CN119490601A

CN119490601A - A method for purifying fusion protein

Info

Publication number: CN119490601A
Application number: CN202411125060.6A
Authority: CN
Inventors: 常甜甜; 陈伟峰; 李磊
Original assignee: Shanghai Maijin Biomedical Technology Co ltd
Current assignee: Shanghai Maijin Biomedical Technology Co ltd
Priority date: 2023-08-16
Filing date: 2024-08-15
Publication date: 2025-02-21

Abstract

本公开涉及一种纯化融合蛋白的方法。具体而言，该方法所述蛋白例如是包含PD‑1结合结构域和细胞因子的融合蛋白。The present disclosure relates to a method for purifying a fusion protein. Specifically, the protein described in the method is, for example, a fusion protein comprising a PD-1 binding domain and a cytokine.

Description

Method for purifying fusion protein

The present disclosure claims priority from China patent application No. 2023110388243, a method of purifying fusion proteins filed on 16/08 of 2023, which is incorporated herein by reference.

Technical Field

The present disclosure relates to a method of purifying a fusion protein, e.g., a fusion protein comprising a PD-1 binding domain and a cytokine.

Background

The antibody can recognize specific epitopes and has good targeting property. Immunocytokines have a variety of roles including immunomodulation and cell growth regulation. The fusion protein obtained by fusion expression of the cytokine and the antibody has targeting and immunoregulation functions, shows better treatment effect than the antibody in a plurality of fields such as tumor treatment, immunoregulation and the like, and has wide application prospect in the biomedical industry. Compared with antibodies, the antibody-cytokine fusion protein has poor stability, and the culture product usually contains various product related impurities such as homodimers, degradation fragments and the like, which brings great challenges to the design of a purification process. How to optimize the purification process, effectively remove the relevant impurities of the product, and improve the purity of the target molecule is an important problem in the current industrial production.

Knob Into Hole (KIH) is a widely used FC construction engineering technique that can be used to promote assembly of heterologous heavy chains and reduce the proportion of misassembled homologous heavy chains, but cannot completely avoid homodimer formation. To solve this problem, researchers have developed various solutions, such as introducing engineered disulfide bonds between heavy chains, optimizing charge distribution at the heavy chain assembly interface, etc., that significantly reduce the proportion of homodimers in the expressed product, but still fail to solve thoroughly. The antibody or antibody fusion protein based on KIH model has homodimer, polymer, degraded fragment and other impurities. The homodimer has smaller physical and chemical property difference with the target molecule, and the removal difficulty is larger. Patent CN115066258a discloses a method for removing bispecific antibody homodimers, which uses the binding force difference between homodimers, target molecules and kappa Select affinity chromatography fillers to remove homodimers, but in this scheme the light chains must be of different subtypes, and the kappa Select affinity chromatography fillers have higher use cost. In another document, it is reported that the use of complex cationic chromatographic fillers (such as Capto MMC IMPRES) can be used to remove the diabodies, homodimers and polymers, but requires significant charge differences between homodimers and 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-ScienceDirect[J].Protein Expression and Purification,167.).

No method for efficiently removing homodimers based on general Pro A affinity chromatography is disclosed in the prior art.

Disclosure of Invention

The present disclosure relates to a method of purifying a fusion protein (e.g., an antibody-cytokine fusion protein) that effectively inhibits the unstable state of protein molecules during purification and effectively removes process-related impurities, particularly effectively reduces the content of homodimers in the fusion protein.

The present disclosure provides a method of purifying a protein (e.g., a fusion protein), the method comprising the steps of:

(a) Loading a sample containing the fusion protein onto an affinity chromatography packing;

(b) The balancing step is to wash the affinity chromatography packing after sample loading by using a balancing buffer solution;

(c) Leaching the affinity chromatography packing by using leaching buffer;

(d) Eluting the fusion protein from the affinity chromatographic packing with eluting buffer, and collecting the eluting solution;

In some embodiments, the fusion protein has an isoelectric Point (PI) of about 4.0 to about 7.5, e.g., an isoelectric point of about 4.5 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 fusion protein has an isoelectric point below 7.0, e.g., an isoelectric point of 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. In some embodiments, the fusion protein has an isoelectric point of 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.

In some embodiments, the fusion protein is a protein that can be chromatographed by an affinity chromatography column, such as an antibody or 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.

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.

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 (+.ltoreq.) about 3.0 units, e.g., about 1.0 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.

In some embodiments, the first rinse buffer pH is from about 3.8 to about 5.0, for example, from about 3.5 to about 4.7, from about 3.5 to about 4.8, from about 3.8 to about 4.5, from about 4.0 to about 4.5, from about 4.1 to about 4.3, or any range between these point values. In some embodiments, the first rinse buffer pH 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.

In some embodiments, the buffer material in the first leaching buffer is selected from one or more of sodium acetate, sodium citrate, tris, sodium dihydrogen phosphate, disodium hydrogen phosphate, acetic acid, citric acid, phosphoric acid. In some embodiments, the buffer material in the first rinse 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 rinse buffer is sodium acetate.

In some embodiments, the concentration of the buffer substance in the first rinse buffer is about 0.03 to about 0.1mol/L, e.g., about 0.03 to about 0.08mol/L, about 0.03 to about 0.07mol/L, about 0.04 to about 0.08mol/L, about 0.04 to about 0.07mol/L, about 0.04 to about 0.06mol/L, or any range between these spot values. In some embodiments, the concentration of the buffer substance in the first rinse buffer is about 0.03mol/L, about 0.04mol/L, about 0.05mol/L, about 0.06mol/L, about 0.07mol/L, about 0.08mol/L, about 0.09mol/L, about 0.1mol/L. In some embodiments, the concentration of the buffer substance in the first rinse buffer is about 0.05mol/L.

In some embodiments, the first leaching buffer further comprises an inorganic salt selected from one or more of sodium chloride, sodium sulfate, ammonium sulfate. In some embodiments, the first leaching buffer comprises sodium chloride.

In some embodiments, the concentration of the inorganic salt in the first rinse buffer is from about 0.1mol/L to about 1.0mol/L, for example, from about 0.1mol/L to about 0.8mol/L, from about 0.1mol/L to about 0.7mol/L, from about 0.1mol/L to about 0.6mol/L, from about 0.1mol/L to about 0.5mol/L, from about 0.3mol/L to about 1.0mol/L, from about 0.4mol/L to about 1.0mol/L, from about 0.3mol/L to about 0.9mol/L, from about 0.3mol/L to about 0.8mol/L, from about 0.3mol/L to about 0.7mol/L, from about 0.4mol/L to about 0.6mol/L, from about 0.4mol/L to about 0.5mol/L, from about 0.3mol/L to about 0.9mol/L, or any value between these ranges. In some embodiments, the concentration of inorganic salts in the first leaching 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 leaching buffer is about 0.4mol/L.

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

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

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

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

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

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

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

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

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

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

In some embodiments, the second rinse buffer pH 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 spot values. In some embodiments, the second rinse buffer pH 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.

In some embodiments, the buffer material in the second leaching buffer is selected from one or more of sodium acetate, sodium citrate, tris, sodium dihydrogen phosphate, disodium hydrogen phosphate, acetic acid, citric acid, phosphoric acid. In some embodiments, the buffer material in the second rinse 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 rinse buffer is sodium acetate.

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

In some embodiments, the second leaching buffer further comprises an inorganic salt selected from one or more of sodium chloride, sodium sulfate, ammonium sulfate. In some embodiments, the second leaching buffer comprises sodium chloride.

In some embodiments, the concentration of inorganic salt in the second rinse buffer is from about 0.2mol/L to about 1.0mol/L, for example, from about 0.3mol/L to about 1.0mol/L, from about 0.4mol/L to about 1.0mol/L, from about 0.3mol/L to about 0.9mol/L, from about 0.3mol/L to about 0.8mol/L, from about 0.3mol/L to about 0.7mol/L, from about 0.4mol/L to about 0.6mol/L, from about 0.4mol/L to about 0.5mol/L, from 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 second rinse buffer is 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 second rinse buffer is about 0.4mol/L.

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

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

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

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

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

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

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

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

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

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

In some embodiments, the pH of the third rinse buffer is about 4.5 to about 8.0, e.g., 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 spot values. In some embodiments, the third rinse buffer pH 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.

In some embodiments, the buffer material in the third leaching buffer is selected from one or more of sodium acetate, sodium citrate, tris, sodium dihydrogen phosphate, disodium hydrogen phosphate, acetic acid, citric acid, phosphoric acid. In some embodiments, the buffer material in the third rinse 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 rinse buffer is sodium acetate.

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

In some embodiments, the third rinse buffer does not comprise an inorganic salt. In some embodiments, the third rinse buffer comprises from about 0.03 to about 0.1mol/L of acetic acid-sodium acetate, e.g., from about 0.04mol/L to about 0.06mol/L of acetic acid-sodium acetate, e.g., about 0.05mol/L of acetic acid-sodium acetate.

In some embodiments, the conductivity of the third rinse buffer is <5.0mS/cm.

In some embodiments, the pH of the equilibration buffer is from about 5.0 to about 8.0, for example, from about 5.5 to about 8.0, from about 6.5 to about 8.0, from about 7.0 to about 7.6, from about 7.2 to about 7.6, from about 6.8 to about 7.8, from about 6.8 to about 8, or any range between these point values. In some embodiments, the third rinse buffer pH 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.

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

In some embodiments, the buffer substance in the equilibration buffer is a phosphate buffer at a concentration of about 0.01mol/L to about 0.1mol/L, for example about 0.01mol/L to about 0.05mol/L, about 0.01mol/L to about 0.06mol/L, about 0.01mol/L to about 0.08mol/L, about 0.02mol/L to about 0.05mol/L, about 0.02mol/L to about 0.06mol/L, about 0.01mol/L to about 0.03mol/L, or any range therebetween. In some embodiments, the phosphate buffer is present at a concentration of about 0.01mol/L, about 0.02mol/L, about 0.03mol/L, about 0.04mol/L, about 0.05mol/L, about 0.06mol/L, about 0.07mol/L, about 0.08mol/L,

In some embodiments, the equilibration buffer further comprises an inorganic salt. In some embodiments, the equilibration buffer further comprises sodium chloride. In some embodiments, the concentration of sodium chloride in the equilibration buffer is from about 0.1mol/L to about 2.0mol/L, such as from about 0.5mol/L to about 1.5mol/L, from about 0.8mol/L to about 2.0mol/L, from about 1.0mol/L to about 1.5mol/L, from 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 equilibration 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.

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

In some embodiments, the equilibration buffer contains 0.02mol/L phosphate, 1.0mol/L sodium chloride, pH7.4.

In some embodiments, the pH of the elution buffer is from about 3.0 to about 5.0, such as from about 3.4 to about 4.0, from about 3.4 to about 3.8, from about 3.0 to about 4.0, from about 3.0 to about 3.8, from about 3.6 to about 5.0, from about 3.0 to about 3.6, from about 3.0 to about 4.0, from about 3.5 to about 5.0, from about 3.8 to about 5.0, from about 3.5 to about 4.5, or any range between these point values. 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.

In some embodiments, the buffer substance of the elution buffer is acetic acid-sodium acetate, citric acid-sodium citrate, glycine-hydrochloric acid, or the like.

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

(c-1) eluting the affinity chromatography packing with a first eluting buffer having a pH of about 3.8 to about 5.0, for example, about 4.0 to about 4.7, about 4.1 to about 4.3.

(C-2) eluting the affinity chromatography packing with a second eluting buffer to increase the pH of the affinity chromatography packing to above 4.5.

(C-3) eluting the affinity chromatography packing with a third eluting buffer to provide an affinity chromatography packing conductivity of 5.0mS/cm or less.

In some embodiments, the first, second, third, and third elution buffers flush 1-10 column volumes.

Illustratively, the first wash buffer washes 2-8 column volumes, the second wash buffer washes 1-5 column volumes, and the third wash buffer washes 1-5 column volumes.

In some embodiments, the affinity chromatography packing is a Protein A affinity chromatography packing, exemplary of which include, but are not limited to, mabSelect Sure, mabselect Sure LX, mabselect PrismA, N Mab, N Mab Pro, or Unimab HC.

The method can effectively remove the homodimer in the fusion protein through affinity chromatography, solves the problem that the homodimer in the fusion protein is difficult to effectively separate only through a simple affinity chromatography step in the prior art, improves the purification efficiency and purity of the fusion protein, and has the advantages of low cost, simple steps and the like.

PD-1 binding Domain

In some embodiments, the amino acid sequences of CDR1, CDR2 and CDR3 of the aforementioned PD-1 binding domain are shown in SEQ ID NO.3, 16, 17, respectively, according to the Kabat numbering system.

In some 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.

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

as shown in SEQ ID NO. 3, 18 and 19,

As shown in SEQ ID NO 3, 4, 5, or

As shown in SEQ ID NO. 3, 4 and 20.

In some embodiments, the immunoglobulin single variable domain of the foregoing PD-1 binding domains is humanized, affinity matured, T cell epitope depleted, antibody deamidation reduced, and/or antibody isomerization engineered. In some embodiments, the immunoglobulin single variable domain is obtained via affinity maturation having one or more changes in one or more CDRs that result in an increase in affinity for PD-1 as compared to the parent.

In some embodiments, the humanization process uses the heavy chain framework region IGHV3-23 x 01 or IGHV3-23 x 04 of a human germline template.

In some embodiments, the amino acid sequence of the immunoglobulin single variable domain in the PD-1 binding domain is as set forth in any one of SEQ ID NOs 2, 8-15, or has at least 80%, at least 85%, at least 90% sequence identity to any one of the foregoing sequences.

