NARA1 or TCB2 antibody was detected respectively by CTLL-2 cell and NK-92 cell proliferation experiments, and the control group was Wild-type hIL-2.
- Table 36 CTLL-2 cell line and NK-92 cell line proliferation assay to detect the biological activity of hIL-2/IL-2 antibody fusion protein constructed based on TCB2 antibody
- hIL-2 and anti-IL-2 antibodies such as NARA1 or TCB2 that specifically recognize the IL2R ⁇ binding site of IL-2
- hIL-2 and anti-IL-2 IL-2 antibody forms an ultra-stable complex structure, inhibits the binding of hIL-2 and IL2R ⁇ , and will not affect the binding of hIL-2 and hIL2R ⁇ / ⁇ .
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Abstract
Description
CDR | Kabat | Chothia | IMGT | Contact |
HCDR1 | 31-35 | 26-32 | 27-38 | 30-35 |
HCDR2 | 50-65 | 52-56 | 56-65 | 47-58 |
HCDR3 | 95-102 | 95-102 | 105-117 | 93-101 |
LCDR1 | 24-34 | 24-34 | 27-38 | 30-36 |
LCDR2 | 50-56 | 50-56 | 56-65 | 46-55 |
LCDR3 | 89-97 | 89-97 | 89-97 | 89-96 |
pI突变位点(EU numbering) | hIgG1 | hIgG2 | hIgG3 | hIgG4 |
第203位 | N | D | N | D |
第274位 | K | Q | Q | Q |
第419位 | Q | Q | Q | E |
抗体∶抗原 | K off(1/s) | K on(1/Ms) | K D(M) | Full R 2 |
chS4B6:hIL2-DM-his | 1.060E-02 | 2.691E05 | 3.939E-08 | 0.9968 |
chS4B6:hIL2-TM-his | 4.761E-03 | 2.104E05 | 2.263E-08 | 0.9955 |
chS4B6:mIL2-his | 1.823E-04 | 1.829E05 | 9.964E-10 | 0.9995 |
样品 | CTLL-2细胞增殖活性(EC50,nM) | NK-92细胞增殖活性(EC50,nM) |
hIL2-DM-his | 0.2739 | 0.03904 |
hIL2-TM-his | 0.209 | 0.04434 |
mIL2-his | 0.4878 | 0.07761 |
hIL-2 | 0.1615 | 0.02033 |
抗体编号 | 重链编号 | 重链Germline | 重链匹配度 | 轻链编号 | 轻链Germline | 轻链匹配度 |
S4B6 | S4B6 | IGHV1-69-2*01 | 62% | S4B6 | IGKV1-39*01 | 66% |
H1K1 | H1 | IGHV4-59*02 | 98% | K1 | IGKV3-20*01 | 98% |
H3K3 | H3 | IGHV2-5*02 | 96% | K3 | IGKV3-11*01 | 98% |
H6K3 | H6 | IGHV1-69-2*01 | 96% | K3 | IGKV3-11*01 | 98% |
H6K7 | H6 | IGHV1-69-2*01 | 96% | K7 | IGKV1-39*01 | 98% |
抗体∶抗原 | K off(1/s) | K on(1/Ms) | K D(M) | Full R 2 |
chS4B6:mIL2-his | 1.823E-04 | 1.829E05 | 9.964E-10 | 0.9995 |
H1K1:mIL2-his | 7.186E-04 | 2.769E04 | 2.595E-08 | 0.9993 |
H3K3:mIL2-his | 2.646E-03 | 2.005E05 | 1.320E-08 | 0.8133 |
H6K3:mIL2-his | 1.799E-04 | 1.792E05 | 1.004E-09 | 0.9993 |
H6K7:mIL2-his | 1.212E-03 | 8.638E05 | 1.403E-09 | 0.9984 |
抗体∶抗原 | K off(1/s) | K on(1/Ms) | K D(M) | Full R 2 |
H6K3:hIL2-DM-his | 1.005E-02 | 2.220E05 | 4.529E-08 | 0.9976 |
H6K3:hIL2-TM-his | 5.688E-03 | 2.372E04 | 2.398E-08 | 0.9963 |
深度人源化抗体编号 | 位置 | 突变情况 |
H6K3-hu01 | H27 | D27Y |
H6K3-hu02 | H29 | I29F |
H6K3-hu03 | H31 | A31D |
H6K3-hu04 | H34 | L34M |
H6K3-hu05 | H48 | I48M |
H6K3-hu06 | H65 | N65S |
H6K3-hu07 | H65 | N65G |
H6K3-hu08 | H76 | N76D |
H6K3-hu09 | L56 | D56S |
H6K3-hu10 | L56 | D56T |
H6K3-hu11 | L66 | R66G |
H6K3-hu12 | L71 | Y71F |
抗体∶抗原 | K off(1/s) | K on(1/Ms) | K D(M) | 结合响应值(nm) |
H6K3-hu01:hIL2-DM-his | / | / | / | 不结合 |
H6K3-hu02:hIL2-DM-his | / | / | / | 不结合 |
H6K3-hu03:hIL2-DM-his | / | / | / | 不结合 |
H6K3-hu04:hIL2-DM-his | 1.486E-02 | 4.298E05 | 3.457E-08 | 0.1429 |
H6K3-hu05:hIL2-DM-his | 1.525E-02 | 3.874E05 | 3.937E-08 | 0.1387 |
H6K3-hu06:hIL2-DM-his | 1.917E-02 | 3.647E05 | 5.255E-08 | 0.1345 |
H6K3-hu07:hIL2-DM-his | 1.811E-02 | 4.001E05 | 4.528E-08 | 0.1296 |
H6K3-hu08:hIL2-DM-his | 2.068E-02 | 3.559E05 | 5.810E-08 | 0.1269 |
H6K3-hu09:hIL2-DM-his | 8.673E-03 | 3.669E05 | 2.364E-08 | 0.1804 |
H6K3-hu10:hIL2-DM-his | 1.055E-02 | 3.725E05 | 2.833E-08 | 0.1716 |
H6K3-hu11:hIL2-DM-his | / | / | / | 不结合 |
H6K3-hu12:hIL2-DM-his | 1.736E-02 | 5.368E05 | 3.233E-08 | 0.1517 |
H6K3:hIL2-DM-his | 1.935E-02 | 4.130E05 | 4.686E-08 | 0.1330 |
抗体∶抗原 | K off(1/s) | K on(1/Ms) | K D(M) | Full R 2 |
hS4B6-H6K3(H):mIL2-his | 2.359E-04 | 9.277E05 | 2.543E-10 | 0.9998 |
hS4B6-H6K3(H):hIL2-DM-his | 3.843E-03 | 2.421E04 | 1.587E-08 | 0.9971 |
hS4B6-H6K3(H):hIL2-TM-his | 2.702E-03 | 2.735E05 | 9.879E-09 | 0.9963 |
样品 | CTLL-2细胞增殖活性(EC50,nM) | NK-92细胞增殖活性(EC50,nM) |
hIL-2 | 0.1615 | 0.03056 |
IC-Cy07 | 0.1494 | 0.01481 |
IC-Cy08 | 1.316 | 0.03399 |
hIL-2 | IC-Cy08 | |
EC50(nM) | 0.006492 | 0.2335 |
样品 | CTLL-2细胞增殖活性(EC50,nM) | NK-92细胞增殖活性(EC50,nM) |
hIL-2 | 0.1469 | 0.01572 |
IC-Cy08 | 3.352 | 0.02635 |
IC-Cy08-DE | 3.36 | 0.02332 |
IC-Cy08-QD | 3.621 | 0.02743 |
IC-Cy08-QE | 3.847 | 0.02612 |
IC-Cy08-QS | 4.64 | 0.03701 |
IC-Cy08-QG | 3.991 | 0.0301 |
IC-Cy08-QK | 3.518 | 0.02691 |
IC-Cy08-QR | 3.061 | 0.0247 |
IC-Cy08突变体样品 | 表达量(mg/L) |
IC-Cy08 | 40 |
IC-Cy08-DE | 93 |
IC-Cy08-QD | 158 |
IC-Cy08-QE | 140 |
IC-Cy08-QS | 113 |
IC-Cy08-QG | 154 |
IC-Cy08-QK | 28 |
IC-Cy08-QR | 47 |
突变体编号 | hIL-2N端前三个氨基酸APT | hIL-2第125位氨基酸残基 |
IC-Cy08(△3,WT) | 删除APT | C |
IC-Cy08(△3,C125A) | 删除APT | A |
IC-Cy08(△3,C125L) | 删除APT | L |
IC-Cy08(△3,C125S) | 删除APT | S |
IC-Cy08(△3,C125Q) | 删除APT | Q |
IC-Cy08(△3,C125V) | 删除APT | V |
样品 | CTLL-2细胞增殖活性(EC50,nM) | NK-92细胞增殖活性(EC50,nM) |
hIL-2 | 0.1956 | 0.008233 |
IC-Cy08 | 2.332 | 0.01564 |
IC-Cy08(△3,WT) | 2.140 | 0.02636 |
IC-Cy08(△3,C125A) | 2.270 | 0.02469 |
IC-Cy08(△3,C125L) | 3.158 | 0.02812 |
IC-Cy08(△3,C125S) | 3.260 | 0.02886 |
IC-Cy08(△3,C125Q) | 4.425 | 0.03354 |
IC-Cy08(△3,C125V) | 2.407 | 0.02878 |
样品 | CTLL-2细胞增殖活性(EC50,nM) | NK-92细胞增殖活性(EC50,nM) |
hIL-2 | 0.1469 | 0.01626 |
IC-Cy08 | 3.159 | 0.03117 |
IC-Cy08-pITM | 3.352 | 0.02929 |
编号 | 氨基酸残基位点 | hIL2序列 | K2G11序列 | K2G11引入半胱氨酸残基突变点位置 |
K2G11-Cy01 | IL:61-L:30 | E61 | S30 | LCDR1 |
K2G11-Cy02 | IL:61-L:31 | E61 | N31 | LCDR1 |
K2G11-Cy03 | IL:61-L:50 | E61 | S50 | LCDR2 |
K2G11-Cy04 | IL:104-L:52 | M104 | T52 | LCDR2 |
K2G11-Cy05 | IL:102-L:67 | T102 | S67 | LFR3 |
K2G11-Cy06 | IL:102-L:68 | T102 | G68 | LFR3 |
K2G11-Cy07 | IL:64-H:97 | K64 | F97 | HCDR3 |
K2G11-Cy08 | IL:65-H:97 | P65 | F97 | HCDR3 |
K2G11-Cy09 | IL:64-L:92 | K64 | Y92 | LCDR3 |
K2G11-Cy10 | IL:42-H:97 | F42 | F97 | HCDR3 |
K2G11-Cy11 | IL:60-L:30 | E60 | S30 | LCDR1 |
K2G11-Cy12 | IL:64-L:32 | K64 | Y32 | LCDR1 |
H6K3(H)-Cy10 | IL:42-H:97 | F42 | F97 | HCDR3 |
样品 | T m1(℃) | T m2(℃) | T m3(℃) | T onset(℃) | T turbility(℃) |
f | s | f | s | f | s | f | s | f | s | |
K2G11-Cy04 | 65.82 | 0.05 | 81.90 | 0.12 | 89.75 | 0.07 | 58.55 | 0.15 | 88.97 | 0.29 |
K2G11-Cy05 | 65.81 | / | 82.29 | / | 88.82 | / | 59.48 | / | 86.9 | / |
K2G11-Cy06 | 65.86 | 0.09 | 83.56 | 0.15 | 87.92 | 0.04 | 57.29 | 0.14 | 84.89 | 0.19 |
编号 | 氨基酸残基位点 | hIL2序列 | TCB2序列 | TCB2引入半胱氨酸突变点位置 |
TCB2-Cy01 | IL:68-H:97 | E68 | A97 | HCDR3 |
TCB2-Cy02 | IL:42-H:99 | F42 | R99 | HCDR3 |
TCB2-Cy03 | IL:68-H:100 | E68 | G100 | HCDR3 |
TCB2-Cy04 | IL:38-H:100A | R38 | F100A | HCDR3 |
TCB2-Cy05 | IL:109-H:54 | D109 | D54 | HCDR2 |
TCB2-Cy06 | IL:34-L:27 | P34 | T27 | LCDR1 |
TCB2-Cy07 | IL:34-L:92 | P34 | D92 | LCDR3 |
TCB2-Cy08 | IL:34-L:93 | P34 | N93 | LCDR3 |
TCB2-Cy09 | IL:36-L:93 | L36 | N93 | LCDR3 |
TCB2-Cy10 | IL:37-L:93 | T37 | N93 | LCDR3 |
TCB2-Cy11 | IL:37-L:94 | T37 | L94 | LCDR3 |
样品 | CTLL-2细胞增殖活性(EC50,nM) | NK-92细胞增殖活性(EC50,nM) |
hIL-2 | 0.2101 | 0.03322 |
NARA1-Cy01 | 2.915 | 0.1279 |
NARA1-Cy04 | 7.464 | 0.1314 |
NARA1-Cy05 | 1.340 | 0.1383 |
NARA1-Cy08 | 21.31 | 0.1201 |
NARA1-Cy09 | 3.359 | 0.1463 |
样品 | CTLL-2细胞增殖活性(EC50,nM) | NK-92细胞增殖活性(EC50,nM) |
hIL-2 | 0.2101 | 0.03322 |
TCB2-Cy01 | 1.338 | 0.05139 |
