Method for purifying fusion protein

Abstract

The present disclosure relates to a method of purifying a fusion protein. In particular, the protein in the method is, for example, a fusion protein comprising a PD-1 binding domain and a cytokine.

Description

[0001] The present disclosure claims priority to the Chinese patent application “A method for purifying fusion proteins” (application number: 2023110388243) filed on August 16, 2023, which is incorporated herein by reference. Technical Field

[0002] The present disclosure relates to a method for purifying a fusion protein, such as a fusion protein comprising a PD-1 binding domain and a cytokine. Background Art

[0003] Antibodies can recognize specific epitopes and have good targeting properties. Immune cytokines have multiple functions including immunoregulation and cell growth regulation. The fusion protein obtained by fusing cytokines with antibodies has both targeting and immunoregulatory effects. It has shown superior therapeutic effects to antibodies in many fields such as tumor treatment and immunoregulation, and has broad application prospects in the biopharmaceutical industry. Compared with antibodies, antibody-cytokine fusion proteins have poor stability, and the culture products usually contain a variety of product-related impurities such as homodimers and degradation fragments, which poses a great challenge to the design of the purification process. How to optimize the purification process, effectively remove product-related impurities, and improve the purity of the target molecule is an important issue in current industrial production.

[0004] Knob into hole (KIH) is a widely used FC structural modification technology that can be used to promote the assembly of heterologous heavy chains and reduce the proportion of misassembly of homologous heavy chains, but it cannot completely avoid the formation of homodimers. To solve this problem, researchers have developed a variety of solutions, such as introducing engineered disulfide bonds between heavy chains and optimizing the charge distribution of the heavy chain assembly interface, which significantly reduced the proportion of homodimers in the expression product, but it still cannot be completely solved. Impurities such as homodimers, aggregates, and degradation fragments exist in antibodies or antibody fusion proteins based on the KIH model. The physical and chemical properties of homodimers and target molecules are slightly different, and it is difficult to remove them. Patent CN115066258A discloses a method for removing bispecific antibody homodimers, which uses the difference in binding force between homodimers, target molecules and κSelect affinity chromatography fillers to remove homodimers, but in this scheme, the light chains must be of different subtypes, and the cost of using κSelect affinity chromatography fillers is relatively high. Other literature reports that composite cationic chromatography materials (such as Capto MMC ImpRes) can be used to remove half antibodies, homodimers and aggregates, but there must be a significant charge difference between the homodimers and the target molecules (Tang J, Zhang X, Chen T, et al. Removal of half antibody, hole-hole homodimer and aggregates during bispecific antibody purification using MOL / LC ImpRes mixed-mode chromatography-Science Direct[J]. Protein Expression and Purification, 167.).

[0005] Currently, the prior art does not disclose a method for effectively removing homodimers based on universal Pro A affinity chromatography. Summary of the invention

[0006] The present disclosure relates to a method for purifying a fusion protein (e.g., a fusion protein of an antibody and a cytokine), which effectively suppresses the unstable state of the protein molecules during the purification process and effectively removes process-related impurities, especially effectively reducing the content of homodimers in the fusion protein.

[0007] The present disclosure provides a method for purifying a protein (e.g., a fusion protein), the method comprising the following steps:

[0008] (a) Sample loading step: loading the sample containing the fusion protein onto the affinity chromatography medium;

[0009] (b) Equilibration step: flushing the affinity chromatography medium after loading with equilibration buffer;

[0010] (c) elution step: eluting the affinity chromatography medium with a elution buffer;

[0011] (d) elution step: eluting the fusion protein from the affinity chromatography medium with an elution buffer and collecting the eluate;

[0012] In some embodiments, the isoelectric point (PI) of the fusion protein is about 4.0 to about 7.5, for example, the isoelectric point is about 4.5 to about 7.5, about 5.0 to about 7.5, about 5.0 to about 7.0, about 5.5 to about 7.0, about 6.0 to about 7.0, about 6.5 to about 7.0, about 6.0 to about 6.8, about 6.5 to about 6.8, or any range between these point values. In some embodiments, the fusion protein is a low isoelectric point antibody. For example, the isoelectric point of the fusion protein is lower than 7.0, for example, the isoelectric point is about 6.0 to about 7.0, about 6.5 to about 7.0, about 5.7 to about 6.8, about 6.5 to about 6.8, or any range between these point values. In some embodiments, the isoelectric point of the fusion protein is about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0.

[0013] In some embodiments, the fusion protein is a protein that can be chromatographed by an affinity chromatography column, such as an antibody or a fusion protein containing an antibody Fc. In some embodiments, the fusion protein is a fusion protein comprising a PD-1 binding domain, a cytokine, and an antibody Fc, wherein the cytokine is selected from IL-2 or a variant thereof.

[0014] In some embodiments, the PD-1 binding domain is located at the N-terminus of Fc, and the cytokine is located at the N-terminus or C-terminus of Fc. In some embodiments, the PD-1 binding domain is located at the N-terminus of Fc, and the cytokine is located at the C-terminus of Fc.

[0015] In some embodiments, the fusion protein comprises a first polypeptide chain and a second polypeptide chain. In some embodiments, the isoelectric point between the first polypeptide chain and the second polypeptide chain differs by no more than (≤) about 3.0 units, for example, about 1.0 to about 3.0 units, about 0.5 to about 3.0 units, about 0.5 to about 1.5 units, about 0.5 to about 2.0 units, about 0.5 to about 2.5 units, about 1.0 to about 1.5 units, about 1.0 to about 2.0 units, about 1.0 to about 2.5 units. For example, about 2.8 units, about 2.5 units, about 2.3 units, about 2.2 units, about 2.0 units, about 1.9 units, about 1.7 units, about 1.5 units, about 1.3 units, about 1.2 units, about 1.1 units, about 1.0 units, about 0.8 units, about 0.5 units. In some embodiments, the isoelectric point between the first polypeptide chain and the second polypeptide chain differs by no more than about 3.0 units. For example, no more than about 2.0 units, or 1.0 units. In some embodiments, the isoelectric point between the first polypeptide chain and the second polypeptide chain differs by no more than about 1.0 units.

[0016] In some embodiments, the pH of the first wash buffer is about 3.8 to about 5.0, for example, about 3.5 to about 4.7, about 3.5 to about 4.8, about 3.8 to about 4.5, about 4.0 to about 4.5, about 4.1 to about 4.3, or any range between these points. In some embodiments, the pH of the first wash buffer is about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5.

[0017] In some embodiments, the buffer substance in the first wash buffer is selected from one or more of sodium acetate, sodium citrate, Tris, sodium dihydrogen phosphate, disodium hydrogen phosphate, acetic acid, citric acid, and phosphoric acid. In some embodiments, the buffer substance in the first wash buffer is selected from acetic acid-sodium acetate, sodium citrate-citric acid, disodium hydrogen phosphate-citric acid, or Tris and disodium hydrogen phosphate-citric acid. In some embodiments, the buffer substance in the first wash buffer is acetic acid-sodium acetate.

[0018] In some embodiments, the concentration of the buffer substance in the first wash buffer is about 0.03 to about 0.1 mol / L, for example, about 0.03 to about 0.08 mol / L, about 0.03 to about 0.07 mol / L, about 0.04 to about 0.08 mol / L, about 0.04 to about 0.07 mol / L, about 0.04 to about 0.06 mol / L, or any range between these point values. In some embodiments, the concentration of the buffer substance in the first wash buffer is about 0.03 mol / L, about 0.04 mol / L, about 0.05 mol / L, about 0.06 mol / L, about 0.07 mol / L, about 0.08 mol / L, about 0.09 mol / L, about 0.1 mol / L. In some embodiments, the concentration of the buffer substance in the first wash buffer is about 0.05 mol / L.

[0019] In some embodiments, the first wash buffer further comprises an inorganic salt, and the inorganic salt is selected from one or more of sodium chloride, sodium sulfate, and ammonium sulfate. In some embodiments, the first wash buffer comprises sodium chloride.

[0020] In some embodiments, the concentration of the inorganic salt in the first wash buffer is about 0.1 mol / L to about 1.0 mol / L, for example, about 0.1 mol / L to about 0.8 mol / L, about 0.1 mol / L to about 0.7 mol / L, about 0.1 mol / L to about 0.6 mol / L, about 0.1 mol / L to about 0.5 mol / L, about 0.3 mol / L to about 1.0 mol / L, about 0.4 mol / L to about 1.0 mol / L, About 0.3mol / L to about 0.9mol / L, about 0.3 to about 0.8mol / L, about 0.3mol / L to about 0.7mol / L, about 0.4mol / L to about 0.7mol / L, about 0.4mol / L to about 0.6mol / L, about 0.4mol / L to about 0.5mol / L, about 0.3mol / L to about 0.5mol / L, about 0.3mol / L to about 0.6mol / L, or any range between these point values. In some embodiments, the concentration of inorganic salts in the first elution buffer is about 0.1mol / L, about 0.2mol / L, about 0.3mol / L, about 0.4mol / L, about 0.5mol / L, about 0.6mol / L, about 0.7mol / L, about 0.8mol / L, about 0.9mol / L. In some embodiments, the concentration of inorganic salts in the first elution buffer is about 0.4mol / L.

[0021] In some embodiments, the first wash buffer contains about 0.03 mol / L to about 0.1 mol / L acetic acid-sodium acetate, and about 0.2 mol / L to about 1.0 mol / L sodium chloride; for example, about 0.03 to about 0.1 mol / L acetic acid-sodium acetate, and about 0.3 mol / L to about 0.7 mol / L sodium chloride; about 0.04 to about 0.06 mol / L acetic acid-sodium acetate, and about 0.4 mol / L to about 0.5 mol / L sodium chloride.

[0022] In some embodiments, the first wash buffer is selected from any one of the following:

[0023] 1) 0.05 mol / L acetic acid-sodium acetate, 0.4 mol / L sodium chloride, pH 4.1;

[0024] 2) 0.05 mol / L acetic acid-sodium acetate, 0.5 mol / L sodium chloride, pH 4.1;

[0025] 3) 0.05 mol / L acetic acid-sodium acetate, 0.5 mol / L sodium chloride, pH 4.3;

[0026] 4) 0.05 mol / L acetic acid-sodium acetate, 0.1 mol / L sodium chloride, pH 4.3;

[0027] 5) 0.05 mol / L acetic acid-sodium acetate, 0.1 mol / L sodium chloride, pH 4.1;

[0028] 6) 0.05 mol / L acetic acid-sodium acetate, 0.5 mol / L sodium chloride, pH 4.1;

[0029] 7) 0.05 mol / L acetic acid-sodium acetate, 0.5 mol / L sodium chloride, pH 4.7;

[0030] 8) 0.05 mol / L acetic acid-sodium acetate, 0.1 mol / L sodium chloride, pH 4.7.

[0031] In some embodiments, the second wash buffer has a pH of about 4.5 to about 8.0, for example, about 4.5 to about 7.5, about 4.5 to about 7.0, about 4.5 to about 6.5, about 4.5 to about 6.0, about 4.0 to about 5.0, about 5.0 to about 6.0, about 4.5 to about 5.5, or any range between these point values. In some embodiments, the second wash buffer has a pH of about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.7, about 5.9, about 6.0, about 6.2, about 6.8, about 6.5, about 7.0, about 7.2, about 7.5, about 7.8.

[0032] In some embodiments, the buffer substance in the second wash buffer is selected from one or more of sodium acetate, sodium citrate, Tris, sodium dihydrogen phosphate, disodium hydrogen phosphate, acetic acid, citric acid, and phosphoric acid. In some embodiments, the buffer substance in the second wash buffer is selected from acetic acid-sodium acetate, sodium citrate-citric acid, disodium hydrogen phosphate-citric acid, or Tris and disodium hydrogen phosphate-citric acid. In some embodiments, the buffer substance in the second wash buffer is acetic acid-sodium acetate.

[0033] In some embodiments, the concentration of the buffer substance in the second wash buffer is about 0.03 to about 0.1 mol / L, for example, about 0.03 to about 0.08 mol / L, about 0.03 to about 0.07 mol / L, about 0.04 to about 0.08 mol / L, about 0.04 to about 0.07 mol / L, about 0.04 to about 0.06 mol / L, or any range between these point values. In some embodiments, the concentration of the buffer substance in the second wash buffer is about 0.03 mol / L, about 0.04 mol / L, about 0.05 mol / L, about 0.06 mol / L, about 0.07 mol / L, about 0.08 mol / L, about 0.09 mol / L, about 0.1 mol / L. In some embodiments, the concentration of the buffer substance in the second wash buffer is about 0.05 mol / L.

[0034] In some embodiments, the second wash buffer further comprises an inorganic salt selected from one or more of sodium chloride, sodium sulfate, and ammonium sulfate. In some embodiments, the second wash buffer comprises sodium chloride.

[0035] In some embodiments, the concentration of the inorganic salt in the second wash buffer is about 0.2 mol / L to about 1.0 mol / L, for example, about 0.3 mol / L to about 1.0 mol / L, about 0.4 mol / L to about 1.0 mol / L, about 0.3 mol / L to about 0.9 mol / L, about 0.3 to about 0.8 mol / L, about 0.3 mol / L to about 0.7 mol / L, about 0.4 mol / L to about 0.7 mol / L, about 0.4 mol / L to about 0.6 mol / L, about 0.4 mol / L to about 0.5 mol / L, about 0.3 mol / L to about 0.5 mol / L, about 0.3 mol / L to about 0.6 mol / L, or any range between these point values. In some embodiments, the concentration of the inorganic salt in the second wash buffer is about 0.2 mol / L, about 0.3 mol / L, about 0.4 mol / L, about 0.5 mol / L, about 0.6 mol / L, about 0.7 mol / L, about 0.8 mol / L, about 0.9 mol / L. In some embodiments, the concentration of the inorganic salt in the second wash buffer is about 0.4 mol / L.

[0036] In some embodiments, the second wash buffer contains about 0.03 mol / L to about 0.1 mol / L of acetic acid-sodium acetate, and about 0.2 mol / L to about 1.0 mol / L of sodium chloride; for example, about 0.03 to about 0.1 mol / L of acetic acid-sodium acetate, and about 0.3 mol / L to about 0.7 mol / L of sodium chloride; about 0.04 to about 0.06 mol / L of acetic acid-sodium acetate, and about 0.4 mol / L to about 0.5 mol / L of sodium chloride. In some embodiments, the second wash buffer contains about 0.03 mol / L to about 0.1 mol / L of acetic acid-sodium acetate, and does not contain sodium chloride.

[0037] In some embodiments, the second wash buffer is selected from any one of the following:

[0038] 1) 0.05 mol / L acetic acid-sodium acetate, 0.4 mol / L sodium chloride, pH 5.0;

[0039] 2) 0.05 mol / L acetic acid-sodium acetate, 0.5 mol / L sodium chloride, pH 5.0;

[0040] 3) 0.05 mol / L acetic acid-sodium acetate, 0.5 mol / L sodium chloride, pH 5.0;

[0041] 4) 0.05 mol / L acetic acid-sodium acetate, pH 4.5;

[0042] 5) 0.05 mol / L acetic acid-sodium acetate, pH 5.0;

[0043] 6) 0.05 mol / L acetic acid-sodium acetate, pH 5.0;

[0044] 7) 0.05 mol / L acetic acid-sodium acetate, pH 4.5;

[0045] 8) 0.05 mol / L acetic acid-sodium acetate, pH 4.5.

[0046] In some embodiments, the pH of the third wash buffer is about 4.5 to about 8.0, for example, about 4.5 to about 7.5, about 4.5 to about 7.0, about 4.5 to about 6.5, about 4.5 to about 6.0, about 4.0 to about 5.0, about 5.0 to about 6.0, about 4.5 to about 5.5, or any range between these point values. In some embodiments, the pH of the third wash buffer is about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.7, about 5.9, about 6.0, about 6.2, about 6.8, about 6.5, about 7.0, about 7.2, about 7.5, about 7.8.

[0047] In some embodiments, the buffer substance in the third wash buffer is selected from one or more of sodium acetate, sodium citrate, Tris, sodium dihydrogen phosphate, disodium hydrogen phosphate, acetic acid, citric acid, and phosphoric acid. In some embodiments, the buffer substance in the third wash buffer is selected from acetic acid-sodium acetate, sodium citrate-citric acid, disodium hydrogen phosphate-citric acid, or Tris and disodium hydrogen phosphate-citric acid. In some embodiments, the buffer substance in the third wash buffer is acetic acid-sodium acetate.

[0048] In some embodiments, the concentration of the buffer substance in the third elution buffer is about 0.03 to about 0.1 mol / L, for example, about 0.03 to about 0.08 mol / L, about 0.03 to about 0.07 mol / L, about 0.04 to about 0.08 mol / L, about 0.04 to about 0.07 mol / L, about 0.04 to about 0.06 mol / L, or any range between these point values. In some embodiments, the concentration of the buffer substance in the third elution buffer is about 0.03 mol / L, about 0.04 mol / L, about 0.05 mol / L, about 0.06 mol / L, about 0.07 mol / L, about 0.08 mol / L, about 0.09 mol / L, about 0.1 mol / L. In some embodiments, the concentration of the buffer substance in the third elution buffer is about 0.05 mol / L.

[0049] In some embodiments, the third wash buffer does not contain inorganic salts. In some embodiments, the third wash buffer contains about 0.03 to about 0.1 mol / L acetic acid-sodium acetate, such as about 0.04 mol / L to about 0.06 mol / L acetic acid-sodium acetate, such as about 0.05 mol / L acetic acid-sodium acetate.

[0050] In some embodiments, the conductivity of the third wash buffer is <5.0 mS / cm.

[0051] In some embodiments, the pH of the equilibration buffer is about 5.0 to about 8.0, for example, about 5.5 to about 8.0, about 6.5 to about 8.0, about 7.0 to about 8.0, about 7.0 to about 7.6, about 7.2 to about 7.6, about 6.8 to about 7.8, about 6.8 to about 8, or any range between these point values. In some embodiments, the pH of the third wash buffer is about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.7, about 5.9, about 6.0, about 6.2, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8.

[0052] In some embodiments, the equilibration buffer is a phosphate buffer, and the concentration of the phosphate buffer is about 0.01 mol / L to about 0.1 mol / L, preferably about 0.01 mol / L to about 0.05 mol / L, and more preferably about 0.01 mol / L to about 0.03 mol / L;

[0053] In some embodiments, the buffer substance in the equilibration buffer is a phosphate buffer, and the concentration of the phosphate buffer is about 0.01 mol / L to about 0.1 mol / L, for example, about 0.01 mol / L to about 0.05 mol / L, about 0.01 mol / L to about 0.06 mol / L, about 0.01 mol / L to about 0.08 mol / L, about 0.02 mol / L to about 0.05 mol / L, about 0.02 mol / L to about 0.06 mol / L, about 0.01 mol / L to about 0.03 mol / L, or any range between these point values. In some embodiments, the concentration of the phosphate buffer is about 0.01 mol / L, about 0.02 mol / L, about 0.03 mol / L, about 0.04 mol / L, about 0.05 mol / L, about 0.06 mol / L, about 0.07 mol / L, about 0.08 mol / L,

[0054] In some embodiments, the equilibrium buffer further comprises an inorganic salt. In some embodiments, the equilibrium buffer further comprises sodium chloride. In some embodiments, the concentration of sodium chloride in the equilibrium buffer is about 0.1mol / L to about 2.0mol / L, such as about 0.5mol / L to about 1.5mol / L, about 0.8mol / L to about 1.5mol / L, about 0.8mol / L to about 2.0mol / L, about 1.0mol / L to about 1.5mol / L, about 1.5mol / L to about 2.0mol / L, or any range between these point values. In some embodiments, the concentration of sodium chloride in the equilibrium buffer is about 0.1mol / L, about 0.2mol / L, about 0.3mol / L, about 0.5mol / L, about 0.7mol / L, about 0.8mol / L, about 1.0mol / L, about 1.2mol / L, about 1.4mol / L, about 1.6mol / L, about 1.8mol / L.

[0055] In some embodiments, the equilibration buffer contains about 0.01 mol / L to about 0.1 mol / L phosphate, and about 0.1 mol / L to about 2.0 mol / L sodium chloride; for example, contains about 0.01 mol / L to about 0.05 mol / L phosphate, and about 0.5 mol / L to about 1.5 mol / L sodium chloride.

[0056] In some embodiments, the equilibration buffer contains 0.02 mol / L phosphate, 1.0 mol / L sodium chloride, pH 7.4.

[0057] In some embodiments, the pH of the elution buffer is about 3.0 to about 5.0, such as about 3.4 to about 4.0, about 3.4 to about 3.8, about 3.0 to about 4.0, about 3.0 to about 3.8, about 3.6 to about 5.0, about 3.0 to about 3.6, about 3.0 to about 4.0, about 3.5 to about 5.0, about 3.8 to about 5.0, about 3.5 to about 4.5, or any range between these points. In some embodiments, the pH of the elution buffer is about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.2, about 4.3, about 4.5, about 4.7, about 4.9.

[0058] In some embodiments, the buffer substance of the elution buffer is acetic acid-sodium acetate, citric acid-sodium citrate, glycine-hydrochloric acid, etc.

[0059] In some embodiments, the (c) rinsing step comprises:

[0060] (c-1) eluting the affinity chromatography medium with a first washing buffer, wherein the pH of the first washing buffer is about 3.8 to about 5.0, for example, about 4.0 to about 4.7, or about 4.1 to about 4.3.

[0061] (c-2) The affinity chromatography medium is eluted with a second elution buffer to increase the pH of the affinity chromatography medium to above 4.5.

[0062] (c-3) The affinity chromatography filler is eluted with a third elution buffer to reduce the conductivity of the affinity chromatography filler to 5.0 mS / cm or less.

[0063] In some embodiments, the first wash buffer, the second wash buffer, and the third wash buffer are washed for 1-10 column volumes.

[0064] Exemplarily, the first washing buffer is used for washing for 2-8 column volumes, the second washing buffer is used for washing for 1-5 column volumes, and the third washing buffer is used for washing for 1-5 column volumes.

[0065] In some embodiments, the affinity chromatography filler is a Protein A affinity chromatography filler; illustratively, the Protein A affinity chromatography filler includes but is not limited to Mabselect Sure, Mabselect Sure LX, Mabselect PrismA, N Mab, N Mab Pro, or Unimab 50HC.

[0066] The method disclosed in the present invention can effectively remove homodimers in fusion proteins through affinity chromatography, which solves the problem in the current prior art that it is difficult to effectively separate homodimers in fusion proteins through only a simple affinity chromatography step, improves the purification efficiency and purity of the fusion protein, and has the advantages of low cost and simple steps.