In some embodiments, the amino acid sequence of the immunoglobulin single variable domain in the PD-1 binding domain is as set forth in SEQ ID NO. 13, or has at least 80%, at least 85%, at least 90% sequence identity to any of the foregoing sequences.

In some embodiments, at least one immunoglobulin single variable domain of the foregoing PD-1 binding domains is a VHH.

In some embodiments, the foregoing PD-1 binding domains comprise one or more (e.g., 2, 3, 4, 5, 6) of the foregoing immunoglobulin single variable domains, which may be the same or different, which may form a dimeric or multimeric molecule.

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 PD-1 or a fragment thereof, such as a camelid antibody, chimeric antibody, humanized antibody, fully human antibody or antigen-binding fragment thereof, such as a recombinant antibody or fragment thereof. In some embodiments, the antigen binding fragments are linear antibodies, single chain antibodies, nanobodies, peptide antibodies peptibody, domain antibodies, and multispecific antibodies (bispecific antibodies, diabody, triabody, and tetrabodies, tandem di-scFv, tandem tri-scFv).

In some embodiments, the foregoing anti-PD-1 antibodies or antigen-binding fragments thereof further comprise a human immunoglobulin F c region, e.g., the Fc region is that of human IgG1, igG2, or IgG 4. The Fc region can have mutations, exemplary mutations are L234A/L235A on IgG1, V234A/G237A/P238A/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 on IgG1, H268Q/V309L/A330S/P331S on IgG2, L234F/L235E/D235A on IgG1, L234A/G235A/P238A/H268A/A330S on IgG1, S214T/E/L234V/L235A/G237A on IgG4, S234A/G237A/L237 on IgG1, and H234A/L235A/L237 on IgG 4. Hybrid IgG2/4Fc domains, such as Fc with residues 117-260 from IgG2 and residues 261-447 from IgG4, may also be used. In some specific embodiments, the Fc region of human IgG4 has a S228P, F234A, L a and/or K447A mutation. In some specific embodiments, the Fc region of human IgG1 has the L234A/L235A or L234A/L235A/P329G mutation.

In some embodiments, the Fc region comprised in the foregoing PD-1 antibodies or antigen-binding fragments thereof may allow the binding protein to form a dimer molecule while extending the in vivo half-life of the binding protein.

In some embodiments, the immunoglobulin single variable domain in the foregoing PD-1 antibodies or antigen-binding fragments thereof is linked to the Fc region by a linker. The linker may be a nonfunctional amino acid sequence 1-20 or more amino acids long, without secondary or more structure. For example, the joint is a flexible joint, such as G₄S、GS、GAP、(G₄S)₂、(G₄S)₃、(G₄S)₄、(G₄S)₅、ASGS or the like.

In some embodiments, the foregoing PD-1 antibodies, or antigen-binding fragments thereof, have an activity selected from at least one of the following:

(a) Binding to human PD-1 or an epitope thereof with a K _D value of 10 or less ^-7;

(b) Inhibit the binding of PD-1 to PD-L1;

(c) Inhibit the binding of PD-1 to PD-L2;

(d) Inducing secretion of IFN-gamma by CD4 ⁺ T cells;

(e) Enhancing activation of PBMCs;

(f) Enhancing activation of T cells;

(g) Inhibit tumor growth.

In some embodiments, the foregoing PD-1 binding domains of the present disclosure may have a K _D value that binds PD-1 of.ltoreq.1X10 ^-7 M, e.g., 1X 10 ^-8 M, or 1X 10 ^-9 M, or 1X 10 ^-10 M.

In some embodiments, the PD-1 binding domains of the present disclosure are capable of specifically binding to human PD-1 and blocking the interaction of PD-1 and PD-L1, and/or PD-1 and PD-L2.

In some embodiments, the foregoing PD-1 binding domains of the present disclosure are capable of inhibiting tumor growth by at least about 10%, e.g., at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%.

In some embodiments, the foregoing PD-1 binding domains of the present disclosure encompass variants, e.g., comprising one or more amino acid substitutions, preferably conservative amino acid substitutions, e.g., comprising 1,2,3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions, as compared to any of SEQ ID NOs 2, 8-15, which substitutions may occur in the CDR region and/or FR region.

In some embodiments, the aforementioned PD-1 binding domains of the present disclosure are resistant to heat treatment or have higher stability. For example, no significant aggregation or degradation was seen for up to 30 days of treatment at 40 ℃, at least stabilization at 60 ℃.

In some embodiments, PD-1 antibodies or antigen-binding fragments thereof are provided that bind or compete for binding to the same epitope as the immunoglobulin single variable domain of the PD-1 binding domains of the foregoing disclosure.

In some embodiments, PD-1 antibodies or antigen-binding fragments thereof are provided that block binding of an immunoglobulin single variable domain of a PD-1 binding domain of the foregoing disclosure to PD-1 (e.g., human PD-1).

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

In some embodiments, the PD-1 binding domains of the foregoing disclosure reduce 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%.

In some embodiments, the disclosure provides PD-1 binding proteins comprising at least one of the aforementioned immunoglobulin single variable domains that bind PD-1 (e.g., VHH).

As previously mentioned, and throughout this disclosure, "at least 90% identical" encompasses 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% identical.

IL-2 or variants thereof

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 the wild type human IL-2 is exemplified by SEQ ID NO. 50. The positions of the IL-2 variants of the present disclosure are numbered according to the wild-type IL-2 amino acid sequence shown in SEQ ID NO. 50.

In some embodiments, the disclosure provides IL-2 variants having reduced affinity for high affinity receptors (IL-2Rα/β/γ) and medium affinity receptors (IL-2Rβ/γ) as compared to wild-type IL-2 and/or having reduced affinity for low affinity receptors (IL-2Rα) as compared to wild-type IL-2. In some embodiments, the IL-2 variant has a greater decrease in affinity for IL-2Rα/β/γ than for IL-2Rβ/γ. The affinity may be detected by conventional methods, such as ELISA, forteBio, SPR, e.g., the SPR detection method provided in example 13 of the present disclosure.

In some embodiments, the disclosure provides IL-2 variants comprising or consisting of mutation (i), mutation (ii), mutation (iii), mutation (iv), or any combination thereof.

Regarding mutation (i):

The present disclosure provides mutations that confer altered (e.g., increased or decreased) affinity for IL-2Rβ/γ to IL-2 variants as compared to wild-type IL-2.

In some embodiments, the IL-2 variant has a mutation (i) that reduces the affinity to IL-2Rβ/γ as compared to wild-type IL-2. In some embodiments, the IL-2 variant has a 1-1000 fold, 2-800 fold, 5-500 fold, 5-200 fold, 10-100 fold, 10-50 fold, 20-200 fold, 20-100 fold, 5-20 fold, 2-20 fold, 50-100 fold, 50-70 fold, e.g., 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, about 500 fold reduced affinity to IL-2R beta/gamma as compared to wild-type IL-2. In some embodiments, the IL-2 variant has a 10-100 fold decrease in affinity for IL-2Rβ/γ as compared to wild-type IL-2. The affinity may be detected by conventional methods, such as the SPR detection method provided in example 13 of the present disclosure.

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

In some embodiments, the aforementioned mutation (I) is selected from position 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 position 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 embodiments, the mutation is selected from 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;, 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.

Regarding mutation (ii):

the present disclosure provides mutations that confer altered (e.g., reduced) affinity for IL-2Rα, or lack binding to IL-2Rα, on IL-2 variants as compared to wild-type IL-2.

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

In some embodiments, the aforementioned mutation (ii) is selected from position 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 position 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,Y45T,Y45Q,Y45E,Y45N,Y45D,Y45R,Y45K,P65R,P65E,P65K,P65H,P65Y,P65Q,P65D,P65N,E68A,E68Q,E68K,E68R,L72G,L72A,L72S,L72T,L72Q,L72E,L72N,L72D,L72R,L72K;, and the mutation (ii) is selected from F42A, Y45A, L72G, F42A/L72G, F42A/Y45A, Y45A/L72G or F42A/Y45A/L72G.

Regarding mutation (iii):

the present disclosure provides mutations (iii) that confer improved stability, expression levels, purity, and drug resistance to IL-2 variants.

The present disclosure incorporates in its entirety the IL-2 variants in WO 2020125743A.

In some embodiments, the IL-2 variant has a mutation (iii) selected from position 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 position 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 the group consisting of N26Q,N29S,N30S,N26Q/N29S/N71Q,N26Q/N29S,N26Q/N30S,N26Q/N30S/Q11C/L132C,N26Q/N29S/L70C/P82C,N26Q/N30S/G27C/F78C.

Regarding mutation (iv):

in some embodiments, the IL-2 variant comprises a mutation (iv) selected from positions 3 and/or 125, in some embodiments the mutation (iv) is selected from positions 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.

I. Exemplary IL-2 variants

The present disclosure provides IL-2 comprising or comprising only the following mutations:

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

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

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

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

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

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

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

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

(i) N26Q and any one of the foregoing (a) - (h);

(j) N29S and any one of the foregoing (a) - (h);

(k) N71Q and any one of the foregoing (a) - (h);

(l) N26Q/N29S for any of the foregoing (a) - (h);

(m) is any one of the foregoing (a) - (h) and N26Q/N29S/N71Q;

(n) is any one of the foregoing (a) - (h) and Q11C/L132C;

(o) is any one of the foregoing (a) - (h) and L70C/P82C;

(p) is any one of the foregoing (a) - (h) and G27C/F78C;

(q) is any one of the foregoing (a) - (h) and T3A;

(r) is any one of the foregoing (a) - (h) and T3A/C125A.

Exemplary IL-2 variants

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。

Exemplary IL-2 variants

T3A/F42A/L72G/N88D/C125A,

T3A/F42A/L72G/N88R/C125A,

T3A/F42A/L72G/D20A/C125A,

T3A/F42A/L72G/D20N/C125A,

T3A/F42A/L72G/V91T/C125A,

T3A/F42A/L72G/Q126D/C125A,

T3A/F42A/L72G/H16A/D84S/C125A,

T3A/F42A/L72G/N26Q/N29S/N71Q/C125A,

T3A/R38E/F42A/V91T/C125A,

T3A/F42A/L72G/N88D/N26Q/N29S/N71Q/C125A,

T3A/F42A/L72G/N88R/N26Q/N29S/N71Q/C125A,

T3A/F42A/L72G/D20A/N26Q/N29S/N71Q/C125A,

T3A/F42A/L72G/D20N/N26Q/N29S/N71Q/C125A,

T3A/F42A/L72G/V91T/N26Q/N29S/N71Q/C125A,

T3A/F42A/L72G/Q126D/N26Q/N29S/N71Q/C125A, or

T3A/F42A/L72G/H16A/D84S/N26Q/N29S/N71Q/C125A。

In some embodiments, the disclosure provides IL-2 variants containing or having an amino acid sequence as set forth in any one of SEQ ID NOS.23-31 or having at least 80%, at least 85%, at least 90% sequence identity thereto.

In some embodiments, any of the foregoing variant IL-2 comprising a mutation (i) has any one or more of 1) -5) relative to wild-type IL-2:

1) Has reduced affinity for high affinity IL-2R receptors (IL-2 Rαβγ);

2) Has reduced affinity for the moderately-affinity IL-2R receptor (IL-2 Rβγ);

3) Has reduced activation of T cells (e.g., CD4 ⁺ and CD8 ⁺ T cells);

4) Has reduced activation of NK cells;

5) Has reduced release of inflammatory factors from IL-2 stimulated T cells and/or NK cells.

In some embodiments, any of the foregoing IL-2 variants containing mutations (i) and (ii) have any one or more of the foregoing 1) -5) and the following 6) -8) relative to wild-type IL-2:

6) Has reduced affinity for IL-2Rα, or does not bind IL-2Rα;

7) The affinity for IL-2Rαβγ is reduced more than for IL-2Rβγ;

8) Has reduced activation of CD25 ⁺ cells (e.g., CD25 ⁺CD8⁺ T cells, treg cells).

In some embodiments, any of the foregoing IL-2 variants containing mutations (i) and (ii) and (iii) and/or (iv) have any one or more of the foregoing 1) -8) and 9) below, relative to wild-type IL-2:

9) Improved stability, patentability, expression level, purity, and/or prolonged in vivo half-life.

Fusion proteins

The present disclosure provides fusion proteins comprising an antigen binding domain, and IL-2 or variants thereof.

In some embodiments, the antigen binding domain in the fusion protein targets an immune effector cell or tumor cell, in some embodiments, the immune effector cell is capable of responding to stimulation or activation of IL-2, in some embodiments, the immune effector cell has IL-2Rβγ thereon, in some embodiments, the immune effector cell is, for example, an NK cell, a CD4+CD25-T cell, a CD8+CD25-T cell.

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, and in some embodiments, the antigen binding domain targets PD-1 (i.e., PD-1 binding domain).

In some embodiments, the PD-1 binding domain is any of the PD-1 binding domains provided in the foregoing disclosure.

Illustratively, in some embodiments, the PD-1 binding domain comprises at least one immunoglobulin single variable domain comprising CDR1, CDR2 and CDR3 of any one of the amino acid sequences shown in SEQ ID NO 2, 8-15, said CDR1, CDR2 and CDR3 being defined according to the Kabat, IMGT, chothia, abM or Contact numbering system.

Illustratively, in some embodiments, the PD-1 binding domain has the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain according to the Kabat numbering system as shown in SEQ ID NOS 3, 16, 17, respectively.