TCB2-Cy02 | 10.73 | 0.08893 |
TCB2-Cy07 | 9.278 | 0.07600 |
TCB2-Cy08 | 13.06 | 0.05778 |
TCB2-Cy09 | 3.763 | 0.07453 |
TCB2-Cy11 | 3.766 | 0.09515 |
Claims (79)
- 一种人白细胞介素-2(hIL-2)变体,其中,所述hIL-2变体包含以下至少一个特性:(1)所述hIL-2变体相对于SEQ ID NO:1所示的野生型hIL-2包含至少一个氨基酸突变;(2)所述hIL-2变体相对于SEQ ID NO:1所示的野生型hIL-2获得了与所述抗IL-2抗体或其抗原结合片段结合的能力;以及(3)所述氨基酸突变对hIL-2的生物学活性没有明显影响,优选地,所述hIL-2变体维持了野生型hIL-2的生物学活性;所述抗IL-2抗体或其抗原结合片段能够与IL2受体α亚基(IL2Rα)竞争性地结合所述hIL-2变体;所述hIL-2变体与所述抗IL-2抗体或其抗原结合片段结合后可以阻断所述hIL-2变体与IL-2Rα的结合,优选地,所述hIL-2变体与SEQ ID NO:1所示的野生型hIL-2相比具有90%以上的氨基酸序列同一性,更优选93%以上的氨基酸序列同一性,优选地,所述hIL-2变体相对于SEQ ID NO:1所示的野生型hIL-2包含一个、或两个、或两个以上氨基酸突变。
- 如权利要求1所述的hIL-2变体,其中,所述hIL-2变体的所述氨基酸突变位于所述hIL-2变体与抗IL-2抗体或其抗原结合片段的结合界面之内,优选地,所述hIL-2变体相对于SEQ ID NO:1所示的野生型hIL-2的氨基酸序列在第64位、第90位和/或第104位包含一种、两种或两种以上氨基酸突变。
- 如权利要求1或2所述的hIL-2变体,其中,相对于如SEQ ID NO:1所示的野生型hIL-2的氨基酸序列,所述hIL-2变体包含如下的至少一种突变:(1)在对应于如SEQ ID NO:1所示的野生型hIL-2的第64位氨基酸残基处的K突变为G、S、T、C、Y、N或Q;(2)在对应于如SEQ ID NO:1所示的野生型hIL-2的第90位氨基酸残基处的N突变为R、K或H;或(3)在对应于如SEQ ID NO:1所示的野生型hIL-2的第104位氨基酸残基处的M突变为E或D。
- 如权利要求1-3中任一项所述的hIL-2变体,其中,所述的hIL-2变体相对于SEQ ID NO:1所示的野生型hIL-2的氨基酸序列包含以下一种、两种、或两种以上氨基酸突变:(1)相对于SEQ ID NO:1所示的野生型hIL-2的氨基酸序列的第64位氨基酸残基处具有氨基酸突变K64G,(2)相对于SEQ ID NO:1所示的野生型hIL-2的氨基酸序列的第90位氨基酸残基处具有氨基酸突变N90R,和(3)相对于如SEQ ID NO:1所示的野生型hIL-2的氨基酸序列的第104位氨基酸残基处具有氨基酸突变M104E;优选地,所述hIL-2变体相对于SEQ ID NO:1所示的野生型hIL-2的氨基酸序列具有氨基酸突变K64G和/或N90R;或优选地,所述hIL-2变体相对于SEQ ID NO:1所示的野生型hIL-2的氨基酸序列具有氨基酸突变K64G、N90R和M104E;更优选地,所述hIL-2变体相对于SEQ ID NO:1所示的野生型hIL-2的氨基酸序列具有氨基酸突变K64G和N90R。
- 如权利要求1-4中任一项所述的hIL-2变体,其中,所述hIL-2变体的所述氨基酸突变位于所述hIL-2变体与抗IL-2抗体或其抗原结合片段的结合界面之外,优选地,所述hIL-2变体相对于SEQ ID NO:1所示的野生型hIL-2的氨基酸序列的N端前三个或前五个氨基酸存在缺失,或相对于SEQ ID NO:1所示的野生型hIL-2的氨基酸序列的N端第三个氨基酸残基具有氨基酸突变,和/或相对于如SEQ ID NO:1所示的野生型hIL-2的氨基酸序列的第125位氨基酸残基处具有氨基酸突变;优选地,所述hIL-2变体的所述氨基酸突变包含对SEQ ID NO:1所示的野生型hIL-2的氨基酸序列的N端前三个进行缺失,和/或相对于如SEQ ID NO:1所示的野生型hIL-2的氨基酸序列的第125位氨基酸残基处具有氨基酸突变C125A、C125L、C125S、C125Q或C125V。
- 一种抗IL-2抗体或其抗原结合片段,其中,所述抗IL-2抗体或其抗原结合片段包含以下至少一个特性:(1)所述抗IL-2抗体或其抗原结合片段能够与IL2Rα竞争性结合权利要求1-5中任一项所述hIL-2变体并且竞争性地阻断权利要求1-5中任一项所述hIL-2变体与IL2Rα或包含有α亚基的IL-2受体聚合体的结合;(2)所述抗IL-2抗体或其抗原结合片段与权利要求1-5中任一项所述hIL-2变体的结合亲和力(K D)值<5×10 -8M,优选地,<1×10 -8M,<5×10 -9M,或<1×10 -9M,或<5×10 -10M,更优选 地,<6×10 -10M;和/或(3)所述抗IL-2抗体或其抗原结合片段与权利要求1-5中任一项所述hIL-2变体的解离常数(K dis)<5×10 -3s -1、1×10 -3s -1、<5×10 -4s -1或<3×10 -4s -1。
- 如权利要求6所述的抗IL-2抗体或其抗原结合片段,其中,所述抗IL-2抗体选自抗小鼠IL-2抗体及其人源化抗体,进一步,所述抗小鼠IL-2抗体选自特异性识别小鼠IL-2(mIL-2)与小鼠IL2Rα(mIL2Rα)结合位点的抗mIL-2抗体,所述抗mIL-2抗体能够与权利要求1-5中任一项所述的hIL-2变体发生交叉反应;优选地,所述抗IL-2抗体或其抗原结合片段包含如下的重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3:(1)X1YYX2H(其中X1=A或D;X2=L或M)所示的HCDR1氨基酸序列,(2)RIDPZ1DDSTKYAENFKX3(其中Z1=E或D;X3=N,S或G)所示的HCDR2氨基酸序列,(3)Z2TFZ3YZ4RELRWFAY(其中Z2=V,I,R或T;Z3=Y,L,H或W;Z4=S,T,A,R,N,D,Q,E,H,I,L,K,F,P,W,Y或V)所示的HCDR3氨基酸序列,优选地,Z2=V或I;Z3=Y或L;Z4=S或T,(4)QASQZ5IZ6NYLS(其中Z5=D,Y,N,H,I,L或F;Z6=G,S,A,R,N,Q,I,L,T或D)所示的LCDR1氨基酸序列,优选地,Z5=D或Y;Z6=G或S,(5)Z7AZ8SLAX4(其中,Z7=S,Q,R,T,W或Y;Z8=T或G;X4=D,S或T)所示的LCDR2氨基酸序列,优选地,Z7=S或Q;Z8=T;X4=S,和(6)氨基酸序列为LQHYSTPYT(如SEQ ID NO:12所示)的LCDR3氨基酸序列。
- 如权利要求6或7所述的抗IL-2抗体或其抗原结合片段,其中,所述抗IL-2抗体或其抗原结合片段包含:如SEQ ID NO:7、SEQ ID NO:13或SEQ ID NO:14所示的HCDR1;如SEQ ID NO:8、SEQ ID NO:15、SEQ ID NO:16或SEQ ID NO:19所示的HCDR2;如SEQ ID NO:9、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:29或SEQ ID NO:34所示的HCDR3;以及如SEQ ID NO:10、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:43或SEQ ID NO:44所示的LCDR1;如SEQ ID NO:11、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:28、SEQ ID NO:32或SEQ ID NO:33所示的LCDR2;如SEQ ID NO:12或SEQ ID NO:38所示的LCDR3。
- 如权利要求6-8中任一项所述的抗IL-2抗体或其抗原结合片段,其中,所述抗IL-2抗体或其抗原结合片段包含如下的重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3:(1)SEQ ID NO:7所示的HCDR1氨基酸序列,SEQ ID NO:8所示的HCDR2氨基酸序列,SEQ ID NO:9所示的HCDR3氨基酸序列,SEQ ID NO:10所示的LCDR1氨基酸序列,SEQ ID NO:11所示的LCDR2氨基酸序列,和SEQ ID NO:12所示的LCDR3氨基酸序列,或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(2)SEQ ID NO:13或14所示的HCDR1氨基酸序列,SEQ ID NO:8所示的HCDR2氨基酸序列,SEQ ID NO:9所示的HCDR3氨基酸序列,SEQ ID NO:10所示的LCDR1氨基酸序列,SEQ ID NO:11所示的LCDR2氨基酸序列,和SEQ ID NO:12所示的LCDR3氨基酸序列,或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(3)SEQ ID NO:7所示的HCDR1氨基酸序列,SEQ ID NO:15或16所示的HCDR2氨基酸序列,SEQ ID NO:9所示的HCDR3氨基酸序列,SEQ ID NO:10所示的LCDR1氨基酸序列,SEQ ID NO:11所示的LCDR2氨基酸序列,SEQ ID NO:12所示的LCDR3氨基酸序列,或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(4)SEQ ID NO:7所示的HCDR1氨基酸序列,SEQ ID NO:8所示的HCDR2氨基酸序列,SEQ ID NO:9所示的HCDR3氨基酸序列,SEQ ID NO:10所示的LCDR1氨基酸序列,SEQ ID NO:17或18所示的LCDR2氨基酸序列,和SEQ ID NO:12所示的LCDR3氨基酸序列,或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85%、86%、87%、 88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(5)SEQ ID NO:14所示的HCDR1氨基酸序列,SEQ ID NO:15所示的HCDR2氨基酸序列,SEQ ID NO:9所示的HCDR3氨基酸序列,SEQ ID NO:10所示的LCDR1氨基酸序列,SEQ ID NO:17所示的LCDR2氨基酸序列,和SEQ ID NO:12所示的LCDR3氨基酸序列,或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列。
- 如权利要求6-9中任一项所述的抗IL-2抗体或其抗原结合片段,其中,所述抗IL-2抗体或其抗原结合片段包含如下的重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3:(1)X1YYX2H(其中X1=A;X2=M)所示的HCDR1氨基酸序列,(2)RIDPZ1DDSTKYAENFKX3(其中Z1=E或D;X3=G)所示的HCDR2氨基酸序列,(3)Z2TFZ3YZ4RELRWFAY(其中Z2=V,I,R或T;Z3=Y,L,H或W;Z4=S,T,A,R,N,D,Q,E,H,I,L,K,F,P,W,Y或V)所示的HCDR3氨基酸序列,优选地,Z2=V或I;Z3=Y或L;Z4=S或T,(4)QASQZ5IZ6NYLS(其中Z5=D,Y,N,H,I,L或F;Z6=G,S,A,R,N,Q,I,L,T或D)所示的LCDR1氨基酸序列,优选地,Z5=D或Y;Z6=G或S,(5)Z7AZ8SLAX4(其中,Z7=S,Q,R,T,W或Y;Z8=T或G;X4=D,S或T)所示的LCDR2氨基酸序列,优选地,Z7=S或Q;Z8=T;X4=S,和(6)氨基酸序列为LQHYSTPYT(SEQ ID NO:12所示)的LCDR3氨基酸序列。
- 如权利要求10所述的抗IL-2抗体或其抗原结合片段,其中,所述抗IL-2抗体或其抗原结合片段包含如下的重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3:(1)SEQ ID NO:14所示的HCDR1氨基酸序列,SEQ ID NO:15所示的HCDR2氨基酸序列,SEQ ID NO:9所示的HCDR3氨基酸序列,SEQ ID NO:26或27所示的LCDR1氨基酸酸序列,SEQ ID NO:17或28所示的LCDR2氨基酸序列,和SEQ ID NO:12所示的LCDR3氨基酸,或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(2)SEQ ID NO:14所示的HCDR1氨基酸序列,SEQ ID NO:15所示的HCDR2氨基酸序列,SEQ ID NO:20、21、22、23、24或25所示的HCDR3氨基酸序列,SEQ ID NO:26或27所示的LCDR1氨基酸酸序列,SEQ ID NO:17或28所示的LCDR2氨基酸序列,和SEQ ID NO:12所示的LCDR3氨基酸,或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(3)SEQ ID NO:14所示的HCDR1氨基酸序列,SEQ ID NO:19所示的HCDR2氨基酸序列,SEQ ID NO:9、20、23、24或25所示的HCDR3氨基酸序列,SEQ ID NO:26或27所示的LCDR1氨基酸酸序列,SEQ ID NO:17或28所示的LCDR2氨基酸序列,和SEQ ID NO:12所示的LCDR3氨基酸,或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或者优选地,所述抗IL-2抗体或其抗原结合片段包含如下的重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3:(1)SEQ ID NO:14所示的HCDR1氨基酸序列,SEQ ID NO:15所示的HCDR2氨基酸序列,SEQ ID NO:24或25所示的HCDR3氨基酸序列,SEQ ID NO:26或27所示的LCDR1氨基酸酸序列,SEQ ID NO:17或28所示的LCDR2氨基酸序列,和SEQ ID NO:12所示的LCDR3氨基酸,或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(2)SEQ ID NO:14所示的HCDR1氨基酸序列,SEQ ID NO:19所示的HCDR2氨基酸序列,SEQ ID NO:24或25所示的HCDR3氨基酸序列,SEQ ID NO:26或27所示的LCDR1氨基酸酸序列,SEQ ID NO:17或28所示的LCDR2氨基酸序列,和SEQ ID NO:12所示的LCDR3氨基酸,或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100% 相同的氨基酸序列;更优选地,所述抗IL-2抗体或其抗原结合片段包含如下的HCDR1、HCDR2和HCDR3以及LCDR1、LCDR2和LCDR3:SEQ ID NO:14所示的HCDR1氨基酸序列、SEQ ID NO:19所示的HCDR2氨基酸序列,SEQ ID NO:24所示的HCDR3氨基酸序列,SEQ ID NO:26所示的LCDR1氨基酸酸序列,SEQ ID NO:17所示的LCDR2氨基酸序列,和SEQ ID NO:12所示的LCDR3氨基酸,或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列。
- 如权利要求6-11中任一项所述的抗IL-2抗体或其抗原结合片段,其中,所述抗IL-2抗体或其抗原结合片段包含:与SEQ ID NO:5、45、47、49、51、52、53、54、55、56、57、58、63、65、66、67、68、69、70、71、72、73、74、75、86或112所示的序列具有至少85%同一性的重链可变区VH;以及与SEQ ID NO:6、46、48、50、59、60、61、62、64、76、77、78、79、80、81、82、83、84、85、87、88、89、90、91、92、93、104、105、106、107、108、109、110或111所示的序列具有至少85%同一性的轻链可变区VL。