[0067] PD-1 binding domain

[0068] In some embodiments, the aforementioned PD-1 binding domain, according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are shown as SEQ ID NO: 3, 16, and 17, respectively.

[0069] In some embodiments, the amino acid sequence of CDR1 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain is shown as SEQ ID NO:3, the amino acid sequence of CDR2 is shown as SEQ ID NO:4 or 18, and the amino acid sequence of CDR3 is shown as any one of SEQ ID NOs:5, 19-20.

[0070] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain are:

[0071] As shown in SEQ ID NO:3, 18, 19,

[0072] As shown in SEQ ID NO:3, 4, 5, or

[0073] As shown in SEQ ID NO:3, 4, 20.

[0074] In some embodiments, the immunoglobulin single variable domain of the aforementioned PD-1 binding domain is humanized, affinity matured, T cell epitopes removed, antibody deamidation reduced and / or antibody isomerization reduced. In some embodiments, the immunoglobulin single variable domain is obtained through affinity maturation, and has one or more changes in one or more CDRs, and the changes result in an increase in affinity for PD-1 compared to the parent.

[0075] In some specific embodiments, the humanization process uses the heavy chain framework region IGHV3-23*01 or IGHV3-23*04 of the human germline template.

[0076] In some embodiments, the amino acid sequence of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain is as shown in any one of SEQ ID NOs: 2, 8-15, or has at least 80%, at least 85%, or at least 90% sequence identity with any of the aforementioned sequences.

[0077] In some embodiments, the amino acid sequence of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain is as shown in SEQ ID NO: 13, or has at least 80%, at least 85%, or at least 90% sequence identity with any of the aforementioned sequences.

[0078] In some embodiments, at least one immunoglobulin single variable domain in the aforementioned PD-1 binding domain is a VHH.

[0079] In some embodiments, the aforementioned PD-1 binding domain comprises one or more (e.g., 2, 3, 4, 5, 6) of the aforementioned immunoglobulin single variable domains, and the immunoglobulin single variable domains may be the same or different and may form a dimer or a multimer molecule.

[0080] In some embodiments, the aforementioned PD-1 binding domain comprises or is an anti-PD-1 antibody or antigen-binding fragment thereof that specifically binds to PD-1 or a fragment thereof; the antibody or antigen-binding fragment thereof is, for example, a camel antibody, a chimeric antibody, a humanized antibody, a fully human antibody or an antigen-binding fragment thereof; the antibody or antigen-binding fragment thereof is, for example, a recombinant antibody or a fragment thereof. In some specific embodiments, the antigen-binding fragment is a linear antibody, a single-chain antibody, a nanobody, a peptide antibody, a domain antibody, and a multispecific antibody (bispecific antibody, diabody, triabody and tetrabody, tandem di-scFv, tandem tri-scFv).

[0081] In some embodiments, the aforementioned anti-PD-1 antibody or antigen-binding fragment thereof further comprises a human immunoglobulin Fc region; for example, the Fc region is the Fc region of human IgG1, IgG2 or IgG4. The Fc region may have mutations, exemplary mutations being L234A / L235A on IgG1, V234A / G237A / P238S / H268A / V309L / A330S / P331S on IgG2, F234A / L235A on IgG4, S228P / F234A / L235A on IgG4, N297A on IgG1, IgG2, IgG3 or IgG4, V234A / G237A on IgG2, K214T / E233P / L234V / L235A / G236 deletion / A327G / P331A / D365E / L358M, H268Q / V309L / A330S / P331S on IgG2, L234F / L235E / D265A on IgG1, L234A / L235A / G237A / P238S / H268A / A330S / P331S on IgG1, S228P / F234A / L235A / G237A / P238S on IgG4, and S228P / F234A / L235A / G236 deletion / G237A / P238S on IgG4. Hybrid IgG2 / 4 Fc domains can also be used, such as an Fc with residues 117-260 from IgG2 and residues 261-447 from IgG4. In some specific embodiments, the Fc region of the human IgG4 has S228P, F234A, L235A and / or K447A mutations. In some specific embodiments, the Fc region of the human IgG1 has L234A / L235A or L234A / L235A / P329G mutations.

[0082] In some embodiments, the Fc region contained in the aforementioned PD-1 antibody or antigen-binding fragment thereof can enable the binding protein to form a dimeric molecule and prolong the in vivo half-life of the binding protein.

[0083] In some embodiments, the immunoglobulin single variable domain in the aforementioned PD-1 antibody or antigen-binding fragment thereof is connected to the Fc region via a linker. The linker may be a non-functional amino acid sequence of 1-20 or more amino acids in length and without secondary or higher structures. For example, the linker is a flexible linker, such as G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, ASGS, etc.

[0084] In some embodiments, the aforementioned PD-1 antibody or antigen-binding fragment thereof has at least one activity selected from the following:

[0085] (a)≤10 -7 K D The value binds to human PD-1 or its epitope;

[0086] (b) inhibiting the binding of PD-1 to PD-L1;

[0087] (c) inhibiting the binding of PD-1 to PD-L2;

[0088] (d) Induction of CD4 + T cells secrete IFN-γ;

[0089] (e) enhance the activation of PBMCs;

[0090] (f) enhancing T cell activation;

[0091] (g) Inhibit tumor growth.

[0092] In some embodiments, the aforementioned PD-1 binding domain of the present disclosure binds to the K D The value can be ≤1×10 -7 M, for example, ≤1×10 -8 M, or ≤1×10 -9 M, or ≤1×10 -10 M.

[0093] In some embodiments, the PD-1 binding domain of the present disclosure can specifically bind to human PD-1 and block the interaction between PD-1 and PD-L1, and / or PD-1 and PD-L2.

[0094] In some embodiments, the aforementioned PD-1 binding domains of the present disclosure are capable of inhibiting tumor growth by at least about 10%, such as at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%.

[0095] In some embodiments, the aforementioned PD-1 binding domains disclosed herein encompass variants, for example, compared with any one of SEQ ID NOs: 2, 8-15, comprising one or more amino acid substitutions, preferably conservative amino acid substitutions, for example, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions, which may occur in the CDR region and / or the FR region.

[0096] In some embodiments, the aforementioned PD-1 binding domain of the present disclosure is resistant to heat treatment or has high stability. For example, no obvious aggregation or degradation is observed after treatment at 40°C for up to 30 days, and the domain is stable at least at 60°C.

[0097] In some embodiments, a PD-1 antibody or an antigen-binding fragment thereof is provided, which binds to or competes for binding to the same epitope as the immunoglobulin single variable domain of the aforementioned PD-1 binding domain of the present disclosure.

[0098] In some embodiments, a PD-1 antibody or an antigen-binding fragment thereof is provided, which blocks the binding of the immunoglobulin single variable domain of the aforementioned PD-1 binding domain of the present disclosure to PD-1 (eg, human PD-1).

[0099] In some embodiments, a PD-1 antibody or an antigen-binding fragment thereof is provided, the binding of which to PD-1 (e.g., human PD-1) is blocked by the immunoglobulin single variable domain in the aforementioned PD-1 antibody or antigen-binding fragment thereof of the present disclosure.

[0100] In some embodiments, the aforementioned PD-1 binding domain of the present disclosure reduces the binding of PD-1 to PD-L1 and / or PD-L2 by at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%.

[0101] In some embodiments, the present disclosure provides a PD-1 binding protein comprising at least one of the aforementioned immunoglobulin single variable domains (such as VHH) that binds to PD-1.

[0102] As previously mentioned, and in this disclosure, "at least 90% identity" includes at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity.

[0103] IL-2 or its variants

[0104] In some embodiments, the cytokine is IL-2 or a variant thereof. The IL-2 is preferably human IL-2, and the amino acid sequence of wild-type human IL-2 is exemplified as SEQ ID NO: 50. The positions of the IL-2 variants disclosed herein are numbered according to the wild-type IL-2 amino acid sequence shown in SEQ ID NO: 50.

[0105] In some embodiments, the present disclosure provides IL-2 variants having reduced affinity for high affinity receptors (IL-2Rα / β / γ) and medium affinity receptors (IL-2Rβ / γ) compared to wild-type IL-2, and / or having an affinity for low affinity receptors (IL-2Rα) eliminated compared to wild-type IL-2. In some specific embodiments, the affinity of the IL-2 variant for IL-2Rα / β / γ is reduced more than the affinity for IL-2Rβ / γ. The affinity can be detected by conventional methods, such as ELISA, ForteBio, SPR, such as the SPR detection method provided in Example 13 of the present disclosure.

[0106] In some embodiments, the present disclosure provides an IL-2 variant comprising or consisting of the following mutation (i), mutation (ii), mutation (iii), mutation (iv) or any combination thereof.

[0107] Regarding mutation (i):

[0108] The present disclosure provides mutations that confer upon IL-2 variants an altered (eg, increased or decreased) affinity for IL-2Rβ / γ compared to wild-type IL-2.

[0109] In some embodiments, the IL-2 variant has a mutation (i) that reduces affinity for IL-2Rβ / γ compared to wild-type IL-2. In some specific embodiments, the IL-2 variant has an affinity for IL-2Rβ / γ that is reduced by 1-1000 times, 2-800 times, 5-500 times, 5-200 times, 10-200 times, 10-100 times, 10-50 times, 20-200 times, 20-100 times, 5-20 times, 2-20 times, 50-100 times, 50-70 times, such as about 5, about 10, about 13, about 15, about 20, about 30, about 40, about 50, about 60, about 63, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 180, about 200, about 250, about 300, about 400, or about 500 times compared to wild-type IL-2. In some specific embodiments, the affinity of the IL-2 variant to IL-2Rβ / γ is reduced by 10-100 times compared to wild-type IL-2. The affinity can be detected by conventional methods, such as the SPR detection method provided in Example 13 of the present disclosure.

[0110] In some embodiments, the aforementioned mutation (i) is located at positions 12-20, positions 84-95, and positions 126-130 of IL-2.

[0111] In some embodiments, the aforementioned mutation (i) is selected from positions 12, 15, 16, 18, 19, 20, 23, 43, 69, 84, 87, 88, 91, 92, 95, 123, 126, 127, 129, 130 or any combination thereof; in some embodiments, the mutation is selected from positions L12, E15, H16, L18, L19, D20, M23, Q22, K43, V69, D84, S87, N88, V91, I92, E95, T123, Q126, S127, I129 and S130 or any combination thereof, such as N88 / D20; in some specific embodiments, the Mutations selected from the group consisting of L12R, L12K, L12E, L12Q, E15Q, E15R, E15A, E15S, H16N, H16A, H16E, H16D, H16G, H16S, H16T, H16V, H16P, D20A, D20H, D20Y, D20N, D20Q, D20E, D20R, D20S, M23A, M23R, M23K, M23G, M23S, M23T, M23V, M23P, S87K, S87A, D84S, D84L, D84N, D84V, D84H, D84Y, D84R, D84K, D84G , D84A, D84T, D84P, N88D, N88A, N88S, N88E, N88T, N88R, N88I, V91A, V91T, V91E, V91I, V91L, V91D, V91N, V91Q, V91S, V91H, I92E, I92T, I92K, I92R, I92L, E95S, E95A, E95R, E95Q, E95G, E95T, E95V, E95P, E95H, E95N, T123A, T123E, T123K, T123Q, Q126D, Q126L, Q126A, Q126S , Q126T, Q126E, S127A, S127E, S127K, S127Q, D84N / E95Q, D84E / E95Q, D84T / E95Q, D84Q / E95Q, D84T / H16T, D84N / V91I, D84T / Q126E, D84N / Q126E, H16T / D84Q, H16T / V91I; in some embodiments, the mutation (i) is selected from N88D, N88R, D20A, D20N, V91T, Q126D or H16A / D84S; in some embodiments, the mutation (i) is N88D or D20A.

[0112] Regarding mutation (ii):

[0113] The present disclosure provides mutations that render IL-2 variants having altered (eg, decreased) affinity for IL-2Rα compared to wild-type IL-2, or not binding to IL-2Rα.

[0114] In some embodiments, the IL-2 variant has a reduced affinity for IL-2Rα compared to wild-type IL-2, or a mutation (ii) that does not bind to IL-2Rα. The affinity can be detected by conventional methods, such as the SPR detection method provided in Example 13 of the present disclosure.

[0115] In some specific embodiments, the aforementioned mutation (ii) is selected from positions 35, 37, 38, 41, 42, 43, 45, 61, 62, 65, 68, 72 or any combination thereof; in some embodiments, the mutation (ii) is selected from positions K35, T37, R38, E62, P65, E68, F42, Y45, L72 or any combination thereof; in some embodiments, the mutation is selected from R38A, R38D, R38E, E62Q, F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S , Y45A, L72G, F42A / L72G, F42A / Y45A, Y45A / L72G or F42A / Y45A / L72G.

[0116] Regarding mutation (iii):

[0117] The present disclosure provides mutations (iii) that allow IL-2 variants to have improved stability, expression level, purity, and drugability.

[0118] The present disclosure incorporates the IL-2 variants in WO2020125743A in its entirety.

[0119] In some embodiments, the IL-2 variant has a mutation (iii), wherein the mutation (iii) is selected from positions 11, 26, 27, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 70, 71, 72, 78, 82, 88, 132 or any combination thereof; in some embodiments, the mutation (iii) is selected from positions N26, N29, N30, N71, Q11, L132, L70, P82, G27, F78 or any combination thereof; in some embodiments, The mutation (iii) is selected from N26Q, N29S, N30S, N71Q, Q11C / L132C, L70C / P82C, G27C / F78C or any combination thereof; in some embodiments, the mutation (iii) is selected from N26Q, N29S, N30S, N26Q / N29S / N71Q, N26Q / N29S, N26Q / N30S, N26Q / N30S / Q11C / L132C, N26Q / N29S / L70C / P82C, N26Q / N30S / G27C / F78C.

[0120] Regarding mutation (iv):

[0121] In some embodiments, the IL-2 variant contains a mutation (iv), wherein the mutation (iv) is selected from position 3 and / or 125; in some embodiments, the mutation (iv) is selected from position T3 and / or C125; in some embodiments, the mutation (iv) is selected from T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, T3P and / or C125A, C125S, C125T, C125V mutations.

[0122] I. Exemplary IL-2 variants

[0123] The present disclosure provides IL-2, which comprises or only comprises the following mutations:

[0124] (a) F42A and N88D, N88A, N88S, N88E, N88T, N88R or N88I;

[0125] (b) F42A and D20A, D20H, D20Y, D20N, D20Q, D20E, D20R or D20S;

[0126] (c) F42A and Q126D, Q126L, Q126A, Q126S, Q126T or Q126E;

[0127] (d) F42A and V91A, V91T, V91E, V91I, V91L, V91D, V91N, V91Q, V91S or V91H;

[0128] (e) F42A / L72G and N88D, N88A, N88S, N88E, N88T, N88R or N88I;

[0129] (f) F42A / L72G and D20A, D20H, D20Y, D20N, D20Q, D20E, D20R or D20S;

[0130] (g) F42A / L72G and Q126D, Q126L, Q126A, Q126S, Q126T or Q126E;

[0131] (h) F42A / L72G and V91A, V91T, V91E, V91I, V91L, V91D, V91N, V91Q, V91S or V91H;

[0132] (i) any one of the above (a) to (h) and N26Q;

[0133] (j) any one of the above (a) to (h) and N29S;

[0134] (k) any one of the above (a) to (h) and N71Q;

[0135] (l) any one of the above (a) to (h) and N26Q / N29S;

[0136] (m) any one of the above (a) to (h) and N26Q / N29S / N71Q;

[0137] (n) any one of the above (a) to (h) and Q11C / L132C;

[0138] (o) any one of the above (a) to (h) and L70C / P82C;

[0139] (p) any one of the above (a)-(h) and G27C / F78C;

[0140] (q) any one of the above (a) to (h) and T3A;

[0141] (r) is any one of the above (a) to (h) and T3A / C125A.

[0142] II. Exemplary IL-2 variants

[0143] The present disclosure provides IL-2, which comprises or only comprises the following mutations:

[0144] 42A / 72G / 88D,

[0145] 42A / 72G / 88R,

[0146] 42A / 72G / 20A,

[0147] 42A / 72G / 20N,

[0148] 42A / 72G / 91T,

[0149] 42A / 72G / 126D,

[0150] 42A / 72G / 16A / 84S,

[0151] 42A / 72G / 26Q / 29S / 71Q,

[0152] 38E / 42A / 91T,

[0153] 42A / 72G / 88D / 26Q / 29S / 71Q,

[0154] 42A / 72G / 88R / 26Q / 29S / 71Q,

[0155] 42A / 72G / 20A / 26Q / 29S / 71Q,

[0156] 42A / 72G / 20N / 26Q / 29S / 71Q,

[0157] 42A / 72G / 91T / 26Q / 29S / 71Q,

[0158] 42A / 72G / 126D / 26Q / 29S / 71Q, or

[0159] 42A / 72G / 16A / 84S / 26Q / 29S / 71Q.

[0160] III. Exemplary IL-2 variants

[0161] The present disclosure provides IL-2, which comprises or only comprises the following mutations:

[0162] T3A / F42A / L72G / N88D / C125A,

[0163] T3A / F42A / L72G / N88R / C125A,

[0164] T3A / F42A / L72G / D20A / C125A,

[0165] T3A / F42A / L72G / D20N / C125A,

[0166] T3A / F42A / L72G / V91T / C125A,

[0167] T3A / F42A / L72G / Q126D / C125A,

[0168] T3A / F42A / L72G / H16A / D84S / C125A,

[0169] T3A / F42A / L72G / N26Q / N29S / N71Q / C125A,

[0170] T3A / R38E / F42A / V91T / C125A,

[0171] T3A / F42A / L72G / N88D / N26Q / N29S / N71Q / C125A,

[0172] T3A / F42A / L72G / N88R / N26Q / N29S / N71Q / C125A,

[0173] T3A / F42A / L72G / D20A / N26Q / N29S / N71Q / C125A,

[0174] T3A / F42A / L72G / D20N / N26Q / N29S / N71Q / C125A,

[0175] T3A / F42A / L72G / V91T / N26Q / N29S / N71Q / C125A,

[0176] T3A / F42A / L72G / Q126D / N26Q / N29S / N71Q / C125A, or

[0177] T3A / F42A / L72G / H16A / D84S / N26Q / N29S / N71Q / C125A.

[0178] In some embodiments, the present disclosure provides an IL-2 variant comprising or having an amino acid sequence as shown in any one of SEQ ID NOs: 23-31 or having at least 80%, at least 85%, or at least 90% sequence identity thereto.

[0179] In some embodiments, any of the aforementioned IL-2 variants containing mutation (i) has any one or more of 1) to 5) relative to wild-type IL-2:

[0180] 1) have reduced affinity for the high-affinity IL-2R receptor (IL-2Rαβγ);

[0181] 2) have reduced affinity for the intermediate affinity IL-2R receptor (IL-2Rβγ);

[0182] 3) have reduced T cells (such as CD4 + and CD8 + T cell) activation;

[0183] 4) has reduced activation of NK cells;

[0184] 5) having reduced release of inflammatory factors from T cells and / or NK cells stimulated by IL-2.

[0185] In some embodiments, any of the aforementioned IL-2 variants containing mutation (i) and mutation (ii) has any one or more of the aforementioned 1)-5) and the following 6)-8) relative to wild-type IL-2:

[0186] 6) have reduced affinity for IL-2Rα, or do not bind to IL-2Rα;

[0187] 7) The affinity for IL-2Rαβγ is reduced more than that for IL-2Rβγ;

[0188] 8) Has reduced expression of CD25 + Cells (e.g. CD25 + CD8 + Activation of T cells, Treg cells).

[0189] In some embodiments, any of the aforementioned IL-2 variants containing mutation (i) and mutation (ii) and mutation (iii) and / or mutation (iv) has any one or more of the aforementioned 1)-8) and the following 9) relative to wild-type IL-2:

[0190] 9) Improved stability, drugability, expression level, purity and / or prolonged in vivo half-life.

[0191] Fusion Protein

[0192] The present disclosure provides fusion proteins comprising an antigen binding domain, and IL-2 or a variant thereof.

[0193] In some embodiments, the antigen binding domain in the fusion protein targets immune effector cells or tumor cells; in some specific embodiments, the immune effector cells are capable of responding to stimulation or activation of IL-2; in some specific embodiments, the immune effector cells have IL-2Rβγ; in some specific embodiments, the immune effector cells are, for example, NK cells, CD4+CD25-T cells, or CD8+CD25-T cells.

[0194] In some embodiments, the antigen binding domain in the fusion protein targets PD-1, CTLA-4, LAG3, TIM3, 4-1BB, OX40, GITR, CD8a, CD8b, CD4, NKp30, NKG2A, TIGIT, TGFβR1, TGFβR2, Fas, NKG2D, NKp46, PD-L1, CD107a, ICOS, TNFR2, FAP, TNC A1, TNC A2, CEA, EDB, MCSP or CD16a; in some specific embodiments, the antigen binding domain targets PD-1 (i.e., PD-1 binding domain).

[0195] In some embodiments, the PD-1 binding domain is any PD-1 binding domain provided hereinabove.

[0196] Exemplarily, in some specific embodiments, the PD-1 binding domain comprises at least one immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises: CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 2, 8-15, wherein the CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems.

[0197] Exemplarily, in some specific embodiments, the PD-1 binding domain, according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NO: 3, 16, and 17, respectively.

[0198] Exemplarily, in some specific embodiments, the amino acid sequence of CDR1 of the immunoglobulin single variable domain in the PD-1 binding domain is shown in SEQ ID NO: 3, the amino acid sequence of CDR2 is shown in SEQ ID NO: 4 or 18, and the amino acid sequence of CDR3 is shown in any one of SEQ ID NO: 5, 19-20.

[0199] Exemplarily, in some embodiments, the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 3, 4 and 5, or as shown in SEQ ID NOs: 3, 18 and 19, or as shown in SEQ ID NOs: 3, 4 and 20.

[0200] Illustratively, in some specific embodiments, the amino acid sequence of the immunoglobulin single variable domain is shown in any one of SEQ ID NOs: 2, 8-15, respectively; or has at least 80% sequence identity with any of the sequences.