Illustratively, in some 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.

Illustratively, in some embodiments, the immunoglobulin single variable domain comprises a CDR1, a CDR2 and a 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.

Illustratively, in some embodiments, the amino acid sequence of the immunoglobulin single variable domain is as set forth in any one of SEQ ID NOs 2, 8-15, respectively, or has at least 80% sequence identity to any one of said sequences.

In some embodiments, the PD-1 binding domain is selected from the group consisting of the PD-1 binding domain of a PD-1 antibody, pembrolizumab (Pembrolizumab), nivolumab (Nivolumab), signal di Li Shan antibody (Sintilimab), tirelizumab (Tislelizumab), cimaprzumab (Cemiplimab), terprin Li Shan antibody (Li Shan), karilizumab (Li Shan), pie Li Shan antibody, sapalimumab, saparix Li Shan antibody (Li Shan), swadazumab (Li Shan), rfed Li Shan antibody (Li Shan), batalimumab (Li Shan), and multi-tarolimumab (Li Shan), in other embodiments, the antigen binding domain of the fusion protein is selected from the group consisting of the antigen binding domain of a PD-L1 antibody (e.g., duvaluzumab (Li Shan), duvaluzumab (Li Shan), ab-Li Shan), ab (1110), ab-2 antibody (Li Shan), triflolizumab (Li Shan), brufiuzumab (Li Shan), and (Li Shan), e.g., TQ-Li Shan, 3, and other embodiments of the fusion protein are selected from the antigen binding domain of antibodies. The antigen binding domain (e.g., PD-1 binding domain) is that portion of the antibody (e.g., PD-1 antibody) that binds an antigen (e.g., PD-1), e.g., fab ', F (ab') 2 thereof, or heavy and light chain variable regions thereof, or domains of HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, LCDR3 of the heavy and light chain variable regions thereof.

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

In some embodiments, the IL-2 or variant thereof in the fusion protein is any of the IL-2 or variants thereof provided in the foregoing disclosure.

Illustratively, in some embodiments, the IL-2 is wild-type human IL-2.

Illustratively, in some embodiments, the IL-2 variants comprise, consist of, or consist of mutation (i), mutation (ii), mutation (iii), mutation (iv), or any combination thereof, provided by the foregoing disclosure.

Illustratively, in some embodiments, the IL-2 variant comprises or only comprises a mutation of any one of (a) - (r) provided by the foregoing disclosure.

Illustratively, in some embodiments, the IL-2 variant comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NOS.23-31 or a sequence having at least 80%, at least 85%, at least 90% identity thereto.

In some embodiments, the fusion protein further comprises a human immunoglobulin Fc region, such as the Fc region of human IgG1, igG2, igG3, or IgG 4.

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

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

In some embodiments, the fusion protein, a fusion protein contains 1 or 2 IL-2 or variants thereof, containing 1,2, 3, 4, 5 or 6 antigen binding domains (e.g., PD-1 binding domains).

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

In some embodiments, the amino acid mutations are included in Fc1 and Fc2 such that Fc1 preferentially pairs with Fc2 or preferentially forms heterodimers over Fc 1. In some embodiments, the mutation is in CH3 of Fc1 and Fc 2. In some embodiments, the amino acid mutations in Fc1 and Fc2 result in greater electrostatic complementarity than a wild-type without the mutation. In some embodiments, the amino acid mutations in Fc1 and Fc2 result in greater spatial complementarity than a wild-type without the mutation.

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 creating a bulge (or knob, 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 creating a recess (or Hole) on the surface of the CH3 domain of Fc2 that interacts with the CH3 domain of Fc 1. 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).

In some embodiments, the Fc1 comprises at least one or at least two amino acid mutations selected from T366S, L A and Y407V (mortar mutation modification), the Fc2 comprises T366W (mortar mutation modification), or the Fc1 comprises T366W (mortar mutation modification), and the Fc2 comprises at least one or at least two amino acid mutations selected from T366S, L A and Y407V (mortar mutation modification).

In some embodiments, the IL-2 or variant thereof is located in the polypeptide chain in which Fc1 resides.

In some embodiments, the natural non-cysteine to cysteine mutation may be included in Fc1 and Fc2, e.g., in CH3, e.g., S354C in Fc1, Y349C in Fc2, or Y349C in Fc1, S354C in Fc 2.

In some embodiments, the Fc1 and Fc2 comprise the following amino acid mutations, or combinations thereof, e.g., within the Fc1 CH3/Fc2 CH3 interface:

T366Y/Y407T;

T366W/Y407A;

T366Y/Y407T;

T394W/F405A;

T366Y/F405A/T394W/Y407T;

T366W/F405W/T394S/Y407A;

F405W/T394S;

D399C/K392C;

T366W/T366S/L368A/Y407V;

T366W/D399C/T366S/L368A/K392C/Y407V;

T366W/K392C/T366S/D399C/L368A/Y407V;

S354C/T366W/Y349C/T366S/L368A/Y407V;

Y349C/T366W/S354C/T366S/L368A/Y407V;

E356C/T366W/Y349C/T366S/L368A/Y407V;

Y349C/T366W/E356C/T366S/L368A/Y407V;

E357C/T366W/Y349C/T366S/L368A/Y407V, and

Y349C/T366W/E357C/T366S/L368A/Y407V。

In some embodiments, the Fc1 and Fc2 further comprise amino acid mutations therein that allow for the formation of an electrostatic interaction interface between Fc1 and Fc2 (e.g., CH3 and CH 3). The amino acid mutations forming the electrostatic interaction interface are for example selected from the following:

K370E/D399K/K439D/D356K/E357K/K409D;

K409D/D399K;

K409E/D399K;

K409E/D399R;

K409D/D399R;

D339K/E356K;

D399K/E356K/K409D/K392D;

D399K/E356K/K409D/K439D;

D399K/E357K/K409D/K370D;

D399K/E356K/E357K/K409D/K392D/K370D;

D399K/E357K/K409D/K392D;

K392D/K409D/D399K, and

K409D/K360D/D399K。

In some embodiments, fc1 and/or Fc2 comprise domains from different antibody subtypes, e.g., from different antibody subtypes CH3.

Furthermore, the present disclosure incorporates by reference the scheme in WO96/27011、WO98/050431、EP1870459、WO2007/110205、WO2007/147901、WO2009/089004、WO2010/129304、WO2011/90754、WO2011/143545、WO2012058768、WO2013157954、WO2013096291 for modification of the CH3 region of the Fc region to enhance heterodimerization.

In some embodiments, fc1 and/or Fc2, e.g., CH3, comprise amino acid mutations that alter effector function. Such antibody effector functions include, but are not limited to, C1q binding and complement dependent cytotoxicity, fc receptor (e.g., fcγr) binding, antibody dependent cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Amino acid mutations that alter effector function are selected, for example, from the following:

S298A/E333A/K334A;

S239D/I332E/A330L;

S239D/I332E/G236A;

G236A/S239D/A330L/I332E;

F243L/R292P/Y300L/V305I/P396L;

K326A/E333A;

K326W/E333S;

K326M/E333S;

C221D/D222C;

S267E/H268F/S324T;

E345R;

S298A/E333A/K334A/N434A;

e294 miss/T307P/N434Y;

T256N/A378V/S383N/N434Y;

T252L/T253S/T254F;

M252Y/S254T/T256E;

M428L/N434S;

L234A/L235A;

S228P/L235E;

L234A/L235A/P331S;

L234A/L235A/P329G;

D265A/E233P;

H268Q/V309L/A330S/P331S;

V234A/G237A/P238S/H268A/V309L/A300S/P331S;

L234A/L235A/G237A/P238S/H268A/V309L/A300S/P331S;

S228P/F234A/L235A;

D270A/P329A;

L234F/L235E;

L234F/L235E/P331S;

F241A/V264A/D265A;

N297G/D265A, and

L234Y/G236W/S298A。

In some embodiments, the Fc1 and/or the Fc2 comprises a mutation that reduces effector function (e.g., ADCC), such as L234A/L235A, L a/L235A/P329G.

In some embodiments, fc1 and Fc2, e.g., CH3 thereof, comprise amino acid mutations for altering half-life (e.g., extending half-life). In some embodiments, the half-life is dependent on FcRn binding affinity. The extension of the half-life may allow for a reduction in the amount of drug administered to the patient, and/or a reduction in the frequency of administration. In some embodiments, the Fc region has the M252Y, S254T and/or T256E mutation. In some embodiments, the fusion protein has a pegylated modification or is conjugated/fused to (human serum) albumin, an albumin binding protein.

In some embodiments, fc1 and/or Fc2, e.g., CH3, comprise one or more allogeneic mutations. In some embodiments, the heterotypic mutation is D356E and L358M.

In some embodiments, the fusion protein comprises a combination of polypeptide chains having the amino acid sequences shown as SEQ ID NO:21 and 22, SEQ ID NO:36 and 37, SEQ ID NO:38 and 39, SEQ ID NO:40 and 41, SEQ ID NO:42 and 43, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any of the polypeptide chains above.

In some specific embodiments, the fusion protein comprises a combination of polypeptide chains of the amino acid sequences shown as SEQ ID NOS 42 and 43 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with any of the above polypeptide chains.

Exemplary fusion proteins

With respect to fusion proteins, the present disclosure provides the following embodiments:

embodiment 1. A fusion protein comprising:

1) PD-1 binding domain, and IL-2 variants containing a mutation selected from N88D or D20A, or

2) PD-1 binding domain, and an IL-2 variant having reduced or eliminated affinity for a high affinity receptor (IL-2Rα/β/γ) as compared to wild-type IL-2, having reduced affinity for a medium affinity receptor (IL-2Rβ/γ) as compared to wild-type IL-2, having reduced affinity for a high affinity receptor as compared to a medium affinity receptor, e.g., the IL-2 variant contains a mutation selected from N88D or D20A.

Embodiment 2. The fusion protein of embodiment 1, which contains a mutation selected from F42, Y45, L72 or any combination thereof;

for example, it contains a mutation selected from F42A, Y45A, L72G or any combination thereof;

More for example, it contains mutations of F42A/L72G, F42A/Y45A, Y45A/L72G, F42A/Y45A/L72G.

Embodiment 3. The fusion protein of embodiment 1 or 2, which contains a mutation selected from the group consisting of N26Q, N29S, N30S, N71Q, Q11C and L132C, L70C and P82C, G27C and F78C, or any combination thereof;

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

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

embodiment 4. A fusion protein comprising:

An antigen binding domain, and an IL-2 or variant thereof, the IL-2 variant comprising a first class of mutation that results in the IL-2 variant having reduced affinity for a medium affinity receptor (IL-2rβ/γ) as compared to wild-type IL-2;

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;

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,E95V,E95P,E95H,E95N,T123A,T123E,T123K,T123Q,Q126D,Q126L,Q126A,Q126S,Q126T,Q126E,S127A,S127E,S127K,S127Q;

As another example, the first type of mutation is selected from the group consisting of N88D, N88R, D20A, D20N, V91T, Q126D, H16A/D84S;

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

Embodiment 5. The fusion protein of embodiment 4, wherein the IL-2 variant comprises a second class of mutations that result in the IL-2 variant having reduced or eliminated affinity for a high affinity receptor (IL-2Rα/β/γ) as compared to wild-type IL-2;

For example, the second class of mutations is selected from R38, E62, P65, E68, F42, Y45, L72, or any combination thereof;

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;

More for example, the second type of mutation is selected from F42A, Y45A, L72G, F42A/L72G, F42A/Y45A, Y45A/L72G or F42A/Y45A/L72G.

Embodiment 6. The fusion protein of embodiment 4 or 5, wherein the IL-2 variant comprises a third class of mutation that confers an increased stability to the IL-2 variant compared to the wild-type IL-2;

For example, the third class of mutations is selected from N26, N29, N30, N71, Q11, L132, L70, P82, G27, F78, or any combination thereof;

For another example, the third class of mutation is selected from the group consisting of N26Q, N29S, N30S, N71Q, Q11C/L132C, L70C/P82C, G27C/F78C, or any combination thereof;

more for example, the third class of mutations 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.

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

the PD-1 binding domain comprises at least one immunoglobulin single variable domain, the immunoglobulin single variable domain comprises:

CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NO 2, 8-15, said CDR1, CDR2 and CDR3 being defined according to the Kabat, IMGT, chothia, abM or Contact numbering system;

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.

For another example, the amino acid sequence of CDR1 of said immunoglobulin single variable 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 NO. 5, 19-20;

More specifically, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NO. 7,18,19, respectively.

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

Group 1) mutations selected from F42A/L72G, F42A/Y45A, Y45A/L72G and F42A/Y45A/L72G;

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

For example, the IL-2 variants contain the following combination of mutations ：F42A/L72G/N88D,F42A/L72G/N88R,F42A/L72G/D20A,F42A/L72G/D20N,F42A/L72G/V91T,F42A/L72G/Q126D,F42A/L72G/H16A/D84S.

Embodiment 9. The fusion protein of embodiment 8, wherein the IL-2 variant further comprises a mutation of group 3), said mutation of group 3) being 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;

For example, the IL-2 variant contains the following combination of mutations:

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,

F42A/L72G/H16A/D84S/N26Q/N29S/N71Q。

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;

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

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 set forth in any one of SEQ ID NOS.23-31.