- 如权利要求6-12中任一项所述的抗IL-2抗体或其抗原结合片段,其中,所述抗IL-2抗体或其抗原结合片段包含如下的具有所述HCDR1、HCDR2和HCDR3的重链可变区,和/或具有所述LCDR1、LCDR2和LCDR3的轻链可变区:(1)分别与SEQ ID NO:45、47或49所示的VH和/或SEQ ID NO:46、48或50所示的VL具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列,优选地,分别与SEQ ID NO:49所示的VH氨基酸序列和/或SEQ ID NO:48或50所示的VL具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(2)分别与SEQ ID NO:54、55、56、57或58所示的VH和/或SEQ ID NO:48所示的VL具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(3)分别与SEQ ID NO:49所示的VH氨基酸序列和/或SEQ ID NO:59、60或62所示的VL具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(4)分别与SEQ ID NO:63所示的VH和/或SEQ ID NO:64所示的VL具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或者,所述抗IL-2抗体或其抗原结合片段包含如下具有所述HCDR1、HCDR2和HCDR3的重链可变区,和/或具有所述LCDR1、LCDR2和LCDR3的轻链可变区:分别与SEQ ID NOs:63和65-75任意一个所示的VH和/或SEQ ID NOs:76-79任意一个所示的VL具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或者,所述抗IL-2抗体或其抗原结合片段包含如下具有所述HCDR1、HCDR2和HCDR3的重链可变区,和/或具有所述LCDR1、LCDR2和LCDR3的轻链可变区:(1)分别与SEQ ID NO:71所示的VH和/或SEQ ID NO:76所示的VL具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(2)分别与SEQ ID NO:72所示的VH和/或SEQ ID NO:77所示的VL具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(3)分别与SEQ ID NO:72或74所示的VH和/或SEQ ID NO:76或79所示的VL具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(4)分别与SEQ ID NO:74或75所示的VH和/或SEQ ID NO:77或78所示的VL具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列。
- 一种hIL-2/IL-2抗体融合蛋白,其包含:(1)如SEQ ID NO:1所示的野生型hIL-2或其变体,(2)抗IL-2抗体或其抗原结合片段,以及(3)所述hIL-2或其变体与所述抗IL-2抗体或其抗原结合片段的抗原-抗体结合界面包含的二硫键,其中,所述抗IL-2抗体或其抗原结合片段能够特异性地结合所述hIL-2或其变体并且竞争性地阻断所述hIL-2或其变体与IL2Rα或包含有α亚基的IL-2受体聚合体(即IL2Rα/β或IL2Rα/β/γ)的结合,以及所述二硫键是通过在所述抗原-抗体结合界面的hIL-2或其变体的序列中引入的至少一个半胱氨酸残基与通过在所述抗原-抗体结合界面的抗IL-2抗体或其抗原结合片段的序列中引入的至少一个半胱氨酸残基所形成的,所述的hIL-2/IL-2抗体融合蛋白能够不与IL2R的α亚基、或α/β二聚体、或α/β/γ三聚体的结合,但是保留了对IL2Rβ和γ受体亚基二聚体(IL2Rβ/γ)的结合以及功能活性。
- 如权利要求14所述的hIL-2/IL-2抗体融合蛋白,其中,所述的抗IL-2抗体或其抗原结合片段与hIL-2变体的结合亲和力(K D)<5×10 -8M,优选地,<1×10 -8M,<5×10 -9M,或<1×10 -9M,或<5×10 -10M,更优选地,<6×10 -10M;和/或所述的抗IL-2抗体或其抗原结合片段与hIL-2变体的解离常数(K dis)<5×10 -3s -1、1×10 -3s -1、<5×10 -4s -1或<3×10 -4s -1。
- 如权利要求14或15所述的hIL-2/IL-2抗体融合蛋白,其中,所述hIL-2或其变体与所述抗IL-2抗体或其抗原结合片段的抗原-抗体结合界面包含了hIL-2或其变体与IL2Rα的结合界面,或所述的抗原-抗体结合界面与hIL-2或其变体与IL2Rα的结合界面存在部分重叠;或者,所述hIL-2或其变体与所述抗IL-2抗体或其抗原结合片段的抗原-抗体结合界面位于所述hIL-2或其变体的AB环、B螺旋、C螺旋、BC环和/或CD环,优选位于所述hIL-2或其变体的AB环、B螺旋、C螺旋和CD环;所述的hIL-2或其变体的IL2Rα结合界面位于hIL-2或其变体的AB环和B螺旋;或者,所述hIL-2或其变体与所述抗IL-2抗体或其抗原结合片段的抗原-抗体结合界面位于所述hIL-2或其变体的第30-45位或其邻近的氨基酸残基,第57-77位或其邻近的氨基酸残基,和/或第90-111位或其邻近的氨基酸残基。
- 如权利要求14-16中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,所述hIL-2变体的氨基酸突变位点位于所述hIL-2变体与抗IL-2抗体或其抗原结合片段的结合界面之内,优选地,所述hIL-2变体相对于SEQ ID NO:1所示的野生型hIL-2的氨基酸序列在第64位、第90位和/或第104位包含一种、两种或两种以上氨基酸突变。
- 如权利要求14-17中任一项所述的hIL-2变体,其中,相对于如SEQ ID NO:1所示的野生型hIL-2的氨基酸序列,所述hIL-2变体包含如下的至少一种突变:(1)在对应于如SEQ ID NO:1所示的野生型hIL-2的第64位氨基酸残基处的K突变为G、S、T、C、Y、N或Q;(2)在对应于如SEQ ID NO:1所示的野生型hIL-2的第90位氨基酸残基处的N突变为R、K或H;或(3)在对应于如SEQ ID NO:1所示的野生型hIL-2的第104位氨基酸残基处的M突变为E或D。
- 如权利要求14-18中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,所述的hIL-2变体相对于SEQ ID NO:1所示的野生型hIL-2的氨基酸序列包含以下一种、两种或两种以上氨基酸突变:(1)相对于SEQ ID NO:1所示的野生型hIL-2的氨基酸序列的第64位氨基酸残基处具有氨基酸突变K64G,(2)相对于SEQ ID NO:1所示的野生型hIL-2的氨基酸序列的第90位氨基酸残基处具有氨基酸突变N90R,和(3)相对于如SEQ ID NO:1所示的野生型hIL-2的氨基酸序列的第104位氨基酸残基处具有氨基酸突变M104E;优选地,所述hIL-2变体相对于SEQ ID NO:1所示的野生型hIL-2的氨基酸序列具有氨基酸突变K64G和/或N90R;或优选地,所述hIL-2变体相对于SEQ ID NO:1所示的野生型hIL-2的氨基酸序列具有氨基酸突变K64G、N90R和M104E(如SEQ ID NO:3所示);更优选地,所述hIL-2变体相对于SEQ ID NO:1所示的野生型hIL-2的氨基酸序列具有氨基酸突变K64G和N90R(如SEQ ID NO:2所示)。
- 如权利要求14-19中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,所述抗IL-2抗体或其抗原结合片段选自抗小鼠IL-2抗体及其人源化抗体,进一步,所述抗小鼠IL-2抗体选自特异性识别小鼠IL-2(mIL-2)与小鼠IL2Rα(mIL2Rα)结合位点的抗mIL-2抗体,所述抗mIL-2抗体能够与 所述的hIL-2变体发生交叉反应;所述抗IL-2抗体也可以是其它能够特异性识别hIL-2上IL2Rα结合位点的抗IL-2人源化抗体或其抗原结合片段,优选的其它抗IL-2人源化抗体选自NARA1和TCB2;优选地,所述抗IL-2抗体或其抗原结合片段包含如下的重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3:(1)X1YYX2H(其中X1=A或D;X2=L或M)所示的HCDR1氨基酸序列,(2)RIDPZ1DDSTKYAENFKX3(其中Z1=E或D;X3=N,S或G)所示的HCDR2氨基酸序列,(3)Z2TFZ3YZ4RELRWFAY(其中Z2=V,I,R或T;Z3=Y,L,H或W;Z4=S,T,A,R,N,D,Q,E,H,I,L,K,F,P,W,Y或V)所示的HCDR3氨基酸序列,优选地,Z2=V或I;Z3=Y或L;Z4=S或T,(4)QASQZ5IZ6NYLS(其中Z5=D,Y,N,H,I,L或F;Z6=G,S,A,R,N,Q,I,L,T或D)所示的LCDR1氨基酸序列,优选地,Z5=D或Y;Z6=G或S,(5)Z7AZ8SLAX4(其中,Z7=S,Q,R,T,W或Y;Z8=T或G;X4=D,S或T)所示的LCDR2氨基酸序列,优选地,Z7=S或Q;Z8=T;X4=S,和(6)氨基酸序列为LQHYSTPYT(SEQ ID NO:12所示)的LCDR3氨基酸序列。
- 如权利要求14-20中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,所述抗IL-2抗体或其抗原结合片段包含:如SEQ ID NO:7、SEQ ID NO:13或SEQ ID NO:14所示的HCDR1;如SEQ ID NO:8、SEQ ID NO:15、SEQ ID NO:16或SEQ ID NO:19所示的HCDR2;如SEQ ID NO:9、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:29或SEQ ID NO:34所示的HCDR3;以及如SEQ ID NO:10、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:35、SEQ ID NO:36或SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:43或SEQ ID NO:44所示的LCDR1;如SEQ ID NO:11、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:28、SEQ ID NO:32或SEQ ID NO:33所示的LCDR2;如SEQ ID NO:12或SEQ ID NO:38所示的LCDR3。
- 如权利要求20或21所述的hIL-2/IL-2抗体融合蛋白,其中,所述抗IL-2抗体或其抗原结合片段包含如下的重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3:(1)SEQ ID NO:7所示的HCDR1氨基酸序列,SEQ ID NO:8所示的HCDR2氨基酸序列,SEQ ID NO:9所示的HCDR3氨基酸序列,SEQ ID NO:10所示的LCDR1氨基酸序列,SEQ ID NO:11所示的LCDR2氨基酸序列,和SEQ ID NO:12所示的LCDR3氨基酸序列,或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(2)SEQ ID NO:13或14所示的HCDR1氨基酸序列,SEQ ID NO:8所示的HCDR2氨基酸序列,SEQ ID NO:9所示的HCDR3氨基酸序列,SEQ ID NO:10所示的LCDR1氨基酸序列,SEQ ID NO:11所示的LCDR2氨基酸序列,和SEQ ID NO:12所示的LCDR3氨基酸序列,或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(3)SEQ ID NO:7所示的HCDR1氨基酸序列,SEQ ID NO:15或16所示的HCDR2氨基酸序列,SEQ ID NO:9所示的HCDR3氨基酸序列,SEQ ID NO:10所示的LCDR1氨基酸序列,SEQ ID NO:11所示的LCDR2氨基酸序列,SEQ ID NO:12所示的LCDR3氨基酸序列,或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(4)SEQ ID NO:7所示的HCDR1氨基酸序列,SEQ ID NO:8所示的HCDR2氨基酸序列,SEQ ID NO:9所示的HCDR3氨基酸序列,SEQ ID NO:10所示的LCDR1氨基酸序列,SEQ ID NO:17或SEQ ID NO:18所示的LCDR2氨基酸序列,和SEQ ID NO:12所示的LCDR3氨基酸序列,或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(5)SEQ ID NO:14所示的HCDR1氨基酸序列,SEQ ID NO:15所示的HCDR2氨基酸序列,SEQ ID NO:9所示的HCDR3氨基酸序列,SEQ ID NO:10所示的LCDR1氨基酸序列,SEQ ID NO:17所示的LCDR2氨基酸序列,和SEQ ID NO:12所示的LCDR3氨基酸序列,或分别 与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;优选地,所述的hIL-2/IL-2抗体融合蛋白中的抗IL-2抗体或其抗原结合片段包含如下的HCDR1、HCDR2和HCDR3以及LCDR1、LCDR2和LCDR3:SEQ ID NO:14所示的HCDR1氨基酸序列、SEQ ID NO:15所示的HCDR2氨基酸序列、SEQ ID NO:9所示的HCDR3氨基酸序列、SEQ ID NO:10所示的LCDR1氨基酸酸序列、SEQ ID NO:17所示的LCDR2氨基酸序列,和SEQ ID NO:12所示的LCDR3氨基酸,或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列。