[0201] In some embodiments, the PD-1 binding domain is selected from the PD-1 binding domains of the following PD-1 antibodies: Pembrolizumab, Nivolumab, Sintilimab, Tislelizumab, Cemiplimab, Toripalimab, Camrelizumab, Penamprolizumab, Sepalimab, Sasanlimab. limab), Spartalizumab, Retifanlimab, Balstilimab, Cetrelimab, Dostarlimab (TSR-042), CS-1003, SCT-I10A, SSI-361, MEDI-0680 (AMP-514), BAT-1306, JTX-4014, JTX-4014, BI-754091, AK-103. In some other embodiments, the antigen binding domain in the fusion protein is selected from the antigen binding domain of the following antibodies: PD-L1 antibody (e.g., Durvalumab, Atezolizumab, Durvalumab, Avelumab, Adebrelimab, Envafolimab, STI-A1110, CS-1001, TQB-2450, KL-A167, IMC-001, CX-072); CTLA-4 antibody (e.g., Tremelimumab, Ipilimumab); 4-1BB antibody (e.g., Urelumab, Utomilumab). The antigen-binding domain (e.g., PD-1 binding domain) is the portion of the antibody (e.g., PD-1 antibody) that binds to the antigen (e.g., PD-1), for example, its Fab', F(ab')2, or its heavy chain variable region and light chain variable region, or a domain containing HCDR1, HCDR2, HCDR3 of its heavy chain variable region and LCDR1, LCDR2, LCDR3 of its light chain variable region.

[0202] In some embodiments, the PD-1 binding domain comprises Pembrolizumab or an antigen-binding fragment thereof (e.g., Fab), for example, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 54, 55, and 56, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 57, 58, and 59. In some specific embodiments, the heavy chain variable region is the heavy chain variable region in the Fab heavy chain of the amino acid sequence shown in SEQ ID NO: 52, and the light chain variable region comprises the light chain variable region in the Fab light chain of the amino acid sequence shown in SEQ ID NO: 53.

[0203] In some embodiments, the IL-2 or variant thereof in the fusion protein is any IL-2 or variant thereof provided in the present disclosure.

[0204] Exemplarily, in some specific embodiments, the IL-2 is wild-type human IL-2.

[0205] Exemplarily, in some specific embodiments, the IL-2 variant comprises mutation (i), mutation (ii), mutation (iii), mutation (iv) or any combination thereof provided herein, or consists of mutation (i), mutation (ii), mutation (iii), mutation (iv) or any combination thereof.

[0206] Exemplarily, in some specific embodiments, the IL-2 variant comprises or only comprises the mutation of any one of (a)-(r) provided hereinabove.

[0207] Exemplarily, in some specific embodiments, the IL-2 variant contains an amino acid sequence as shown in any one of SEQ ID NOs: 23-31, or a sequence having at least 80%, at least 85%, or at least 90% identity thereto, or consists of the above sequence.

[0208] In some embodiments, the fusion protein further comprises a human immunoglobulin Fc region, such as the Fc region of human IgG1, IgG2, IgG3 or IgG4.

[0209] In some embodiments, the antigen binding domain (e.g., PD-1 binding domain) in the fusion protein is located at the N-terminus of the Fc region, and the IL-2 or its variant is located at the N-terminus or C-terminus of the Fc region. In some specific embodiments, the antigen binding domain is located at the N-terminus of the Fc, and the IL-2 or its variant is located at the C-terminus of the Fc region.

[0210] In some embodiments, the valency ratio of the antigen binding domain (e.g., PD-1 binding domain) to IL-2 or its variant in the fusion protein is between 6:1 and 1:3 (e.g., 4:1 to 1:2), specifically 4:1, 2:1 or 1:1.

[0211] In some embodiments, the fusion protein contains 1 or 2 IL-2 or variants thereof and 1, 2, 3, 4, 5 or 6 antigen binding domains (eg, PD-1 binding domains) in one fusion protein.

[0212] In some embodiments, the fusion protein further comprises an Fc region, wherein the Fc region comprises a first subunit Fc1 and a second subunit Fc2 capable of associating with each other.

[0213] In some embodiments, amino acid mutations are included in Fc1 and Fc2, so that Fc1 preferentially pairs with Fc2 or preferentially forms heterodimers compared to Fc1. In some embodiments, the mutations are located in CH3 of Fc1 and Fc2. In some embodiments, the amino acid mutations in Fc1 and Fc2 produce greater electrostatic complementarity than the wild type without the mutations. In some embodiments, the amino acid mutations in Fc1 and Fc2 produce greater steric complementarity than the wild type without the mutations.

[0214] In some embodiments, in Fc1 and Fc2, for example, within the CH3 / CH3 interface, one or more amino acid residues in the CH3 domain of Fc1 are mutated with one or more amino acid residues having a larger side chain volume, thereby generating a protrusion (or knob) on the surface of the CH3 domain of Fc1, and one or more, preferably two or three amino acid residues in the CH3 domain of Fc2 that interact with the CH3 domain of Fc1 are mutated with amino acid residues having a smaller side chain volume, thereby generating a depression (or hole) on the surface of the CH3 domain of Fc2 that interacts with the CH3 domain of Fc1. In some embodiments, the input residue with a larger side chain volume is phenylalanine (F), tyrosine (Y), arginine (R) or tryptophan (W). In some embodiments, the input residue with a smaller side chain volume is serine (S), alanine (A), valine (V) or threonine (T).

[0215] In some specific embodiments, the Fc1 comprises at least one or at least two amino acid mutations selected from T366S, L368A and Y407V (hole mutation modification), and the Fc2 comprises T366W (knob mutation modification); or the Fc1 comprises T366W (knob mutation modification), and the Fc2 comprises at least one or at least two amino acid mutations selected from T366S, L368A and Y407V (hole mutation modification).

[0216] In some specific embodiments, the IL-2 or variant thereof is located in the polypeptide chain where Fc1 is located.

[0217] In some specific embodiments, Fc1 and Fc2, for example, CH3, may contain a natural non-cysteine ​​to cysteine ​​mutation, such as S354C in Fc1 and Y349C in Fc2; or Y349C in Fc1 and S354C in Fc2.

[0218] In some embodiments, Fc1 and Fc2, for example, within the Fc1 CH3 / Fc2 CH3 interface, comprise the following amino acid mutations or a combination thereof:

[0219] T366Y / Y407T;

[0220] T366W / Y407A;

[0221] T366Y / Y407T;

[0222] T394W / F405A;

[0223] T366Y / F405A / T394W / Y407T;

[0224] T366W / F405W / T394S / Y407A;

[0225] F405W / T394S;

[0226] D399C / K392C;

[0227] T366W / T366S / L368A / Y407V;

[0228] T366W / D399C / T366S / L368A / K392C / Y407V;

[0229] T366W / K392C / T366S / D399C / L368A / Y407V;

[0230] S354C / T366W / Y349C / T366S / L368A / Y407V;

[0231] Y349C / T366W / S354C / T366S / L368A / Y407V;

[0232] E356C / T366W / Y349C / T366S / L368A / Y407V;

[0233] Y349C / T366W / E356C / T366S / L368A / Y407V;

[0234] E357C / T366W / Y349C / T366S / L368A / Y407V; and

[0235] Y349C / T366W / E357C / T366S / L368A / Y407V.

[0236] In some specific embodiments, Fc1 and Fc2 further comprise amino acid mutations that form an electrostatic interaction interface between Fc1 and Fc2 (e.g., CH3 and CH3). The amino acid mutations that form the electrostatic interaction interface are, for example, selected from the following:

[0237] K370E / D399K / K439D / D356K / E357K / K409D;

[0238] K409D / D399K;

[0239] K409E / D399K;

[0240] K409E / D399R;

[0241] K409D / D399R;

[0242] D339K / E356K;

[0243] D399K / E356K / K409D / K392D;

[0244] D399K / E356K / K409D / K439D;

[0245] D399K / E357K / K409D / K370D;

[0246] D399K / E356K / E357K / K409D / K392D / K370D;

[0247] D399K / E357K / K409D / K392D;

[0248] K392D / K409D / D399K; and

[0249] K409D / K360D / D399K.

[0250] In some embodiments, Fc1 and / or Fc2 comprise domains from different antibody subtypes, such as CH3 from different antibody subtypes.

[0251] In addition, the present disclosure cites WO96 / 27011, WO98 / 050431, EP1870459, WO2007 / 110205, WO2007 / 147901, WO2009 / 089004, WO2010 / 129304, WO2011 / 90754, WO2011 / 143545, WO2012058768, WO2013157954, and WO2013096291 for modifying the CH3 region of the Fc region to enhance heterodimerization.

[0252] In some embodiments, Fc1 and / or Fc2, such as CH3, contain amino acid mutations for changing effector functions. The antibody effector functions include, but are not limited to: C1q binding and complement-dependent cytotoxicity, Fc receptor (e.g., FcγR) binding, antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Amino acid mutations that change effector functions are, for example, selected from the following:

[0253] S298A / E333A / K334A;

[0254] S239D / I332E / A330L;

[0255] S239D / I332E / G236A;

[0256] G236A / S239D / A330L / I332E;

[0257] F243L / R292P / Y300L / V305I / P396L;

[0258] K326A / E333A;

[0259] K326W / E333S;

[0260] K326M / E333S;

[0261] C221D / D222C;

[0262] S267E / H268F / S324T;

[0263] E345R;

[0264] S298A / E333A / K334A / N434A;

[0265] E294 deletion / T307P / N434Y;

[0266] T256N / A378V / S383N / N434Y;

[0267] T252L / T253S / T254F;

[0268] M252Y / S254T / T256E;

[0269] M428L / N434S;

[0270] L234A / L235A;

[0271] S228P / L235E;

[0272] L234A / L235A / P331S;

[0273] L234A / L235A / P329G;

[0274] D265A / E233P;

[0275] H268Q / V309L / A330S / P331S;

[0276] V234A / G237A / P238S / H268A / V309L / A300S / P331S;

[0277] L234A / L235A / G237A / P238S / H268A / V309L / A300S / P331S;

[0278] S228P / F234A / L235A;

[0279] D270A / P329A;

[0280] L234F / L235E;

[0281] L234F / L235E / P331S;

[0282] F241A / V264A / D265A;

[0283] N297G / D265A; and

[0284] L234Y / G236W / S298A.

[0285] In some specific embodiments, the Fc1 and / or the Fc2 comprise mutations that reduce effector function (eg, ADCC), such as L234A / L235A, L234A / L235A / P329G.

[0286] In some embodiments, Fc1 and Fc2, for example, in their CH3, contain amino acid mutations for changing the half-life (e.g., extending the half-life). In some embodiments, the half-life depends on the FcRn binding affinity. The extension of the half-life can allow a reduction in the amount of the drug given to the patient, and / or reduce the frequency of administration. In some specific embodiments, the Fc region has M252Y, S254T and / or T256E mutations. In some embodiments, the fusion protein has a PEGylated modification or is conjugated / fused to (human serum) albumin or an albumin-binding protein.

[0287] In some embodiments, Fc1 and / or Fc2, such as CH3, comprises one or more allotypic mutations. In some embodiments, the allotypic mutations are D356E and L358M.

[0288] In some embodiments, the fusion protein comprises a polypeptide chain combination of the following amino acid sequences: SEQ ID NOs: 21 and 22; SEQ ID NOs: 36 and 37; SEQ ID NOs: 38 and 39; SEQ ID NOs: 40 and 41; SEQ ID NOs: 42 and 43; or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the above polypeptide chain combinations.

[0289] In some specific embodiments, the fusion protein comprises a polypeptide chain combination of the following amino acid sequences: SEQ ID NO: 42 and 43 or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the above polypeptide chain combinations.

[0290] Exemplary Fusion Proteins

[0291] Regarding fusion proteins, the present disclosure provides the following embodiments:

[0292] Embodiment 1. A fusion protein comprising:

[0293] 1) a PD-1 binding domain, and an IL-2 variant comprising a mutation selected from N88D or D20A; or

[0294] 2) a PD-1 binding domain, and an IL-2 variant, wherein the IL-2 variant has a reduced or eliminated affinity for high-affinity receptors (IL-2Rα / β / γ) compared to wild-type IL-2, and has a reduced affinity for medium-affinity receptors (IL-2Rβ / γ) compared to wild-type IL-2, and the affinity for high-affinity receptors is more reduced than the affinity for medium-affinity receptors; for example, the IL-2 variant contains a mutation selected from N88D or D20A.

[0295] Embodiment 2. The fusion protein of embodiment 1, comprising a mutation selected from F42, Y45, L72 or any combination thereof;

[0296] For example, it contains a mutation selected from F42A, Y45A, L72G or any combination thereof;

[0297] More specifically, it contains mutations of F42A / L72G, F42A / Y45A, Y45A / L72G, and F42A / Y45A / L72G.

[0298] Embodiment 3. The fusion protein of embodiment 1 or 2, comprising a mutation selected from N26Q, N29S, N30S, N71Q, Q11C and L132C, L70C and P82C, G27C and F78C or any combination thereof;

[0299] For example, it contains a mutation or combination selected from the group consisting of:

[0300] N26Q, N29S, N30S, N26Q / N29S / N71Q, N26Q / N29S, N26Q / N30S, N26Q / N30S / Q11C / L132C, N26Q / N29S / L70C / P82C, N26Q / N30S / G27C / F78C.

[0301] Embodiment 4. A fusion protein comprising:

[0302] An antigen binding domain, and IL-2 or a variant thereof, wherein the IL-2 variant contains a first type of mutation, wherein the first type of mutation causes the IL-2 variant to have a reduced affinity for the medium affinity receptor (IL-2Rβ / γ) compared to wild-type IL-2;

[0303] For example, the first class of mutations is selected from the group consisting of: L12, E15, H16, L18, L19, D20, M23, Q22, K43, V69, D84, S87, N88, V91, I92, E95, T123, Q126, S127, I129, and S130;

[0304] For another example, the first type of mutation is selected from: L12R, L12K, L12E, L12Q, E15Q, E15R, E15A, E15S, H16A, H16E, H16D, H16G, H16S, H16T, H16V, H16P, D20A, D20H, D20Y, D20N, M23A, M23R, M23K, M23G, M23S, M23T, M23V, M23P, S87K, S87A, D84S, D84L, D84N, D84V, D84H, D84Y, D84R, D84K, D84G, D84A, D84T, D84P, N88D, N88A, N88S, N88T, N88R, N88I, V91A, V91T, V91E, I92A, E95S, E95A, E95R, E95Q, E95G, E95T, E9 5V, E95P, E95H, E95N, T123A, T123E, T123K, T123Q, Q126D, Q126L, Q126A, Q126S, Q126T, Q126E, S127A, S127E, S127K, S127Q;

[0305] For another example, the first type of mutation is selected from: N88D, N88R, D20A, D20N, V91T, Q126D, H16A / D84S;

[0306] More for example, the first type of mutation is selected from N88D or D20A.

[0307] Embodiment 5. A fusion protein as described in embodiment 4, wherein the IL-2 variant contains a second type of mutation, wherein the second type of mutation causes the IL-2 variant to have a reduced or eliminated affinity for the high affinity receptor (IL-2Rα / β / γ) compared to wild-type IL-2;

[0308] For example, the second type of mutation is selected from R38, E62, P65, E68, F42, Y45, L72 or any combination thereof;

[0309] For another example, the second type of mutation is selected from R38A, R38D, R38E, E62Q, P65R, P65E, P65K, P65H, P65Y, P65Q, P65D, P65N, E68A, E68Q, E68K, E68R, F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, L72K;

[0310] More specifically, the second type of mutation is selected from F42A, Y45A, L72G, F42A / L72G, F42A / Y45A, Y45A / L72G or F42A / Y45A / L72G.

[0311] Embodiment 6. The fusion protein of embodiment 4 or 5, wherein the IL-2 variant contains a third type of mutation, and the third type of mutation makes the IL-2 variant have increased stability compared with wild-type IL-2;

[0312] For example, the third type of mutation is selected from N26, N29, N30, N71, Q11, L132, L70, P82, G27, F78 or any combination thereof;

[0313] For another example, the third type of mutation is selected from N26Q, N29S, N30S, N71Q, Q11C / L132C, L70C / P82C, G27C / F78C or any combination thereof;

[0314] For example, the third type of mutation is selected from: N26Q, N29S, N30S, N26Q / N29S / N71Q, N26Q / N29S, N26Q / N30S, N26Q / N30S / Q11C / L132C, N26Q / N29S / L70C / P82C, N26Q / N30S / G27C / F78C.

[0315] Embodiment 7. A fusion protein comprising: a PD-1 binding domain, and IL-2 or a variant thereof;

[0316] The PD-1 binding domain comprises at least one immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises:

[0317] CDR1, CDR2 and CDR3 in the amino acid sequence of any one of SEQ ID NOs: 2, 8-15; said CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems;

[0318] For example, according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NOs: 3, 16 and 17, respectively.

[0319] For another example, the amino acid sequence of CDR1 of the immunoglobulin single variable domain is shown in SEQ ID NO: 3, the amino acid sequence of CDR2 is shown in SEQ ID NO: 4 or 18, and the amino acid sequence of CDR3 is shown in any one of SEQ ID NO: 5, 19-20;

[0320] For example, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NOs: 7, 18, and 19, respectively.

[0321] Embodiment 8. The fusion protein according to any one of embodiments 1 to 7, wherein the IL-2 variant contains a combination of mutations selected from group 1) and group 2):

[0322] Group 1) mutations are selected from the group consisting of: F42A / L72G, F42A / Y45A, Y45A / L72G and F42A / Y45A / L72G;

[0323] Group 2) mutations are selected from the group consisting of: N88R, N88G, N88I, N88D, D20H, D20Y, V91T, Q126L, Q126D, H16A / D84S;

[0324] For example, the IL-2 variant contains the following mutation combinations: F42A / L72G / N88D, F42A / L72G / N88R, F42A / L72G / D20A, F42A / L72G / D20N, F42A / L72G / V91T, F42A / L72G / Q126D, F42A / L72G / H16A / D84S.

[0325] Embodiment 9. The fusion protein of embodiment 8, wherein the IL-2 variant further comprises a mutation of group 3), wherein the mutation of group 3) is selected from the group consisting of: N26Q, N29S, N30S, N26Q / N29S / N71Q, N26Q / N29S, N26Q / N30S, N26Q / N30S / Q11C / L132C, N26Q / N29S / L70C / P82C and N26Q / N30S / G27C / F78C;

[0326] For example, the IL-2 variant contains the following mutation combination:

[0327] F42A / L72G / N88D / N26Q / N29S / N71Q,

[0328] F42A / L72G / N88R / N26Q / N29S / N71Q,

[0329] F42A / L72G / D20A / N26Q / N29S / N71Q,

[0330] F42A / L72G / D20N / N26Q / N29S / N71Q,

[0331] F42A / L72G / V91T / N26Q / N29S / N71Q,

[0332] F42A / L72G / Q126D / N26Q / N29S / N71Q,

[0333] F42A / L72G / H16A / D84S / N26Q / N29S / N71Q.

[0334] Embodiment 10. The fusion protein of any one of embodiments 1 to 9, comprising a mutation selected from T3 and / or C125 or a combination thereof;

[0335] For example, T3A and / or C125A, C125S, C125T, C125V mutations.

[0336] Embodiment 11. The fusion protein according to any one of embodiments 1 to 10, wherein the IL-2 variant has an amino acid sequence as shown in any one of SEQ ID NOs: 23-31.

[0337] Embodiment 12. In the fusion protein according to any one of embodiments 4 to 6, 8 to 10,

[0338] 1) The antigen is selected from:

[0339] 1-1) Antigens on tumor cells or immune cells in the tumor microenvironment;

[0340] 1-2) PD-1, CTLA-4, LAG3, TIM3, 4-1BB, OX40, GITR, CD8a, CD8b, CD4, NKp30, NKG2A, TIGIT, TGFβR1, TGFβR2, Fas, NKG2D, NKp46, PD-L1, CD107a, ICOS, TNFR2, FAP, TNC A1, TNCA2, CEA, EDB, MCSP or CD16a;

[0341] 1-3) Immune checkpoint antigens, such as PD-1, PD-L1, and CTLA-4;

[0342] 2) The antigen binding domain comprises a VH domain and a VL domain of an antibody selected from the following, or HCDR1, HCDR2, HCDR3 of VH and LCDR1, LCDR2, LCDR3 of VL:

[0343] PD-1 antibodies (e.g., pembrolizumab, nivolumab, sintilimab, tislelizumab, cemiplimab, toripalimab, camrelizumab, penamprolizumab, sepalizumab, sasanlimab, spartalizumab) artalizumab, retifanlimab, balstilimab, cetrelimab, dostarlimab (TSR-042), CS-1003, SCT-I10A, SSI-361, MEDI-0680 (AMP-514), BAT-1306, JTX-4014, JTX-4014, BI-754091, AK-103);

[0344] CTLA-4 antibodies (eg, Tremelimumab, Ipilimumab);

[0345] 4-1BB antibodies (eg, Urelumab, Utomilumab);

[0346] PD-L1 antibodies (e.g., Durvalumab, Atezolizumab, Durvalumab, Avelumab, Adebrelimab, Envafolimab, STI-A1110, CS-1001, TQB-2450, KL-A167, IMC-001, CX-072).

[0347] Embodiment 13. The fusion protein according to any one of Embodiments 1 to 3 and 12, wherein the PD-1 binding domain comprises at least one immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises:

[0348] CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 2, 8-15, wherein the CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems;

[0349] For example, according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NOs: 3, 16 and 17, respectively;

[0350] For another example, the amino acid sequence of CDR1 of the immunoglobulin single variable domain is shown in SEQ ID NO: 3, the amino acid sequence of CDR2 is shown in SEQ ID NO: 4 or 18, and the amino acid sequence of CDR3 is shown in any one of SEQ ID NO: 5, 19-20;

[0351] For example, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NOs: 3, 18, and 19, 3, 4, and 5, or 3, 4, and 20, respectively.

[0352] Embodiment 14. A fusion protein according to embodiment 7 or 13, wherein the immunoglobulin single variable domain is:

[0353] 1) Humanization, affinity maturation, removal of T cell epitopes, reduction of antibody deamidation and / or reduction of antibody isomerization; for example, the heavy chain framework region of the human germline template used in the humanization process is IGHV3-23*01 or IGHV3-23*04;

[0354] 2) Camelid antibodies, chimeric antibodies, humanized antibodies, fully human antibodies or antigen-binding fragments thereof.

[0355] Embodiment 15. A fusion protein as described in embodiment 13 or 14, wherein the amino acid sequence of the immunoglobulin single variable domain is as follows:

[0356] Any one of SEQ ID NO:2, 8-15; or

[0357] Having at least 90% sequence identity with any of the aforementioned sequences.