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

1) The antigen is selected from the group consisting of:

1-1) an antigen on a tumor cell or on an immune cell in a tumor microenvironment;

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、TNC A2、CEA、EDB、MCSP Or CD16a;

1-3) immune checkpoint antigens, such as PD-1, PD-L1, CTLA-4;

2) The antigen binding domain comprises a VH domain and a VL domain of an antibody selected from the group consisting of HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 of VH:

PD-1 antibodies (e.g., pembrolizumab (Pembrolizumab), nivolumab, xindi Li Shan antibody (Sintilimab), tirelimumab (Tislelizumab), cimaprepitant Li Shan antibody (Cemiplimab), terlipendant Li Shan antibody (Toripalimab), carlizumab (Camrelizumab), pe An Puli antibody, saparhimumab, sarat Li Shan antibody (Sasanlimab), stdazumab (Spartalizumab), raf Li Shan antibody (Retifanlimab), baterimumab (Balstilimab), cetirimumab (Cetrelimab), doralimumab (Dostarlimab;TSR-042),CS-1003,SCT-I10A,SSI-361,MEDI-0680(AMP-514),BAT-1306,JTX-4014,JTX-4014,BI-754091,AK-103);

CTLA-4 antibodies (e.g., tremelimumab, ipilimumab (Ipilimumab));

4-1BB antibodies (e.g., wu Ruilu mab (Urelumab), wu Tuolu mab (Utomilumab));

PD-L1 antibodies (e.g., duvaluzumab (Durvalumab), ab-zumab (Atezolizumab), duvalli You Shan antibody (Durvalumab), avizumab (Avelumab), aldbilimab (Adebrelimab), enfra Li Shan antibody (envafolimab), STI-A1110, CS-1001, TQB-2450, KL-A167, IMC-001, CX-072).

Embodiment 13. The fusion protein of any one of embodiments 1 to 3,12, wherein the PD-1 binding domain comprises at least one immunoglobulin single variable domain comprising:

CDR1, CDR2 and CDR3 in the amino acid sequences shown in any one of SEQ ID nos. 2, 8-15, said CDR1, CDR2 and CDR3 being defined according to the Kabat, IMGT, chothia, abM or Contact numbering system;

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, 17, respectively;

more specifically, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are shown as SEQ ID NO. 3, 18, and 19, respectively, as SEQ ID NO. 3,4, and 5, or as SEQ ID NO. 3,4, and 20.

Embodiment 14. The fusion protein of embodiment 7 or 13, wherein the immunoglobulin single variable domain is:

1) Humanized, affinity maturation, removal of T cell epitopes, reduction of antibody deamidation and/or reduction of antibody isomerisation engineering, e.g.IGHV 3-23 x 01 or IGHV3-23 x 04 for the heavy chain framework region of the human germline template used in the humanized engineering process;

2) Camelid antibodies, chimeric antibodies, humanized antibodies, fully human antibodies or antigen binding fragments thereof.

Embodiment 15. The fusion protein of embodiment 13 or 14, wherein the amino acid sequence of the immunoglobulin single variable domain is as in each

SEQ ID NO. 2, 8-15, or

Has at least 90% sequence identity to any one of the preceding sequences.

Embodiment 17. The fusion protein of any of the preceding embodiments further comprising a human immunoglobulin Fc region, e.g., the Fc region is a human IgG1, igG2, igG3, or IgG4 Fc region.

Embodiment 18. The fusion protein of embodiment 17, wherein the Fc region of human IgG1 comprises one or more amino acid mutations that reduce binding to Fc receptors (FcR, e.g., fcγr), and/or reduce effector function;

For example, the Fc region of human IgG1 contains one or more amino acid mutations that reduce binding to fcγ receptors (fcγr), and/or reduce ADCC effects;

For another example, the Fc region of human IgG1 comprises a mutation of L234, L235, P329, or any combination thereof;

more for example, the Fc region of human IgG1 contains the L234A/L235A or L234A/L235A/P329G mutation.

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

for example, 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 create a bulge in the CH3 region of the first subunit, the bulge can be placed in a cavity within 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 create a cavity within the CH3 region of the second subunit, the bulge in the CH3 region of the first subunit can be placed, and for example, the amino acid residues having a larger side chain volume can be selected from R, F, Y, W and the amino acid residues having a smaller side chain volume can be selected from A, S, T, V.

Embodiment 20. The fusion protein of embodiment 19, wherein

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

the first subunit of the Fc region contains a mutation of S354 or E356 and the second subunit contains a mutation selected from Y349, or

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

For example, the number of the cells to be processed,

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

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

The first subunit of the Fc region contains the S354C/T366W mutation and the second subunit contains the Y349C/T366S/L368A/Y407V mutation.

Embodiment 21. The fusion protein according to embodiment 19 or 20, wherein the second subunit of the Fc region further comprises a H435 and/or R436 mutation, preferably a H435R/Y436F, H435R, H435K mutation.

Embodiment 22. The fusion protein of any one of embodiments 17 to 21, wherein the Fc region of human IgG1 comprises a mutation that enhances FcRn-mediated recycling, and/or prolongs half-life;

for example, the Fc region of human IgG1 contains mutations M252 and/or M428;

For example, the Fc region of human IgG1 contains the M252Y/M428V, M252Y/M428L mutation.

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

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

Embodiment 24. The fusion protein according to any of the preceding embodiments, wherein the valence ratio of the PD-1 binding domain or antigen binding domain to IL-2 or variant thereof is between 6:1 and 1:3 (e.g. 4:1 and 1:2), preferably 4:1, 2:1 or 1:1, or wherein one fusion protein comprises 1 or 2 IL-2 or variants thereof and 1,2, 3, 4, 5 or 6 PD-1 binding domains or antigen binding domains.

Embodiment 25. The fusion protein of any one of embodiments 1 to 3,7,11 to 23 comprising a combination of polypeptide chains as set forth in any one of the following I) -VI):

A first polypeptide chain [ PD-1 binding domain 1] - [ Fc region first subunit ] - [ linker 1]a- [ IL-2 or variant thereof ],

A second polypeptide chain [ PD-1 binding domain 1] - [ Fc region second subunit ];

Wherein, -represents a peptide bond, the linker is a polypeptide capable of performing a linking function, a is selected from 0 or 1, [ PD-1 binding domain 1] is selected from the PD-1 binding domains of any of embodiments 11 to 13;

for example, the linker is (G _xS)_y linker, wherein x is an integer from 1 to 5 and y is an integer from 1 to 6; for example, linker 1 is (G ₄S)₃, linker 2 is (G ₄S)₂, linker 3 is G ₄ S).

Some other linkers include, but are not limited to, amino acid sequences as shown in (G _mS_n)_h or (GGNGT) _h or (YGNGT) _h or (EPKSS) _h, where 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).

The above polypeptide chains are in the order from N-terminus to C-terminus.

Embodiment 26. The fusion protein of any of the preceding embodiments, comprising a combination of polypeptide chains of the amino acid sequences shown below:

SEQ ID NOS 42 and 43;

SEQ ID NOS.21 and 22;

SEQ ID NOS 36 and 37;

SEQ ID NOS 38 and 39, or,

SEQ ID NOS.40 and 41.

Or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity in combination with any of the above polypeptide chains.

Unless otherwise specified, the mutation sites of the above Fc region are encoded according to the EU numbering system.

The present disclosure provides a method of purifying a fusion protein comprising the steps of:

(c) Leaching the affinity chromatography packing by using leaching buffer;

(c-1) rinsing the affinity chromatography packing with a first rinsing buffer;

(c-2) rinsing the affinity chromatography packing with a second rinsing buffer;

(c-3) rinsing the affinity chromatography packing with a third rinsing buffer.

(D) The elution step comprises eluting the fusion protein from the affinity chromatography packing with an elution buffer, e.g., having a pH of about 3.0 to about 5.0, e.g., about 3.4 to about 4.0, e.g., about 3.4 to about 3.8, and collecting the eluate

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

In some embodiments, the pH of the second leaching 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

In some embodiments, the pH of the third leaching 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.

In some embodiments, the first wash buffer contains from about 0.03 to about 0.1mol/L of sodium acetate-acetate, and from about 0.1 to about 0.6mol/L of sodium chloride, e.g., from about 0.04mol/L to about 0.06mol/L of sodium acetate-acetate, and from about 0.4mol/L to about 0.6mol/L of sodium chloride;

In some embodiments, the second wash buffer contains from about 0.03 to about 0.1mol/L of sodium acetate-acetate, and from about 0.3 to about 0.7mol/L of sodium chloride, e.g., from about 0.04mol/L to about 0.06mol/L of sodium acetate-acetate, and from about 0.4mol/L to about 0.5mol/L of sodium chloride;

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

In some embodiments, the elution buffer contains from about 0.01mol/L to about 0.5mol/L of sodium acetate-acetate, for example from about 0.05mol/L to about 0.1mol/L of sodium acetate-acetate;

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

In some embodiments, the affinity chromatography packing is a Protein A affinity chromatography packing, e.g., mabSelect Sure, mabselect Sure LX, mabselect PrismA, N Mab, N Mab Pro, or Unimab 50HC;

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

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 fusion protein comprises asymmetric first and second polypeptide chains, wherein the PI of the fusion protein differs by no more than about 3.0 units from any two of the PI of the first polypeptide chain and the PI of the second polypeptide chain. For example, no more than about 2.0 units, or 1.0 units.

In some embodiments, the PD-1 binding domain in the fusion protein comprises at least one immunoglobulin single variable domain comprising CDR1, CDR2 and CDR3 of any one of the amino acid sequences shown in SEQ ID NO 2, 8-15, said CDR1, CDR2 and CDR3 being defined according to the Kabat, IMGT, chothia, abM or Contact numbering system.

In some embodiments, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NOS 3, 16, 17, respectively.

In some embodiments, the amino acid sequences of CDR1, CDR2, CDR3 of the immunoglobulin single variable domain in the PD-1 binding domain are shown as SEQ ID NO 3, 18, 19, 3,4, 5, or 3,4, 20, respectively.

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 shown in any one of SEQ ID NOS.23-31 or having at least 90% sequence identity thereto.

In some embodiments, the fusion protein comprises a combination of polypeptide chains of the amino acid sequences shown below:

SEQ ID NOS 42 and 43;

SEQ ID NOS.21 and 22;

SEQ ID NOS 36 and 37;

SEQ ID NOS 38 and 39, or,

SEQ ID NOS.40 and 41.

(c) Leaching the affinity chromatography packing by using leaching buffer;

(c-1) eluting the affinity chromatography packing with 2-8 column volumes of a first eluting buffer,

(C-2) eluting the affinity chromatography packing with a second eluting buffer of 1-5 column volumes to raise the pH of the affinity chromatography packing to above 4.5;

(c-3) eluting the affinity chromatography packing with a third eluting buffer of 1-5 column volumes to reduce the conductivity of the affinity chromatography packing to below 5.0 mS/cm;

In some embodiments, the pH of the first eluent is from about 3.8 to about 5.0, such as from about 4.0 to about 4.7, illustratively from about 4.1 to about 4.3;

In some embodiments, the pH of the second leaching buffer is about 4.5 to about 8.0, for example about 4.5 to about 6.5, illustratively about 4.5 to about 5.5;

In some embodiments, the pH of the third leaching buffer is about 4.5 to about 8.0, for example about 4.5 to about 6.5, illustratively about 4.5 to about 5.5;

In some embodiments, the pH of the elution buffer is from about 3.0 to about 5.0, for example from about 3.4 to about 4.0, more typically from about 3.4 to about 3.8.

In some embodiments, the first wash buffer contains from about 0.03 to about 0.1mol/L of sodium acetate-acetate, and from about 0.1 to about 0.6mol/L of sodium chloride, and illustratively from about 0.04mol/L to about 0.06mol/L of sodium acetate-acetate, and from about 0.4mol/L to about 0.6mol/L of sodium chloride.

In some embodiments, the second wash buffer contains from about 0.03 to about 0.1mol/L of sodium acetate-acetate, and from about 0.3 to about 0.7mol/L of sodium chloride, and illustratively from about 0.04mol/L to about 0.06mol/L of sodium acetate-acetate, and from about 0.4mol/L to about 0.5mol/L of sodium chloride.

In some embodiments, the third rinse buffer contains from about 0.03 to about 0.1mol/L of acetic acid-sodium acetate, illustratively from about 0.04mol/L to about 0.06mol/L of acetic acid-sodium acetate, illustratively free of sodium chloride.

In some embodiments, the elution buffer contains from about 0.01mol/L to about 0.5mol/L of sodium acetate-acetate, illustratively from about 0.05mol/L to about 0.1mol/L of sodium acetate-acetate.

In some embodiments, the equilibration buffer contains from about 0.01mol/L to about 0.1mol/L phosphate, from about 0.1mol/L to about 2.0mol/L sodium chloride, illustratively from about 0.01mol/L to about 0.05mol/L Phosphate (PB), from about 0.5mol/L to about 1.5mol/L sodium chloride.

In some embodiments, the affinity chromatography packing is a Protein A affinity chromatography packing, illustratively from MabSelect Sure, mabselect Sure LX, mabselect PrismA, N Mab, N Mab Pro, or Unimab HC.

In some embodiments, the fusion protein is a fusion protein comprising an antibody Fc, illustratively 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.

In some embodiments, the fusion protein comprises asymmetric first and second polypeptide chains, wherein the PI of the fusion protein differs by no more than about 3.0 units from any two of the PI of the first polypeptide chain and the PI of the second polypeptide chain. For example, about 2.0 units, or 1.0 units.

SEQ ID NOS 42 and 43;

SEQ ID NOS.21 and 22;

SEQ ID NOS 36 and 37;

SEQ ID NOS 38 and 39, or,

SEQ ID NOS.40 and 41.