- 如权利要求20-22中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,所述抗IL-2抗体或其抗原结合片段包含如下的重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3:(1)X1YYX2H(其中X1=A;X2=M)所示的HCDR1氨基酸序列,(2)RIDPZ1DDSTKYAENFKX3(其中Z1=E或D;X3=G)所示的HCDR2氨基酸序列,(3)Z2TFZ3YZ4RELRWFAY(其中Z2=V,I,R或T;Z3=Y,L,H或W;Z4=S,T,A,R,N,D,Q,E,H,I,L,K,F,P,W,Y或V)所示的HCDR3氨基酸序列,优选地,Z2=V或I;Z3=Y或L;Z4=S或T,(4)QASQZ5IZ6NYLS(其中Z5=D,Y,N,H,I,L或F;Z6=G,S,A,R,N,Q,I,L,T或D)所示的LCDR1氨基酸序列,优选地,Z5=D或Y;Z6=G或S,(5)Z7AZ8SLAX4(其中,Z7=S,Q,R,T,W或Y;Z8=T或G;X4=D,S或T)所示的LCDR2氨基酸序列,优选地,Z7=S或Q;Z8=T;X4=S,和(6)LQHYSTPYT所示的LCDR3氨基酸序列;优选地,所述抗IL-2抗体或其抗原结合片段包含如下的重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3:(1)SEQ ID NO:14所示的HCDR1氨基酸序列,SEQ ID NO:15所示的HCDR2氨基酸序列,SEQ ID NO:9所示的HCDR3氨基酸序列,SEQ ID NO:26或27所示的LCDR1氨基酸酸序列,SEQ ID NO:17或28所示的LCDR2氨基酸序列,和SEQ ID NO:12所示的LCDR3氨基酸,或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(2)SEQ ID NO:14所示的HCDR1氨基酸序列,SEQ ID NO:15所示的HCDR2氨基酸序列,SEQ ID NO:20、21、22、23、24或25所示的HCDR3氨基酸序列,SEQ ID NO:26或27所示的LCDR1氨基酸酸序列,SEQ ID NO:17或28所示的LCDR2氨基酸序列,和SEQ ID NO:12所示的LCDR3氨基酸,或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(3)SEQ ID NO:14所示的HCDR1氨基酸序列,SEQ ID NO:19所示的HCDR2氨基酸序列,SEQ ID NO:9、20、23、24或25所示的HCDR3氨基酸序列,SEQ ID NO:26或27所示的LCDR1氨基酸酸序列,SEQ ID NO:17或28所示的LCDR2氨基酸序列,和SEQ ID NO:12所示的LCDR3氨基酸,或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或者,所述抗IL-2抗体或其抗原结合片段包含如下的重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3:(1)SEQ ID NO:14所示的HCDR1氨基酸序列,SEQ ID NO:15所示的HCDR2氨基酸序列,SEQ ID NO:24或25所示的HCDR3氨基酸序列,SEQ ID NO:26或27所示的LCDR1氨基酸酸序列,SEQ ID NO:17或28所示的LCDR2氨基酸序列,和SEQ ID NO:12所示的LCDR3氨基酸,或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(2)SEQ ID NO:14所示的HCDR1氨基酸序列,SEQ ID NO:19所示的HCDR2氨基酸序列,SEQ ID NO:24或25所示的HCDR3氨基酸序列,SEQ ID NO:26或27所示的LCDR1氨基酸酸序列,SEQ ID NO:17或28所示的LCDR2氨基酸序列,和SEQ ID NO:12所示的LCDR3氨基酸,或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85%、 86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;优选地,所述抗IL-2抗体或其抗原结合片段包含如下的HCDR1、HCDR2和HCDR3以及LCDR1、LCDR2和LCDR3:SEQ ID NO:14所示的HCDR1氨基酸序列、SEQ ID NO:19所示的HCDR2氨基酸序列,SEQ ID NO:24所示的HCDR3氨基酸序列,SEQ ID NO:26所示的LCDR1氨基酸酸序列,SEQ ID NO:17所示的LCDR2氨基酸序列,和SEQ ID NO:12所示的LCDR3氨基酸,或分别与所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列。
- 如权利要求14-23中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,所述抗IL-2抗体或其抗原结合片段包含:与SEQ ID NO:5、45、47、49、51、52、53、54、55、56、57、58、63、65、66、67、68、69、70、71、72、73、74、75、86或112所示的序列具有至少85%同一性的重链可变区VH;以及与SEQ ID NO:6、46、48、50、59、60、61、62、64、76、77、78、79、80、81、82、83、84、85、87、88、89、90、91、92、93、104、105、106、107、108、109、110或111所示的序列具有至少85%同一性的轻链可变区VL。
- 如权利要求14-24中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,所述抗IL-2抗体或其抗原结合片段包含如下的具有所述HCDR1、HCDR2和HCDR3的重链可变区,和具有所述LCDR1、LCDR2和LCDR3的轻链可变区:(1)分别与SEQ ID NO:45、47或49所示的VH和SEQ ID NO:46、48或50所示的VL具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列,优选地,分别与SEQ ID NO:49所示的VH氨基酸序列和SEQ ID NO:48或50所示的VL具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(2)分别与SEQ ID NO:54、55、56、57或58所示的VH和SEQ ID NO:48所示的VL具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(3)分别与SEQ ID NO:49所示的VH氨基酸序列和SEQ ID NO:59、60或62所示的VL具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(4)分别与SEQ ID NO:63所示的VH和SEQ ID NO:64所示的VL具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或者,所述抗IL-2抗体或其抗原结合片段包含如下的具有所述HCDR1、HCDR2和HCDR3的重链可变区,和具有所述LCDR1、LCDR2和LCDR3的轻链可变区:分别与SEQ ID NOs:63和65-75任意一个所示的VH和SEQ ID NOs:76-79任意一个所示的VL具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或者,所述抗IL-2抗体或其抗原结合片段包含如下具有所述HCDR1、HCDR2和HCDR3的重链可变区,和具有所述LCDR1、LCDR2和LCDR3的轻链可变区:(1)分别与SEQ ID NO:71所示的VH和SEQ ID NO:76所示的VL具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(2)分别与SEQ ID NO:72所示的VH和SEQ ID NO:77所示的VL具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(3)分别与SEQ ID NO:72或74所示的VH和SEQ ID NO:76或79所示的VL具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(4)分别与SEQ ID NO:74或75所示的VH和SEQ ID NO:77或78所示的VL具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列。
- 如权利要求14-25中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,所述hIL-2/IL-2抗体融合蛋白中的hIL-2变体与抗IL-2抗体或其抗原结合片段的抗原-抗体结合界面包含二硫键,所述抗原-抗体结合界面与所述hIL-2变体与IL2Rα的结合界面存在重叠,进一步,所述hIL-2变体与所述抗IL-2抗体或其抗原结合片段的抗原-抗体结合界面位于所述hIL-2变体的AB环、B螺旋、C螺旋和CD环,优选位于所述hIL-2变体的第41-45位或其邻近的氨基酸残基、第57-68位或其邻近的氨基酸残基以及第90-107位或其邻近的氨基酸残基;所述二硫键是通过在所述抗原-抗体结合界面的抗IL-2抗体或其抗原结合片段的序列中引入的至少一个半胱氨酸残基与通过在所述抗原-抗体结合界面的hIL-2变体的序列中引入的至少一个半胱氨酸残基所形成;优选地,所述的hIL-2/IL-2抗体融合蛋白中的hIL-2变体与所述抗IL-2抗体或其抗原结合片段的抗原-抗体结合界面包含两个或两个以上半胱氨酸残基以形成所述二硫键。
- 如权利要求26所述的hIL-2/IL-2抗体融合蛋白,其中,所述hIL-2变体相对于SEQ ID NO:1所示的野生型hIL-2的氨基酸序列在第42位、第60位、第61位、第64位、第65位、第102位和第104位氨基酸残基处具有一个、或两个、或两个以上氨基酸突变,以引入第一个半胱氨酸残基。
- 如权利要求26所述的hIL-2/IL-2抗体融合蛋白,其中,所述抗IL-2抗体或其抗原结合片段包括:(1)氨基酸序列为X1YYX2H(X1=A;X2=M)的HCDR1,(2)氨基酸序列为RIDPZ1DDSTKYAENFKX3(Z1=E或D;X3=G)的HCDR2,(3)氨基酸序列为Z2TFZ3YZ4RELRWFAY(Z2=V或I;Z3=Y或L;Z4=S或T)的HCDR3,(4)氨基酸序列为QASQZ5IZ6NYLS(Z5=D或Y;Z6=G或S)的LCDR1,(5)氨基酸序列为Z7AZ8SLAX4(Z7=S或Q;Z8=T;X4=S)的LCDR2,和(6)氨基酸序列为LQHYSTPYT(SEQ ID NO:12所示)的LCDR3,并且所述抗IL-2抗体或其抗原结合片段在HCDR3的第3位氨基酸残基处、LCDR1的第7位、第8位和/或第9位氨基酸残基处、LCDR2的第1位、第3位氨基酸残基处、或LCDR3第4位氨基酸残基处具有一个、或两个、或两个以上氨基酸突变,以引入第二个半胱氨酸残基。
- 如权利要求26所述的hIL-2/IL-2抗体融合蛋白,其中,所述抗IL-2抗体或其抗原结合片段包含如下的具有所述HCDR1、HCDR2和HCDR3的重链可变区和/或具有所述LCDR1、LCDR2和LCDR3的轻链可变区:(1)分别与SEQ ID NO:63所示的VH和/或SEQ ID NO:64所示的VL具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列,或(2)分别与SEQ ID NOs:63和65-75任意一个所示的VH和/或SEQ ID NOs:76-79任意一个所示的VL具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列,并且所述抗IL-2抗体或其抗原结合片段在其轻链可变区的框架区具有一个、或两个、或两个以上氨基酸突变,以引入第二个半胱氨酸残基,所述氨基酸突变位于所述轻链可变区的第67和/或68位氨基酸残基处。
- 如权利要求26-29中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,在所述hIL-2/IL-2抗体融合蛋白的抗原-抗体结合界面的hIL-2变体通过氨基酸突变的方式引入第一半胱氨酸残基,在所述抗原-抗体结合界面的抗IL-2人源化抗体或其抗原结合片段通过氨基酸突变的方式引入第二半胱氨酸残基,优选的氨基酸突变是氨基酸取代、插入或添加,且在所述hIL-2变体引入第一半胱氨酸残基所在的位点和在所述抗IL-2人源化抗体或抗原结合片段引入第二半光氨酸残基所在的位点能够正确配对,所述正确配对的位点位于所述hIL-2变体与所述抗IL-2人源化抗体或其抗原结合片段的抗原-抗体结合界面,并且配对氨基酸残基之间的空间距离小于10埃,优选地,所述空间距离小于5埃。
- 如权利要求26-30中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,在所述hIL-2/IL-2抗体融合蛋白中的hIL-2变体与抗IL-2人源化抗体或其抗原结合片段的抗原-抗体结合界面形成二硫键的第一半胱氨酸残基和第二半胱氨酸残基的配对选自以下一个、或两个、或两个以上配对氨基酸突变的组合:(1)所述第一半胱氨酸残基是所述hIL-2变体相对于如SEQ ID NO:1所示的野生型hIL-2的氨基酸序列的第42位氨基酸残基处具有的氨基酸突变F42C,或第64位氨基酸残基处具有的氨基酸突变K64C,或第65位氨基酸残基处具有的氨基酸突变P65C,和所述第二半胱氨酸残基是所述抗IL-2人源化抗体或其抗原结合片段通过在氨基酸序列为Z2TFZ3YZ4RELRWFAY(Z2=V或I;Z3=Y或L;Z4=S或T)的HCDR3第3位上引入氨基酸突变F3C所获得;和/或(2)所述第一半胱氨酸残基是所述hIL-2变体相对于如SEQ ID NO:1所示的野生型hIL-2 的氨基酸序列的第60位氨基酸残基处具有的氨基酸突变E60C,和所述第二半胱氨酸残基是所述抗IL-2人源化抗体或其抗原结合片段通过在其LCDR1第7位上引入氨基酸突变G7C或S7C所获得;和/或(3)所述第一半胱氨酸残基是所述hIL-2变体相对于如SEQ ID NO:1所示的野生型hIL-2的氨基酸序列的第61位氨基酸残基处具有的氨基酸突变E61C,和所述第二半胱氨酸残基是所述抗IL-2人源化抗体或其抗原结合片段通过在氨基酸序列为QASQZ5IZ6NYLS(Z5=D或Y;Z6=G或S)的LCDR1第7位上引入氨基酸突变G7C或S7C所获得,或所述第二半胱氨酸残基是所述抗IL-2人源化抗体或其抗原结合片段通过在氨基酸序列为QASQZ5IZ6NYLS(Z5=D或Y;Z6=G或S)的LCDR1第8位上引入氨基酸突变N8C所获得,或所述第二半胱氨酸残基是所述抗IL-2人源化抗体或其抗原结合片段通过在氨基酸序列为Z7AZ8SLAX4(Z7=S或Q;Z8=T;X4=S)的LCDR2第1位上引入氨基酸突变S1C或Q1C获得;和/或(4)所述第一半胱氨酸残基是所述hIL-2变体相对于如SEQ ID NO:1所示的野生型hIL-2的氨基酸序列的第104位氨基酸残基处具有的氨基酸突变M104C,和所述第二半胱氨酸残基是所述抗IL-2人源化抗体或其抗原结合片段通过在氨基酸序列为Z7AZ8SLAX4(Z7=S或Q;Z8=T;X4=S)的LCDR2第3位上引入氨基酸突变T3C所获得;和/或(5)所述第一半胱氨酸残基是所述hIL-2变体相对于如SEQ ID NO:1所示的野生型hIL-2的氨基酸序列的第102位氨基酸残基处具有的氨基酸突变T102C,所述第二半胱氨酸残基是所述抗IL-2人源化抗体或其抗原结合片段通过在其轻链可变区第67位上引入氨基酸突变S67C所获得,或所述第二半胱氨酸残基是所述抗IL-2人源化抗体或其抗原结合片段通过在其轻链可变区第68位上引入氨基酸突变G68C所获得;和/或(6)所述第一半胱氨酸残基是所述hIL-2变体相对于如SEQ ID NO:1所示的野生型hIL-2的氨基酸序列的第64位氨基酸残基处具有的氨基酸突变K64C,和所述第二半胱氨酸残基是所述抗IL-2人源化抗体或其抗原结合片段通过在氨基酸序列为QASQZ5IZ6NYLS(Z5=D或Y;Z6=G或S)的LCDR1第9位上引入氨基酸突变Y9C所获得,或所述第二半胱氨酸残基是所述抗IL-2人源化抗体或其抗原结合片段通过在氨基酸序列为LQHYSTPYT(SEQ ID NO:12)的LCDR3第4位上引入氨基酸突变Y4C所获得;或者,在所述hIL-2/IL-2抗体融合蛋白中的hIL-2变体与抗IL-2人源化抗体或其抗原结合片段的抗原-抗体结合界面形成二硫键的第一半胱氨酸残基和第二半胱氨酸残基的配对选自以下一个、或两个、或两个以上配对氨基酸突变的组合:(1)所述第一半胱氨酸残基是所述hIL-2变体相对于如SEQ ID NO:1所示的野生型hIL-2的氨基酸序列的第104位氨基酸残基处具有的氨基酸突变M104C,和所述第二半胱氨酸残基是所述抗IL-2人源化抗体或其抗原结合片段通过在其LCDR2第3位上引入氨基酸突变T3C所获得,所述抗IL-2人源化抗体或其抗原结合片段包含SEQ ID NO:14所示的HCDR1氨基酸序列、SEQ ID NO:19所示的HCDR2氨基酸序列、SEQ ID NO:24所示的HCDR3氨基酸序列和SEQ ID NO:26所示的LCDR1氨基酸序列、SEQ ID NO:17所示的LCDR2氨基酸序列和SEQ ID NO:12所示的LCDR3氨基酸序列;和/或(2)所述第一半胱氨酸残基是所述hIL-2变体相对于如SEQ ID NO:1所示的野生型hIL-2的氨基酸序列的第102位氨基酸残基处具有的氨基酸突变T102C,所述第二半胱氨酸残基是所述抗IL-2人源化抗体或其抗原结合片段通过在其轻链可变区第67位上引入氨基酸突变S67C所获得,所述抗IL-2人源化抗体或其结合片段包含与SEQ ID NO:77所示的VL具有85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列,或所述第二半胱氨酸残基是所述抗IL-2人源化抗体或其抗原结合片段通过在其轻链可变区第68位上引入氨基酸突变G68C所获得,所述抗IL-2人源化抗体或其结合片段包含与SEQ ID NO:77所示的VL具有85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;和/或(3)所述第一半胱氨酸残基是所述hIL-2变体相对于如SEQ ID NO:1所示的野生型hIL-2的氨基酸序列的第42位氨基酸残基处具有的氨基酸突变F42C,或第64位氨基酸残基处具有的氨基酸突变K64C,或第65位氨基酸残基处具有的氨基酸突变P65C,和所述第二半胱氨酸 残基是所述抗IL-2人源化抗体或其抗原结合片段通过在氨基酸序列为Z2TFZ3YZ4RELRWFAY(Z2=V或I;Z3=Y或L;Z4=S或T)的HCDR3第3位上引入氨基酸突变F3C所获得,所述抗IL-2人源化抗体或其抗原结合片段包含SEQ ID NO:14所示的HCDR1氨基酸序列、SEQ ID NO:15所示的HCDR2氨基酸序列、SEQ ID NO:9所示的HCDR3氨基酸序列和SEQ ID NO:10所示的LCDR1氨基酸序列、SEQ ID NO:17所示的LCDR2氨基酸序列和SEQ ID NO:12所示的LCDR3氨基酸序列。