[0358] Embodiment 17. The fusion protein of any of the preceding embodiments, further comprising a human immunoglobulin Fc region; for example, the Fc region is the Fc region of human IgG1, IgG2, IgG3 or IgG4.

[0359] Embodiment 18. The fusion protein of embodiment 17, wherein the Fc region of human IgG1 contains one or more amino acid mutations that reduce binding to Fc receptors (FcR, such as FcγR), and / or reduce effector function;

[0360] For example, the Fc region of human IgG1 contains one or more amino acid mutations that reduce binding to Fcγ receptor (FcγR) and / or reduce ADCC effect;

[0361] For another example, the Fc region of the human IgG1 contains mutations in L234, L235, P329 or any combination thereof;

[0362] For example, the Fc region of human IgG1 contains L234A / L235A or L234A / L235A / P329G mutations.

[0363] Embodiment 19. The fusion protein of embodiment 17 or 18, wherein the Fc region comprises a mutation that promotes association (or heterodimerization) of the first and second subunits of the Fc region;

[0364] For example, the amino acid residues in the CH3 region of the first subunit of the Fc region are replaced with amino acid residues having a larger side chain volume to generate a protrusion in the CH3 region of the first subunit, and the protrusion can be placed in the cavity in the CH3 region of the second subunit, and the amino acid residues in the CH3 region of the second subunit of the Fc region are replaced with amino acid residues having a smaller side chain volume to generate a cavity in the CH3 region of the second subunit, in which the protrusion in the CH3 region of the first subunit can be placed; for another example, the amino acid residues with a larger side chain volume are selected from R, F, Y, and W, and the amino acid residues with a smaller side chain volume are selected from A, S, T, and V.

[0365] Embodiment 20. A fusion protein as described in embodiment 19, wherein

[0366] The first subunit of the Fc region contains a T366 mutation, and the second subunit contains a mutation selected from T366, L368, Y407 or any combination thereof;

[0367] The first subunit of the Fc region contains a mutation in S354 or E356, and the second subunit contains a mutation selected from Y349; or

[0368] The first subunit of the Fc region contains S354 and T366 mutations, and the second subunit contains Y349, T366, L368 and Y407 mutations;

[0369] For example,

[0370] The first subunit of the Fc region contains a T366W mutation, and the second subunit contains a mutation selected from T366S, L368A, Y407V or any combination thereof;

[0371] The first subunit of the Fc region contains a S354C or E356C mutation, and the second subunit contains a mutation selected from Y349C; or

[0372] The first subunit of the Fc region contains S354C / T366W mutations, and the second subunit contains Y349C / T366S / L368A / Y407V mutations.

[0373] Embodiment 21. A fusion protein as described in Embodiment 19 or 20, wherein the second subunit of the Fc region further contains H435 and / or R436 mutations, preferably H435R / Y436F, H435R, H435K mutations.

[0374] Embodiment 22. The fusion protein of any one of embodiments 17 to 21, wherein the Fc region of human IgG1 contains mutations that enhance FcRn-mediated recycling and / or extend half-life;

[0375] For example, the Fc region of the human IgG1 contains M252 and / or M428 mutations;

[0376] For example, the Fc region of human IgG1 contains M252Y / M428V, M252Y / M428L mutations.

[0377] Embodiment 23. The fusion protein according to any of the preceding embodiments, wherein the PD-1 binding domain or the antigen binding domain is located at the N-terminus of the Fc region, and the IL-2 variant is located at the N-terminus or the C-terminus of the Fc region;

[0378] For example, the PD-1 binding domain or the antigen binding domain is located at the N-terminus of the Fc region, and the IL-2 variant is located at the C-terminus of the Fc region.

[0379] Embodiment 24. A fusion protein as described in any of the preceding embodiments, wherein the valence ratio of the PD-1 binding domain or antigen binding domain to IL-2 or its variant is between 6:1 and 1:3 (e.g., 4:1 to 1:2), preferably 4:1, 2:1 or 1:1; or, a fusion protein contains 1 or 2 IL-2 or its variants and 1, 2, 3, 4, 5 or 6 PD-1 binding domains or antigen binding domains.

[0380] Embodiment 25. A fusion protein as described in any one of embodiments 1 to 3, 7, 11 to 23, comprising a polypeptide chain combination as shown in any one of the following I)-VI):

[0381] First polypeptide chain: [PD-1 binding domain 1]-[Fc region first subunit]-[Linker 1]a-[IL-2 or its variant],

[0382] Second polypeptide chain: [PD-1 binding domain 1]-[Fc region second subunit];

[0383] Wherein, - represents a peptide bond, the linker is a polypeptide capable of realizing the connection function, a is selected from 0 or 1; [PD-1 binding domain 1] is selected from any PD-1 binding domain in Embodiments 11 to 13;

[0384] For example, the linker is (G x S) y Linker, wherein x is selected from an integer of 1-5, and y is selected from an integer of 1-6; for another example, linker 1 is (G4S)3, linker 2 is (G4S)2, and linker 3 is G4S.

[0385] Some other linkers include, but are not limited to, (G m S n ) h or (GGNGT) h or (YGNGT) h or (EPKSS) h The amino acid sequence shown, wherein m, n are each independently selected from an integer of 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and h is independently selected from an integer of 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

[0386] The order of the above polypeptide chains is from N-terminus to C-terminus.

[0387] Embodiment 26. A fusion protein as described in any of the preceding embodiments, comprising a polypeptide chain combination of the following amino acid sequences:

[0388] SEQ ID NO:42 and 43;

[0389] SEQ ID NO: 21 and 22;

[0390] SEQ ID NO:36 and 37;

[0391] SEQ ID NO:38 and 39; or,

[0392] SEQ ID NO:40 and 41.

[0393] Or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the above polypeptide chains in combination.

[0394] Unless otherwise specified, the mutation sites in the above Fc regions are coded according to the EU numbering system.

[0395] The present disclosure provides a method for purifying a fusion protein, which comprises the following steps:

[0396] (a) Sample loading step: loading the sample containing the fusion protein onto the affinity chromatography medium;

[0397] (b) Equilibration step: flushing the affinity chromatography medium after loading with equilibration buffer;

[0398] (c) elution step: eluting the affinity chromatography medium with a elution buffer;

[0399] (c-1) eluting the affinity chromatography medium with a first elution buffer;

[0400] (c-2) eluting the affinity chromatography medium with a second elution buffer;

[0401] (c-3) Eluting the affinity chromatography medium with a third elution buffer.

[0402] (d) Elution step: eluting the fusion protein from the affinity chromatography medium with an elution buffer, and collecting the eluate; the pH of the elution buffer is, for example, about 3.0 to about 5.0, for example, about 3.4 to about 4.0, for example, about 3.4 to about 3.8

[0403] In some embodiments, the pH of the first eluent is about 3.8 to about 5.0, such as about 4.0 to about 4.7, such as about 4.1 to about 4.3.

[0404] In some embodiments, the pH of the second wash buffer is about 4.5 to about 8.0, such as about 4.5 to about 6.5, such as about 4.5 to about 5.5.

[0405] In some embodiments, the pH of the third wash buffer is about 4.5 to about 8.0, such as about 4.5 to about 6.5, such as about 4.5 to about 5.5.

[0406] In some embodiments, the first wash buffer contains about 0.03 to about 0.1 mol / L of acetic acid-sodium acetate, and about 0.1 to about 0.6 mol / L of sodium chloride; for example, about 0.04 to about 0.06 mol / L of acetic acid-sodium acetate, and about 0.4 to about 0.6 mol / L of sodium chloride;

[0407] In some embodiments, the second wash buffer contains about 0.03 to about 0.1 mol / L of acetic acid-sodium acetate, and about 0.3 to about 0.7 mol / L of sodium chloride; for example, about 0.04 to about 0.06 mol / L of acetic acid-sodium acetate, and about 0.4 to about 0.5 mol / L of sodium chloride;

[0408] In some embodiments, the third wash buffer contains about 0.03 to about 0.1 mol / L of acetic acid-sodium acetate. In some embodiments, the third wash buffer contains about 0.04 to about 0.06 mol / L of acetic acid-sodium acetate. In some embodiments, the third wash buffer does not contain sodium chloride;

[0409] In some embodiments, the elution buffer contains about 0.01 mol / L to about 0.5 mol / L acetic acid-sodium acetate, for example, about 0.05 mol / L to about 0.1 mol / L acetic acid-sodium acetate;

[0410] In some embodiments, the equilibration buffer contains about 0.01 mol / L to about 0.1 mol / L phosphate, and about 0.1 mol / L to about 2.0 mol / L sodium chloride; for example, about 0.01 mol / L to about 0.05 mol / L phosphate, and about 0.5 mol / L to about 1.5 mol / L sodium chloride;

[0411] In some embodiments, the affinity chromatography medium is a Protein A affinity chromatography medium; for example, Mabselect Sure, Mabselect Sure LX, Mabselect PrismA, N Mab, N Mab Pro, or Unimab50HC;

[0412] In some embodiments, the fusion protein is a fusion protein comprising an antibody Fc; in some embodiments, the fusion protein comprises a fusion protein of a PD-1 binding domain, a cytokine and an antibody Fc, wherein the cytokine is selected from IL-2 or a variant thereof.

[0413] In some embodiments, the PD-1 binding domain is located at the N-terminus of Fc, and the cytokine is located at the N-terminus or C-terminus of Fc.

[0414] In some embodiments, the fusion protein comprises an asymmetric first polypeptide chain and a second polypeptide chain, wherein the difference between any two of the PI of the fusion protein, the PI of the first polypeptide chain and the PI of the second polypeptide chain is no greater than about 3.0 units, for example, no greater than about 2.0 units, or 1.0 units.

[0415] In some embodiments, the PD-1 binding domain in the fusion protein comprises at least one immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises: CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 2, 8-15, wherein the CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems.

[0416] In some embodiments, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NO: 3, 16 and 17, respectively.

[0417] In some embodiments, the amino acid sequence of CDR1 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain is shown as SEQ ID NO:3, the amino acid sequence of CDR2 is shown as SEQ ID NO:4 or 18, and the amino acid sequence of CDR3 is shown as any one of SEQ ID NOs:5, 19-20.

[0418] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain are respectively as shown in SEQ ID NOs: 3, 18, and 19, as shown in SEQ ID NOs: 3, 4, and 5, or as shown in SEQ ID NOs: 3, 4, and 20.

[0419] In some embodiments, the cytokine is IL-2 or a variant thereof. In some specific embodiments, the IL-2 variant comprises an amino acid sequence as shown in any one of SEQ ID NOs: 23-31 or having at least 90% sequence identity thereto.

[0420] In some embodiments, the fusion protein comprises a polypeptide chain combination of the following amino acid sequences:

[0421] SEQ ID NO:42 and 43;

[0422] SEQ ID NO: 21 and 22;

[0423] SEQ ID NO:36 and 37;

[0424] SEQ ID NO:38 and 39; or,

[0425] SEQ ID NO:40 and 41.

[0426] The present invention discloses a method for purifying a fusion protein, which comprises the following steps:

[0427] (a) Sample loading step: loading the sample containing the fusion protein onto the affinity chromatography medium;

[0428] (b) Equilibration step: flushing the affinity chromatography medium after loading with equilibration buffer;

[0429] (c) elution step: eluting the affinity chromatography medium with a elution buffer;

[0430] (c-1) eluting the affinity chromatography medium with 2-8 column volumes of the first elution buffer,

[0431] (c-2) eluting the affinity chromatography medium with 1 to 5 column volumes of a second elution buffer to raise the pH of the affinity chromatography medium to above 4.5;

[0432] (c-3) eluting the affinity chromatography filler with 1 to 5 column volumes of a third elution buffer to reduce the conductivity of the affinity chromatography filler to less than 5.0 mS / cm;

[0433] (d) elution step: eluting the fusion protein from the affinity chromatography medium with an elution buffer and collecting the eluate;

[0434] In some embodiments, the pH of the first eluent is about 3.8 to about 5.0, for example, about 4.0 to about 4.7, exemplarily about 4.1 to about 4.3;

[0435] In some embodiments, the pH of the second wash buffer is about 4.5 to about 8.0, for example, about 4.5 to about 6.5, exemplarily about 4.5 to about 5.5;

[0436] In some embodiments, the pH of the third wash buffer is about 4.5 to about 8.0, for example, about 4.5 to about 6.5, exemplarily about 4.5 to about 5.5;

[0437] In some embodiments, the pH of the elution buffer is about 3.0 to about 5.0, for example, about 3.4 to about 4.0, more exemplarily about 3.4 to about 3.8.

[0438] In some embodiments, the first wash buffer contains about 0.03 to about 0.1 mol / L of acetic acid-sodium acetate, and about 0.1 to about 0.6 mol / L of sodium chloride; illustratively, it contains about 0.04 to about 0.06 mol / L of acetic acid-sodium acetate, and about 0.4 to about 0.6 mol / L of sodium chloride.

[0439] In some embodiments, the second wash buffer contains about 0.03 to about 0.1 mol / L of acetic acid-sodium acetate, and about 0.3 to about 0.7 mol / L of sodium chloride; illustratively, it contains about 0.04 to about 0.06 mol / L of acetic acid-sodium acetate, and about 0.4 to about 0.5 mol / L of sodium chloride.

[0440] In some embodiments, the third wash buffer contains about 0.03 to about 0.1 mol / L acetic acid-sodium acetate, illustratively about 0.04 to about 0.06 mol / L acetic acid-sodium acetate, and illustratively does not contain sodium chloride.

[0441] In some embodiments, the elution buffer contains about 0.01 mol / L to about 0.5 mol / L acetic acid-sodium acetate, illustratively about 0.05 mol / L to about 0.1 mol / L acetic acid-sodium acetate.

[0442] In some embodiments, the equilibration buffer contains about 0.01 mol / L to about 0.1 mol / L phosphate, and about 0.1 mol / L to about 2.0 mol / L sodium chloride; illustratively, it contains about 0.01 mol / L to about 0.05 mol / L phosphate (PB), and about 0.5 mol / L to about 1.5 mol / L sodium chloride.

[0443] In some embodiments, the affinity chromatography medium is a Protein A affinity chromatography medium; exemplarily selected from Mabselect Sure, Mabselect Sure LX, Mabselect PrismA, N Mab, N Mab Pro, or Unimab50HC.

[0444] In some embodiments, the fusion protein is a fusion protein comprising an antibody Fc; exemplarily a fusion protein comprising a PD-1 binding domain, a cytokine and an antibody Fc, wherein the cytokine is selected from IL-2 or a variant thereof.

[0445] In some embodiments, the PD-1 binding domain is located at the N-terminus of Fc, and the cytokine is located at the N-terminus or C-terminus of Fc.

[0446] In some embodiments, the fusion protein comprises an asymmetric first polypeptide chain and a second polypeptide chain, wherein the difference between any two of the PI of the fusion protein, the PI of the first polypeptide chain and the PI of the second polypeptide chain is no greater than about 3.0 units, for example, about 2.0 units, or 1.0 units.

[0447] In some embodiments, the PD-1 binding domain in the fusion protein comprises at least one immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises: CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 2, 8-15, wherein the CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems.

[0448] In some embodiments, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NO: 3, 16 and 17, respectively.

[0449] In some embodiments, the amino acid sequence of CDR1 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain is shown as SEQ ID NO:3, the amino acid sequence of CDR2 is shown as SEQ ID NO:4 or 18, and the amino acid sequence of CDR3 is shown as any one of SEQ ID NOs:5, 19-20.

[0450] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain are respectively as shown in SEQ ID NOs: 3, 18, and 19, as shown in SEQ ID NOs: 3, 4, and 5, or as shown in SEQ ID NOs: 3, 4, and 20.

[0451] In some embodiments, the cytokine is IL-2 or a variant thereof. In some specific embodiments, the IL-2 variant comprises an amino acid sequence as shown in any one of SEQ ID NOs: 23-31 or having at least 90% sequence identity thereto.

[0452] In some embodiments, the fusion protein comprises a polypeptide chain combination of the following amino acid sequences:

[0453] SEQ ID NO:42 and 43;

[0454] SEQ ID NO: 21 and 22;

[0455] SEQ ID NO:36 and 37;

[0456] SEQ ID NO:38 and 39; or,

[0457] SEQ ID NO:40 and 41. BRIEF DESCRIPTION OF THE DRAWINGS

[0458] Figure 1To detect the degree of T cell activation by A17m09_hIgG4 through the release of cytokines in DC cell:T cell mixed lymphoid reaction, Pembrolizumab was used as a positive control and hIgG4 as a negative control.

[0459] Figure 2 The fusion protein of anti-PD-1 antibody and IL-2 mutant was constructed. The anti-PD-1 nanobody A17m0902 was coupled to the N-terminus of the Knob and Hole chains through the hinge region of the IgG1 heavy chain, and the IL-2 mutant was connected to the C-terminus of the Knob chain of Fc through a (G4S)3 linker.

[0460] FIG3 shows the results of the proliferation activity test of the same form of PD-1-IL-2v fusion protein with different IL-2 mutants on M07e and M07e-hPD-1 cell lines. Figure 3A The results of the proliferation activity of PD-1-IL-2v fusion protein on M07e cells. Figure 3B This is the result of the proliferation activity of PD-1-IL-2v fusion protein on M07e-hPD-1 cells.

[0461] Figure 4 These are the results of the proliferation-promoting activity test of PD-1-IL-2v or IgG-IL-2v on M07e-hPD-1 cells.

[0462] FIG5 shows the results of the detection of the proliferation activity of unstimulated T cells by PD-1-IL-2v with different IL-2 mutants. Figure 5A For CD4 + T cell proliferation results, Figure 5B For CD8 + T cell proliferation results.

[0463] FIG6 shows the results of detecting the proliferation activity of T cells stimulated by anti-CD3 antibodies by PD-1-IL-2v with different IL-2 mutants. Fig. 6A For CD4 + T cell proliferation results, Figure 6B For CD8 + T cell proliferation results. DETAILED DESCRIPTION

[0464] the term

[0465] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present disclosure belongs.

[0466] The term "impurities" refers to substances that are different from the desired antibody product. Impurities include, but are not limited to: host cell substances, such as Chinese hamster ovary protein (CHP); leached protein A; nucleic acids; variants, fragments, aggregates or derivatives of the desired antibody; other polypeptides; endotoxins; viral contaminants; cell culture medium components.

[0467] The term "buffer" refers to a solution that resists changes in pH by the action of its acid-base pair components. A variety of buffers that can be used depending on, for example, the desired buffer pH are described in Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., Ed. Calbiochem Corporation (1975).

[0468] The term "equilibration buffer" in the present disclosure refers to a buffer used to equilibrate an ion exchange material prior to loading a composition comprising an antibody of interest and one or more contaminants onto the ion exchange material.

[0469] The term "elution buffer" refers to a buffer containing a primary species such as sodium citrate or sodium acetate, which is used to elute the target product, such as an antibody, protein, etc., from a chromatography column.

[0470] The term "conductivity" refers to the ability of an aqueous solution to conduct an electric current between two electrodes. In a solution, electric current flows by ion transport, and as the amount of ions present in the aqueous solution increases, the solution will have a higher conductivity. The basic unit of measurement for conductivity is Siemens (or Ohm), Ohm (mS / cm), and can be measured using a conductivity meter, such as various models of Orion conductivity meters. Because electrolytic conductivity is the ability of ions in a solution to carry an electric current, the conductivity of a solution can be changed by changing the ion concentration therein. For example, the concentration of the buffer in the solution and / or the concentration of a salt (e.g., sodium oxide, sodium acetate, or potassium oxide) can be changed to achieve the desired conductivity.

[0471] The term “UV 280 " refers to the absorbance value of the sample at a wavelength of 280 nanometers. Protein molecules usually contain amino acids such as tyrosine, tryptophan, and phenylalanine, which have a maximum absorption peak at a wavelength of 280 nanometers, and the absorbance value is proportional to the protein concentration.

[0472] The term "about" refers to a value within an acceptable error range of a specific value determined by a person of ordinary skill in the art, which value depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" or "substantially comprising" can mean a range of up to 20%, such as between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% and 5%, between 0.5% and 1%, and in this disclosure, each case in which a number or numerical range is preceded by the term "about" also includes embodiments of the given number. In addition, particularly for biological systems or processes, the term can mean up to an order of magnitude or up to 5 times the value. Unless otherwise stated, when a specific value appears in the application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value.

[0473] The three letter codes and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0474] "Programmed death 1", "programmed cell death 1", "protein PD-1", "PD-1", "PDCD1" and "hPD-1" are used interchangeably and include variants, isoforms, species homologs of human PD-1, and analogs having at least one common epitope with PD-1. The complete PD-1 sequence can be found from GenBank Accession No. U64863. "Programmed death ligand-1 (PD-L1)" is one of the two cell surface glycoprotein ligands of PD-1 (the other is PD-L2), which downregulates T cell activation and cytokine secretion when bound to PD-1. "PD-L1" as used herein includes human PD-L1 (hPD-L1), variants, isoforms, and interspecies homologs of hPD-L1, and analogs having at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found using GenBank Accession No. Q9NZQ7.

[0475] "PD-1 binding protein" means any protein that can specifically bind to PD-1 or any molecule comprising the protein. PD-1 binding protein may include an antibody, an antigen binding fragment thereof, or a conjugate thereof as defined in the present disclosure for PD-1. PD-1 binding protein also encompasses immunoglobulin superfamily antibodies (IgSF) or CDR-grafted molecules. The "PD-1 binding protein" of the present disclosure may include at least one immunoglobulin single variable domain (such as VHH) that binds to PD-1. In some embodiments, the "PD-1 binding protein" may include 2, 3, 4 or more immunoglobulin single variable domains (such as VHH) that bind to PD-1. In addition to the immunoglobulin single variable domain comprising PD-1, the PD-1 binding protein of the present disclosure may also include a linker and / or a portion having an effector function, such as a half-life extension portion (such as an immunoglobulin single variable domain that binds serum albumin), and / or a fusion partner (such as serum albumin) and / or a conjugated polymer (such as PEG) and / or an Fc region. In some embodiments, the “PD-1 binding protein” of the present disclosure also encompasses bi / multispecific antibodies, which contain immunoglobulins that bind to different antigens (e.g., a first antibody that binds to a first antigen (e.g., PD-1) and a second antibody that binds to a second antigen (e.g., 4-1BB), optionally, a third antibody that binds to a third antigen, and further optionally, a fourth antibody that binds to a fourth antigen.