Drawings

FIG. 1 shows the detection of the activation of T cells by cytokine release in a DC cell/T cell mixed lymphocyte reaction using Pembrolizumab as positive control and hIgG4 as negative control.

FIG. 2 is the construction of fusion proteins of anti-PD-1 antibodies and IL-2 mutants. Wherein, the anti-PD-1 nanobody A17m0902 is coupled with N terminal of Knob and Hole chain respectively through IgG1 heavy chain hinge region, IL-2 mutant is connected with C terminal of Knob chain of Fc through G ₄S)₃ linker.

FIG. 3 shows the results of the proliferation activity of the same form of PD-1-IL-2v fusion protein with different IL-2 mutants on M07e and M07e-hPD-1 cell lines. Wherein FIG. 3A shows the result of the proliferation activity of the PD-1-IL-2v fusion protein on M07e cells, and FIG. 3B shows the result of the proliferation activity of the PD-1-IL-2v fusion protein on M07e-hPD-1 cells.

FIG. 4 shows the results of the measurement of the pro-proliferative activity of PD-1-IL-2v or IgG-IL-2v on M07e-hPD-1 cells.

FIG. 5 shows the results of the proliferation activity assay of PD-1-IL-2v with different IL-2 mutants on unstimulated T cells. Wherein, FIG. 5A shows the proliferation result of CD4 ⁺ T cells, and FIG. 5B shows the proliferation result of CD8 ⁺ T cells.

FIG. 6 shows the results of the detection of the proliferation activity of PD-1-IL-2v with different IL-2 mutants on T cells stimulated with anti-CD 3 antibodies. Wherein, FIG. 6A shows the proliferation result of CD4 ⁺ T cells, and FIG. 6B shows the proliferation result of CD8 ⁺ T cells.

Detailed Description

Terminology

For easier understanding of the present disclosure, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein within this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The term "impurity" refers to a substance that is different from the desired antibody product. Impurities include, but are not limited to, host cell material such as chinese hamster ovary protein (CHP), leached protein a, nucleic acids, variants, fragments, aggregates or derivatives of desired antibodies, other polypeptides, endotoxins, viral contaminants, cell culture media components.

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

The term "equilibration buffer" refers in this disclosure 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.

The term "elution buffer" refers to a buffer containing a major substance (A PRIMARYSPECIES), such as sodium citrate or sodium acetate, which is used to elute a target product, such as an antibody, protein, or the like, from a chromatography column.

The term "conductivity" refers to the ability of an aqueous solution to conduct electrical current between two electrodes. In a solution, where the current flows through ion transport, the solution will have a higher conductivity as the amount of ions present in the aqueous solution increases. The basic unit of measure of conductivity is Siemens (or ohms), ohms (mS/cm), and can be measured using a conductivity meter, such as an Orion conductivity meter of various types. Because electrolytic conductivity is the ability of ions in a solution to carry an electrical current, the conductivity of a solution can be altered by changing the concentration of ions therein. For example, the concentration of buffer and/or the concentration of salt (e.g., sodium oxide, sodium acetate, or potassium oxide) in the solution may be varied to achieve the desired conductivity.

The term "UV ₂₈₀" refers to the absorbance value of a sample at a wavelength of 280 nm. Protein molecules generally contain amino acids such as tyrosine, tryptophan, phenylalanine and the like, have a maximum absorption peak at a wavelength of 280 nanometers, and have an absorbance value proportional to the protein concentration.

The term "about" means that the value is within an acceptable error range for the particular value being determined by one of ordinary skill in the art, which value depends in part on how the measurement or determination is made (i.e., the limits of the measurement system). For example, "about" may mean within 1 or exceeding a standard deviation of 1. Or "about" or "substantially comprising" may mean a range of up to 20%, for example, between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% and 1%, in this disclosure, each instance preceded by the term "about" by the numerical value or numerical range also includes the given number of embodiments. Furthermore, the term may mean at most one order of magnitude or at most 5 times the value, especially for biological systems or processes. Unless otherwise indicated, when a particular value is found in the present disclosure and claims, the meaning of "about" or "consisting essentially of" should be assumed to be within the acceptable error range for that particular value.

The amino acid three-letter codes and one-letter codes used in the present disclosure are as described in J.biol. Chem,243, p3558 (1968).

"Programmed death 1", "programmed cell death 1", "protein 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 epitope in common with PD-1. The complete PD-1 sequence can be found from GenBank accession U64863. "programmed death ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands for PD-1 (the other is PD-L2), which down-regulates T cell activation and cytokine secretion upon binding 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 number Q9 NZQ.

By "PD-1 binding protein" is meant any protein or any molecule comprising said protein capable of specifically binding to PD-1. The PD-1 binding protein may comprise an antibody, antigen-binding fragment thereof, or conjugate thereof as defined herein, directed against PD-1. PD-1 binding proteins also encompass immunoglobulin superfamily antibodies (IgSF) or CDR-grafted molecules. The "PD-1 binding proteins" of the present disclosure may comprise at least one immunoglobulin single variable domain (e.g., VHH) that binds PD-1. In some embodiments, a "PD-1 binding protein" may comprise 2,3, 4 or more immunoglobulin single variable domains (e.g., VHH) that bind PD-1. The PD-1 binding proteins of the present disclosure may also comprise, in addition to an immunoglobulin single variable domain of PD-1, a linker and/or a moiety with effector function, such as a half-life extending moiety (e.g., an immunoglobulin single variable domain that binds serum albumin), and/or a fusion partner (e.g., serum albumin) and/or a conjugated polymer (e.g., PEG) and/or an Fc region. In some embodiments, the "PD-1 binding proteins" of the present disclosure also encompass bispecific antibodies comprising an immunoglobulin that binds to a different antigen (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., 4-1 BB), optionally including a third antibody that binds to a third antigen, further optionally including a fourth antibody that binds to a fourth antigen.

The "PD-1 binding proteins" or "PD-1 antibodies or antigen-binding fragments thereof" of the present disclosure may comprise one or more effector molecules, e.g., by conjugation. Such "effector molecules" include, for example, antineoplastic agents, drugs, toxins, bioactive 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 analysis. In some embodiments, the effector molecule is a cytokine (e.g., IL-2, IL-15, or variants thereof). In other embodiments, the effector molecule is a polymer, which may generally be a synthetic or naturally occurring polymer, 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. Conjugation of the polymer to the PD-1 binding protein or PD-1 antibody can be accomplished by conventional methods.

A "cytokine" is a protein released by one cell population that acts as an intercellular mediator on other cells. Examples of such cytokines include lymphokines, monokines, chemokines, and traditional polypeptide hormones. Exemplary cytokines include human IL-2, IL-15, IFN-gamma, IL-6, TNF alpha, IL-17, and IL-5, and variants thereof.

"Interleukin-2" or "IL-2" refers to any native 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 that results from processing in a cell. The term also encompasses naturally occurring variants of IL-2, such as splice variants or allelic variants. An exemplary wild-type human IL-2 has the amino acid sequence shown in SEQ ID NO. 87. Unprocessed human IL-2 additionally comprises an N-terminal 20 amino acid signal peptide (shown as SEQ ID NO.272 in WO 2012107417) that is absent from the mature IL-2 molecule.

"Mutant IL-2" or "IL-2 variant" refers to a polypeptide that differs from the amino acid sequence of wild-type IL-2 by a mutation (e.g., substitution, deletion, addition) at least one amino acid position.

In the present disclosure, when referring to IL-2 variants, single amino acid substitutions are described in the following manner [ original amino acid residues/positions/substituted amino acid residues ]. For example, the substitution of phenylalanine at position 42 for alanine may be represented as F42A. Individual single amino acid substitutions can be joined by "/" to represent combinatorial mutations at a plurality of given positions. For example, the combined mutation at positions F42A, L G and N88R may be expressed as F42A/L72G/N88R.

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 known as wt IL-2). The corresponding amino acid positions on other IL-2 proteins or polypeptides (including full length sequences or truncated fragments) can be identified by amino acid sequence alignment with SEQ ID NO. 87. Thus, in the present disclosure, unless otherwise indicated, the amino acid position of an 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 phenylalanine residue F at position 42 of SEQ ID NO. 40, or an amino acid residue aligned at a corresponding position on other IL-2 polypeptide sequences. Sequence alignment for amino acid position determination may be performed using Basic Local ALIGNMENT SEARCH Tool available from https:// blast.ncbi.lm.nih.gov/blast.cgi, using default parameters.

"CD25" or "alpha subunit of the 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 as well as 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.

"High affinity IL-2 receptor" refers to a heterotrimeric form of the IL-2 receptor that consists of a receptor gamma subunit (also known as the universal cytokine receptor gamma subunit, yc or CD 132), a receptor beta subunit (also known as CD122 or p 70) and a receptor alpha subunit (also known as CD25 or p 55). In contrast, a "medium affinity IL-2 receptor" refers to an IL-2 receptor comprising only the gamma and beta subunits without the alpha subunit (see, e.g., olejniczak and Kasprzak, medSci Monit, RA179-189 (2008)).

"Antibody" is used in its broadest sense and covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies and antibody fragments (or antigen-binding fragments, or antigen-binding portions), so long as they exhibit the desired antigen-binding activity. Antibodies may refer to immunoglobulins which are tetrapeptide chain structures formed by joining two identical heavy chains and two identical light chains together by interchain disulfide bonds. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be classified into five classes, or isotypes of immunoglobulins, igM, igD, igG, igA and IgE, with their respective heavy chains being the μ, δ, γ, α and ε chains, respectively. The same class of Ig can be further classified into different subclasses according to the amino acid composition of the hinge region and the number and position of disulfide bonds of the heavy chain, e.g., igG can be classified into IgG1, igG2, igG3, and IgG4. Light chains are classified by the difference in constant regions as either kappa chains or lambda chains. Each of the five classes of Ig may have either a kappa chain or a lambda chain. The sequences of the heavy and light chains of the antibody near the N-terminus vary widely, being the variable region (V region), and the remaining amino acid sequences near the C-terminus are relatively stable, being the constant region (C region). The variable region comprises 3 hypervariable regions (CDRs) and 4 Framework Regions (FR) of relatively conserved sequence. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the light chain variable region (VL) and heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from amino terminus to carboxyl terminus. The 3 CDR regions of the light chain are referred to as LCDR1, LCDR2 and LCDR3, and the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3.

"Antigen binding fragments" encompass single chain antibodies (i.e., full length heavy and light chains), fab, modified Fab, fab ', modified Fab', F (ab ') 2, 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 foregoing (see, e.g., Holliger and Hudson,2005,Nature Biotech.23(9)：1126-1136;Adair and Lawson,2005,Drug Design Reviews-Online2(3),209-217). in some embodiments, antigen binding fragments are VHH. Methods of producing and preparing such antigen binding fragments are well known in the art (see, e.g., verma et al, 1998,Journal ofImmunological Methods,216,165-181). Fab-Fv forms are first disclosed in WO2009/040562, disulfide stabilized forms thereof Fab-dsFv are first disclosed in WO2010/035012. The antigen binding fragments of the present disclosure also include Fab and Fab' fragments described in WO2005/003169, WO2005/003170, and WO 2005/003171. Multivalent antibodies may comprise multispecific or may be monospecific (see, e.g., WO 92/83 and WO 05/113605), the latter one being either as described in WO92/22583 or as illustrated in WO 22522583.

An "Fc domain" or "Fc region" is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. It includes a native sequence Fc region and a variant Fc region. Although the boundaries of the Fc region of an IgG heavy chain may vary slightly, a human IgG heavy chain Fc region is generally defined as extending from Cys226 or Pro230 to the carboxy-terminus of the heavy chain. However, antibodies produced by the host cell may undergo post-translational cleavage, by cleaving one or more, especially one or two, amino acids from the C-terminus of the heavy chain. Thus, an antibody produced by a host cell by expression of a particular nucleic acid molecule encoding a full-length heavy chain may comprise a full-length heavy chain, or it may comprise a cut variant of a full-length heavy chain (also referred to as a "cut 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 according to the Kabat EU index). Thus, the C-terminal lysine (K447), or C-terminal glycine (G446) and lysine (K447) of the Fc region may be present or absent. The amino acid sequence of the heavy chain comprising the Fc region (or a subunit of the Fc region as defined in the present disclosure) represents in the present disclosure a C-terminal-free glycine-lysine dipeptide, if not otherwise indicated. In one embodiment, the heavy chain of one subunit of the Fc region comprised in the immunoconjugate comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to the EU index of Kabat). In one embodiment, the heavy chain of one subunit of the Fc region comprised in the immunoconjugate comprises an additional C-terminal glycine residue (G446, numbering according to the EU index of Kabat). A composition (e.g., a pharmaceutical composition) of the present disclosure comprises a population of immunoconjugates of the present disclosure. The population of immunoconjugates may comprise molecules with full length heavy chains and molecules with cleaved variant heavy chains. The population of immunoconjugates may consist of a mixture of molecules having full length heavy chains and molecules having cleaved variant heavy chains, wherein at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the immunoconjugates have cleaved variant heavy chains, which may be C-terminal lysine (K447) or C-terminal glycine (G446) and lysine (K447) of the Fc region are absent. Unless otherwise specified in the present disclosure, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al Sequences of Proteins of Immunological Interest, 5 th edition Public HEALTH SERVICE, NATIONALINSTITUTES of Health, bethesda, MD,1991.