- 如权利要求14-20中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,在所述野生型hIL-2与所述其它特异性识别hIL-2的IL2Rα结合位点的抗IL-2人源化抗体或其抗原结合片段形成的抗原-抗体复合物的结合界面引入二硫键,所述抗原-抗体复合物选自hIL-2∶NARA1抗原-抗体复合物和hIL-2∶TCB2抗原-抗体复合物,其中,所述NARA1或其抗原结合片段包含SEQ ID NO:142所示的HCDR1氨基酸序列、SEQ ID NO:143所示的HCDR2氨基酸序列、SEQ ID NO:144所示的HCDR3氨基酸序列,以及SEQ ID NO:145所示的LCDR1氨基酸序列、SEQ ID NO:146所示的LCDR2氨基酸序列、和SEQ ID NO:147所示的LCDR3氨基酸序列;TCB2或其抗原结合片段包含SEQ ID NO:170所示的HCDR1氨基酸序列、SEQ ID NO:171所示的HCDR2氨基酸序列、SEQ ID NO:172所示的HCDR3氨基酸序列,以及SEQ ID NO:173所示的LCDR1氨基酸序列、SEQ ID NO:174所示的LCDR2氨基酸序列、和SEQ ID NO:175所示的LCDR3氨基酸序列。
- 如权利要求32所述的hIL-2/IL-2抗体融合蛋白,其中,所述hIL-2∶NARA1抗原-抗体复合物的结合界面包含了hIL-2与IL2Ra的结合界面,所述的hIL-2∶NARA1抗原-抗体复合物的结合界面位于所述hIL-2的AB环、B螺旋和BC环,进一步,所述hIL-2∶NARA1抗原-抗体复合物的结合界面位于所述hIL-2的第30-43位或其邻近的氨基酸残基和第71-77位或其邻近的氨基酸残基。
- 如权利要求32或33所述的hIL-2/IL-2抗体融合蛋白,其中,所述hIL-2∶NARA1抗原-抗体复合物的结合界面包含两个或两个以上半胱氨酸残基以形成所述二硫键,其中,所述hIL-2相对于SEQ ID NO:1所示的野生型hIL-2的氨基酸序列在第34位、第37位、第41位、第42位、第68位、第73位、第75位、第77位和第111位氨基酸残基处具有一个、或两个、或两个以上氨基酸突变,以引入第一个半胱氨酸残基;在NARA1抗体或其抗原结合片段的CDR区中特定氨基酸残基处具有一个、或两个、或两个以上氨基酸突变,以引入第二个半胱氨酸残基,所述氨基酸突变包括位于NARA1或其抗原结合片段的HCDR1第1位,和/或位于HCDR2第2位、第6位和/或第8位,和/或位于HCDR3第3位和/或第8位,和/或位于LCDR1第9位,和/或位于LCDR3第3位、第4位和/或第6位氨基酸残基的突变。
- 如权利要求32-34中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,在所述hIL-2∶NARA1抗原-抗体复合物的结合界面或相邻位置形成二硫键的第一半胱氨酸残基和第二半胱氨酸残基的配对选自以下一个、或两个、或两个以上配对氨基酸突变的组合:(1)所述第一半胱氨酸残基是所述hIL-2的氨基酸序列的第34位氨基酸残基处具有的氨基酸突变P34C,和所述第二半胱氨酸残基是所述NARA1抗体或其抗原结合片段通过在其HCDR3第8位上引入氨基酸突变A8C所获得,或所述第二半胱氨酸残基是所述抗NARA1抗体或其抗原结合片段通过在LCDR3第3位或第4位分别引入氨基酸突变S3C或N4C所获得;和/或(2)所述第一半胱氨酸残基是所述hIL-2的氨基酸序列的第37位氨基酸残基处具有的氨基酸突变T37C,和所述第二半胱氨酸残基是所述NARA1抗体或其抗原结合片段通过在其LCDR3第6位上引入氨基酸突变D6C所获得;和/或(3)所述第一半胱氨酸残基是所述hIL-2的氨基酸序列的第41位氨基酸残基处具有的氨基酸突变T41C,和所述第二半胱氨酸残基是所述NARA1抗体或其抗原结合片段通过在其HCDR2第2位上引入氨基酸突变I2C所获得;和/或(4)所述第一半胱氨酸残基是所述hIL-2的氨基酸序列的第42位氨基酸残基处具有的氨基酸突变F42C,和所述第二半胱氨酸残基是所述NARA1抗体或其抗原结合片段通过在其HCDR2第8位上引入氨基酸突变G8C所获得;和/或(5)所述第一半胱氨酸残基是所述hIL-2的氨基酸序列的第68位氨基酸残基处具有的氨基酸突变E68C,和所述第二半胱氨酸残基是所述NARA1或其抗原结合片段通过在其HCDR1第1位上引入氨基酸突变N1C所获得;和/或(6)所述第一半胱氨酸残基是所述hIL-2的氨基酸序列的第73位氨基酸残基处具有的氨基酸 突变A73C,和所述第二半胱氨酸残基是所述NARA1或其抗原结合片段通过在其HCDR3第3位上引入氨基酸突变G3C所获得;和/或(7)所述第一半胱氨酸残基是所述hIL-2的氨基酸序列的第75位或第77位氨基酸残基处分别具有的氨基酸突变S75C或N77C,和所述第二半胱氨酸残基是所述NARA1或其抗原结合片段通过在其LCDR1第9位上引入氨基酸突变D9C所获得;和/或(8)所述第一半胱氨酸残基是所述hIL-2的氨基酸序列的第111位氨基酸残基处具有的氨基酸突变T111C,和所述第二半胱氨酸残基是所述NARA1抗体或其抗原结合片段通过在其HCDR2第6位上引入氨基酸突变S6C所获得;优选地,在所述hIL-2∶NARA1抗原-抗体复合物的结合界面形成二硫键的第一半胱氨酸残基和第二半胱氨酸残基的配对选自以下一个、或两个、或两个以上配对氨基酸突变的组合:(1)所述第一半胱氨酸残基是所述hIL-2的氨基酸序列的第37位氨基酸残基处具有的氨基酸突变T37C,和所述第二半胱氨酸残基是所述NARA1抗体或其抗原结合片段通过在其LCDR3第6位上引入氨基酸突变D6C所获得;或(2)所述第一半胱氨酸残基是所述hIL-2的氨基酸序列的第68位氨基酸残基处具有的氨基酸突变E68C,和所述第二半胱氨酸残基是所述NARA1抗体或其抗原结合片段通过在其HCDR1第1位上引入氨基酸突变N1C所获得;或(3)所述第一半胱氨酸残基是所述hIL-2的氨基酸序列的第75位或第77位氨基酸残基处具有的氨基酸突变S75C或N77C,和所述第二半胱氨酸残基是所述NARA1抗体或其抗原结合片段通过在其LCDR1第9位上引入氨基酸突变D9C所获得;和/或(4)所述第一半胱氨酸残基是所述hIL-2的氨基酸序列的第111位氨基酸残基处具有的氨基酸突变T111C,和所述第二半胱氨酸残基是所述NARA1抗体或其抗原结合片段通过在其HCDR2第6位上引入氨基酸突变S6C所获得。
- 如权利要求32所述的hIL-2/IL-2抗体融合蛋白,其中,所述hIL-2∶TCB2抗原-抗体复合物的结合界面包含了hIL-2与IL2Rα的结合界面,所述的hIL-2∶TCB2抗原-抗体复合物的结合界面位于所述hIL-2的AB环、B螺旋和CD环,进一步,所述hIL-2∶TCB2抗原-抗体复合物的结合界面位于所述hIL-2的第34-45位或其邻近的氨基酸残基、第62-76位或其邻近的氨基酸残基和第111位或其邻近的氨基酸残基。
- 如权利要求32或36所述的hIL-2/IL-2抗体融合蛋白,其中,所述hIL-2∶TCB2抗原-抗体复合物的结合界面包含两个或两个以上半胱氨酸残基以形成所述二硫键,其中,所述hIL-2相对于SEQ ID NO:1所示的野生型hIL-2的氨基酸序列在第34位、第36位、第37位、第38位、第42位、第68位和第109位氨基酸残基处具有一个、或两个、或两个以上氨基酸突变,以引入第一个半胱氨酸残基,在TCB2抗体或其抗原结合片段的CDR区中特定氨基酸残基处具有一个、或两个、或两个以上氨基酸突变,以引入第二个半胱氨酸残基,所述氨基酸突变包括位于TCB2或其抗原结合片段的HCDR2第6位,和/或位于HCDR3第3位、第5位、第6位、和/或第7位,和/或位于LCDR1第4位,位于LCDR3第4位、第5位、和/或第6位氨基酸残基的突变。
- 如权利要求32、36和37中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,在所述hIL-2∶TCB2抗原-抗体复合物的结合界面形成二硫键的第一半胱氨酸残基和第二半胱氨酸残基的配对选自以下一个、或两个、或两个以上配对氨基酸突变的组合:(1)所述第一半胱氨酸残基是所述hIL-2的氨基酸序列的第34位氨基酸残基处具有的氨基酸突变P34C,和所述第二半胱氨酸残基是所述TCB2或其抗原结合片段通过在其LCDR1第4位上引入氨基酸突变T4C所获得,或所述第二半胱氨酸是所述TCB2或其抗原结合片段通过在其LCDR3第4位或第5位上分别引入氨基酸突变D4C或N5C所获得;和/或(2)所述第一半胱氨酸残基是所述hIL-2的氨基酸序列的第36位氨基酸残基处具有的氨基酸突变L36C,和所述第二半胱氨酸残基是所述TCB2或其抗原结合片段通过在其LCDR3第5位上引入氨基酸突变N5C所获得;和/或(3)所述第一半胱氨酸残基是所述hIL-2的氨基酸序列的第37位氨基酸残基处具有的氨基酸突变T37C,和所述第二半胱氨酸残基是所述TCB2或其抗原结合片段通过在其LCDR3第5位或第6位上分别引入氨基酸突变N5C或L6C所获得;和/或(4)所述第一半胱氨酸残基是所述hIL-2的氨基酸序列的第38位氨基酸残基处具有的氨基酸突变R38C,和所述第二半胱氨酸残基是所述TCB2或其抗原结合片段通过在其HCDR3第7位上引入氨基酸突变F7C所获得;和/或(5)所述第一半胱氨酸残基是所述hIL-2的氨基酸序列的第42位氨基酸残基处具有的氨基酸突变F42C,和所述第二半胱氨酸残基是所述TCB2或其抗原结合片段通过在其HCDR3第5位上引入氨基酸突变R5C所获得;和/或(6)所述第一半胱氨酸残基是所述hIL-2的氨基酸序列的第68位氨基酸残基处具有的氨基酸突变E68C,和所述第二半胱氨酸残基是所述TCB2或其抗原结合片段通过在其HCDR3第3位或第6位上分别引入氨基酸突变A3C或G6C所获得;和/或(7)所述第一半胱氨酸残基是所述hIL-2的氨基酸序列的第109位氨基酸残基处具有的氨基酸突变D109C,和所述第二半胱氨酸残基是所述TCB2或其抗原结合片段通过在其HCDR2第6位上引入氨基酸突变D6C所获得;优选地,在所述hIL-2∶TCB2抗原-抗体复合物的结合界面形成二硫键的第一半胱氨酸残基和第二半胱氨酸残基的配对选自以下一个、或两个、或两个以上配对氨基酸突变的组合:(1)所述第一半胱氨酸残基是所述hIL-2的氨基酸序列的第34位氨基酸残基处具有的氨基酸突变P34C,和所述第二半胱氨酸是所述TCB2或其抗原结合片段通过在其LCDR3第4位或第5位上分别引入氨基酸突变D4C或N5C所获得;或(2)所述第一半胱氨酸残基是所述hIL-2的氨基酸序列的第36位氨基酸残基处具有的氨基酸突变L36C,和所述第二半胱氨酸残基是所述TCB2或其抗原结合片段通过在其LCDR3第5位上引入氨基酸突变N5C所获得;或(3)所述第一半胱氨酸残基是所述hIL-2的氨基酸序列的第37位氨基酸残基处具有的氨基酸突变T37C,和所述第二半胱氨酸残基是所述TCB2或其抗原结合片段通过在其LCDR3第6位上分别引入氨基酸突变L6C所获得;或(4)所述第一半胱氨酸残基是所述hIL-2的氨基酸序列的第42位氨基酸残基处具有的氨基酸突变F42C,和所述第二半胱氨酸残基是所述TCB2或其抗原结合片段通过在其HCDR3第5位上引入氨基酸突变R5C所获得;和/或(5)所述第一半胱氨酸残基是所述hIL-2的氨基酸序列的第68位氨基酸残基处具有的氨基酸突变E68C,和所述第二半胱氨酸残基是所述TCB2或其抗原结合片段通过在其HCDR3第3位上分别引入氨基酸突变A3C所获得。
- 如权利要求14-31中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,所述hIL-2/IL-2抗体融合蛋白包含重链可变区的HCDR1、HCDR2、HCDR3和轻链可变区的LCDR1、LCDR2、LCDR3,所述的HCDR3、LCDR1、LCDR2和/或LCDR3包含一个、或两个、或两个以上半胱氨酸残基突变,所述重链可变区的HCDR1、HCDR2、HCDR3和轻链可变区的LCDR1、LCDR2、LCDR3包括:(1)氨基酸序列为X1YYX2H(X1=A;X2=M,如SEQ ID NO:14所示)的HCDR1,(2)氨基酸序列为RIDPZ1DDSTKYAENFKX3(Z1=E或D;X3=G)的HCDR2,(3)氨基酸序列为Z2TX ⅡZ3YZ4RELRWFAY(Z2=V或I;X Ⅱ=F或C;Z3=Y或L;Z4=S或T)的HCDR3,(4)氨基酸序列为QASQZ5IZ6X ⅠX ⅣLS(Z5=D或Y;Z6=G,S或C;X Ⅰ=N或C;X Ⅳ=Y或C)的LCDR1,(5)氨基酸序列为Z7AZ8SLAX4(Z7=S,Q或C;Z8=T或C;X4=S)的LCDR2,和(6)氨基酸序列为LQHX ⅢSTPYT(X Ⅲ=Y或C)的LCDR3。
- 如权利要求39所述的hIL-2/IL-2抗体融合蛋白,其中,所述hIL-2/IL-2抗体融合蛋白包含重链可变区的HCDR1、HCDR2、HCDR3和轻链可变区的LCDR1、LCDR2、LCDR3,所述的HCDR3、LCDR1、LCDR2和/或LCDR3包含一个、或两个、或两个以上半胱氨酸残基突变,所述重链可变区的HCDR1、HCDR2、HCDR3和轻链可变区的LCDR1、LCDR2、LCDR3包括:(1)氨基酸序列如SEQ ID NO:14所示的HCDR1,氨基酸序列如SEQ ID NO:15所示的HCDR2,氨基酸序列如SEQ ID NO:9所示的HCDR3,氨基酸序列如SEQ ID NO:30或31所示的LCDR1,氨基酸序列如SEQ ID NO:17所示的LCDR2,氨基酸序列如SEQ ID NO:12所示的LCDR3;和/或(2)氨基酸序列如SEQ ID NO:14所示的HCDR1,氨基酸序列如SEQ ID NO:15所示的HCDR2,氨基酸序列如SEQ ID NO:9所示的HCDR3,氨基酸序列如SEQ ID NO:10所示的LCDR1,氨基酸序列如SEQ ID NO:17、32或33所示的LCDR2,氨基酸序列如SEQ ID NO:12所示的LCDR3;和/或(3)氨基酸序列如SEQ ID NO:14所示的HCDR1,氨基酸序列如SEQ ID NO:15所示的HCDR2,氨基酸序列如SEQ ID NO:29所示的HCDR3,氨基酸序列如SEQ ID NO:10所示的LCDR1,氨基酸序列如SEQ ID NO:17所示的LCDR2,氨基酸序列如SEQ ID NO:12所示的LCDR3;和/或(4)氨基酸序列如SEQ ID NO:14所示的HCDR1,氨基酸序列如SEQ ID NO:19所示的HCDR2,氨基酸序列如SEQ ID NO:24所示的HCDR3,氨基酸序列如SEQ ID NO:35或36所示的LCDR1,氨基酸序列如SEQ ID NO:17所示的LCDR2,氨基酸序列如SEQ ID NO:12所示的LCDR3;和/或(5)氨基酸序列如SEQ ID NO:14所示的HCDR1,氨基酸序列如SEQ ID NO:19所示的HCDR2,氨基酸序列如SEQ ID NO:24所示的HCDR3,氨基酸序列如SEQ ID NO:26所示的LCDR1,氨基酸序列如SEQ ID NO:17、32或33所示的LCDR2,氨基酸序列如SEQ ID NO:12所示的LCDR3;和/或(6)氨基酸序列如SEQ ID NO:14所示的HCDR1,氨基酸序列如SEQ ID NO:19所示的HCDR2,氨基酸序列如SEQ ID NO:34所示的HCDR3,氨基酸序列如SEQ ID NO:26所示的LCDR1,氨基酸序列如SEQ ID NO:17所示的LCDR2,氨基酸序列如SEQ ID NO:12所示的LCDR3;和/或(7)氨基酸序列如SEQ ID NO:14所示的HCDR1,氨基酸序列如SEQ ID NO:19所示的HCDR2,氨基酸序列如SEQ ID NO:24所示的HCDR3,氨基酸序列如SEQ ID NO:26所示的LCDR1,氨基酸序列如SEQ ID NO:17所示的LCDR2,氨基酸序列如SEQ ID NO:38所示的LCDR3;和/或(8)氨基酸序列如SEQ ID NO:14所示的HCDR1,氨基酸序列如SEQ ID NO:19所示的HCDR2,氨基酸序列如SEQ ID NO:24所示的HCDR3,氨基酸序列如SEQ ID NO:37所示的LCDR1,氨基酸序列如SEQ ID NO:17所示的LCDR2,氨基酸序列如SEQ ID NO:12所示的LCDR3,优选地,所述hIL-2/IL-2抗体融合蛋白包含如下的重链可变区的HCDR1、HCDR2、HCDR3和轻链可变区的LCDR1、LCDR2、LCDR3:(1)氨基酸序列如SEQ ID NO:14所示的HCDR1,氨基酸序列如SEQ ID NO:15所示的HCDR2,氨基酸序列如SEQ ID NO:29所示的HCDR3,氨基酸序列如SEQ ID NO:10所示的LCDR1,氨基酸序列如SEQ ID NO:17所示的LCDR2,氨基酸序列如SEQ ID NO:12所示的LCDR3;和/或(2)氨基酸序列如SEQ ID NO:14所示的HCDR1,氨基酸序列如SEQ ID NO:19所示的HCDR2,氨基酸序列如SEQ ID NO:24所示的HCDR3,氨基酸序列如SEQ ID NO:26所示的LCDR1,氨基酸序列如SEQ ID NO:17或33所示的LCDR2,氨基酸序列如SEQ ID NO:12所示的LCDR3。