[0476] The "PD-1 binding protein" or "PD-1 antibody or antigen-binding fragment thereof" of the present disclosure may contain one or more effector molecules, for example, in a conjugated manner. The "effector molecule" includes, for example, anti-tumor agents, drugs, toxins, biologically active proteins (e.g., enzymes), other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof (e.g., DNA, RNA and fragments thereof), radionuclides, in particular radioiodides, radioisotopes, chelated metals, nanoparticles, and reporter groups such as fluorescent compounds or compounds detectable by NMR or ESR spectroscopy. In some embodiments, the effector molecule is a cytokine (e.g., IL-2, IL-15 or a variant thereof). In other embodiments, the effector molecule is a polymer, which may generally be a synthetic or naturally occurring polymer, and specific examples of synthetic polymers include optionally substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol) or derivatives thereof, and specific naturally occurring polymers include lactose, amylose, dextran, glycogen or derivatives thereof. In other embodiments, the polymer is albumin or a fragment thereof, such as human serum albumin or a fragment thereof. The conjugation of the polymer to the PD-1 binding protein or PD-1 antibody can be achieved by conventional methods.

[0477] "Cytokines" are proteins released by a cell population that act as intercellular mediators on other cells. Examples of such cytokines include lymphokines, monokines, chemokines, and traditional polypeptide hormones. Exemplary cytokines include: human IL-2, IL-15, IFN-γ, IL-6, TNFα, IL-17, and IL-5 and variants thereof.

[0478] "Interleukin-2" or "IL-2" refers to any natural IL-2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses unprocessed IL-2 as well as any form of IL-2 derived from processing in cells. The term also encompasses naturally occurring IL-2 variants, such as splice variants or allelic variants. The amino acid sequence of an exemplary wild-type human IL-2 is shown in SEQ ID NO: 87. Unprocessed human IL-2 additionally includes a signal peptide of 20 amino acids at the N-terminus (as shown in SEQ ID NO.272 in WO2012107417), which is lacking in mature IL-2 molecules.

[0479] "Mutated IL-2" or "IL-2 variant" refers to a polypeptide that differs from the wild-type IL-2 amino acid sequence by a mutation (eg, substitution, deletion, addition) at at least one amino acid position.

[0480] In the present disclosure, when referring to IL-2 variants, single amino acid substitutions are described in the following manner: [original amino acid residue / position / substituted amino acid residue]. For example, the substitution of phenylalanine at position 42 to alanine can be represented as F42A. Single amino acid substitutions can be connected by " / " to represent combined mutations at multiple given positions. For example, a combined mutation at the positions of F42A, L72G and N88R can be represented as: F42A / L72G / N88R.

[0481] In the present disclosure, when referring to the amino acid position in the IL-2 protein or IL-2 sequence segment, it is determined by reference to the amino acid sequence SEQ ID NO: 87 of the wild-type human IL-2 protein (also referred to as wt IL-2). The corresponding amino acid position on other IL-2 proteins or polypeptides (including full-length sequences or truncated fragments) can be identified by comparing the amino acid sequence with SEQ ID NO: 87. Therefore, in the present disclosure, unless otherwise specified, the amino acid position of the IL-2 protein or polypeptide is the amino acid position numbered according to SEQ ID NO: 87. For example, when referring to "F42", it refers to the 42nd phenylalanine residue F of SEQ ID NO: 40, or the amino acid residue at the corresponding position on other IL-2 polypeptide sequences after comparison. The sequence alignment implemented for the determination of the amino acid position can be performed using the Basic Local Alignment Search Tool available from https: / / blast.ncbi.nlm.nih.gov / Blast.cgi, using default parameters.

[0482] "CD25" or "α subunit of IL-2 receptor" refers to any native CD25 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), including "full-length" unprocessed CD25 and any form of CD25 derived from processing in cells, and also includes naturally occurring CD25 variants, such as splice variants or allelic variants. In certain embodiments, CD25 is human CD25.

[0483] "High affinity IL-2 receptor" refers to the heterotrimeric form of the IL-2 receptor, which is composed of a receptor γ subunit (also known as the universal cytokine receptor γ subunit, γc or CD132), a receptor β subunit (also known as CD122 or p70) and a receptor α subunit (also known as CD25 or p55). In contrast, a "medium affinity IL-2 receptor" refers to an IL-2 receptor that contains only γ and β subunits without an α subunit (see, e.g., Olejniczak and Kasprzak, MedSci Monit 14, RA179-189 (2008)).

[0484] "Antibody" is used in the broadest sense, covering various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (such as bispecific antibodies), full-length antibodies and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. Antibodies may refer to immunoglobulins, which are tetrapeptide chains composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. Based on this, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain, respectively. The same class of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of the heavy chain disulfide bonds, such as IgG can be divided into IgG1, IgG2, IgG3, IgG4. Light chains are divided into κ chains or λ chains according to the differences in the constant region. Each of the five types of Ig can have a kappa chain or a lambda chain. The sequences of about 110 amino acids near the N-terminus of the antibody heavy chain and light chain vary greatly, which is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region (C region). The variable region includes three hypervariable regions (CDRs) and four relatively conservative framework regions (FRs). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2 and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2 and HCDR3.

[0485] "Antigen binding fragment" encompasses single chain antibodies (i.e., full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g., VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope binding fragments of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). In some embodiments, the antigen binding fragment is VHH. Methods for generating and preparing these antigen binding fragments are well known in the art (see, e.g., Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). The Fab-Fv format was first disclosed in WO2009 / 040562, and its disulfide-stabilized form Fab-dsFv was first disclosed in WO2010 / 035012. Antigen-binding fragments of the present disclosure also include Fab and Fab' fragments described in WO2005 / 003169, WO2005 / 003170 and WO2005 / 003171. Multivalent antibodies may comprise multispecifics such as bispecifics or may be monospecifics (see, for example, WO92 / 22583 and WO05 / 113605), an example of the latter being Tri-Fab (or TFM) described in WO 92 / 22583.

[0486] "Fc domain" or "Fc region" is used to define the C-terminal region of at least a portion of the constant region in the immunoglobulin heavy chain. It includes a native sequence Fc region and a variant Fc region. Although the boundaries of the Fc region of the IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or Pro230 to the carboxyl end of the heavy chain. However, the antibody produced by the host cell may undergo post-translational cleavage, and one or more, particularly one or two amino acids are removed from the C-terminus of the heavy chain. Therefore, the antibody produced by the host cell by expressing a specific nucleic acid molecule encoding a full-length heavy chain may include a full-length heavy chain, or it may include a cleavage variant of the full-length heavy chain (also referred to as a "cleavage variant heavy chain" in the present disclosure). This may be the case when the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering is according to the Kabat EU index). Therefore, the C-terminal lysine (K447) of the Fc region, or the C-terminal glycine (G446) and lysine (K447) may be present or absent. If not otherwise specified, the amino acid sequence of the heavy chain comprising the Fc region (or the subunit of the Fc region defined in the present disclosure) is represented in the present disclosure without the C-terminal glycine-lysine dipeptide. In one embodiment, the heavy chain of a subunit of the Fc region included in the immunoconjugate comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering is according to the EU index of Kabat). In one embodiment, the heavy chain of a subunit of the Fc region included in the immunoconjugate comprises an additional C-terminal glycine residue (G446, numbering is according to the EU index of Kabat). The composition (e.g., pharmaceutical composition) of the present disclosure comprises a population of immunoconjugates of the present disclosure. The population of immunoconjugates may include molecules with full-length heavy chains and molecules with cleavage variant heavy chains. The population of immunoconjugates can be composed of a mixture of molecules with full-length heavy chains and molecules with cleavage variant heavy chains, wherein at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the immunoconjugates have cleavage variant heavy chains, which can be the C-terminal lysine (K447) of the Fc region, or the C-terminal glycine (G446) and lysine (K447) are absent. Unless otherwise specified in the present disclosure, the numbering of amino acid residues in the Fc region or constant region is in accordance with the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0487] The "subunit" of the Fc region refers to one of the two polypeptides that form the dimeric Fc region, i.e., a polypeptide comprising the C-terminal constant region of the immunoglobulin heavy chain that is capable of stabilizing its own association. For example, the subunits of the IgG Fc region comprise the IgG CH2 and IgG CH3 constant domains. In the present disclosure, the use of "first subunit" or Fc1, "second subunit" or Fc2 is not intended to confer a particular order or orientation, but is used for ease of distinction.

[0488] "Promoting the modification (or mutation) of the first subunit and the second subunit of the Fc domain to unite" is to reduce or prevent the polypeptide comprising the Fc domain subunit from uniting with the same polypeptide to form a peptide backbone operation or a post-translational modification of the Fc domain subunit. As used herein, specifically, the modification promoting the combination includes a separate modification of each of the two Fc domain subunits (i.e., the first subunit and the second subunit of the Fc domain) that are desired to be combined, wherein the modifications are complementary to each other, thereby promoting the combination of the two Fc domain subunits. For example, the modification promoting the combination can change the structure or charge of one or both Fc domain subunits, thereby promoting their combination in three dimensions or electrostatically, respectively. In this way, (hetero) dimerization occurs between a polypeptide comprising the first Fc domain subunit and a polypeptide comprising the second Fc domain subunit, which may be different in the sense that the other components (e.g., antigen binding modules) fused to each subunit are different. In some embodiments, the modification promoting the combination is included in an amino acid mutation in the Fc domain, specifically an amino acid substitution.

[0489] "Complementarity" refers to the combination of interactions that affect heavy chain / light chain pairing at the interface of, for example, CH1 and CL (or CH3 and CH3) of an antigen binding protein described herein. "Steric complementarity" or "conformational complementarity" refers to the compatibility of the three-dimensional structures at the interacting surfaces of, for example, CH1 and CL (or CH3 and CH3). "Electrostatic complementarity" refers to the compatibility of the placement of negatively and / or positively charged atoms at the interacting surfaces of, for example, CH1 and CL (or CH3 and CH3). Methods for measuring electrostatic complementarity at protein / protein interfaces are known in the art and are described, for example, in McCoy et al. (1997) J Mol Biol 268, 570-584; Lee et al., (2001) Protein Sci. 10, 362-377; and Chau et al. (1994) J Comp Mol Des 8, 51325. Methods for measuring steric complementarity at protein / protein interfaces are known in the art and are described, for example, in Lawrence et al. (1993) J Mol Biol 234, 946-950; Walls et al. (1992) J Mol Biol 228, 277-297; and Schueler-Furman et al. (2005) Proteins 60, 187-194.

[0490] "Effector functions" when referring to the use of antibodies refer to those biological activities that can be attributed to the Fc region of the antibody and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC), Fc receptor (FcR) binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, cell surface receptor (e.g., B cell receptor) downregulation and B cell activation. ADCC is an immune mechanism that leads to the lysis of antibody-coated target cells by immune effector cells. Target cells are cells that are specifically bound by antibodies or derivatives thereof containing Fc regions, generally via the protein portion of the N-terminus of the Fc region. "Reduced ADCC" is defined as a reduction in the number of target cells lysed in a given time with a given concentration of antibody in the medium surrounding the target cells by the ADCC mechanism defined above, and / or an increase in the concentration of antibody in the medium surrounding the target cells required to achieve the lysis of a given number of target cells in a given time by the ADCC mechanism. The reduction in ADCC is relative to ADCC mediated by the same antibody produced by the same type of host cell but not engineered using the same standard production, purification, formulation and storage methods known to those skilled in the art. For example, the reduction in ADCC mediated by an antibody comprising an amino acid substitution that reduces ADCC in its Fc domain is relative to ADCC mediated by the same antibody without such amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, for example, WO2006 / 082515 or WO2012 / 130831).

[0491] An "activating Fc receptor" is an Fc receptor that, upon engagement of the Fc domain of an antibody, initiates a signaling event that stimulates cells bearing the receptor to perform effector functions. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0492] "Immunoconjugate" refers to a polypeptide molecule comprising at least one IL-2 molecule and at least one antibody. As described herein, the IL-2 molecule can be linked to the antibody through a variety of interactions and in a variety of configurations. In a specific embodiment, the IL-2 molecule is fused to the antibody via a peptide linker. A specific immunoconjugate according to the present invention consists essentially of an IL-2 molecule and an antibody connected by one or more linker sequences.

[0493] "Decreased affinity" or "decreased binding", e.g., decreased binding to IL-2Rα, refers to a decrease in the affinity of the corresponding interaction, e.g., as measured by SPR. The term also includes a decrease in affinity to zero or below the detection limit of the analytical method, i.e., complete elimination of the interaction. Conversely, "decreased affinity" or "increased binding" refers to an increase in the binding affinity of the corresponding interaction.

[0494] For the determination or definition of CDRs, the deterministic depiction of CDRs and the identification of residues comprising the binding site of the antibody can be accomplished by resolving the structure of the antibody and / or resolving the structure of the antibody-ligand complex. This can be accomplished by any of the various techniques known to those skilled in the art, such as X-ray crystallography. A variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definitions, and conformational definitions.

[0495] The Kabat numbering system is a standard for numbering residues in antibodies and is often used to identify CDR regions (see, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8). The Chothia numbering system is similar to the Kabat numbering system, but the Chothia numbering system takes into account the location of certain structural loop regions. (See, e.g., Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83). The AbM numbering system uses an integrated suite of computer programs produced by the Oxford Molecular Group that model antibody structure (see, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd). The AbM numbering system uses a combination of knowledge databases and ab initio approaches to model the tertiary structure of antibodies from primary sequence (see those described by Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach" in PROTEINS, Structure, Function and Genetics Suppl., 3: 194-198). Contact definitions are based on analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5: 732-45). In conformational definitions, positions of CDRs can be identified as residues that make enthalpic contributions to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166). Still other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental results that a particular residue or group of residues does not significantly affect antigen binding. As used in the present disclosure, CDR may refer to CDR defined by any method known in the art (including a combination of methods). The correspondence between various numbering systems is well known to those skilled in the art, and an exemplary one is shown in Table 1 below.

[0496] Table 1. Relationships between CDR numbering systems

[0497] CDR IMGT Kabat AbM Chothia Contact HCDR1 27-38 31-35 26-35 26-32 30-35 HCDR2 56-65 50-65 50-58 52-56 47-58 HCDR3 105-117 95-102 95-102 95-102 93-101 LCDR1 27-38 24-34 24-34 24-34 30-36 LCDR2 56-65 50-56 50-56 50-56 46-55 LCDR3 105-117 89-97 89-97 89-97 89-96

[0498] The CDR amino acid residues of the VL and VH regions of the antibodies of the present disclosure conform in number and position to the well-known Kabat numbering system.

[0499] The antibodies disclosed herein may be polyclonal, monoclonal, xenogeneic, allogeneic, isogenic or modified forms thereof, wherein monoclonal antibodies are particularly suitable for use in a number of embodiments. In general, the antibodies disclosed herein are recombinant antibodies. As used herein, "recombinant" refers generally to products such as cells or nucleic acids, proteins or vectors, indicating that the cells, nucleic acids, proteins or vectors have been modified by the introduction of heterologous nucleic acids or proteins or by altering natural nucleic acids or proteins, or that the cells are derived from cells so modified. For example, recombinant cells express genes that are not present in the natural (non-recombinant) cell form or express natural genes that are abnormally expressed, underexpressed or not expressed at all.

[0500] A "domain" of a polypeptide or protein refers to a folded protein structure that is able to maintain its tertiary structure independently of the rest of the protein. In general, a domain is responsible for a single functional property of a protein and in many cases can be added, removed or transferred to other proteins without loss of the function of the rest of the protein and / or the domain.

[0501] "Immunoglobulin domain" refers to a globular region of an antibody chain (e.g., a chain of a conventional tetrapeptide chain structure antibody or a chain of a heavy chain antibody), or refers to a polypeptide consisting essentially of such a globular region. An immunoglobulin domain is characterized in that it maintains the immunoglobulin fold characteristic of an antibody molecule, which consists of a two-layer sandwich of about 7 antiparallel beta-pleated strands arranged in two beta-pleated sheets, optionally stabilized by conserved disulfide bonds.

[0502] An "immunoglobulin variable domain" refers to an immunoglobulin domain that is essentially composed of four "framework regions" referred to in the art and hereinafter as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", respectively, wherein the framework regions are separated by three "complementarity determining regions" or "CDRs" referred to in the art and hereinafter as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3". Therefore, the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An immunoglobulin variable domain confers specificity to an antigen by having an antigen binding site.

[0503] "Immunoglobulin single variable domain" is generally used to refer to an immunoglobulin variable domain (which may be a heavy chain or light chain domain, including a VH, VHH or VL domain) that can form a functional antigen binding site without interacting with other variable domains (e.g., without the required VH / VL interaction between the VH and VL domains of conventional four-chain monoclonal antibodies). Examples of "immunoglobulin single variable domains" include nanobodies (including VHH, humanized VHH and / or camelized VH, such as camelized human VH), IgNAR, domains, (single domain) antibodies (e.g., dAbsTM) that are VH domains or derived from VH domains, and (single domain) antibodies (e.g., dAbsTM) that are VL domains or derived from VL domains. Immunoglobulin single variable domains based on and / or derived from heavy chain variable domains (e.g., VH or VHH domains) are generally preferred. A specific example of an immunoglobulin single variable domain is a "VHH domain" (or simply "VHH") as defined below.

[0504] "VHH domain", also known as heavy chain single domain antibody, VHH, VHH antibody fragment, VHH antibody, nanobody, is a variable domain of an antigen-binding immunoglobulin called a "heavy chain antibody" (i.e., "antibody lacking a light chain") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). "VHH domain" is used to distinguish the variable domain from the heavy chain variable domain (which is referred to as "VH domain" in the present disclosure) and the light chain variable domain (which is referred to as "VL domain" in the present disclosure) present in conventional tetrapeptide chain structure antibodies. The VHH domain specifically binds to an epitope without the need for other antigen binding domains (this is in contrast to the VH or VL domains in conventional tetrapeptide chain antibodies, in which case the epitope is recognized by the VL domain together with the VH domain). The VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain. "Heavy chain single domain antibody", "VHH domain", "VHH", "VHH antibody fragment", "VHH antibody" and "domain" ("Nanobody" is a trademark of Ablynx NV, Ghent, Belgium) are used interchangeably. "VHH domain" includes but is not limited to natural antibodies produced by camelids, antibodies produced by camelids that are then humanized, or antibodies obtained by phage display technology. In some embodiments, the VHH domain comprises three CDRs and four framework regions, designated as FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. In some embodiments, the VHH domain may be truncated at the N-terminus or C-terminus so that it comprises only part of FR1 and / or FR4, or lacks one or both of those framework regions, as long as the VHH substantially retains antigen binding and specificity. The total number of amino acid residues in a VHH domain will typically be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described in the present disclosure.

[0505] As is known in the art for VH domains and VHH domains, the total number of amino acid residues in each CDR may be different and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by the Kabat numbering). This means that in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. Other numbering systems or coding conventions include Chothia, IMGT, AbM.

[0506] "Humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting non-human CDR sequences into the variable region framework of a human antibody. The strong immune response induced by chimeric antibodies due to carrying a large amount of non-human protein components can be overcome. In order to avoid a decrease in activity while reducing immunogenicity, the variable region of the fully human antibody can be subjected to minimal reverse mutation to maintain activity. Examples of "humanization" include "humanization" of a VHH domain derived from Camelidae by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at corresponding positions in the VH domain of a human conventional tetrapeptide chain structure antibody (also referred to as "sequence optimization" in this disclosure, in addition to humanization, "sequence optimization" may also cover other modifications to the sequence by one or more mutations that provide improved properties of VHH, such as removing potential post-translational modification sites). The humanized VHH domain may contain one or more completely human framework region sequences, and in some specific embodiments, may contain the human framework region sequence of IGHV3. Humanization methods such as protein surface amino acid humanization (resurfacing) and antibody humanization universal framework transplantation (CDR grafting to a universal framework), that is, CDR "transplantation" to other "scaffolds" (including but not limited to human scaffolds or non-immunoglobulin scaffolds). Scaffolds and techniques suitable for the CDR transplantation are known in the art. For example, the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database, as well as in Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. The humanized antibodies disclosed in the present invention also include humanized antibodies after affinity maturation of CDR by phage display. In addition, in order to avoid the decrease in immunogenicity and the decrease in activity caused, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity.

[0507] An "affinity matured" antibody refers to an antibody that has one or more changes in one or more hypervariable regions (HVRs) compared to a parent antibody that does not have such changes, such changes result in an improvement in the affinity of the antibody for the antigen. For example, an "affinity matured" PD-1 antibody has one or more changes in one or more CDRs that result in an increase in affinity for the antigen compared to its parent antibody. Affinity matured antibodies can be prepared by methods known in the art, for example, as described in Marks et al., 1992, Biotechnology 10:779-783 or Barbas et al., 1994, Proc. Nat. Acad. Sci, USA 91:3809-3813.; Shier et al., 1995, Gene 169:147-155; Yelton et al., 1995, Immunol. 155:1994-2004; Jackson et al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al., 1992, J. MoI. Biol. 226(3):889896; KS Johnson and RE Hawkins, "Affinity maturation of antibodies using phage display", Oxford University Press 1996.

[0508] Typically, the PD-1 antibodies, immunoconjugates of the present disclosure will be expressed as preferably 10 -7 Up to 10 -10 mol / L (M), more preferably 10 -8 Up to 10 -10 mol / L, even more preferably 10 -9 Up to 10 -10 or lower dissociation constant (KD), and / or at least 10 -7 M, preferably at least 10 -8 M, more preferably at least 10 -9 M, more preferably at least 10 -10 The association constant (KA) of M binds to the antigen to be bound (i.e., PD-1). Any value greater than 10 -4KD values ​​of M are generally considered to be indicative of non-specific binding. Specific binding of an antigen binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competitive assays) as described in the present disclosure.

[0509] "Antigen" refers to a molecule of an immunologically active vertebrate used for immunization to produce antibodies that recognize the antigen, or to screen an expression library (e.g., especially a phage, yeast, or ribosome display library). In the present disclosure, antigens are more broadly defined to include target molecules specifically recognized by antibodies, as well as a portion or mimetic of a molecule used in an immunization process for producing antibodies or in a library screening for selecting antibodies. For example, for antibodies that bind to human PD-1 of the present disclosure, monomers and multimers (e.g., dimers, trimers, etc.) of human PD-1, as well as truncated variants and other variants of human PD-1 are all referred to as PD-1 antigens.