"Subunit" of an Fc region refers to one of two polypeptides forming a dimeric Fc region, i.e., a polypeptide comprising a C-terminal constant region in an immunoglobulin heavy chain capable of stabilizing self association. For example, the subunits of the IgG Fc region comprise IgG CH2 and IgG CH3 constant domains. In this disclosure, the use of "first subunit" or Fc1, "second subunit" or Fc2 is not intended to impart a particular order or orientation, and is used for ease of distinction.

A "modification (or mutation) that facilitates the association of a first subunit and a second subunit of an Fc domain" is a peptide backbone manipulation or post-translational modification of an Fc domain subunit that reduces or prevents association of a polypeptide comprising the Fc domain subunit with the same polypeptide to form a homodimer. As used herein, in particular, modifications that promote association include separate modifications to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) that are desired to be associated, wherein the modifications are complementary to each other, thereby promoting association of the two Fc domain subunits. For example, modifications that promote association may alter the structure or charge of one or both Fc domain subunits, thereby sterically or electrostatically promoting their association, respectively. As such, (heterodimerization occurs between a polypeptide comprising a first Fc domain subunit and a polypeptide comprising a second Fc domain subunit, which may be different in the sense that the other components fused to each subunit (e.g., antigen binding moiety) are different. In some embodiments, the modification that facilitates association comprises an amino acid mutation, specifically an amino acid substitution, in the Fc domain.

"Complementarity" refers to, for example, the combination of interactions at the interface of CH1 and CL (or CH3 and CH 3) of an antigen binding protein described herein that affect heavy/light chain pairing. "spatial complementarity" or "conformational complementarity" refers to the compatibility of three-dimensional structures at the interaction surface of, for example, CH1 and CL (or CH3 and CH 3). "electrostatic complementarity" refers to the compatibility of placing negatively and/or positively charged atoms at the interacting surface of, for example, CH1 and CL (or CH3 and CH 3). Methods for measuring electrostatic complementarity at a Protein/Protein interface 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 Des8, 51325. Methods for measuring spatial 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 Biol228,277-297; and Schueler-Furman et al (2005) proteins60, 187-194.

"Effector function" when referring to antibody use refers to those biological activities attributable to the Fc region of the antibody and which 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 Cell Phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation. ADCC is an immune mechanism that results in the lysis of antibody-coated target cells by immune effector cells. The target cell is a cell to which an antibody or derivative thereof comprising an Fc region typically specifically binds via the N-terminal protein portion of the Fc region. "reduced ADCC" is defined as a decrease in the number of target cells lysed in a given time by an ADCC mechanism as defined above at a given concentration of antibody in the target cell's surrounding medium, and/or an increase in the concentration of antibody in the target cell's surrounding medium required for lysis of a given number of target cells in a given time by an ADCC mechanism. The decrease in ADCC is relative to ADCC mediated by the same antibody produced by the same type of host cell but not yet engineered, using the same standard production, purification, formulation and storage methods known to those skilled in the art. For example, the decrease in ADCC mediated by an antibody comprising an amino acid substitution in its Fc domain that decreases ADCC 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 e.g. WO2006/082515 or WO 2012/130831).

An "activated Fc receptor" is an Fc receptor that, upon engagement of the Fc domain of an antibody, initiates a signaling event that stimulates cells carrying the receptor to perform effector functions. Human activated Fc receptors include fcyriiia (CD 16 a), fcyri (CD 64), fcyriia (CD 32) and fcyri (CD 89).

An "immunoconjugate" refers to a polypeptide molecule comprising at least one IL-2 molecule and at least one antibody. As described herein, IL-2 molecules can be linked to antibodies through a variety of interactions and in a variety of configurations. In certain embodiments, the IL-2 molecule is fused to the antibody via a peptide linker. A particular immunoconjugate according to the invention essentially consists of one IL-2 molecule and an antibody linked by one or more linker sequences.

"Reduced avidity" or "reduced binding", e.g., reduced binding to IL-2Rα, refers to a reduced avidity of the corresponding interaction, e.g., as measured by SPR. The term also includes affinity decreases to 0 or below the detection limit of the assay method, i.e. the interaction is completely eliminated. Conversely, "reduced avidity" or "increased binding" refers to an increase in binding avidity of the corresponding interaction.

For determination or definition of CDRs, deterministic delineation of CDRs and identification of residues comprising the binding site of an antibody can be accomplished by resolving the structure of the antibody and/or resolving the structure of the antibody-ligand complex. This may be accomplished by any of a variety of 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, kabat numbering system, chothia numbering system, abM numbering system, IMGT numbering system, contact definition, conformational definition.

The Kabat numbering system is a standard for numbering residues in antibodies and is commonly 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 considers the position 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 a computer program integration suite produced by Oxford Molecular Group to model the antibody Structure (see, e.g., martin et al ,1989,ProcNatl Acad Sci(USA),86：9268-9272;"AbMTM,A Computer Program for ModelingVariable Regions of Antibodies,"Oxford,UK;Oxford Molecular,Ltd).AbM numbering system using a combination of knowledge database and ab initio methods; additionally other CDR boundary definitions may not strictly follow one of the methods described above, but still overlap at least a portion of the Kabat CDRs, although predictions or experimental results of antigen binding are not significantly affected according to a particular residue or group of residues, which may be shortened or lengthened as used in the present disclosure) based on analysis of available complex crystal structures (see, e.g., macCallum et al, 1996, J.mol. Biol., 5:732-45). In conformational definitions, the positions of CDRs may be identified as residues contributing to enthalpy binding for antigen binding (see, e.g., makabe et al, 2008,Journal ofBiological Chemistry,283:1156-1166). In addition, other CDR boundary definitions may not strictly follow one of the methods described above.

TABLE 1 relationship between CDR numbering systems

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

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 known Kabat numbering system.

Antibodies of the present disclosure may be polyclonal, monoclonal, xenogenic, allogeneic, syngeneic, or modified versions thereof, with monoclonal antibodies being particularly useful in a variety of embodiments. Generally, the antibodies of the present disclosure are recombinant antibodies. "recombinant" as used herein refers broadly to products such as cells or nucleic acids, proteins or vectors, which means that the cells, nucleic acids, proteins or vectors have been modified by the introduction of heterologous nucleic acids or proteins or alteration of native 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 native (non-recombinant) cell forms or express native genes that are otherwise abnormally expressed, under expressed, or not expressed at all.

A "domain" of a polypeptide or protein refers to a folded protein structure that is capable of maintaining its tertiary structure independent 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 losing the function of the remainder of the protein and/or the domain.

An "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 to a polypeptide consisting essentially of such globular region. The immunoglobulin domain is characterized in that it maintains the immunoglobulin folding characteristics of the antibody molecule, consisting of a 2-layer sandwich of about 7 antiparallel β -sheet strands, optionally stabilized by a conserved disulfide bond, arranged in two β -sheets.

"Immunoglobulin variable domain" refers to an immunoglobulin domain consisting essentially 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", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be expressed as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Immunoglobulin variable domains confer specificity for antigens by having antigen binding sites.

An "immunoglobulin single variable domain" is generally used to refer to an immunoglobulin variable domain (which may be a heavy or light chain domain, including a VH, VHH, or VL domain) that can form a functional antigen binding site without interaction with other variable domains (e.g., without VH/VL interactions as required between VH and VL domains of a conventional four chain monoclonal antibody). Examples of "immunoglobulin single variable domains" include nanobodies (including VHHs, humanized VHHs, and/or camelized VH, e.g., camelized human VH), igNAR, domains, (single domain) antibodies that are VH domains or are derived from VH domains (e.g., dAbsTM), and (single domain) antibodies that are VL domains or are derived from VL domains (e.g., dAbsTM). 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.

"VHH domain", also known as a heavy chain single domain antibody, VHH antibody fragment, VHH antibody, nanobody, is an antigen-binding immunoglobulin variable domain (Hamers-Casterman C,Atarhouch T,Muyldermans S,Robinson G,Hamers C,Songa EB,Bendahman N,Hamers R.："Naturally occurring antibodies devoid of light chains";Nature363,446-448(1993))., known as a "heavy chain antibody" (i.e., an "antibody lacking a light chain") is used to distinguish the variable domain from the heavy chain variable domain (which is referred to as a "VH domain" in the present disclosure) and the light chain variable domain (which is referred to as a "VL domain" in the present disclosure) present in conventional tetrapeptide chain structural antibodies. The VHH domain specifically binds to the epitope without the need for additional antigen binding domains (this is in contrast to VH or VL domains in conventional tetrapeptide chain structural antibodies, in which case the epitope is recognized by the VL domain along with the VH domain). VHH domains are small stable and efficient antigen recognition units formed from a single immunoglobulin domain. "heavy chain single domain antibody", "VHH domain", "VHH antibody fragment", "VHH antibody" and "domain" ("Nanobody" is a trademark of Ablynx n.v. company, ghent, belgium) are used interchangeably. "VHH domains" include, but are not limited to, natural antibodies produced by camelids, either humanized or screened by phage display techniques. In some embodiments, a VHH domain comprises three CDRs and four framework regions designated FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, the VHH domain may be truncated at the N-or C-terminus such that it comprises only a portion of FR1 and/or FR4, or lacks one or both of those framework regions, so long as the VHH substantially retains antigen binding and specificity. The total number of amino acid residues in the VHH domain will typically range from 110 to 120, often between 112 and 115. It should be noted, however, that smaller and longer sequences may also be suitable for the purposes described in this disclosure.

As is well 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 Kabat numbering (i.e., one or more positions according to Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering). This means that in general, 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 rules include Chothia, IMGT, abM.

"Humanized antibody (humanized antibody)", also known as a CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting non-human CDR sequences into the variable region framework of a human antibody. The strong immune response induced by chimeric antibodies due to the large number of non-human protein components can be overcome. To avoid a decrease in activity while simultaneously decreasing immunogenicity, the fully human antibody variable region may be subjected to minimal back mutations to maintain activity. Examples of "humanisation" include that a camelidae derived VHH domain may be "humanised" 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 human conventional tetrapeptide chain structural antibody VH domain (also referred to herein as "sequence optimisation", which may also encompass, in addition to humanisation, other modifications to the sequence by one or more mutations that provide improved properties of the VHH, for example removal of potential post-translational modification sites). The humanized VHH domain may contain one or more fully human framework region sequences and in some embodiments may contain human framework region sequences of IGHV 3. Methods of humanization such as protein surface amino acid humanization (resurfacing) and antibody humanization universal frame grafting (CDR GRAFTING to a universal framework) are used, i.e., the CDRs are "grafted" onto other "scaffolds (including but not limited to human scaffolds or non-immunoglobulin scaffolds). Scaffolds and techniques suitable for such CDR grafting are known in the art. Germline DNA sequences for human heavy and light chain variable region genes can be found, for example, in the "VBase" human germline sequence database, and in Kabat, E.A. et al, 1991Sequences of Proteins of Immunological Interest, 5 th edition. Humanized antibodies of the present disclosure also include humanized antibodies that are further affinity matured from phage display to CDRs. In addition, to avoid a decrease in immunogenicity while at the same time causing a decrease in activity, the human antibody variable region framework sequences may be subjected to minimal reverse or back-mutations to maintain activity.

An "avidity mature" antibody refers to an antibody having one or more alterations in one or more hypervariable regions (HVRs) that result in improved avidity of the antibody for an antigen as compared to a parent antibody that does not possess such alterations. For example, a "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 as compared to its parent antibody. Affinity matured antibodies can be prepared, for example, by methods known in the art as described by Marks et al 1992,Biotechnology 10:779-783 or Barbas et al 1994,Proc.Nat.Acad.Sci,USA 91:3809-3813, by thier et al 1995,Gene 169:147-155, by Yelton et al 1995, by immunol.155:1994-2004, by Jackson et al 1995, J.immunol.154 (7): 3310-9, and by 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.

Typically, the PD-1 antibodies, immunoconjugates of the disclosure will bind to the antigen to be bound (i.e., PD-1) with a dissociation constant (KD) of preferably 10 ^-7 to 10 ^-10 moles/liter (M), more preferably 10 ^-8 to 10 ^-10 moles/liter, even more preferably 10 ^-9 to 10 ^-10 or less, and/or with an association constant (KA) of at least 10 ^-7 M, preferably at least 10 ^-8 M, more preferably at least 10 ^-9 M, more preferably at least 10 ^-10 M, as measured in a Biacore or KinExA or Fortibio assay. Any KD value greater than 10 ^-4 M is generally considered to indicate 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.

"Antigen" refers to a molecule used to immunize an immunocompetent vertebrate to produce antibodies that recognize the antigen, or to screen an expression library (e.g., a phage, yeast, or ribosome display library, among others). In the present disclosure, antigens are defined more broadly, including target molecules specifically recognized by antibodies, as well as including portions or mimics of the molecules used in the immunization process for producing antibodies or in library screening for selection of antibodies. For example, for antibodies of the present disclosure that bind to human PD-1, monomers and multimers (e.g., dimers, trimers, etc.) of human PD-1, as well as truncated and other variants of human PD-1, are referred to as PD-1 antigens.

An "epitope" refers to a site on an antigen that binds to an immunoglobulin or antibody. Epitopes can be formed by contiguous amino acids, or non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are typically maintained after exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost after treatment with denaturing solvents. Epitopes typically comprise at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation detection assays, among others. Methods of determining the spatial conformation of an epitope include techniques in the art and techniques described in this disclosure, such as X-ray crystallography, two-dimensional nuclear magnetic resonance, and the like.