- 如权利要求14-31、39或40中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,所述hIL-2/IL-2抗体融合蛋白在所述IL-2抗体的轻链可变区第一位或LCDR1第4位还可以具有氨基酸突变,所述氨基酸突变包括D1E、Q4D、Q4E、Q4S、Q4G、Q4K或Q4R,优选地,所述氨基酸突变包括D1E、Q4D、Q4E、Q4S或Q4G。
- 如权利要求14-20、32-35中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,所述hIL-2/IL-2抗体融合蛋白包含重链可变区的HCDR1、HCDR2、HCDR3和轻链可变区的LCDR1、LCDR2、LCDR3,所述的HCDR1、HCDR2、HCDR3、LCDR1、和/或LCDR3包含一个、或两个、或两个以上半胱氨酸残基突变,所述重链可变区的HCDR1、HCDR2、HCDR3和轻链可变区的LCDR1、LCDR2、LCDR3包括:(1)氨基酸序列如SEQ ID NO:142或148所示的HCDR1,(2)氨基酸序列如SEQ ID NO:143、149、150或151所示的HCDR2,(3)氨基酸序列如SEQ ID NO:144、152或153所示的HCDR3,(4)氨基酸序列如SEQ ID NO:145或154所示的LCDR1,(5)氨基酸序列如SEQ ID NO:146所示的LCDR2,(6)氨基酸序列如SEQ ID NO:147、155、156或157所示的LCDR3,进一步,所述重链可变区的HCDR1、HCDR2、HCDR3和轻链可变区的LCDR1、LCDR2、LCDR3包括:(1)氨基酸序列如SEQ ID NO:142所示的HCDR1,氨基酸序列如SEQ ID NO:149所示的HCDR2,氨基酸序列如SEQ ID NO:144所示的HCDR3,氨基酸序列如SEQ ID NO:145所示的LCDR1,氨基酸序列如SEQ ID NO:146所示的LCDR2,氨基酸序列如SEQ ID NO:147所示的LCDR3;和/或(2)氨基酸序列如SEQ ID NO:142所示的HCDR1,氨基酸序列如SEQ ID NO:143所示的HCDR2,氨基酸序列如SEQ ID NO:152或153所示的HCDR3,氨基酸序列如SEQ ID NO:145所示的LCDR1,氨基酸序列如SEQ ID NO:146所示的LCDR2,氨基酸序列如SEQ ID NO:147所示的LCDR3;和/或(3)氨基酸序列如SEQ ID NO:142所示的HCDR1,氨基酸序列如SEQ ID NO:143所示的HCDR2,氨基酸序列如SEQ ID NO:144所示的HCDR3,氨基酸序列如SEQ ID NO:154所示的LCDR1,氨基酸序列如SEQ ID NO:146所示的LCDR2,氨基酸序列如SEQ ID NO:147所示的LCDR3;和/或(4)氨基酸序列如SEQ ID NO:142所示的HCDR1,氨基酸序列如SEQ ID NO:143所示的HCDR2,氨基酸序列如SEQ ID NO:144所示的HCDR3,氨基酸序列如SEQ ID NO:145所示的LCDR1,氨基酸序列如SEQ ID NO:146所示的LCDR2,氨基酸序列如SEQ ID NO:155、156或157所示的LCDR3;和/或(5)氨基酸序列如SEQ ID NO:148所示的HCDR1,氨基酸序列如SEQ ID NO:143所示的HCDR2,氨基酸序列如SEQ ID NO:144所示的HCDR3,氨基酸序列如SEQ ID NO:145所示的 LCDR1,氨基酸序列如SEQ ID NO:146所示的LCDR2,氨基酸序列如SEQ ID NO:147所示的LCDR3;和/或(6)氨基酸序列如SEQ ID NO:142所示的HCDR1,氨基酸序列如SEQ ID NO:150或151所示的HCDR2,氨基酸序列如SEQ ID NO:144所示的HCDR3,氨基酸序列如SEQ ID NO:145所示的LCDR1,氨基酸序列如SEQ ID NO:146所示的LCDR2,氨基酸序列如SEQ ID NO:147所示的LCDR3;优选地,所述的hIL-2/IL-2抗体融合蛋白包含如下的所述重链可变区的HCDR1、HCDR2、HCDR3和轻链可变区的LCDR1、LCDR2、LCDR3:(1)氨基酸序列如SEQ ID NO:142所示的HCDR1,氨基酸序列如SEQ ID NO:149所示的HCDR2,氨基酸序列如SEQ ID NO:144所示的HCDR3,氨基酸序列如SEQ ID NO:145所示的LCDR1,氨基酸序列如SEQ ID NO:146所示的LCDR2,氨基酸序列如SEQ ID NO:147所示的LCDR3;和/或(2)氨基酸序列如SEQ ID NO:142所示的HCDR1,氨基酸序列如SEQ ID NO:143所示的HCDR2,氨基酸序列如SEQ ID NO:144所示的HCDR3,氨基酸序列如SEQ ID NO:154所示的LCDR1,氨基酸序列如SEQ ID NO:146所示的LCDR2,氨基酸序列如SEQ ID NO:147所示的LCDR3;和/或(3)氨基酸序列如SEQ ID NO:142所示的HCDR1,氨基酸序列如SEQ ID NO:143所示的HCDR2,氨基酸序列如SEQ ID NO:144所示的HCDR3,氨基酸序列如SEQ ID NO:145所示的LCDR1,氨基酸序列如SEQ ID NO:146所示的LCDR2,氨基酸序列如SEQ ID NO:157所示的LCDR3;和/或(4)氨基酸序列如SEQ ID NO:148所示的HCDR1,氨基酸序列如SEQ ID NO:143所示的HCDR2,氨基酸序列如SEQ ID NO:144所示的HCDR3,氨基酸序列如SEQ ID NO:145所示的LCDR1,氨基酸序列如SEQ ID NO:146所示的LCDR2,氨基酸序列如SEQ ID NO:147所示的LCDR3。
- 如权利要求14-20、32、36-38中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,所述hIL-2/IL-2抗体融合蛋白包含重链可变区的HCDR1、HCDR2、HCDR3和轻链可变区的LCDR1、LCDR2、LCDR3,所述的HCDR2、HCDR3、LCDR1、和/或LCDR3包含一个、或两个、或两个以上半胱氨酸残基突变,所述重链可变区的HCDR1、HCDR2、HCDR3和轻链可变区的LCDR1、LCDR2、LCDR3包括:(1)氨基酸序列如SEQ ID NO:170所示的HCDR1,(2)氨基酸序列如SEQ ID NO:171或176所示的HCDR2,(3)氨基酸序列如SEQ ID NO:172、177、178、179或180所示的HCDR3,(4)氨基酸序列如SEQ ID NO:173或181所示的LCDR1,(5)氨基酸序列如SEQ ID NO:174所示的LCDR2,(6)氨基酸序列如SEQ ID NO:175、182、183或184所示的LCDR3,进一步,所述重链可变区的HCDR1、HCDR2、HCDR3和轻链可变区的LCDR1、LCDR2、LCDR3包括:(1)氨基酸序列如SEQ ID NO:170所示的HCDR1,氨基酸序列如SEQ ID NO:171所示的HCDR2,氨基酸序列如SEQ ID NO:177、178、179或180所示的HCDR3,氨基酸序列如SEQ ID NO:173所示的LCDR1,氨基酸序列如SEQ ID NO:174所示的LCDR2,氨基酸序列如SEQ ID NO:175所示的LCDR3;和/或(2)氨基酸序列如SEQ ID NO:170所示的HCDR1,氨基酸序列如SEQ ID NO:176所示的HCDR2,氨基酸序列如SEQ ID NO:172所示的HCDR3,氨基酸序列如SEQ ID NO:173所示的LCDR1,氨基酸序列如SEQ ID NO:174所示的LCDR2,氨基酸序列如SEQ ID NO:175所示的LCDR3;和/或(3)氨基酸序列如SEQ ID NO:170所示的HCDR1,氨基酸序列如SEQ ID NO:171所示的HCDR2,氨基酸序列如SEQ ID NO:172所示的HCDR3,氨基酸序列如SEQ ID NO:181所示的LCDR1,氨基酸序列如SEQ ID NO:174所示的LCDR2,氨基酸序列如SEQ ID NO:175所示的LCDR3;和/或(4)氨基酸序列如SEQ ID NO:170所示的HCDR1,氨基酸序列如SEQ ID NO:171所示的HCDR2,氨基酸序列如SEQ ID NO:172所示的HCDR3,氨基酸序列如SEQ ID NO:173所示的LCDR1,氨基酸序列如SEQ ID NO:174所示的LCDR2,氨基酸序列如SEQ ID NO:182、183或184所示的LCDR3;优选地,所述的hIL-2/IL-2抗体融合蛋白包含如下的所述重链可变区的HCDR1、HCDR2、HCDR3和轻链可变区的LCDR1、LCDR2、LCDR3:(1)氨基酸序列如SEQ ID NO:170所示的HCDR1,氨基酸序列如SEQ ID NO:171所示 的HCDR2,氨基酸序列如SEQ ID NO:177或178所示的HCDR3,氨基酸序列如SEQ ID NO:173所示的LCDR1,氨基酸序列如SEQ ID NO:174所示的LCDR2,氨基酸序列如SEQ ID NO:175所示的LCDR3;和/或(2)氨基酸序列如SEQ ID NO:170所示的HCDR1,氨基酸序列如SEQ ID NO:171所示的HCDR2,氨基酸序列如SEQ ID NO:172所示的HCDR3,氨基酸序列如SEQ ID NO:173所示的LCDR1,氨基酸序列如SEQ ID NO:174所示的LCDR2,氨基酸序列如SEQ ID NO:182、183或184所示的LCDR3。
- 如权利要求14-31、39、40中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,所述hIL-2/IL-2抗体融合蛋白包含具有所述HCDR1、HCDR2和HCDR3的重链可变区和/或具有所述LCDR1、LCDR2和LCDR3的轻链可变区,所述重链可变区包含分别与SEQ ID NO:63、86、74或112所示的序列具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列,和/或轻链可变区包含分别与SEQ ID NO:64、77、80、81、82、83、84、85、87、88、89、90、91、92、93、104、105、106、107、108、109、110或111所示的序列具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列,优选地,所述重链可变区包含分别与SEQ ID NO:86或74所示的序列具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列,和/或轻链可变区包含分别与SEQ ID NO:64、87、88、89、90、91、107、108或109所示的序列具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或者,所述hIL-2/IL-2抗体融合蛋白包含重链可变区和轻链可变区,所述重链可变区和轻链可变区包括:(1)分别与SEQ ID NO:86所示的重链可变区和SEQ ID NO:64、87、88、89、90或91所示的轻链可变区具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(2)分别与SEQ ID NO:74所示的重链可变区和SEQ ID NO:107、108或109所示的轻链可变区具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列。
- 如权利要求14-20、32-35和42中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,所述hIL-2/IL-2抗体融合蛋白包含具有所述HCDR1、HCDR2和HCDR3的重链可变区和/或具有所述LCDR1、LCDR2和LCDR3的轻链可变区,所述重链可变区包含分别与SEQ ID NO:140、158、159、160、161、162或163所示的序列具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列,和/或轻链可变区包含分别与SEQ ID NO:141、164、165、166或167所示的序列具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列,进一步,所述hIL-2/IL-2抗体融合蛋白包含重链可变区和轻链可变区,所述重链可变区和轻链可变区包括:(1)分别与SEQ ID NO:158、159、160、161、162或163所示的重链可变区和SEQ ID NO:141所示的轻链可变区具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(2)分别与SEQ ID NO:140所示的重链可变区和SEQ ID NO:164、165、166或167所示的轻链可变区具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;优选地,所述hIL-2/IL-2抗体融合蛋白包含如下的重链可变区和轻链可变区:(1)分别与SEQ ID NO:158或161所示的重链可变区和SEQ ID NO:141所示的轻链可变区具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(2)分别与SEQ ID NO:140所示的重链可变区和SEQ ID NO:164或167所示的轻链可变区具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列。