[0510] "Epitope" refers to a site on an antigen that binds to an immunoglobulin or antibody. An epitope can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by tertiary folding of a protein. Epitopes formed by adjacent amino acids are usually retained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are usually lost after treatment with a denaturing solvent. An epitope usually includes at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitope is bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation detection analysis, etc. Methods for determining the spatial conformation of an epitope include techniques in the art and the techniques described in the present disclosure, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, etc.

[0511] "Specific binding" and "selective binding" refer to the binding of an antibody to an epitope on a predetermined antigen. For example, when human PD-1 or its epitope is used as an analyte and an antibody is used as a ligand, the antibody binds to the antigen at a specific binding rate of less than 10 -7 M or even smaller equilibrium dissociation constant (K D ) binds to a predetermined antigen or an epitope thereof, and its affinity for binding to the predetermined antigen or an epitope thereof is at least twice its affinity for binding to a non-specific antigen (such as BSA, etc.) other than the predetermined antigen (or its epitope) or a closely related antigen. "Antibodies that recognize antigens" can be used interchangeably with "antibodies that specifically bind" in the present disclosure.

[0512] "Binding affinity" or "avidity" is used in the present disclosure as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or portion thereof and an antigen). The binding affinity between two molecules can be quantified by determining the dissociation constant (KD). KD can be determined by measuring the kinetics of complex formation and dissociation using, for example, a surface plasmon resonance (SPR) method (Biacore). The rate constants corresponding to the association and dissociation of a monovalent complex are referred to as the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. K D Through the equation K D = kd / ka is related to ka and kd. The value of the dissociation constant can be determined directly by well-known methods and can be calculated even for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9: 340-362). For example, K can be determined using a double filtration nitrocellulose filter binding assay such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). D Other standard assays for assessing the binding ability of an antibody to a target antigen are known in the art, including, for example, ELISA, Western blot, RIA, and flow cytometry analysis, as well as other assays exemplified elsewhere in this disclosure. The binding kinetics and binding affinity of an antibody may also be determined by standard assays known in the art, such as surface plasmon resonance (SPR), for example, by using Biacore TM The K of each antibody / antigen complex can be compared. D The K values ​​can be used to compare the binding affinities associated with different molecular interactions, for example, comparison of the binding affinities of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared for the K values ​​of the interaction of interest (e.g., a specific interaction between an antibody and an antigen). D The K values ​​are compared with those of non-target interactions (e.g., control antibodies known not to bind CD40). D Value is evaluated.

[0513] "Conservative substitution" refers to substitution with another amino acid residue having a property similar to the original amino acid residue. For example, lysine, arginine and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine ​​and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine and methionine have similar properties in that they have non-polar side chains. In addition, tyrosine, phenylalanine, tryptophan and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when replacing an amino acid residue in a group showing similar properties as described above, it will not show a specific change in properties.

[0514] "Homology", "identity" or "sequence identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two compared sequences are occupied by the same nucleotide or amino acid monomer, for example, if every position of the two DNA molecules is occupied by the same nucleotide, then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, when the sequences are optimally aligned, if 6 out of 10 positions in the two sequences are matched or homologous, then the two sequences are 60% homologous. In general, comparison is made when the two sequences are aligned to obtain the maximum percentage of homology.

[0515] Example

[0516] The present disclosure is further described below in conjunction with examples, but these examples are not intended to limit the scope of the present disclosure. Experimental methods in the examples of the present disclosure that do not specify specific conditions are usually performed under conventional conditions, such as the Cold Spring Harbor Antibody Technology Laboratory Manual and the Molecular Cloning Manual; or under the conditions recommended by the raw material or product manufacturer. Reagents that do not specify specific sources are conventional reagents purchased from the market.

[0517] Example 1. Screening, preparation and modification of anti-PD-1 nanobodies

[0518] 1. Antibody Screening

[0519] The present invention uses human PD-1 with a His tag as an immune antigen (AcroBiosystems, PD1-H5221) to immunize alpacas, construct a nanoantibody (VHH) yeast display library, and screen antibodies. Human PD-1 and cynomolgus monkey PD-1 labeled with biotin are used as screening antigens (AcroBiosystems, PD1-H82E4; PD1-C82E6), and 18 unique VHH sequences cross-linked with human and monkey PD-1 antigens are obtained through monoclonal identification, sequencing, and sequence analysis.

[0520] The sequence of human PD-1 protein (NP_005009.2) is as follows, where the underlined portion is the extracellular region sequence, and the amino acids start and end at Leu25-Gln 167.

[0521] > Human PD-1 amino acid sequence

[0522] MQIPQAPWPVVWAVLQLGWRPGWF LDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWY RMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRV TERRAEVPTAHPSPSPRPAGQFQ TLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL(SEQ IDNO:1)

[0523] The A17 sequence is shown below, and the CDRs follow the Kabat numbering convention.

[0524] >A17 variable region

[0525] EVQVVESGGGLVQPGGSLRLSCVASGLTFS DYSMS WYRQAPGKERELVA IISGSGVIAHYVDSVKG RFTISRDNAKSTVYLQMVSLKPEDRGVYYCRA VSDW WGQGTQVTVSS (SEQ ID NO: 2)

[0526] >A17 CDR1

[0527] DYSMS (SEQ ID NO: 3)

[0528] >A17 CDR2

[0529] IISGSGVIAHYVDSVKG(SEQ ID NO:4)

[0530] >A17 CDR3

[0531] VSDWDDY (SEQ ID NO: 5)

[0532] A17 was linked to a human IgG4 Fc fragment (including the hinge region and with S228P, according to the EU numbering system) to construct a VHH-Fc antibody. The plasmid was constructed, transiently transfected into 293 cells, expressed, purified using a protein A column, washed with PBS, eluted with 0.1M glycine buffer, pH 2.5. Dialyzed into PBS pH7.4 buffer. After detection, the target protein was obtained.

[0533] >hIgG4

[0534] ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:6)

[0535] >A17_hIgG4

[0536] EVQVVESGGGLVQPGGSLRLSCVASGLTFS DYSMS WYRQAPGKERELVA IISGSGVIAHYVDSVKG RFTISRDNAKSTVYLQMVSLKPEDRGVYYCRA VSDW WGQGTQVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKG LPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0537] (SEQ ID NO:7)

[0538] 2. Humanization and back mutation

[0539] Antibody humanization was performed by the CDR-grafting method. The parental PD-1 antibody was compared with the full human germline genes in the IMGT or NCBI / igblast database through the IMGT or NCBI website, and the human germline gene IGHV 3-23 (IGHV3-23*01; IGHV3-23*04) highly homologous to the PD-1 nanobody was selected as the humanization template, and the CDR was transplanted to retain the four key residues (Y37, E44, R45, L47) in the FR2 of the nanobody. The key core residues close to the CDR region and the Vernier position residues that interact with the CDR were reverse mutated. Humanized antibodies A17h1, A17h2, and A17h3 were obtained.

[0540] >A17h1 variable region

[0541] EVQLQESGGGLVQPGGSLRLSCTASGLTFS DYSMS WYRQAPGKGRELVA IISGSGVIAHYVDSVKG RFTISRDNSKNTVYLQMRSLTPEDRAVYYCRA VSDW WGQGTQVTVSS (SEQ ID NO: 8)

[0542] >A17h2 variable region

[0543] EVQLVESGGGLVQPGGSLRLSCAASGLTFS DYSMS WVRQAPGKGLEWVA IISGSGVIAHYVDSVKG RFTISRDNSKNTVYLQMNSLRAEDTAVYYCRA VSDW WGQGTQVTVSS (SEQ ID NO:9)

[0544] >A17h3 variable region

[0545] EVQLVESGGGLVQPGGSLRLSCAASGLTFS DYSMS WVRQAPGKGLEWVA IISGSGVIAHYVDSVKG RFTISRDNSKNTVYLQMNSLRAEDTAVYYCRA VSDWDEY WGQGTQVTVSS (SEQ ID NO: 10)

[0546] 3. Remove TCE (T cell epitope), reduce antibody deamidation, and reduce antibody isomerization to obtain the following sequence:

[0547] >A17m09 variable region

[0548] EVQLQESGGGLVQPGGSLRLSCTASGLTFS DYSMS WYRQAPGKGRELVA IISGSGVITHYVDSVKG RTISRDNAKNTVYLQMRSLTPEDRAVYYCRA VSDWEDY WGQGTQVTVSS (SEQ ID NO: 11)

[0549] A17m09 was further selected to germline the antibody sequence to improve the degree of humanization, and the following sequence was obtained:

[0550] >A17m0901 variable region

[0551] EVQLVESGGGLVQPGGSLRLSCAASGFTFS DYSMS WYRQAPGKGRELVA IISGSGVITHYVDSVKG RFTISRDNAKNTLYLQMRSLRAEDTAVYYCRA VSDWEDY WGQGTQVTVSS (SEQ ID NO: 12)

[0552] >A17m0902 variable region

[0553] EVQLVESGGGLVQPGGSLRLSCAASGLTFS DYSMS WYRQAPGKGRELVA IISGSGVITHYVDSVKG RFTISRDNAKNTLYLQMRSLRAEDTAVYYCRA VSDWEDY WGQGTQVTVSS (SEQ ID NO: 13)

[0554] >A17m0903 variable region

[0555] EVQLVESGGGLVQPGGSLRLSCAASGLTFS DYSMS WYRQAPGKGRELVA IISGSGVITHYVDSVKG RFTISRDNAKNTVYLQMRSLRAEDTAVYYCRA VSDWEDY WGQGTQVTVSS (SEQ ID NO: 14)

[0556] >A17m0905 variable region

[0557] EVQLVESGGGLVQPGGSLRLSCAASGLTFS DYSMS WYRQAPGKGRELVA IISGSGVITHYVDSVKG RFTISRDNAKNTVYLQMRSLRAEDRAVYYCRA VSDWEDYWGQGTQVTVSS (SEQ ID NO: 15) That is, A17 of the present disclosure has the following general sequence:

[0558] CDR1: DYSMS (SEQ ID NO: 3)

[0559] CDR2: IISGSGVIX1HYVDSVKG, wherein X1 is selected from T or A (SEQ ID NO: 16)

[0560] CDR3: VSDWX4X5Y, wherein X4 is selected from D or E, and X5 is selected from D or E (SEQ ID NO: 17)

[0561] Specifically, CDR2 may be:

[0562] IISGSGVIAHYVDSVKG(SEQ ID NO:4)

[0563] IISGSGVITHYVDSVKG(SEQ ID NO:18)

[0564] CDR3 can be:

[0565] VSDWDDY (SEQ ID NO: 5)

[0566] VSDWEDY (SEQ ID NO: 19)

[0567] VSDWDEY (SEQ ID NO: 20)

[0568] Example 2. Anti-PD-1 Nanobody Functional Detection

[0569] 1.ELISA

[0570] Use PBS to dissolve human PD-1 protein (his tag) to 1μg / mL, add 100uL / well to a 96-well plate, and coat the antigen at 4℃ overnight. Wash three times with PBST (PBS+0.05% tween20) solution. Add PBST / 1% BSA solution and incubate at 37℃ for 1h for blocking. After washing three times with PBST solution, add different concentrations of PD-1 candidate VHH-Fc antibodies (diluted with PBST / 1% BSA solution) and incubate at 37℃ for 1.5 hours. Wash three times with PBST solution. Add 100μL HRP-conjugated anti-human IgG4 Fc secondary antibody (Thermo) solution and incubate at 37℃ for 1 hour. After washing three times with PBST solution, add 100μL / well TMB solution, react at room temperature for 5min, add 50μL stop solution, and then use an enzyme reader to read the 450nM value and calculate the EC 50,Table 4 shows that A17_hIgG4 has a strong binding affinity with PD-1 antigen.

[0571] Table 4. EC of anti-PD-1 nanobody detected by ELISA 50 value

[0572] Antibody ID <![CDATA[EC 50 (nM)]]> A17_hIgG4 0.06099

[0573] 2. FACS

[0574] To detect the binding ability of anti-PD-1 nanobodies to PD-1 expressed on the cell membrane, different concentrations of candidate anti-PD-1 VHH-Fc antibodies were added to 10 5 / well of Jurkat cells overexpressing human PD-1 (Cat: J1250, Promega), incubated at room temperature for 20 min, washed twice with PBS, added 100 μL of anti-human IgG Fc fluorescent secondary antibody (Biolegend), incubated at room temperature for 20 min, washed twice with PBS, resuspended in 250 μL PBS, detected the fluorescent signal by FACS, and calculated EC 50 Pembrolizumab (purchased from Bio-Bio) was used as a control. Table 5 shows the EC values ​​of A17_hIgG4 50 The value was 0.3568 nM, which was significantly better than other antibodies obtained in the simultaneous screening (results not shown).

[0575] Table 5. EC of anti-PD-1 nanobody binding to cell surface PD-1 detected by FACS 50 value

[0576] Antibody ID <![CDATA[EC 50 (nM) <!-- 36 -->]]> A17_hIgG4 0.3568 Pembrolizumab 0.2169

[0577] 3. PD-1 / PD-L1 reporter gene system to detect the blocking of PD-1 by anti-PD-1 nanoantibodies

[0578] CHO-K1 cells overexpressing PD-L1 were diluted to 4 × 10 5 / mL, add 100 μL to each well of a 96-well plate and culture overnight. After aspirating the culture medium, quickly add 40 μL of 1.25×10 6 / mL PD-1 Jurkat cells and 40μL anti-PD-1 nanoantibody solution (1640+2% FBS solution dilution) of different concentrations were added. Incubate at 37℃ for 6 hours, return to room temperature, add 40μL Bright-glo reagent (Cat: E2620, Promega) to each well, shake at 350rpm in the dark for 5 minutes, keep in the dark, and stand at room temperature for 5 minutes. Then use an enzyme reader to read the fluorescence value and calculate the IC 50Pembrolizumab and hIgG4 isotype antibodies were used as controls. As shown in Table 6, the biological functional activity IC 50 The value is 1.199 nM, which is significantly better than other antibodies screened simultaneously in the present disclosure (e.g., A3_hIgG4 is 7.62, A13_hIgG4 is 3.208; the sequences of A3_hIgG4 and A13_hIgG4 are not shown).

[0579] Table 6. IC values ​​of anti-PD-1 nanoantibodies detected by PD-1 / PD-L1 NFAT reporter gene system 50 value

[0580] Antibody ID <![CDATA[IC 50 (nM)]]> A17_hIgG4 1.199 Pembrolizumab 0.5126 hIgG4 -

[0581] The above method was used to verify the function of anti-PD-1 nanoantibody. See Table 7 for the IC of A17h1_hIgG4. 50 The activity is similar to that of the positive antibody Pembrolizumab. Among the candidate molecules with sequence modification, the biological activity IC 50 The values ​​were 0.5307nM and 0.4352nM, respectively, which were better than Pembrolizumab.

[0582] Table 7. IC values ​​of modified anti-PD-1 nanoantibodies detected by PD-1 / PD-L1 NFAT reporter gene system 50

[0583] Antibody ID <![CDATA[IC 50 (nM)]]> A17h1_hIgG4 1.039 A17m09_hIgG4 1.083 A17m0901_hIgG4 0.5307 A17m0902_hIgG4 0.4352 A17m0903_hIgG4 1.284 A17m0905_hIgG4 0.5588 Pembrolizumab 0.9756

[0584] 4. Identification of the binding affinity between the modified anti-PD-1 nanobody and the antigen PD-1

[0585] The affinity of anti-PD-1 nanoantibodies to PD-1 antigens was detected by surface plasmon resonance (SPR) using Biacore 8k (GE Healthcare). The CM5 sensor chip was used in the experiment, and the mobile phase was HBS-EP+ buffer solution (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20). The anti-human IgG (Fc) antibody was prepared into a 30μg / mL solution with 10mM sodium acetate buffer (pH 5.0), and the Immobilization program was selected to automatically couple the anti-human IgG (Fc) antibody channel to amino groups. Each antibody to be tested was prepared as a ligand with HBS-EP+ buffer solution, and captured with the anti-human IgG (Fc) antibody on the chip channel. Human antigen protein (Sino Biological, 10377-H08H; 90311-C08H) was used as the analyte and prepared with HBS-EP+ buffer solution. The analyte was diluted 2 times in a gradient and flowed through the experimental channel and the reference channel at a flow rate of 30 μL / min. The binding time was 1 min and the dissociation time was 15 min. The regeneration buffer 10 mM Glycine pH 1.5 (GE Healthcare, BR-1003-54) was run at a flow rate of 10 μL / min for 30 s, and the binding rate Ka and dissociation rate Kd, as well as the dissociation constant (i.e., affinity K) were calculated. D ). See Table 8 for the results.

[0586] Table 8. SPR affinity data of anti-PD-1 nanobodies binding to human PD-1 antigen

[0587] Antibody ID ka(1 / Ms) Kd(1 / s) <![CDATA[K D (M)]]> A17h1_hIgG4 4.30E+05 8.68E-04 2.02E-09 A17m09_hIgG4 4.58E+05 1.63E-03 3.56E-09 A17m0901_hIgG4 2.97E+05 1.92E-03 6.44E-09 A17m0902_hIgG4 2.91E+05 1.77E-03 6.09E-09 Pembrolizumab 1.25E+06 3.89E-03 3.11E-09

[0588] The results showed that the humanized antibody A17h1_hIgG4 and the subsequently modified and optimized antibodies A17m09_hIgG4, A17m0901_hIgG4, and A17m0902_hIgG4 had similar K values ​​to Pembrolizumab. D The dissociation time of A17h1_hIgG4, A17m09_hIgG4, A17m0901_hIgG4, and A17m0902_hIgG4 were slower than that of Pembrolizumab.

[0589] 5. DC:T mixed lymphocyte reaction to test the in vitro efficacy of modified anti-PD-1 nanoantibodies (MLR)

[0590] Co-culture of mature dendritic cells (DC) with allogeneic T cells can activate T cells and promote T cell secretion of cytokines. This process is inhibited by PD-1 / PD-L1 signals, and the use of PD-1 antibodies can relieve inhibition and promote T cell activation. Therefore, the DC:T mixed lymphocyte reaction experiment can be used to test the in vitro efficacy of PD-1 antibodies.

[0591] Monocytes were isolated from fresh peripheral blood (PBMC) of a healthy person using a sorting kit (cat: 19359, stemcell). After seven days of culture, monocytes were induced to differentiate into mature DC cells using a DC cell induction kit (cat: 10985, stemcell). Allogeneic T cells were then isolated from fresh PBMC of another healthy person using a sorting kit (cat: 17951, stemcell). 5,000 mature DC cells were co-cultured with 50,000 allogeneic T cells, and candidate antibodies of corresponding concentrations were added. After six days of co-culture, the cell supernatant was aspirated, and the IFN-γ concentration was detected using the Cisbio-HTRF IFN-γ detection kit (Cat: 62HIFNGPEH, Perkinelmer), and the EC was calculated. 50 .

[0592] like Figure 1 As shown in Figure 2, A17m09_hIgG4 showed in vitro efficacy that was basically consistent with Pembrolizumab in the MLR experiment. 50 The EC of pembrolizumab is 0.2931 nM. 50 It is 0.3271nM.

[0593] Example 3. Construction and activity detection of fusion protein of anti-PD-1 antibody and IL-2 mutant (PD-1-IL-2v)

[0594] 1. Molecular Construction

[0595] according to Figure 2 The schematic diagram of the molecular format shown is to construct a fusion protein of anti-PD-1 antibody and IL-2 mutant. Figure 2The Fc of the middle molecule adopts Knob-in-Hole (KIH) technology, that is, S354C, T366W (constituting Knob chain) and Y349C, T366S, L368A, Y407V (Eu numbering system) (constituting Hole chain) mutations are introduced into the two Fcs to promote the formation of heterodimers; for the convenience of purification, H435R, Y436F (Eu numbering system) are additionally introduced into the Hole chain; in addition, L234A, L235A and P329G mutations are introduced into both the Knob and Hole chains to eliminate the ADCC effect; for the heavy chain without light chain pairing, the C220S mutation is introduced into the hinge region to remove the free, unpaired cysteine. IL-2 uses IL-2v (V91T) including T3A / R38E / F42A / V91T / C125A mutations.

[0596] like Figure 2 In the molecule A-V91T of the structure shown, the anti-PD-1 nanobody A17m0902 is coupled to the N-terminus of the Knob and Hole chains respectively through the hinge region of the IgG1 heavy chain; IL-2v(V91T) is connected to the C-terminus of the Knob chain of Fc through a (G4S)3 linker.

[0597] The sequences of each molecule are as follows, with the Fc region underlined and the linker in italics.

[0598] >Knob chain of A-V91T

[0599] EVQLQESGGGLVQPGGSLRLSCTASGLTFSDYSMSWYRQAPGKGRELVAIISGSGVIAHYVDSVKGRFTISRDNSKNTVYLQMRSLTPEDRAVYYCRAVSDWDDYWGQGTQVTVSS EPKSSDKTHTCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG GGGGSGGGGSGGGGSAPASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINTIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:21)

[0600] >Hole chain of A-V91T

[0601] EVQLQESGGGLVQPGGSLRLSCTASGLTFSDYSMSWYRQAPGKGRELVAIISGSGVIAHYVDSVKGRFTISRDNSKNTVYLQMRSLTPEDRAVYYCRAVSDWDDYWGQGTQVTVSS EPKSSDKTHTCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSP (SEQ ID NO:22)

[0602] >IL-2v (containing T3A / R38E / F42A / V91T / C125A mutations)

[0603] APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINTIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:23)

[0604] The above fusion protein was expressed in HEK293 cells and purified by Protein A. The molecular weight of the purified product was confirmed by SDS-PAGE to be consistent with the expectation, proving that the target protein was prepared.

[0605] 2. Detection of PD-1-IL-2v-promoting M07e cell proliferation activity

[0606] M07e is a human cell line that only expresses IL-2Rβ and IL-2Rγ, but not IL-2Rα. In order to detect the activity of the above different forms of PD-1-IL2v(V91T) fusion protein and its dependence on PD-1 expression, we overexpressed human PD-1 (SEQ ID NO: 1) in the M07e cell line to obtain the M07e-hPD-1 cell line, and detected the effect of the above fusion protein on cell proliferation in M07e and M07e-hPD-1 cell lines, respectively.

[0607] Complete culture medium: RPMI 1640 + 2mM L-glutamine + 1mM sodium pyruvate + 10% fetal bovine serum + 15ng / mL GM-CSF; basal culture medium: RPMI 1640 + 2mM L-glutamine + 1mM sodium pyruvate + 10% fetal bovine serum.