"Specifically bind", "selectively bind" refers to the binding of an antibody to an epitope on a predetermined antigen. For example, when human PD-1 or an epitope thereof is used as an analyte and an antibody is used as a ligand, the antibody binds to a predetermined antigen or epitope thereof with an equilibrium dissociation constant (K _D) of about less than 10 ^-7 M or even less, and its affinity for binding to the predetermined antigen or epitope thereof is at least twice that of its affinity for binding to non-specific antigens other than the predetermined antigen (or epitope thereof) or closely related antigen (e.g., BSA, etc.), as determined by Surface Plasmon Resonance (SPR) techniques in an instrument. "antibody that recognizes an antigen" may be used interchangeably in this disclosure with "antibody that specifically binds".

"Binding avidity" 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, the Surface Plasmon Resonance (SPR) method (Biacore). The rate constants corresponding to the binding and dissociation of the monovalent complex are referred to as the binding rate constant ka (or kon) and dissociation rate constant kd (or koff), respectively. K _D is related to ka and kd by the equation K _D =kd/ka. The values of the dissociation constants 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 _D may 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). Other standard assays for assessing the binding capacity of an antibody to a target antigen are known in the art, including, for example, ELISA, western blot, RIA and flow cytometry assays, as well as other assays exemplified elsewhere in the disclosure. The binding kinetics and binding avidity of the antibodies can also be assessed by standard assays known in the art, such as Surface Plasmon Resonance (SPR), for example by using the Biacore ^TM system or KinExA. Binding affinities associated with different molecular interactions, e.g., binding affinities of different antibodies for a given antigen, can be compared by comparing the K _D values of the individual antibody/antigen complexes. Similarly, the specificity of an interaction can be assessed by determining and comparing the K _D value for a desired interaction (e.g., a specific interaction between an antibody and an antigen) with the K _D value for a non-desired interaction (e.g., a control antibody known not to bind CD 40).

"Conservative substitution" refers to the substitution of another amino acid residue that has similar properties 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 nonpolar side chains. In addition, tyrosine, phenylalanine, tryptophan and histidine have similar properties in that they have aromatic side chains. Thus, it will be apparent to those skilled in the art that even when amino acid residues in groups exhibiting similar properties as described above are substituted, it will not exhibit a particular change in properties.

"Homology", "identity" or "sequence identity" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both comparison sequences is occupied by the same nucleotide or amino acid monomer, for example if each position of two DNA molecules is occupied by the same nucleotide, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matched or homologous positions shared by the two sequences divided by the number of positions compared x 100%. For example, in the optimal alignment of sequences, if there are 6 matches or homologies at 10 positions in the two sequences, then the two sequences are 60% homologous. In general, a comparison is made when two sequences are aligned to give the greatest percent homology.

Examples

The present disclosure is further described below in connection with the examples, which are not intended to limit the scope of the present disclosure. The experimental methods for which specific conditions are not noted in the examples of the present disclosure are generally conducted according to conventional conditions such as a laboratory manual of antibody technology in cold spring harbor, a manual of molecular cloning, or according to conditions recommended by raw materials or commercial manufacturers. The reagents of specific origin are not noted and are commercially available conventional reagents.

Example 1 screening, preparation and engineering of anti-PD-1 nanobodies

1. Antibody screening

The present disclosure uses His-tagged human PD-1 as an immunizing antigen (AcroBiosystems, PD-H5221) to immunize alpaca, constructs a nanobody (VHH) yeast display library, screens antibodies. The biotin-labeled human PD-1 and cynomolgus PD-1 are used as screening antigens (AcroBiosystems, PD-H82E 4; PD1-C82E 6), and 18 VHH unique sequences for cross-binding of human and monkey PD-1 antigens are obtained through monoclonal identification, sequencing and sequence analysis.

The sequence of the human PD-1 protein (NP-005009.2) is shown below, in which the extracellular domain sequence is underlined and the amino acids start and stop Leu25-Gln 167.

Human PD-1 amino acid sequence

MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL(SEQ ID NO:1)

The A17 sequence is shown below, with the CDRs using the Kabat numbering convention.

Variable region of A17

EVQVVESGGGLVQPGGSLRLSCVASGLTFSDYSMSWYRQAPGKERELVAIISGSGVIAHYVDSVKGRFTISRDNAKSTVYLQMVSLKPEDRGVYYCRAVSDWDDYWGQGTQVTVSS(SEQ ID NO:2)

>A17 CDR1

DYSMS(SEQ ID NO:3)

>A17 CDR2

IISGSGVIAHYVDSVKG(SEQ ID NO:4)

>A17 CDR3

VSDWDDY(SEQ ID NO:5)

A17 was ligated to a human IgG4 Fc (comprising the hinge region and carrying S228P according to the EU numbering system) fragment to construct a VHH-Fc antibody. Plasmid construction, transient 293 cells, expression, purification using protein A column, PBS wash column, elution with 0.1M glycine buffer, pH 2.5. Dialyzed into PBS pH7.4 buffer. And detecting to obtain the target protein.

>hIgG4

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:6)

>A17_hIgG4

EVQVVESGGGLVQPGGSLRLSCVASGLTFSDYSMSWYRQAPGKERELVAIISGSGVIAHYVDSVKGRFTISRDNAKSTVYLQMVSLKPEDRGVYYCRAVSDWDDYWGQGTQVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

(SEQ ID NO:7)

2. Humanization and back mutation

Antibody humanization was performed by CDR-grafting method. The parental PD-1 antibodies were aligned with the fully human germline genes in the IMGT or NCBI/igblast database via the IMGT or NCBI website, and human germline genes IGHV3-23 (IGHV 3-23 x 01; IGHV3-23 x 04) that were highly homologous to the PD-1 nanobody were selected as humanized templates, the CDRs were grafted, and 4 key residues (Y37, E44, R45, L47) in the FR2 of the nanobody were retained. And back-mutating the critical core residues adjacent to the CDR regions and the Vernier position residues for CDR interaction. Humanized antibodies A17h1, A17h2, A17h3 were obtained.

A17h1 variable region

EVQLQESGGGLVQPGGSLRLSCTASGLTFSDYSMSWYRQAPGKGRELVAIISGSGVIAHYVDSVKGRFTISRDNSKNTVYLQMRSLTPEDRAVYYCRAVSDWDDYWGQGTQVTVSS(SEQ ID NO:8)

A17h2 variable region

EVQLVESGGGLVQPGGSLRLSCAASGLTFSDYSMSWVRQAPGKGLEWVAIISGSGVIAHYVDSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCRAVSDWDDYWGQGTQVTVSS(SEQ ID NO:9)

A17h3 variable region

EVQLVESGGGLVQPGGSLRLSCAASGLTFSDYSMSWVRQAPGKGLEWVAIISGSGVIAHYVDSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCRAVSDWDEYWGQGTQVTVSS(SEQ ID NO:10)

3. TCE (T cell epitope) removal, reduction of antibody deamidation, reduction of antibody isomerization engineering to obtain the following sequences:

variable region of A17m09

EVQLQESGGGLVQPGGSLRLSCTASGLTFSDYSMSWYRQAPGKGRELVAIISGSGVITHYVDSVKGRTISRDNAKNTVYLQMRSLTPEDRAVYYCRAVSDWEDYWGQGTQVTVSS(SEQ ID NO:11)

The A17m09 is further selected to reconstruct the antibody sequence in a embryogenic way so as to improve the humanization degree, and the following sequences are obtained:

A17m0901 variable region

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYSMSWYRQAPGKGRELVAIISGSGVITHYVDSVKGRFTISRDNAKNTLYLQMRSLRAEDTAVYYCRAVSDWEDYWGQGTQVTVSS(SEQ ID NO:12)

A17m0902 variable region

EVQLVESGGGLVQPGGSLRLSCAASGLTFSDYSMSWYRQAPGKGRELVAIISGSGVITHYVDSVKGRFTISRDNAKNTLYLQMRSLRAEDTAVYYCRAVSDWEDYWGQGTQVTVSS(SEQ ID NO:13)

A17m0903 variable region

EVQLVESGGGLVQPGGSLRLSCAASGLTFSDYSMSWYRQAPGKGRELVAIISGSGVITHYVDSVKGRFTISRDNAKNTVYLQMRSLRAEDTAVYYCRAVSDWEDYWGQGTQVTVSS(SEQ ID NO:14)

A17m0905 variable region

EVQLVESGGGLVQPGGSLRLSCAASGLTFSDYSMSWYRQAPGKGRELVAIISGSGVITHYVDSVKGRFTISRDNAKNTVYLQMRSLRAEDRAVYYCRAVSDWEDYWGQGTQVTVSS(SEQ ID NO:15) That is, a17 of the present disclosure has the following general formula sequence:

CDR1:DYSMS(SEQ ID NO:3)

CDR2: IISGSGVIX ₁ HYVDSVKG, wherein X ₁ is selected from T or A (SEQ ID NO: 16)

CDR3: VSDWX ₄X₅ Y, wherein X ₄ is selected from D or E and X ₅ is selected from D or E (SEQ ID NO: 17)

Specifically, CDR2 may be:

IISGSGVIAHYVDSVKG(SEQ ID NO:4)

IISGSGVITHYVDSVKG(SEQ ID NO:18)

CDR3 may be:

VSDWDDY(SEQ ID NO:5)

VSDWEDY(SEQ ID NO:19)

VSDWDEY(SEQ ID NO:20)

example 2 anti-PD-1 nanobody function assay

1.ELISA

Human PD-1 protein (his tag) was dissolved to 1. Mu.g/mL using PBS, 100 uL/well was added to 96-well plates, and antigen was coated at 4℃overnight. The wash was performed three times with PBST (PBS+0.05% tween 20) solution. Blocking was performed by adding PBST/1% BSA solution, incubating for 1h at 37 ℃. After three washes with PBST solution, PD-1 candidate VHH-Fc antibodies (PBST/1% BSA solution dilution) were added at different concentrations and incubated at 37℃for 1.5 hours. The PBST solution was washed three times. mu.L of HRP-conjugated anti-human IgG4 Fc secondary antibody (Thermo) solution was added and incubated at 37℃for 1 hour. After washing with PBST solution three times, 100. Mu.L/well TMB solution was added, and after 5min reaction at room temperature, 50. Mu.L of stop solution was added, and then 450nM was read using an ELISA plate to calculate EC ₅₀, and Table 4 shows that A17_hIgG4 has a strong binding force with PD-1 antigen.

TABLE 4 ELISA detection of EC ₅₀ values of anti-PD-1 nanobodies

Antibody numbering	EC₅₀(nM)
		A17_hIgG4	0.06099

2.FACS

To examine the binding capacity of anti-PD-1 nanobody to PD-1 expressed on cell membrane, candidate anti-PD-1 VHH-Fc antibodies at different concentrations were added to 10 ⁵/well of Jurkat cells overexpressing human PD-1 (Cat: J1250, promega), incubated at room temperature for 20min, washed twice with PBS, then added with 100. Mu.L of anti-human IgG Fc fluorescent secondary antibody (bioleged), incubated at room temperature for 20min, washed twice with PBS, resuspended in 250. Mu.L of PBS, and the fluorescence signal was detected using FACS to calculate EC ₅₀. Pembrolizumab (purchased from the Baiying organism) was used as a control. Table 5 shows that the EC ₅₀ value of A17_hIgG4 is 0.3568nM, significantly superior to other antibodies obtained from the simultaneous screening (results not shown).

TABLE 5 FACS detection of EC ₅₀ values for anti-PD-1 nanobody binding to cell surface PD-1

Antibody numbering	EC₅₀(nM)
		A17_hIgG4	0.3568
Pembrolizumab	0.2169

PD-1/PD-L1 reporter System detection of blocking of PD-1 by anti-PD-1 nanobodies

CHO-K1 cells overexpressing PD-L1 were diluted to 4×10 ⁵/mL and incubated overnight in 96-well plates with 100 μl/well. After aspiration of the medium, 40. Mu.L of 1.25X10 ⁶/mL PD-1Jurkat cells and different concentrations of 40. Mu.L of anti-PD-1 nanobody solution (1640+2% FBS solution dilution) were added rapidly. After incubation at 37℃for 6 hours, the temperature was returned to room temperature, 40. Mu.L of Bright-glo reagent (Cat: E2620, promega) was added to each well, shaking was performed at 350rpm in the dark for 5min, and after standing at room temperature for 5min in the dark, the fluorescence value was read using an ELISA reader, and IC ₅₀ was calculated. Pembrolizumab and hIgG4 isotype antibodies were used as controls. As shown in table 6, the a17_higg4 biological functional activity IC ₅₀ value was 1.199nM, significantly superior to other antibodies screened synchronously by the present disclosure (e.g., a3_higg4 was 7.62, a13_higg4 was 3.208, a3_higg4 and a13_higg4 did not show sequences).

TABLE 6 IC ₅₀ values for detection of Activity of anti-PD-1 nanobodies by PD-1/PD-L1 NFAT reporter System

Antibody numbering	IC₅₀(nM)
		A17_hIgG4	1.199
Pembrolizumab	0.5126
		hIgG4	-

Functional verification of anti-PD-1 nanobodies was performed as described above, see table 7, with IC ₅₀ activity of a17h1_higg4 being similar to positive antibody Pembrolizumab. In the sequence modified candidate molecules, the biological activity IC ₅₀ values of the A17m0901_hIgG4 and the A17m0902_hIgG4 antibodies are 0.5307nM and 0.4352nM respectively, which is better than Pembrolizumab.