- 如权利要求14-20、32、36-38和43中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,所述hIL-2/IL-2抗体融合蛋白包含具有所述HCDR1、HCDR2和HCDR3的重链可变区和/或具有所述 LCDR1、LCDR2和LCDR3的轻链可变区,所述重链可变区包含分别与SEQ ID NO:168、185、186、187、188或189所示的序列具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列,和/或轻链可变区包含分别与SEQ ID NO:169、190、191、192或193所示的序列具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列,进一步,所述hIL-2/IL-2抗体融合蛋白包含如下的重链可变区和轻链可变区:(1)分别与SEQ ID NO:185、186、187、188或189所示的重链可变区和SEQ ID NO:169所示的轻链可变区具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(2)分别与SEQ ID NO:168所示的重链可变区和SEQ ID NO:190、191、192或193所示的轻链可变区具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;优选地,所述hIL-2/IL-2抗体融合蛋白包含如下的重链可变区和轻链可变区:(1)分别与SEQ ID NO:185或186所示的重链可变区和SEQ ID NO:169所示的轻链可变区具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;(2)分别与SEQ ID NO:168所示的重链可变区和SEQ ID NO:191、192或193所示的轻链可变区具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列。
- 如权利要求14-46中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,所述抗IL-2抗体或其抗原片段与hIL-2或其变体可以通过连接子连接,所述连接子选自包括(G4S)4、(G4S)3、(GAF)2,优选地,所述的连接子为(G4S)4。
- 如权利要求14-47中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,所述hIL-2或其变体进一步包含一种、或两种或其以上氨基酸突变:相对于SEQ ID NO:1所示的野生型hIL-2的氨基酸序列的N端前三个或前五个氨基酸存在缺失;或相对于SEQ ID NO:1所示的野生型hIL-2的氨基酸序列的N端第三个氨基酸残基具有氨基酸突变;和/或相对于如SEQ ID NO:1所示的野生型hIL-2的第氨基酸序列的125位氨基酸残基处具有氨基酸突变C125A、C125L、C125S、C125Q或C125V,优选地,所述的hIL-2或其变体相对于SEQ ID NO:1所示的野生型hIL-2的氨基酸序列的N端前三个氨基酸存在缺失,以及相对于如SEQ ID NO:1所示的野生型hIL-2的氨基酸序列在第125位氨基酸残基处具有氨基酸突变C125A、C125L、C125S、C125Q或C125V。
- 如权利要求14-48中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,所述hIL-2/IL-2抗体融合蛋白包含第一多肽,所述第一多肽与SEQ ID NO:95、96、97、98、99、100、101、102、103、113、114、115、116、117、118、119、120、133、134、135、136、137、138、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225或226所示的序列具有至少85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99%或100%相同的氨基酸序列。
- 如权利要求14-49中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,所述hIL-2/IL-2抗体融合蛋白包含第一多肽和第二多肽,所述第一多肽包含具有半胱氨酸残基突变的hIL-2或其变体可操作地连接至来源于所述抗IL-2抗体或其抗原结合片段的一个可变区,第二多肽包含所述抗IL-2抗体或其抗原结合片段的另一个可变区;进一步,所述第一多肽包含具有半胱氨酸残基突变的hIL-2或其变体、连接子和所述抗IL-2抗体或其抗原结合片段的一个可变区,可以选择在所述第一多肽中的抗IL-2抗体或其抗原结合片段的一个可变区和/或第二多肽中的抗IL-2抗体或其抗原结合片段的另一个可变区进行半胱氨酸残基突变,由此可与具有半胱氨酸残基突变的hIL-2或其变体形成正确配对且稳定的二硫键,且所述二硫键位于所述hIL-2或其变体与所述抗IL-2抗体或其抗原结合片段的抗原-抗体结合界面。
- 如权利要求49或50所述的hIL-2/IL-2抗体融合蛋白,其中,所述的第一多肽包含具有半胱氨酸残基突变的hIL-2变体、连接子和抗IL-2抗体或其抗原结合片段的重链可变区,第二多 肽包含所述抗IL-2抗体或其抗原结合片段的轻链可变区,其中,所述第一多肽包含与SEQ ID NO:95-103、113-120、或133-138任意一个所示的序列具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列,和/或所述第二多肽包含与SEQ ID NO:64、77、80-85、87-93、或104-111任意一个所示的序列具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列。
- 如权利要求51所述的hIL-2/IL-2抗体融合蛋白,其中,所述hIL-2/IL-2抗体融合蛋白包含如下的第一多肽和第二多肽:(1)分别与SEQ ID NO:95所示的第一多肽和SEQ ID NO:80所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(2)分别与SEQ ID NO:96所示的第一多肽和SEQ ID NO:81所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(3)分别与SEQ ID NO:97所示的第一多肽和SEQ ID NO:82所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(4)分别与SEQ ID NO:98所示的第一多肽和SEQ ID NO:83所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(5)分别与SEQ ID NO:99所示的第一多肽和SEQ ID NO:84所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(6)分别与SEQ ID NO:100所示的第一多肽和SEQ ID NO:85所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(7)分别与SEQ ID NO:101、102、103、133-138任意一个所示的第一多肽和SEQ ID NO:64所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(8)分别与SEQ ID NO:102所示的第一多肽和SEQ ID NO:87-93任意一个所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(9)分别与SEQ ID NO:113所示的第一多肽和SEQ ID NO:104、105或106所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(10)分别与SEQ ID NO:114所示的第一多肽和SEQ ID NO:107所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(11)分别与SEQ ID NO:115所示的第一多肽和SEQ ID NO:108或109所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(12)分别与SEQ ID NO:116、117或119所示的第一多肽和SEQ ID NO:77所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(13)分别与SEQ ID NO:118所示的第一多肽和SEQ ID NO:110所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(14)分别与SEQ ID NO:120所示的第一多肽和SEQ ID NO:104所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(15)分别与SEQ ID NO:118所示的第一多肽和SEQ ID NO:111所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;优选地,所述hIL-2/IL-2抗体融合蛋白包含如下的第一多肽和第二多肽:(1)分别与SEQ ID NO:101、102、103、133-138任意一个所示的第一多肽和SEQ ID NO:64所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(2)分别与SEQ ID NO:102所示的第一多肽和SEQ ID NO:87-91任意一个所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(3)分别与SEQ ID NO:114所示的第一多肽和SEQ ID NO:107所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(4)分别与SEQ ID NO:115所示的第一多肽和SEQ ID NO:108或109所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列。
- 如权利要求49或50所述的hIL-2/IL-2抗体融合蛋白,其中,所述的第一多肽包含具有半胱氨酸残基突变的hIL-2变体、连接子和抗IL-2抗体或其抗原结合片段的轻链可变区,第二多肽包含所述抗IL-2抗体或其抗原结合片段的轻链可变区,其中,所述第一多肽包含与SEQ ID NOs:206-226任意一个所示的序列具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列,和/或所述第二多肽包含与SEQ ID NO:140、158-163、168、或185-189任意一个所示的序列具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列。
- 如权利要求53所述的hIL-2/IL-2抗体融合蛋白,其中,所述hIL-2/IL-2抗体融合蛋白包含如下的第一多肽和第二多肽:(1)分别与SEQ ID NO:206所示的第一多肽和SEQ ID NO:158所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(2)分别与SEQ ID NO:207所示的第一多肽和SEQ ID NO:159所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(3)分别与SEQ ID NO:208所示的第一多肽和SEQ ID NO:160所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(4)分别与SEQ ID NO:209-213任意一个所示的第一多肽和SEQ ID NO:140所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(5)分别与SEQ ID NO:214所示的第一多肽和SEQ ID NO:161所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(6)分别与SEQ ID NO:215所示的第一多肽和SEQ ID NO:162所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(7)分别与SEQ ID NO:216所示的第一多肽和SEQ ID NO:163所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(8)分别与SEQ ID NO:217所示的第一多肽和SEQ ID NO:185或187所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(9)分别与SEQ ID NO:218所示的第一多肽和SEQ ID NO:186所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(10)分别与SEQ ID NO:219所示的第一多肽和SEQ ID NO:188所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(11)分别与SEQ ID NO:220任意一个所示的第一多肽和SEQ ID NO:189所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(12)分别与SEQ ID NO:221-226任意一个所示的第一多肽和SEQ ID NO:168所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;优选地,所述hIL-2/IL-2抗体融合蛋白包含如下的第一多肽和第二多肽:(1)分别与SEQ ID NO:206所示的第一多肽和SEQ ID NO:158所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(2)分别与SEQ ID NO:209、210或213所示的第一多肽和SEQ ID NO:140所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(3)分别与SEQ ID NO:214所示的第一多肽和SEQ ID NO:161所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(4)分别与SEQ ID NO:217所示的第一多肽和SEQ ID NO:185所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(5)分别与SEQ ID NO:218所示的第一多肽和SEQ ID NO:186所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(6)分别与SEQ ID NO:222、223、224或226所示的第一多肽和SEQ ID NO:168所示的第二多肽具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列。
- 一种hIL-2/IL-2抗体融合蛋白,其包含:(1)如权利要求1-5中任一项所述的hIL-2变体,(2)如权利要求6-13中任一项所述的抗IL-2抗体或其抗原结合片段,以及(3)所述hIL-2变体与所述抗IL-2抗体或其抗原结合片段的抗原-抗体结合界面包含二硫键,其中,所述抗IL-2抗体或其抗原结合片段能够与IL2Rα竞争性结合所述hIL-2变体并且能够阻断所述hIL-2变体与IL2Rα或包含有α亚基的IL-2受体聚合体(即IL2Rα/β或IL2Rα/β/γ)的结合,以及所述的抗原-抗体结合界面与hIL-2变体与IL2Rα的结合界面存在部分重叠,所述二硫键是通过在所述抗原-抗体结合界面的hIL-2变体的序列中引入的至少一个半胱氨酸残基与通过在所述抗原-抗体结合界面的抗IL-2抗体或其抗原结合片段的序列中引入的至少一个半胱氨酸残基所形成的,所述的hIL-2/IL-2抗体融合蛋白不与IL2R的α亚基,或α/β二聚体,或α/β/γ三聚体结合,但是保留了对IL2Rβ和γc受体亚基二聚体(IL2Rβ/γ)的结合以及功能活性。
- 如权利要求55所述的hIL-2/IL-2抗体融合蛋白,其中,所述hIL-2变体与所述抗IL-2抗体或其抗原结合片段的抗原-抗体结合界面位于所述hIL-2变体的AB环、B螺旋、C螺旋和CD环,优选位于所述hIL-2变体的AB环、B螺旋、C螺旋和CD环;所述的hIL-2变体的IL2Ra结合界面位于hIL-2变体的AB环和B螺旋。