[0608] M07e cell proliferation assay: M07e cells or M07e-hPD1 were cultured in complete culture medium at 37°C and 5% CO2. The cells were passaged and collected by centrifugation after 1-2 days. The cells were washed three times with PBS and resuspended in basal culture medium to prepare 6.0×10 5 The cell suspension of 10 cells was placed in a 96-well plate with a volume of 50 μL per well. 50 μL of the test drug prepared with basal culture medium at 2× corresponding concentration was added. After 72 hours of cell culture, 100 μL of lysis solution CELLTITER-Glo reagent (Promega) was added to each well, mixed, and the absorbance was measured at a wavelength of 570 nm using an ELISA reader with 630 nm as the reference wavelength.

[0609] The proliferation activity data of PD-1-IL-2v (V91T) fusion protein on M07e and M07e-hPD-1 cells are shown in Table 12. A PEGylated IL-2 mutant (PEG-IL-2v) was used as a positive control, wherein the IL-2 mutant had T3A, F42A, L72G, N26Q, N29S, N71Q and C125A mutations, which did not bind to IL-2Rα, but could bind to the affinity receptor IL-2Rβγ in IL-2.

[0610] The results showed that PEG-IL-2v had comparable activity against M07e and M07e-hPD-1 cell proliferation, and that various forms of PD-1-IL-2 (V91T) had significantly stronger activity against M07e-hPD-1 than against M07e cell proliferation, indicating that anti-PD-1 antibodies can increase the selectivity of IL-2 mutants for PD-1-positive cells. In addition, various forms of PD-1-IL-2 (V91T) had comparable activity against M07e proliferation, but C-V91T was slightly stronger than A-V91T against M07e-hPD-1 proliferation, and A-V91T was stronger than B-V91T and E-V91T.

[0611] Table 9. Proliferative activity of PD-1-IL-2v(V91T) on M07e and M07e-hPD-1 cells

[0612]

[0613] Example 4. Construction of PD-1-IL-2v carrying different IL-2 mutants

[0614] Although PD-1-IL-2v(V91T) was less active than PEG-IL-2v in M07e without PD-1 expression, it could still stimulate cell proliferation, indicating that IL-2v(V91T) could still strongly bind to the IL-2 medium-affinity receptor IL-2Rβγ, and PD-1-IL-2(V91T) could still systemically activate NK and CD8 + T cells and cause toxicity. In order to further reduce the toxicity of PD-1-IL-2v and make the activity of IL-2 more dependent on the expression of PD-1, we constructed PD-1-IL-2v with different IL-2 mutants based on the A-V91T molecular form. All IL-2 mutants carry mutations of F42A and L72G to eliminate the binding of IL-2 to IL-2Rα; in addition, for stability considerations, N26Q, N29S and N71Q are added to the IL-2 mutants to remove the possible deamidation modification in IL-2 and increase the stability of IL-2. In addition, they also carry their own unique mutations as shown in Table 10, which reduce the affinity of IL-2 to the medium affinity receptor IL-2Rβγ to varying degrees.

[0615] Table 10. IL-2 mutations in PD-1-IL-2v

[0616]

[0617] >IL-2 sequence in A-PC

[0618] APASSSTKKTQLQLEHLLLDLQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:24)

[0619] >IL-2 sequence in A-V91T-02

[0620] APASSSTKKTQLQLEHLLLDLQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRDLISNINTIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:25)

[0621] >IL-2 sequence in A-D20A

[0622] APACSTKKTQLQLEHLLLQMILQGISNYKNPKLTRMLTTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:26)

[0623] >A-D20N in IL-2

[0624] APASSSTKKTQLQLEHLLLNLQMILQGISNYKNPKLTRMLTRMLTAKFYMPKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:27)

[0625] >A-N88D in the IL-2

[0626] APASSSTKKTQLQLEHLLLDLQMILQGISNYKNPKLTRMLTTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:28)

[0627] >A-N88R in the IL-2

[0628] APACSTKKTQLQLEHLLLDLQMILQGISNYKNPKLTRMLTTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:29)

[0629] >A-Q126D in IL-2

[0630] APASSSTKKTQLQLEHLLLDLQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFADSIISTLT(SEQ ID NO:30)

[0631] >IL-2 sequence in A-H16A / D84S

[0632] APASSSTKKTQLQLEALLLDLQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRSLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:31)

[0633] L234A and L235A mutations are introduced into Fc to eliminate the ADCC effect; S354C, T366W (constituting Knob chain) and Y349C, T366S, L368A, Y407V (Eu numbering system) (constituting Hole chain) mutations are introduced to promote the formation of heterodimers; and C220S mutation is introduced into the hinge region. For the convenience of purification, the antibody may contain H435R, Y436F (Eu numbering system) mutations in the Hole chain. In addition, the Hole chain of the Fc region of these antibodies may be D356, L358 or E356, M358 (Eu numbering system) at amino acids 356 and 358, respectively. They appear in different alleles and do not affect the function of the antibody.

[0634] >Fc Knob chain (D356, L358)

[0635] EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:32)

[0636] >Hole chain of Fc (D356, L358)

[0637] EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP(SEQ ID NO:33)

[0638] >Knob chain of Fc (E356, M358)

[0639] EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:34)

[0640] >Hole chain of Fc (E356, M358)

[0641] EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:35)

[0642] Exemplarily, the full-length sequences of A-D20A and A-N88D are given, with Fc underlined and the linker italicized.

[0643] >Knob chain of A-D20A (D356, L358)

[0644] EVQLVESGGGLVQPGGSLRLSCAASGLTFSDYSMSWYRQAPGKGRELVAIISGSGVITHYVDSVKGRFTISRDNAKNTLYLQMRSLRAEDTAVYYCRAVSDWEDYWGQGTQVTVSS EPKSSDKTHTCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG GGGGSGGGGSGGGGSAPASSSTKKTQLQLEHLLLALQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:36)

[0645] >Hole chain of A-D20A (D356, L358)

[0646] EVQLVESGGGLVQPGGSLRLSCAASGLTFSDYSMSWYRQAPGKGRELVAIISGSGVITHYVDSVKGRFTISRDNAKNTLYLQMRSLRAEDTAVYYCRAVSDWEDYWGQGTQVTVSS EPKSSDKTHTCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSP (SEQ ID NO:37)

[0647] >Knob chain of A-D20A (E356, M358)

[0648] EVQLVESGGGLVQPGGSLRLSCAASGLTFSDYSMSWYRQAPGKGRELVAIISGSGVITHYVDSVKGRFTISRDNAKNTLYLQMRSLRAEDTAVYYCRAVSDWEDYWGQGTQVTVSS EPKSSDKTHTCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSAPASSSTKKTQLQLEHLLLALQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:38)

[0649] >Hole chain of A-D20A (E356, M358)

[0650] EVQLVESGGGLVQPGGSLRLSCAASGLTFSDYSMSWYRQAPGKGRELVAIISGSGVITHYVDSVKGRFTISRDNAKNTLYLQMRSLRAEDTAVYYCRAVSDWEDYWGQGTQVTVSS EPKSSDKTHTCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:39)

[0651] >Knob chain of A-N88D (D356, L358) EVQLVESGGGLVQPGGSLRLSCAASGLTFSDYSMSWYRQAPGKGRELVAIISGSGVITHYVDSVKGRFTISRDNAKNTLYLQMRSLRAEDTAVYYCRAVSDWEDYWGQGTQVTVSS EP KSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G GGGGSGGGGSGGGGSAPASSSTKKTQLQLEHLLLDLQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQID NO:40)

[0652] >Hole chain of A-N88D (D356, L358)

[0653] EVQLVESGGGLVQPGGSLRLSCAASGLTFSDYSMSWYRQAPGKGRELVAIISGSGVITHYVDSVKGRFTISRDNAKNTLYLQMRSLRAEDTAVYYCRAVSDWEDYWGQGTQVTVSS EPKSSDKTHTCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO:41)

[0654] >Knob chain of A-N88D (E356, M358)

[0655] EVQLVESGGGLVQPGGSLRLSCAASGLTFSDYSMSWYRQAPGKGRELVAIISGSGVITHYVDSVKGRFTISRDNAKNTLYLQMRSLRAEDTAVYYCRAVSDWEDYWGQGTQVTVSS EPKSSDKTHTCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG GGGGSGGGGSGGGGSAPASSSTKKTQLQLEHLLLDLQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:42)

[0656] >Hole chain of A-N88D (E356, M358)

[0657] EVQLVESGGGLVQPGGSLRLSCAASGLTFSDYSMSWYRQAPGKGRELVAIISGSGVITHYVDSVKGRFTISRDNAKNTLYLQMRSLRAEDTAVYYCRAVSDWEDYWGQGTQVTVSS EPKSSDKTHTCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:43)

[0658] In addition, to prove the dependence of PD-1-IL-2v on PD-1, we replaced the anti-PD-1 antibody part in part of PD-1-IL-2v with antibodies against unrelated antigens, while retaining the mutation of the IL-2 part, and obtained IgG-PC, and the corresponding IgG-V91T containing V91T on IL-2, and the corresponding IgG-D20A containing D20A on IL-2, and the corresponding IgG-N88D containing N88D on IL-2. The difference between IgG-PC and IgG-V91T, IgG-D20A, and IgG-N88D is only in the IL-2 part. The sequence is as follows:

[0659] > Knob chain of IgG-PC

[0660] (SEQ ID NO:44)

[0661] > Knob chain of IgG-V91T

[0662] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNSWIGWFRQAPGQGLEWIGDIYPGGGYTNYNEIFKGKATMTADTSTNTAYMELSSLRSEDTAVYYCSRGIPGYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSAPASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINTIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:45)

[0663] >Knob chain of IgG-N88D

[0664] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNSWIGWFRQAPGQGLEWIGDIYPGGGYTNYNEIFKGKATMTADTSTNTAYMELSSLRSEDTAVYYCSRGIPGYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSAPASSSTKKTQLQLEHLLLDLQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:46)

[0665] >Knob chain of IgG-D20A

[0666] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNSWIGWFRQAPGQGLEWIGDIYPGGGYTNYNEIFKGKATMTADTSTNTAYMELSSLRSEDTAVYYCSRGIPGYAMDYWGQGTLVTSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGGSGGGSGGGGSAPASSSTKKTQLQLEHLLLALQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT

[0667] (SEQ ID NO:47)

[0668] >Hole chain of IgG-PC, IgG-V91T, IgG-D20A or IgG-N88D QVQLVQSGAEVKKPGASVKVSCKASGYTFTNSWIGWFRQAPGQGLEWIGDIYPGGGYTNYNEIFKGKATMTADTSTNTAYME LSSLSEDTAVYYCSRGIPGYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSP(SEQ ID NO:48)

[0669] >Light chain DIQMTQSPSSSLSASVGDRVTMSCKSSQSLLNSGDQKNYLTWYQQKPGKAPKLLIYWASTGESGVPSRFSGSGSGTDFTFTISSLQPEDIATYY of >IgG-PC, IgG-V91T, IgG-D20A or IgG-N88D CQNDYSYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:49)

[0670] The above fusion protein was expressed in HEK293 cells and purified by Protein A. The molecular weight of the purified product was confirmed by SDS-PAGE to be consistent with the expectation, proving that the target protein was obtained.

[0671] In addition, the wild-type IL-2 sequence of the present disclosure is:

[0672] >wt IL-2

[0673] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT(SEQ ID NO:50)

[0674] Example 5. Detection of PD-1-IL-2v promoting M07e cell proliferation activity

[0675] In order to detect the activity of the above-mentioned PD-1-IL-2v fusion proteins with different IL-2 mutants and their dependence on PD-1 expression, we detected the effect of the fusion proteins in Example 4 on cell proliferation in M07e and M07e-hPD-1 cell lines. The experimental method is shown in Example 3. The relative cell proliferation activity of PD-1-IL-2v was calculated as follows:

[0676]

[0677] in, represents the activity of PD-1-IL-2v at the concentration of conc, Represents the luminescence intensity of cell lysate when stimulated by PD-1-IL-2v at the concentration of conc, It represents the luminescence intensity of the cell lysate after stimulating the cells with the positive control molecule A-PC at a concentration of 1000 nM in M07e or M07e-hPD-1 cell lines. It represents the background luminescence intensity when only the culture medium was added to the M07e or M07e-hPD-1 cell line without any PD-1-IL2v or PEG-IL-2v stimulation.

[0678] The experimental results are shown in Table 11 and Figure 3A and Figure 3BThe results showed that 1) in M07e cells, A-PC and PEG-IL-2v had comparable activities, indicating that in the absence of PD-1 overexpression, the activities of IL-2 bound to antibodies and IL-2 coupled to PEG were consistent; the activities of A-V91T and IgG-V91T were comparable and weaker than A-PC, indicating that V91T did reduce the activity of IL-2 but could still stimulate the proliferation of M07e; other mutants, including D20A, D20N, N88R, N88D and Q126D, basically did not activate M07e cells, and only A-N88D had weak activity at a concentration of 1000nM. 2) In M07e-hPD-1 cells, due to the overexpression of PD-1 on the cells, the activity of each PD-1-IL-2v at lower concentrations far exceeded that of IgG-V91T and PEG-IL-2v, indicating that anti-PD-1 antibodies greatly increased the dependence of PD-1-IL-2v's cell proliferation-promoting activity on PD-1 expression; compared with A-PC and A-V91T, the maximum activity of other PD-1-IL-2v on M07e-hPD-1 cell proliferation was slightly lower. 3) The activities of various PD-1-IL-2v in M07e and M07e-hPD-1 cell proliferation experiments were compared. PD-1-IL2v with D20A, D20N, N88R, N88D or Q126D mutations basically did not activate M07e cells, but could strongly stimulate the proliferation of M07e-hPD-1 cells, indicating that PD-1-IL-2v reduced the activation of PD-1-negative cells while retaining the selective activation of PD-1-positive cells; while A-PC and A-V91T, although they have selective activation of PD-1-positive cells, can also activate PD-1-negative cells.

[0679] Table 11. Proliferative activity of PD-1-IL-2v with different IL-2 mutations on M07e and M07e-hPD-1 cells

[0680]

[0681] In order to better demonstrate the dependence of PD-1-IL-2v(N88D) and PD-1-IL-2v(D20A) on PD-1, we compared the effects of A-N88D and IgG-N88D, A-D20A and IgG-D20 on cell proliferation in the M07e-hPD-1 cell line. The experimental results are shown in Tables 12 and Figure 4The results showed that at higher concentrations (1000 nM), IgG-N88D and IgG-D20A still could not activate the proliferation of M07e-hPD-1 cells, but A-N88D and A-D20A could activate the proliferation of M07e-hPD-1 cells at lower concentrations.

[0682] Table 12. Proliferative activity of PD-1-IL-2v or IgG-IL-2v on M07e-hPD-1 cells

[0683]

[0684] Example 6. Detection of PD-1-IL-2v T cell proliferation activity

[0685] Unstimulated T cells do not express or low-express PD-1, while T cells stimulated with CD3 antibodies highly express PD-1. To test whether the above-mentioned PD-1-IL-2v fusion proteins with different IL-2 mutants can activate T cells in human peripheral blood cells (PBMCs) with low and high PD-1 expression, we isolated T cells from PBMCs, stimulated the isolated T cells with CD3 antibodies, and tested the effects of the above-mentioned fusion proteins on the proliferation of unstimulated and stimulated T cells.

[0686] T cell proliferation experiment stimulated by CD3 antibody: After the cryopreserved human PBMCs were revived, T cells were isolated using Pan T Cell Isolation Kit (Miltenyi, 130-096-535) to obtain T cells. CD3 antibody was diluted to 0.1 μg / mL with PBS, added to a 10 cm culture dish, incubated at 37°C for 2 hours for coating, and washed with PBS for later use. T cells were cultured at 2×10 6 Cells / mL were plated in a 10 cm culture dish pre-coated with CD3 antibody and stimulated for 24 h at 37°C and 5% CO2. Stimulated T cells were collected, washed, counted, and the cell density was adjusted to 5×10 5 cells / mL, 200 μL per well was plated in a 96-well plate, the samples to be tested were gradiently diluted with culture medium to make 5× working solution, 50 μL of each concentration of the samples to be tested were added to the stimulated T cells and mixed, and cultured for 4 days. The cells treated with drugs were washed twice with FACS buffer (PBS + 2% FBS), and the cell life and death were marked with Fixable Viability Dye (eBioscience, 65-0865-14). After staining with CD3 (BD, 565491), CD4 (BD, 558116), CD8 (BD, 565310), Ki-67 (eBioscience, 12-5698-82)) flow cytometry antibodies, the cell proliferation was detected by FACS.

[0687] Unstimulated T cell proliferation assay: After separation, the T cells were counted and counted as 5×10 5 Cells / mL were plated in 96-well plates, 200 μL per well, and the samples to be tested were diluted with culture medium to make 5× working solution. 50 μL of each concentration of the sample to be tested was added to the stimulated T cells, mixed, and cultured for 4 days. The cells treated with drugs were washed twice with FACS buffer, and the cell death and viability were marked with FVD780. After flow cytometry antibody staining, the cell proliferation was detected by FACS.

[0688] The experimental results are shown in Tables 13, 14 and Figure 5A , Figure 5B , Fig. 6A , Figure 6B The results showed that 1) in unstimulated T cells, A-PC showed activity to activate T proliferation (CD8 + T cell proliferation EC 50 =21.46nM, CD4 + T cell proliferation EC 50 =8.61nM); A-V91T is less active than A-PC, indicating that V91T does reduce the activity of IL-2, but can still promote the proliferation of unstimulated T cells; other mutants, including D20A, D20N, N88R, N88D, and Q126D, did not show activation of T cells when the concentration reached 100nM. 2) In T cells stimulated with CD3 antibodies, each PD-1-IL-2v showed significant activity in promoting T cell proliferation at a lower concentration (0.1nM). Compared with A-PC and A-V91T, the maximum activity of PD-1-IL-2v carrying other mutations on the proliferation of pan T cells stimulated with CD3 antibodies was slightly lower. 3) The activities of each fusion protein in the T cell proliferation experiments without stimulation and with CD3 antibody stimulation were compared. PD-1-IL-2v with D20A, D20N, N88R, N88D, or Q126D mutations did not activate the proliferation of unstimulated T cells, but could strongly stimulate the proliferation of T cells stimulated with CD3 antibody; while A-PC and A-V91T, although they could more strongly activate the proliferation of T cells stimulated with CD3 antibody, could also strongly activate T cells without CD3 antibody stimulation.

[0689] Table 13. Proliferative activity of PD-1-IL-2v carrying different IL-2 mutations on unstimulated T cells

[0690]

[0691]

[0692] Table 14. Proliferative activity of PD-1-IL-2v with different IL-2 mutations on T cells stimulated with CD3 antibodies

[0693]

[0694] Example 7. Identification of the binding affinity of PD-1-IL2v to antigens PD-1 and IL-2Rα or IL-2Rβ / γ

[0695] The surface plasmon resonance (SPR) technique was used to detect the binding affinity of the above-mentioned PD-1-IL-2v fusion proteins with different IL-2 mutants to PD-1 and human IL-2Rα or IL-2Rβ / γ. The experiment was detected using a Biacore 8K (GE Healthcare) instrument. A CM5 sensor chip was selected, and the mobile phase was HBS-EP+ buffer solution (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20). The anti-human IgG (Fc) antibody was prepared into a 25μg / mL solution with 10mM sodium acetate buffer (pH 5.0), and the Immobilization program was selected to automatically couple the anti-human IgG (Fc) antibody channel to amino groups. Each PD-1-IL2v to be tested was prepared as a ligand with HBS-EP+ buffer solution, and captured with the anti-human IgG (Fc) antibody on the chip channel. Human PD-1 antigen protein (Sino Biological, 10377-H08H), human IL-2Rα receptor protein (Sino Biological, 10165-H08H) or human IL-2Rβ / γ receptor heterodimer protein (Acrosystem, ILG-H5283) were used as analytes and prepared with HBS-EP+ buffer solution. The analytes were diluted 2-fold from 200nM, with a total of 7 concentration points, and flowed through the experimental channel and reference channel at a flow rate of 30μL / min. The binding time was 140s and the dissociation time was 600s. The regeneration buffer 10mM Glycine pH 1.5 (GE Healthcare, 29238268-AA) was run at a flow rate of 10μL / min for 30s. The binding rate Ka and dissociation rate Kd, as well as the dissociation constant (i.e., affinity K D The result is as follows.

[0696] Table 15. SPR affinity of PD-1-IL2v binding to human PD-1 antigen

[0697] Antibody ID ka(1 / Ms) Kd(1 / s) <![CDATA[K D (M)]]> A-PC 9.37E+04 1.82E-03 1.94E-08 A-N88D 9.98E+04 1.75E-03 1.76E-08 A-D20A 9.06E+04 1.68E-03 1.85E-08

[0698] Table 16. SPR binding affinity of anti-PD-1 antibody and IL-2 mutant fusion proteins binding to human IL-2Rα

[0699] Antibody ID ka(1 / Ms) Kd(1 / s) <![CDATA[K D (M)]]> A-PC NB NB NB A-N88D NB NB NB A-D20A NB NB NB

[0700] (NB: not combined)

[0701] Table 17. SPR binding affinity of anti-PD-1 antibody and IL-2 mutant fusion proteins binding to human IL-2Rβ / γ

[0702] Antibody ID ka(1 / Ms) Kd(1 / s) <![CDATA[K D (M)]]> A-PC 8.81E+04 2.23E-05 2.53E-10 A-N88D 6.37E+04 2.15E-04 3.38E-09 A-D20A 4.27E+04 6.81E-04 1.60E-08

[0703] The results of binding to human PD-1 antigen showed that A-PC, A-N88D and A-D20A had similar binding affinity to human PD-1, and were similar to the binding affinity of A17m0902_hIgG4 to human PD-1 (Table 8). The results of binding to human IL-2Rα (Table 16) showed that A-PC, A-N88D and A-D20A did not bind to human IL-2Rα. The results of binding to human IL-2Rβ / γ (Table 17) showed that A-PC had the strongest affinity to human IL-2Rβ / γ, and the affinity of A-N88D and A-D20A to human IL-2Rβ / γ was reduced by about 13 times and about 63 times, respectively.

[0704] The following purification was performed on PD-1-IL2v (A-N88D, Knob chain as shown in SEQ ID NO:42, Hole chain as shown in SEQ ID NO:43).