- 如权利要求55或56所述的hIL-2/IL-2抗体融合蛋白,其中,所述hIL-2变体与所述抗IL-2抗体或其抗原结合片段的抗原-抗体结合界面位于所述hIL-2变体的第30-45位或其邻近的氨基酸残基,第57-77位或其邻近的氨基酸残基,和/或第90-111位或其邻近的氨基酸残基,优选地,所述hIL-2变体与所述抗IL-2抗体或其抗原结合片段的抗原-抗体结合界面位于所述hIL-2变体的第41-45位或其邻近的氨基酸残基、第57-68位或其邻近的氨基酸残基以及第90-107位或其邻近的氨基酸残基。
- 如权利要求55-57中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,所述的hIL-2/IL-2抗体融合蛋白中的hIL-2变体与所述抗IL-2抗体或其抗原结合片段的抗原-抗体结合界面包含两个或两个以上由以下所述的半胱氨酸残基形成所述的二硫键:(1)所述hIL-2变体相对于SEQ ID NO:1所示的野生型hIL-2的氨基酸序列在第42位、第64位、第65位、第102位和第104位氨基酸残基处具有一个、或两个、或两个以上氨基酸突变,以引入第一个半胱氨酸残基;和(2)如权利要求6-13中任一项所述抗IL-2抗体或其抗原结合片段在HCDR3的第3位氨基酸残基处和LCDR2的第3位氨基酸残基处具有一个或两个氨基酸突变,以引入第二个半胱氨酸残基;或如权利要求13所述抗IL-2抗体或其抗原结合片段在其轻链可变区的框架区具有一个或两个氨基酸突变,以引入第二个半胱氨酸残基,所述氨基酸突变位于所述轻链可变区的第67和/或68位氨基酸残基处;在所述hIL-2变体引入第一个半胱氨酸残基所在的位点和在所述抗IL-2人源化抗体或抗原结合片段引入第二个半光氨酸残基所在的位点能够正确配对,所述正确配对的位点位于所述hIL-2变体与所述抗IL-2抗体或其抗原结合片段的抗原-抗体结合界面,并且配对氨基酸残基之间的空间距离小于10埃,优选地,所述空间距离小于5埃。
- 如权利要求55-58中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,在所述hIL-2/IL-2抗体融合蛋白中的hIL-2变体与抗IL-2抗体或其抗原结合片段的抗原-抗体结合界面形成二硫键的第一半胱氨酸残基和第二半胱氨酸残基的配对选自以下一个、或两个、或两个以上配对氨基酸突变的组合:(1)所述第一半胱氨酸残基是所述hIL-2变体相对于如SEQ ID NO:1所示的野生型hIL-2的氨基酸序列的第104位氨基酸残基处具有的氨基酸突变M104C,和所述第二半胱氨酸残基是所述抗IL-2抗体或其抗原结合片段通过在其LCDR2第3位上引入氨基酸突变T3C所获得,所述抗IL-2抗体或其抗原结合片段包含SEQ ID NO:14所示的HCDR1氨基酸序列、SEQ ID NO:19所示的HCDR2氨基酸序列、SEQ ID NO:24所示的HCDR3氨基酸序列和SEQ ID NO:26所示的LCDR1氨基酸序列、SEQ ID NO:17所示的LCDR2氨基酸序列和SEQ ID NO:12所示的LCDR3氨基酸序列;和/或(2)所述第一半胱氨酸残基是所述hIL-2变体相对于如SEQ ID NO:1所示的野生型hIL-2的氨基酸序列的第102位氨基酸残基处具有的氨基酸突变T102C,所述第二半胱氨酸残基是所述抗IL-2人源化抗体或其抗原结合片段通过在其轻链可变区第67位上引入氨基酸突变S67C所获得,所述抗IL-2抗体或其结合片段包含与SEQ ID NO:77所示的VL具有85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列,或所述第二半胱氨酸残基是所述抗IL-2抗体或其抗原结合片段通过在其轻链可变区第68位上引入氨基酸突变G68C所获得,所述抗IL-2抗体或其结合片段包含与SEQ ID NO:77所示的VL具有85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;和/或(3)所述第一半胱氨酸残基是所述hIL-2变体相对于如SEQ ID NO:1所示的野生型hIL-2的氨基酸序列的第42位氨基酸残基处具有的氨基酸突变F42C,或第64位氨基酸残基处具有的氨基酸突变K64C,或第65位氨基酸残基处具有的氨基酸突变P65C,和所述第二半胱氨酸残基是所述抗IL-2抗体或其抗原结合片段通过在其HCDR3第3位上引入氨基酸突变F3C所获得,所述抗IL-2抗体或其抗原结合片段包含SEQ ID NO:14所示的HCDR1氨基酸序列、SEQ ID NO:15所示的HCDR2氨基酸序列、SEQ ID NO:9所示的HCDR3氨基酸序列和SEQ ID NO:10或39-42任一项所示的LCDR1氨基酸序列、SEQ ID NO:17所示的LCDR2氨基酸序列和SEQ ID NO:12所示的LCDR3氨基酸序列。
- 如权利要求55-59中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,所述hIL-2/IL-2抗体融合蛋白包含重链可变区的HCDR1、HCDR2、HCDR3和轻链可变区的LCDR1、LCDR2、LCDR3,所述的HCDR3和/或LCDR2包含一个半胱氨酸残基突变,所述重链可变区的HCDR1、HCDR2、HCDR3和轻链可变区的LCDR1、LCDR2、LCDR3包括:(1)氨基酸序列如SEQ ID NO:14所示的HCDR1,氨基酸序列如SEQ ID NO:15所示的HCDR2,氨基酸序列如SEQ ID NO:29所示的HCDR3,氨基酸序列如SEQ ID NO:10或39-42任一项所示的LCDR1,氨基酸序列如SEQ ID NO:17所示的LCDR2,氨基酸序列如SEQ ID NO:12所示的LCDR3;或(2)氨基酸序列如SEQ ID NO:14所示的HCDR1,氨基酸序列如SEQ ID NO:19所示的HCDR2,氨基酸序列如SEQ ID NO:24所示的HCDR3,氨基酸序列如SEQ ID NO:26所示的LCDR1,氨基酸序列如SEQ ID NO:17或33所示的LCDR2,氨基酸序列如SEQ ID NO:12所示的LCDR3。
- 如权利要求55-60中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,所述hIL-2/IL-2抗体融合蛋白包含具有所述HCDR1、HCDR2和HCDR3的重链可变区,和/或具有所述LCDR1、LCDR2和LCDR3的轻链可变区,所述重链可变区包含分别与SEQ ID NO:86或74所示的序列具有至少85%、86%、87%、88%、 89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列,和/或轻链可变区包含分别与SEQ ID NO:64、87-91、107、108或109任一项所示的序列具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列。进一步,所述重链可变区和轻链可变区包括:(1)分别与SEQ ID NO:86所示的重链可变区和SEQ ID NO:64或87-91任一项所示的轻链可变区具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列;或(2)分别与SEQ ID NO:74所示的重链可变区和SEQ ID NO:107、108或109所示的轻链可变区具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同的氨基酸序列。
- 如权利要求14-61中任一项所述的hIL-2/IL-2抗体融合蛋白,其中,所述hIL-2/IL-2抗体融合蛋白还包括恒定区,所述恒定区包括抗体重链恒定区和轻链恒定区,其中,所述重链恒定区包括人IgG重链恒定区Fc区的天然和突变蛋白形式,还包括含有促进二聚体形成的铰链区的多肽截短形式,优选的Fc区来源于人IgG,包括IgG1、IgG2、IgG3和IgG4,进一步,所述的Fc区包含工程化的人IgG1 Fc区,其包含以下一种、或两种或其以上氨基酸取代:(1)L234F/L235E/P331S(EU编号系统),以降低Fc区与FcγRs和C1q的结合;和/或(2)N203D/K274Q/Q419E(EU编号系统),以延长所述hIL-2/IL-2融合蛋白在体内的半衰期。
- 一种核酸,其编码如权利要求1-5中任一项所述的hIL-2变体、权利要求6-13中任一项所述的抗IL-2抗体或其抗原结合片段、或权利要求14-62任一项所述的hIL-2/IL-2抗体融合蛋白的核酸。
- 一种表达载体,其能够表达权利要求63所述的核酸。
- 一种宿主细胞,包含权利要求63所述的核酸或权利要求64所述的表达载体。
- 一种使用权利要求65所述的宿主细胞制备权利要求1-5中任一项所述的hIL-2变体、权利要求6-13中任一项所述的抗IL-2抗体或其抗原结合片段、或权利要求14-62任一项所述的hIL-2/IL-2抗体融合蛋白的方法,包括:(i)在所述的宿主细胞中表达所述hIL-2变体、所述抗IL-2抗体或其抗原结合片段、或所述hIL-2/IL-2抗体融合蛋白,和(ii)从所述的宿主细胞或细胞培养物中分离所述hIL-2变体、所述抗IL-2抗体或其抗原结合片段、或所述hIL-2/IL-2抗体融合蛋白。
- 一种双特异性分子,包含如权利要求6-13中任一项所述的抗IL-2抗体或其抗原结合片段、或如权利要求14-62中任一项所述的hIL-2/IL-2抗体融合蛋白,所述双特异性抗体的两个臂之一包含所述抗IL-2抗体或其抗原结合片段、或hIL-2/IL-2抗体融合蛋白,而另一个臂包含对hIL-2以外的抗原(优选与癌症相关的抗原或免疫检查点蛋白抗原)具有特异性的抗体,或与免疫效应器细胞相关的抗原特异性结合的抗体或其抗原结合片段,所述与癌症相关的抗原可以选自HER2、EGFR、VEGF、VEGFR、CD19、CD20、CD30、CD33、CD52、BCMA、SLAMF7、MUC1、MUC16、EphB2、E-选择素、EpCam、CEA、PSMA、PSA、ERBb3、c-MET、ILT3、ILT7、5T4、GPC-3、DLL3等,所述免疫检查点蛋白抗原可以选自PD-1、PD-L1、PD-L2、TIM3、CTLA-4、LAG-3、CEACAM-1、CEACAM-5、VISTA、BTLA、TIGIT、LAIR1、CD40、CD40L、OX40、CD160、2B4或TGFR。
- 一种免疫缀合物、嵌合抗原受体、工程化T细胞受体、或溶瘤病毒,包含权利要求6-13中任一项所述的抗IL-2抗体或其抗原结合片段、或权利要求14-62中任一项所述hIL-2/IL-2抗体融合蛋白。
- 一种药物组合物,包含如权利要求6-13中任一项所述的抗IL-2抗体或其抗原结合片段、权利要求14-62中任一项所述的hIL-2/IL-2抗体融合蛋白、权利要求67所述的双特异性分子、或权利要求68所述的免疫缀合物、嵌合抗原受体、工程化T细胞受体、或溶瘤病毒,以及药学上可接受的载体。
- 一种药盒,包含权利要求69所述的药物组合物、任选的另一种抗癌剂和/或免疫调节剂,所述抗癌剂包括微管破坏物、抗代谢物、拓扑异构酶抑制剂、DNA嵌入剂、烷化剂、激素治疗物、激酶抑制剂(例如,酪氨酸激酶抑制剂包括EGFR抑制剂、HER2抑制剂、HER3抑制剂、IGFR抑制剂和Met抑制剂)、受体拮抗剂、肿瘤细胞凋亡的激活剂(包括IAP抑制剂、Bcl2抑制剂、MC11抑制剂、TRAIL抑制剂、CHK抑制剂)、或任何抗血管生成的药物;所述免疫调节剂包括PD-1、PD-L1、PD-L2、TIM3、CTLA-4、LAG-3、CEACAM-1、CEACAM-5、VISTA、BTLA、TIGIT、LAIR1、CD160、 2B4或TGFR抑制剂。
- 一种试剂盒,包括有效量的如权利要求6-13中任一项所述的抗IL-2抗体或其抗原结合片段、或权利要求14-62中任一项所述的hIL-2/IL-2抗体融合蛋白、或权利要求69所述的药物组合物。
- 如权利要求6-13中任一项所述的抗IL-2抗体或其抗原结合片段、或权利要求14-62中任一项所述的hIL-2/IL-2抗体融合蛋白在用于制备治疗疾病的药物组合物或制剂中的用途,所述疾病包括增殖性疾病或感染、免疫缺陷疾病,所述增殖性疾病包括癌症和其它细胞增殖病症。
- 如权利要求72所述的用途,其中,所述癌症包括黑色素瘤、肺癌、结直肠癌、前列腺癌、乳腺癌、卵巢癌、宫颈癌、肾癌、肝癌、脑癌、食道癌、胆囊癌、胰腺癌、胃癌、甲状腺癌、膀胱癌和淋巴瘤;所述免疫缺陷疾病包括HIV阳性、免疫抑制疾病、慢性感染。
- 一种通过提高宿主免疫响应来治疗疾病、病状或病症的方法,包括:向受试者施用有效量的如权利要求6-13所述的抗IL-2抗体或其抗原结合片段、或权利要求14-62所述的hIL-2/IL-2抗体融合蛋白、或权利要求69所述的药物组合物、或权利要求70所述的药盒、或权利要求71所述的试剂盒来特异性激活表达IL2Rβ/γ效应细胞(例如,CD8 +T细胞和NK细胞)的生物学活性,从而改善疾病状况。
- 一种刺激免疫系统的方法,包括:向受试者施用有效量的如权利要求6-13所述的抗IL-2抗体或其抗原结合片段、或权利要求14-62所述的hIL-2/IL-2抗体融合蛋白、或权利要求69所述的药物组合物、或权利要求70所述的药盒、或权利要求71所述的试剂盒来刺激免疫系统,包括免疫功能的一般升高、T细胞功能升高、B细胞功能升高、淋巴细胞功能的恢复、IL2Rβ/γ表达升高、T细胞应答性升高、天然杀伤细胞活性或淋巴因子激活的杀伤细胞活性升高、受试者体内CD8 +T细胞与CD4 +T细胞比率升高。
- 一种治疗、缓解或预防对宿主免疫系统的刺激是有益的疾病状况的方法,包括:向所述受试者施用治疗有效量的如权利要求6-13所述的抗IL-2抗体或其抗原结合片段、或权利要求14-62所述的hIL-2/IL-2抗体融合蛋白、或权利要求69所述的药物组合物、或权利要求70所述的药盒、或权利要求71所述的试剂盒,以通过增强所述受试者细胞免疫应答来改善疾病状况,所述疾病状况包括宿主免疫应答不足或免疫缺陷,进一步所述疾病状况是增殖性疾病或感染、免疫缺陷疾病,所述增殖性疾病包括癌症和其它细胞增殖病症。
- 如权利要求76所述的方法,其中,所述癌症选自鳞状细胞癌(例如上皮鳞状细胞癌)、肺癌(包括小细胞肺癌、非小细胞肺癌、肺的腺癌、和肺的鳞癌)、腹膜癌、肝细胞癌、胃癌(包括胃肠癌和胃肠基质癌)、胰腺癌、成胶质细胞瘤、宫颈癌、卵巢癌、肝癌、膀胱癌、尿道癌、肝瘤、乳腺癌、结肠癌、直肠癌、结肠直肠癌、子宫内膜癌或子宫癌、唾液腺癌、肾癌、前列腺癌、外阴癌、甲状腺癌、肝癌,肛门癌、阴茎癌、黑素瘤、浅表扩散性黑素瘤、恶性雀斑样痣黑素瘤、肢端黑素瘤、结节性黑素瘤、多发性骨髓瘤和B细胞淋巴瘤、非霍奇金淋巴瘤(Non-Hodgkin Lymphoma)、慢性淋巴细胞性白血病(CLL)、急性成淋巴细胞性白血病(ALL)、毛细胞性白血病、慢性成髓细胞性白血病、和移植后淋巴增殖性病症(PTLD)、以及与瘢痣病、水肿(例如与脑瘤有关的)和梅格斯氏综合征(Meigs syndrome)有关的异常血管增殖、脑瘤和脑癌、以及头颈癌。
- 如权利要求76所述的方法,进一步包含施用免疫检查点抑制剂、抗原特异性免疫治疗剂、化疗试剂、表观遗传修饰剂、细胞因子、生长因子、抑制剂、靶向肿瘤抗原的抗体、肿瘤疫苗、和/或其他癌症疗法,其中,所述的免疫检查点包括PD-1、PD-L1、PD-L2、TIM3、CTLA-4、LAG-3、CEACAM-1、CEACAM-5、VISTA、BTLA、TIGIT、LAIR1、CD160、2B4或TGFR;所述的抗原特异性免疫治疗剂包含嵌合抗原受体T细胞(CAR-T细胞)、嵌合抗原受体NK细胞(CAR-NK细胞)、和/或肿瘤浸润淋巴细胞(TIL);所述的其他癌症疗法包括外科手术、化学疗法、细胞毒性剂、光动力疗法、免疫调节或放射疗法。
- 如权利要求76所述的方法,其中,所述免疫缺陷疾病包括HIV阳性、免疫抑制疾病、慢性感染。
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CA3234995A CA3234995A1 (en) | 2021-10-14 | 2022-08-03 | Novel antibody-cytokine fusion protein, preparation method therefor and use thereof |
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