[0705] Example 8: Affinity chromatography and protein detection

[0706] The cell clarification fluid containing the PD-1-IL2v fusion protein is subjected to affinity chromatography, and the specific steps are as follows:

[0707] Step 1: Load the cell clarification solution onto the equilibrated affinity chromatography column, and the affinity chromatography filler is N Mab;

[0708] Step 2: Use the equilibration buffer to flush the loaded column until UV 280 , pH, and conductivity are stable, and the equilibrium buffer is 0.02mol / L phosphate, 1.0mol / L sodium chloride, pH7.4;

[0709] Step 3: Use the first elution buffer to wash 5-8 column volumes. See Table 18 for the solution types.

[0710] Step 4: Use the second washing buffer to wash 2-3 column volumes to raise the pH of the affinity chromatography column to above 4.5. See Table 18 for the solution types;

[0711] Step 5: flushing with a third elution buffer for 2-3 column volumes, the solution type is shown in Table 18, and the conductivity of the third elution buffer is less than 5 mS / cm;

[0712] Step 6: Elute with 0.05 mol / L sodium acetate-acetic acid, pH 3.6 solution, and collect the eluate;

[0713] Step 7: Calculate the chromatography yield and detect the fusion protein in the eluate obtained by chromatography.

[0714] Table 18. Affinity chromatography and protein detection

[0715]

[0716] Example 9: Affinity chromatography and protein detection

[0717] Affinity chromatography of PD-1-IL2v was performed according to the conditions described in No. 1 in Example 8, and the first elution volume was taken as the upper limit (8 volumes). After obtaining the fusion protein of affinity chromatography, anion exchange chromatography and cation exchange chromatography were continued. The SEC-HPLC purity, RP-HPLC purity and NRCE purity of the fusion protein obtained in each step were detected. The results are shown in Table 19.

[0718] Table 19 Preparation of purified samples and detection of fusion proteins

[0719]

[0720] Example 10: Affinity chromatography and protein detection

[0721] The cell clarification fluid containing PD-1-IL2v is subjected to affinity chromatography, and the specific steps are as follows:

[0722] Step 1: Load the cell clarification solution onto the equilibrated affinity chromatography column, and the affinity chromatography filler is N Mab;

[0723] Step 2: Use the equilibration buffer to flush the loaded column until UV 280 , pH, and conductivity are stable, and the equilibrium buffer is 0.02mol / L phosphate, 1.0mol / L sodium chloride, pH7.4;

[0724] Step 3: Rinse with 0.05 mol / LM acetic acid-sodium acetate, pH 5.0 solution until UV280, pH, and conductivity are stable;

[0725] Step 4: Elute with 0.05 mol / L sodium acetate-acetic acid, pH 3.6 solution, and collect the eluate;

[0726] The SEC-HPLC purity of the fusion protein in the obtained affinity chromatography eluate was 82.07%, and the RP-HPLC purity was 81.14%.

[0727] Example 11: Affinity chromatography and protein detection

[0728] The fusion protein was prepared according to the affinity chromatography method described in Example 10; after obtaining the fusion protein of affinity chromatography, anion exchange chromatography and cation exchange chromatography were continued. The SEC-HPLC purity, RP-HPLC purity and NRCE purity of the fusion protein in each step of chromatography were detected. The results are shown in Table 20.

[0729] Table 20. Preparation of purified samples and detection of fusion proteins

[0730]

[0731] Example 12: Affinity chromatography and protein detection

[0732] The cell clarification fluid containing PD-1-IL2v is subjected to affinity chromatography, and the specific steps are as follows:

[0733] Step 1: Load the cell clarification solution onto the equilibrated affinity chromatography column, and the affinity chromatography filler is N Mab;

[0734] Step 2: Use the equilibration buffer to flush the loaded column until UV 280 , pH, and conductivity are stable, and the equilibrium buffer is 0.02mol / L phosphate, 1.0mol / L sodium chloride, pH7.4;

[0735] Step 3: Use the first washing buffer to wash 5-8 column volumes. See Table 21 for the solution types.

[0736] Step 4: Use the second washing buffer to wash 2-3 column volumes. See Table 21 for the solution types.

[0737] Step 5: Use the third elution buffer to wash for 2-3 column volumes. See Table 21 for the solution types.

[0738] Step 6: Elute with 0.05 mol / L sodium acetate-acetic acid, pH 3.6 solution, and collect the eluate;

[0739] Step 7: Calculate the chromatography yield and detect the fusion protein in the obtained eluate;

[0740] Table 21 Affinity chromatography and intermediate detection

[0741]

[0742]

Claims

1. A method for purifying a fusion protein, the method comprising the following steps: (a) Sample loading step: loading the sample containing the fusion protein onto the affinity chromatography medium; (b) Equilibration step: flushing the affinity chromatography medium after loading with equilibration buffer; (c) elution step: eluting the affinity chromatography medium with a elution buffer; (d) elution step: eluting the fusion protein from the affinity chromatography medium with an elution buffer and collecting the eluate; Wherein, the fusion protein comprises a PD-1 binding domain and a cytokine; Preferably, the isoelectric point (PI) of the fusion protein is about 4.0 to about 7.5, preferably about 5.0 to about 7.0, more preferably about 6.0 to about 7.

0.

2. The method according to claim 1, wherein: The fusion protein is a fusion protein comprising an antibody Fc; preferably, the fusion protein is a fusion protein comprising a PD-1 binding domain, a cytokine and an antibody Fc, wherein the cytokine is selected from IL-2 or a variant thereof; Preferably, the PD-1 binding domain is located at the N-terminus of Fc, and the cytokine is located at the N-terminus or C-terminus of Fc; Preferably, the fusion protein comprises a first polypeptide chain and a second polypeptide chain; wherein the isoelectric point between the first polypeptide chain and the second polypeptide chain differs by no more than about 3.0 units.

3. The method according to any one of claims 1 to 2, wherein: The elution step comprises sequentially rinsing with a first elution buffer, a second elution buffer and a third elution buffer; The pH of the first washing buffer is about 3.8 to about 5.0, preferably about 4.0 to about 4.7, more preferably about 4.1 to about 4.3; The pH of the second washing buffer is about 4.5 to about 8.0, preferably about 4.5 to about 6.5, more preferably about 4.5 to about 5.5; The pH of the third washing buffer is about 4.5 to about 8.0, preferably about 4.5 to about 6.5, more preferably about 4.5 to about 5.0; The pH of the equilibration buffer is about 5.0 to about 8.0, preferably about 6.5 to about 8.0, more preferably about 7.0 to about 8.0; and / or, The pH of the elution buffer is about 3.0 to about 5.0, preferably about 3.4 to about 4.0, and more preferably about 3.4 to about 3.

8.

4. The method according to claim 3, wherein: The buffer substances in the first washing buffer, the second washing buffer and the third washing buffer are independently selected from one or more of sodium acetate, sodium citrate, Tris, sodium dihydrogen phosphate, disodium hydrogen phosphate, acetic acid, citric acid and phosphoric acid; Preferably, the buffer substances in the first washing buffer, the second washing buffer and the third washing buffer are each independently selected from acetic acid-sodium acetate, sodium citrate-citric acid, disodium hydrogen phosphate-citric acid, or Tris and disodium hydrogen phosphate-citric acid, more preferably acetic acid-sodium acetate; Preferably, the concentration of the buffer substance in the first wash buffer is about 0.03 to about 0.1 mol / L, preferably about 0.04 to about 0.06 mol / L; The concentration of the buffer substance in the second washing buffer is about 0.03 to about 0.1 mol / L, preferably about 0.04 to about 0.06 mol / L; and / or, The concentration of the buffer substance in the third washing buffer is about 0.03 to about 0.1 mol / L, preferably about 0.04 to about 0.06 mol / L; preferably, the conductivity of the third washing buffer is less than 5.0 mS / cm; Preferably, The first washing buffer further comprises an inorganic salt, and the inorganic salt is selected from one or more of sodium chloride, sodium sulfate, and ammonium sulfate; preferably sodium chloride, or sodium chloride and sodium sulfate, and more preferably sodium chloride; preferably, the concentration of the inorganic salt in the first washing buffer is about 0.1 mol / L to about 1.0 mol / L, preferably about 0.1 mol / L to about 0.6 mol / L, and preferably about 0.4 mol / L to about 0.6 mol / L; The second washing buffer further comprises an inorganic salt, and the inorganic salt is selected from one or more of sodium chloride, sodium sulfate, and ammonium sulfate; preferably sodium chloride, or sodium chloride and sodium sulfate, and more preferably sodium chloride; preferably, the concentration of the inorganic salt in the second washing buffer is about 0.2 to about 1.0 mol / L, preferably about 0.3 mol / L to about 0.7 mol / L, and preferably about 0.4 mol / L to about 0.6 mol / L; and / or, The buffer substance in the equilibration buffer is a phosphate buffer, and the concentration of the phosphate buffer is about 0.01 mol / L to about 0.1 mol / L, preferably about 0.01 mol / L to about 0.05 mol / L, more preferably about 0.01 mol / L to about 0.03 mol / L; preferably, the equilibration buffer further comprises an inorganic salt, preferably sodium chloride, more preferably about 0.1 mol / L to about 2.0 mol / L of sodium chloride, more preferably about 0.5 mol / L to about 1.5 mol / L of sodium chloride.

5. The method according to any one of claims 3 to 4, wherein: The (c) elution step comprises: (c-1) eluting the affinity chromatography medium with a first elution buffer, wherein the pH of the first elution buffer is about 3.8 to about 5.0, more preferably about 4.0 to about 4.7, and more preferably about 4.1 to about 4.3; (c-2) eluting the affinity chromatography filler with a second elution buffer to increase the pH of the affinity chromatography filler to above 4.5; (c-3) eluting the affinity chromatography filler with a third elution buffer to reduce the conductivity of the affinity chromatography filler to less than 5.0 mS / cm; Preferably, the first elution buffer, the second elution buffer, and the third elution buffer are washed for 1-10 column volumes; more preferably, the first elution buffer is washed for 2-8 column volumes, the second elution buffer is washed for 1-5 column volumes, and the third elution buffer is washed for 1-5 column volumes; Preferably, the affinity chromatography filler is a Protein A affinity chromatography filler; more preferably, the Protein A affinity chromatography filler is selected from Mabselect Sure, Mabselect Sure LX, Mabselect PrismA, N Mab, N Mab Pro or Unimab 50HC; Preferably, the method reduces or removes homodimers in the sample.

6. A method for purifying a fusion protein, comprising the following steps: (a) Sample loading step: loading the sample containing the fusion protein onto the affinity chromatography medium; (b) Equilibration step: flushing the affinity chromatography medium after loading with equilibration buffer; (c) elution step: eluting the affinity chromatography medium with a elution buffer; (c-1) eluting the affinity chromatography medium with a first elution buffer, wherein the pH of the first elution buffer is preferably about 3.8 to about 5.0, more preferably about 4.0 to about 4.7, and more preferably about 4.1 to about 4.3; (c-2) eluting the affinity chromatography medium with a second elution buffer, wherein the pH of the second elution buffer is preferably about 4.5 to about 8.0, more preferably about 4.5 to about 6.5, and more preferably about 4.5 to about 5.5; (c-3) eluting the affinity chromatography medium with a third elution buffer, wherein the pH of the third elution buffer is preferably about 4.5 to about 8.0, more preferably about 4.5 to about 6.5, and more preferably about 4.5 to about 5.5; (d) an elution step: eluting the fusion protein from the affinity chromatography medium with an elution buffer, and collecting the eluate; the pH of the elution buffer is preferably about 3.0 to about 5.0, more preferably about 3.4 to about 4.0, and more preferably about 3.4 to about 3.8; Preferably, the first washing buffer contains about 0.03 to about 0.1 mol / L of acetic acid-sodium acetate, and about 0.1 to about 0.6 mol / L of sodium chloride; preferably contains about 0.04 to about 0.06 mol / L of acetic acid-sodium acetate, and about 0.4 to about 0.6 mol / L of sodium chloride; The second washing buffer contains about 0.03 to about 0.1 mol / L of acetic acid-sodium acetate, and about 0.3 to about 0.7 mol / L of sodium chloride; preferably contains about 0.04 to about 0.06 mol / L of acetic acid-sodium acetate, and about 0.4 to about 0.5 mol / L of sodium chloride; The third washing buffer contains about 0.03 to about 0.1 mol / L of acetic acid-sodium acetate, preferably about 0.04 to about 0.06 mol / L of acetic acid-sodium acetate, and preferably does not contain sodium chloride; The elution buffer contains about 0.01 mol / L to about 0.5 mol / L acetic acid-sodium acetate, preferably about 0.05 mol / L to about 0.1 mol / L acetic acid-sodium acetate; The equilibration buffer contains about 0.01 mol / L to about 0.1 mol / L of phosphate, and about 0.1 mol / L to about 2.0 mol / L of sodium chloride; preferably contains about 0.01 mol / L to about 0.05 mol / L of phosphate, and about 0.5 mol / L to about 1.5 mol / L of sodium chloride; Preferably, the affinity chromatography filler is a Protein A affinity chromatography filler; preferably selected from Mabselect Sure, Mabselect Sure LX, Mabselect PrismA, N Mab, N Mab Pro or Unimab 50HC; Preferably, the fusion protein is a fusion protein comprising an antibody Fc; preferably a fusion protein comprising a PD-1 binding domain, a cytokine and an antibody Fc, wherein: The cytokine is selected from IL-2 or a variant thereof; Preferably, the PD-1 binding domain is located at the N-terminus of Fc, and the cytokine is located at the N-terminus or C-terminus of Fc; Preferably, the fusion protein comprises an asymmetric first polypeptide chain and a second polypeptide chain; wherein the difference between any two of the PI of the fusion protein, the PI of the first polypeptide chain and the PI of the second polypeptide chain is no greater than about 3.0 units; or: (a) Sample loading step: loading the sample containing the fusion protein onto the affinity chromatography medium; (b) Equilibration step: flushing the affinity chromatography medium after loading with equilibration buffer; (c) elution step: eluting the affinity chromatography medium with a elution buffer; (c-1) eluting the affinity chromatography medium with 2-8 column volumes of a first elution buffer, wherein the pH of the first elution buffer is preferably about 3.8 to about 5.0, more preferably about 4.0 to about 4.7, and more preferably about 4.1 to about 4.3; (c-2) eluting the affinity chromatography medium with 1-5 column volumes of a second elution buffer to raise the pH of the affinity chromatography medium to above 4.5; the pH of the second elution buffer is preferably about 4.5 to about 8.0, more preferably about 4.5 to about 6.5, and more preferably about 4.5 to about 5.5; (c-3) eluting the affinity chromatography filler with 1-5 column volumes of a third elution buffer to reduce the conductivity of the affinity chromatography filler to below 5.0 mS / cm; the pH of the third elution buffer is preferably about 4.5 to about 8.0, more preferably about 4.5 to about 6.5, and more preferably about 4.5 to about 5.5; (d) an elution step: eluting the fusion protein from the affinity chromatography medium with an elution buffer, and collecting the eluate; the pH of the elution buffer is preferably about 3.0 to about 5.0, more preferably about 3.4 to about 4.0, and more preferably about 3.4 to about 3.8; Preferably, the first washing buffer contains about 0.03 to about 0.1 mol / L of acetic acid-sodium acetate, and about 0.1 to about 0.6 mol / L of sodium chloride; preferably contains about 0.04 to about 0.06 mol / L of acetic acid-sodium acetate, and about 0.4 to about 0.6 mol / L of sodium chloride; The second washing buffer contains about 0.03 to about 0.1 mol / L of acetic acid-sodium acetate, and about 0.3 to about 0.7 mol / L of sodium chloride; preferably contains about 0.04 to about 0.06 mol / L of acetic acid-sodium acetate, and about 0.4 to about 0.5 mol / L of sodium chloride; The third washing buffer contains about 0.03 to about 0.1 mol / L of acetic acid-sodium acetate, preferably about 0.04 to about 0.06 mol / L of acetic acid-sodium acetate, and preferably does not contain sodium chloride; The elution buffer contains about 0.01 mol / L to about 0.5 mol / L acetic acid-sodium acetate, preferably about 0.05 mol / L to about 0.1 mol / L acetic acid-sodium acetate; The equilibration buffer contains about 0.01 mol / L to about 0.1 mol / L phosphate, and about 0.1 mol / L to about 2.0 mol / L sodium chloride; preferably contains about 0.01 mol / L to about 0.05 mol / L phosphate (PB), and about 0.5 mol / L to about 1.5 mol / L sodium chloride; Preferably, the affinity chromatography filler is a Protein A affinity chromatography filler; preferably selected from Mabselect Sure, Mabselect Sure LX, Mabselect PrismA, N Mab, N Mab Pro, or Unimab 50HC; Preferably, the fusion protein is a fusion protein comprising an antibody Fc; preferably a fusion protein comprising a PD-1 binding domain, a cytokine and an antibody Fc, wherein the cytokine is selected from IL-2 or a variant thereof; Preferably, the PD-1 binding domain is located at the N-terminus of Fc, and the cytokine is located at the N-terminus or C-terminus of Fc; Preferably, the fusion protein comprises an asymmetric first polypeptide chain and a second polypeptide chain; wherein the difference between any two of the PI of the fusion protein, the PI of the first polypeptide chain and the PI of the second polypeptide chain is no greater than about 3.0 units.

7. The method according to any one of claims 1 to 6, wherein: The fusion protein comprises a PD-1 binding domain and a cytokine; The PD-1 binding domain comprises an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 2, 8-15, or the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NOs: 3, 16 and 17, respectively, and the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems; Preferably, the CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are As shown in SEQ ID NO:3, 18, 19, As shown in SEQ ID NO:3, 4, 5, or As shown in SEQ ID NO:3, 4, 20; Preferably, the PD-1 binding domain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 2, 8-15, or having at least 90% sequence identity thereto; more preferably, the PD-1 binding domain comprises an amino acid sequence as shown in SEQ ID NO: 13, or having at least 90% sequence identity thereto.

8. The method according to claim 7, wherein: The cytokine is IL-2 or a variant thereof; preferably, the IL-2 variant contains the following mutation: 42A / 72G / 88D, 42A / 72G / 88R, 42A / 72G / 20A, 42A / 72G / 20N, 42A / 72G / 91T, 42A / 72G / 126D, 42A / 72G / 16A / 84S, 42A / 72G / 26Q / 29S / 71Q, 38E / 42A / 91T, 42A / 72G / 88D / 26Q / 29S / 71Q, 42A / 72G / 88R / 26Q / 29S / 71Q, 42A / 72G / 20A / 26Q / 29S / 71Q, 42A / 72G / 20N / 26Q / 29S / 71Q, 42A / 72G / 91T / 26Q / 29S / 71Q, 42A / 72G / 126D / 26Q / 29S / 71Q, or 42A / 72G / 16A / 84S / 26Q / 29S / 71Q; Preferably, the mutations shown below are included: F42A / L72G / N88D, F42A / L72G / N88R, F42A / L72G / D20A, F42A / L72G / D20N, F42A / L72G / V91T, F42A / L72G / Q126D, F42A / L72G / H16A / D84S, F42A / L72G / N26Q / N29S / N71Q, R38E / F42A / V91T, F42A / L72G / N88D / N26Q / N29S / N71Q, F42A / L72G / N88R / N26Q / N29S / N71Q, F42A / L72G / D20A / N26Q / N29S / N71Q, F42A / L72G / D20N / N26Q / N29S / N71Q, F42A / L72G / V91T / N26Q / N29S / N71Q, F42A / L72G / Q126D / N26Q / N29S / N71Q, or F42A / L72G / H16A / D84S / N26Q / N29S / N71Q; Preferably, the IL-2 variant contains mutations at positions 3 and / or 125, wherein the mutation at position 3 is preferably T3A; the mutation at position 125 is preferably C125A or C125S; More preferably, the IL-2 variant contains or has an amino acid sequence as shown in any one of SEQ ID NOs: 23-31, or a sequence having at least 90% identity thereto.

9. The method according to any one of claims 1 to 8, wherein: The fusion protein further comprises an immunoglobulin Fc region; preferably, the Fc region is the Fc region of human IgG1, IgG2 or IgG4; Preferably, the Fc region of human IgG1 contains one or more amino acid mutations that reduce binding to Fcγ receptor (FcγR) and / or reduce ADCC effect; More preferably, the Fc region of human IgG1 contains mutations of L234, L235, P329 or any combination thereof; Most preferably, the Fc region of the human IgG1 contains L234A / L235A, L234A / L235A / P329G mutations; The mutations in the Fc region are numbered according to the EU numbering system.

10. The method of claim 9, wherein: The Fc region comprises a mutation that promotes association of the first and second subunits of the Fc region; Preferably, amino acid residues in the CH3 region of the first subunit of the Fc region are replaced with amino acid residues having a larger side chain volume to generate a protuberance in the CH3 region of the first subunit, which protuberance can be placed in a cavity in the CH3 region of the second subunit, and amino acid residues in the CH3 region of the second subunit of the Fc region are replaced with amino acid residues having a smaller side chain volume to generate a cavity in the CH3 region of the second subunit in which the protuberance in the CH3 region of the first subunit can be placed; Preferably, the first subunit of the Fc region contains a T366W mutation, and the second subunit contains a mutation selected from T366S, L368A, Y407V or any combination thereof; the first subunit of the Fc region contains a S354C or E356C mutation, and the second subunit contains a mutation selected from Y349C; or, the first subunit of the Fc region contains a S354C / T366W mutation, and the second subunit contains a Y349C / T366S / L368A / Y407V mutation; Preferably, the second subunit of the Fc region further contains H435R / Y436F, H435R, or H435K mutation; Preferably, the PD-1 binding domain is located at the N-terminus of the Fc region, and the cytokine is located at the N-terminus or C-terminus of the Fc region; more preferably, the cytokine is located at the polypeptide chain where the first subunit of the Fc region is located; Preferably, the fusion protein comprises the following structure: 1) The first polypeptide chain comprises, from N-terminus to C-terminus: [PD-1 binding domain 1]-[Fc region first subunit]-[linker 1]a-[IL-2 or its variant], 2) The second polypeptide chain comprises from the N-terminus to the C-terminus: [PD-1 binding domain 1]-[Fc region second subunit]; Wherein, a is selected from 0 or 1, and the structure of the linker is (G x S) y , wherein x is selected from an integer of 1-5, and y is selected from an integer of 1-6; preferably, x is 4, and y is 1, 2 or 3; More preferably, the fusion protein comprises a polypeptide chain combination of the following amino acid sequence: SEQ ID NO:42 and 43; SEQ ID NO: 21 and 22; SEQ ID NO:36 and 37; SEQ ID NO:38 and 39; or, SEQ ID NO:40 and 41.

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