TABLE 7 detection of Activity of engineered anti-PD-1 nanobodies by PD-1/PD-L1 NFAT reporter System IC ₅₀

Antibody numbering	IC₅₀(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

4. Binding affinity identification of engineered anti-PD-1 nanobodies to antigen PD-1

Affinity of anti-PD-1 nanobodies to PD-1 antigen was detected using surface plasmon resonance (surface plasmon resonance, SPR) using Biacore 8k (GE Healthcare). The experiment selects CM5 sensor chip, and the mobile phase adopts HBS-EP+ buffer solution (10mM HEPES,150mM NaCl,3mM EDTA,0.05%surfactant P20). Anti-human IgG (Fc) antibodies were amino-coupled immobilized using a 10mM sodium acetate buffer (pH 5.0) to prepare a 30. Mu.g/mL solution, and the Immobilisation program was selected for automation. The antibody to be detected is respectively prepared by using HBS-EP+ buffer solution as ligand, and the anti-human IgG (Fc) antibody on the chip channel is used for capturing. Human antigenic protein (Sino Biological,10377-H08H; 90311-C08H) was used as the analyte, and the analyte was formulated with HBS-EP+ buffer solution, diluted by a 2-fold gradient, and flowed through the experimental and reference channels at a flow rate of 30. Mu.L/min, combined for 1min, and dissociated for 15min. Regeneration buffer 10mM Glycine pH 1.5 (GE HEALTHCARE, BR-1003-54) was run at a flow rate of 10. Mu.L/min for 30s, and the binding rate Ka and dissociation rate Kd, as well as the dissociation constant (i.e., affinity K _D) were calculated. The results are shown in Table 8.

TABLE 8 SPR affinity data for binding of anti-PD-1 nanobodies to human PD-1 antigen

Antibody numbering	ka(1/Ms)	Kd(1/s)	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

The results show that the data obtained from the above-mentioned method, humanized antibody A17h1_hIgG4 and subsequent engineering the optimized antibody A17m09_hIgG4 engineering optimized antibodies a17m09_hIgG 4. A17h1_hIgG4 a17m09_hIgG4 A17m0901_hIgG4 A17m0902_hIgG4 has a slower dissociation time than Pembrolizumab

DC:T mixed lympho-reaction to examine in vitro efficacy (MLR) of engineered anti-PD-1 nanobodies

Mature dendritic cells (DENDRITIC CELLS, DC) are co-cultured with allogeneic T cells to activate the T cells and promote the secretion of cytokines by the T cells. The process is inhibited by PD-1/PD-L1 signals, and the inhibition can be relieved by using a PD-1 antibody to promote T cell activation. Thus, in vitro efficacy of PD-1 antibodies can be tested using DC:T mixed lymphoresponse experiments.

Monocytes were sorted 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 sorted from fresh PBMC of another healthy person using a sorting kit (cat: 17951, stemcell). 5000 mature DC cells were co-cultured with 50000 allogeneic T cells, and candidate antibodies were added at corresponding concentrations, after six days of co-culture, cell supernatants were aspirated, IFN-gamma concentrations were detected using a Cisbio-HTRF IFN-gamma detection kit (Cat: 62 HIFUCH, perkinelmer), and EC ₅₀ was calculated.

As shown in FIG. 1, A17m09_hIgG4 showed in vitro potency substantially consistent with Pembrolizumab in the MLR assay. Wherein, the EC ₅₀ of A17m09_hIgG4 is 0.2931nM and the EC ₅₀ of Pembrolizumab is 0.3271nM.

EXAMPLE 3 construction and Activity detection of fusion proteins of anti-PD-1 antibodies with IL-2 mutants (PD-1-IL-2 v)

1. Molecular building

Fusion proteins of anti-PD-1 antibodies and IL-2 mutants were constructed according to the molecular format schematic shown in FIG. 2. The Fc of the molecules of FIG. 2 all employed the Knob-in-Hole (KIH) technique, i.e., mutations of S354C, T366W (constituting Knob chain) and Y349C, T366S, L368A, Y407V (constituting Hole chain) were introduced into the two Fc chains, respectively, to promote the formation of heterodimers, H435R, Y436F (Eu numbering system) were additionally introduced into the Hole chain for ease of purification, and furthermore, L234A, L235A and P329G mutations were introduced into the Knob and Hole chains, respectively, to eliminate ADCC effects, and C220S mutations were introduced into the heavy chain, hinge region, paired with no light chain, to eliminate free, unpaired cysteines. IL-2V (V91T) comprising a T3A/R38E/F42A/V91T/C125A mutation was used.

In the molecule A-V91T with the structure shown in FIG. 2, the anti-PD-1 nanobody A17m0902 is coupled to N-terminal of Knob and Hole chains respectively through IgG1 heavy chain hinge region, and IL-2V (V91T) is linked to C-terminal of Knob chain of Fc through (G ₄S)₃ linker).

The sequence of each molecule is as follows, underlined as Fc region, italicized as linker.

Knob chain of A-V91T

EVQLQESGGGLVQPGGSLRLSCTASGLTFSDYSMSWYRQAPGKGRELVAIISGSGVIAHYVDSVKGRFTISRDNSKNTVYLQMRSLTPEDRAVYYCRAVSDWDDYWGQGTQVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSAPASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINTIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:21)

Hole chain of A-V91T

EVQLQESGGGLVQPGGSLRLSCTASGLTFSDYSMSWYRQAPGKGRELVAIISGSGVIAHYVDSVKGRFTISRDNSKNTVYLQMRSLTPEDRAVYYCRAVSDWDDYWGQGTQVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSP(SEQ ID NO:22)

IL-2V (comprising a T3A/R38E/F42A/V91T/C125A mutation)

APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINTIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:23)

The fusion Protein is expressed in HEK293 cells, and purified by Protein A, and the molecular weight of the purified product is confirmed to meet the expectations by SDS-PAGE, thus proving that the target Protein is prepared.

Detection of proliferation Activity of PD-1-IL-2v on M07 e-promoting cells

M07e is a human cell line, and only expresses IL-2Rbeta and IL-2Rgamma, but does not express IL-2Ralpha. To examine the activity of the different forms of PD-1-IL2V (V91T) fusion proteins and the dependence on PD-1 expression, we overexpressed human PD-1 (SEQ ID NO: 1) in the M07e cell line to give M07e-hPD-1 cell lines, and examined the effect of the fusion proteins on cell proliferation in the M07e and M07e-hPD-1 cell lines, respectively.

The complete culture solution comprises RPMI 1640+2mM L-glutamine+1 mM sodium pyruvate+10% fetal bovine serum+15 ng/mL GM-CSF, and the basal culture medium comprises RPMI 1640+2mM L-glutamine+1 mM sodium pyruvate+10% fetal bovine serum.

M07e cell proliferation assay M07e cells or M07e-hPD1 were cultured in complete medium at 37℃under 5% CO ₂, passaged for 1-2 days, collected by centrifugation, washed 3 times with PBS, resuspended in basal medium to prepare a cell suspension containing 6.0X10. 10 ⁵ cells per mL, and plated in 96-well plates at a volume of 50. Mu.L per well. After cells were cultured for 72 hours by adding 50. Mu.L of a test drug prepared with a basal medium at a concentration of 2X, 100. Mu. L CELLTITER-Glo reagent (Promega) was added to each well, and the mixture was mixed and absorbance was measured at a wavelength of 570nm using a microplate reader with a reference wavelength of 630 nm.

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

The results show that PEG-IL-2V has comparable activity on M07e and M07e-hPD-1 cell proliferation, and that various forms of PD-1-IL-2 (V91T) have significantly greater activity on M07e-hPD-1 than on M07e cell proliferation, indicating that anti-PD-1 antibodies can increase the selectivity of IL-2 mutants for PD-1 positive cells. In addition, the activity on M07E proliferation was comparable to that of the various forms of PD-1-IL-2 (V91T), but the activity on M07E-hPD-1 proliferation was slightly stronger for C-V91T than for A-V91T, and stronger for A-V91T than for B-V91T and E-V91T.

TABLE 9 proliferation Activity of PD-1-IL-2V (V91T) on M07e and M07e-hPD-1 cells

EXAMPLE 4 construction of PD-1-IL-2v carrying different IL-2 mutants

PD-1-IL-2V (V91T), in M07e without PD-1 expression, although less active than PEG-IL-2V, still stimulated cell proliferation, suggesting that IL-2V (V91T) may still bind strongly to the intermediate affinity receptor IL-2Rβγ in IL-2, and PD-1-IL-2 (V91T) may still systematically activate NK and CD8 ⁺ T cells and cause toxicity. To further reduce the toxicity of PD-1-IL-2V, making the activity of IL-2 more dependent on the expression of PD-1, we constructed PD-1-IL-2V with different IL-2 mutants on the basis of the A-V91T molecular form. All IL-2 mutants carry F42A and L72G mutations to eliminate binding of IL-2 to IL-2Rα, and in addition, N26Q, N S and N71Q are added to the IL-2 mutants for stability to remove deamidation modifications that may be present in IL-2 and to increase the stability of IL-2. And they also each bear a different unique mutation as shown in Table 10, each of which reduces the affinity of IL-2 to the intermediate affinity receptor IL-2Rβγ to a different extent.

TABLE 10 IL-2 mutations in PD-1-IL-2v

IL-2 sequence in A-PC

APASSSTKKTQLQLEHLLLDLQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:24)

IL-2 sequence in A-V91T-02

APASSSTKKTQLQLEHLLLDLQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRDLISNINTIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:25)

IL-2 sequence in A-D20A

APASSSTKKTQLQLEHLLLALQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:26)

IL-2 sequence in A-D20N

APASSSTKKTQLQLEHLLLNLQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:27)

IL-2 sequence in A-N88D

APASSSTKKTQLQLEHLLLDLQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:28)

IL-2 sequence in A-N88R

APASSSTKKTQLQLEHLLLDLQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:29)

IL-2 sequence in A-Q126D

APASSSTKKTQLQLEHLLLDLQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFADSIISTLT(SEQ ID NO:30)

IL-2 sequence in A-H16A/D84S

APASSSTKKTQLQLEALLLDLQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRSLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:31)

The L234A and L235A mutations were introduced on Fc to eliminate ADCC effects, S354C, T366W (constituting Knob chain) and Y349C, T366S, L368A, Y407V (Eu numbering system) (constituting Hole chain) mutations were introduced to promote heterodimer formation, and the hinge region introduced C220S mutation. For ease of purification, the antibodies 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, which occur in different alleles, respectively, and do not affect the function of the antibody.

Knob chain of Fc (D356, L358)

EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:32)

Hole chain of Fc (D356, L358)

EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP(SEQ ID NO:33)

Knob chain of Fc (E356, M358)

EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:34)

Hole chain of Fc (E356, M358)

EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:35)

Illustratively, the full length sequences of A-D20A and A-N88D are given, underlined as Fc, italicized as linker.

Knob chain of A-D20A (D356, L358)

EVQLVESGGGLVQPGGSLRLSCAASGLTFSDYSMSWYRQAPGKGRELVAIISGSGVITHYVDSVKGRFTISRDNAKNTLYLQMRSLRAEDTAVYYCRAVSDWEDYWGQGTQVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSAPASSSTKKTQLQLEHLLLALQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:36)

Hole chain of A-D20A (D356, L358)

EVQLVESGGGLVQPGGSLRLSCAASGLTFSDYSMSWYRQAPGKGRELVAIISGSGVITHYVDSVKGRFTISRDNAKNTLYLQMRSLRAEDTAVYYCRAVSDWEDYWGQGTQVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSP(SEQ ID NO:37)

Knob chain of A-D20A (E356, M358)

EVQLVESGGGLVQPGGSLRLSCAASGLTFSDYSMSWYRQAPGKGRELVAIISGSGVITHYVDSVKGRFTISRDNAKNTLYLQMRSLRAEDTAVYYCRAVSDWEDYWGQGTQVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSAPASSSTKKTQLQLEHLLLALQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:38)

Hole chain of A-D20A (E356, M358)

EVQLVESGGGLVQPGGSLRLSCAASGLTFSDYSMSWYRQAPGKGRELVAIISGSGVITHYVDSVKGRFTISRDNAKNTLYLQMRSLRAEDTAVYYCRAVSDWEDYWGQGTQVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:39)

Knob chain of A-N88D (D356,L358)EVQLVESGGGLVQPGGSLRLSCAASGLTFSDYSMSWYRQAPGKGRELVAIISGSGVITHYVDSVKGRFTISRDNAKNTLYLQMRSLRAEDTAVYYCRAVSDWEDYWGQGTQVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSAPASSSTKKTQLQLEHLLLDLQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:40)

Hole chain of A-N88D (D356, L358)

EVQLVESGGGLVQPGGSLRLSCAASGLTFSDYSMSWYRQAPGKGRELVAIISGSGVITHYVDSVKGRFTISRDNAKNTLYLQMRSLRAEDTAVYYCRAVSDWEDYWGQGTQVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP(SEQ ID NO:41)

Knob chain of A-N88D (E356, M358)

EVQLVESGGGLVQPGGSLRLSCAASGLTFSDYSMSWYRQAPGKGRELVAIISGSGVITHYVDSVKGRFTISRDNAKNTLYLQMRSLRAEDTAVYYCRAVSDWEDYWGQGTQVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSAPASSSTKKTQLQLEHLLLDLQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:42)

Hole chain of A-N88D (E356, M358)

EVQLVESGGGLVQPGGSLRLSCAASGLTFSDYSMSWYRQAPGKGRELVAIISGSGVITHYVDSVKGRFTISRDNAKNTLYLQMRSLRAEDTAVYYCRAVSDWEDYWGQGTQVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:43)

Furthermore, to demonstrate the dependence of PD-1-IL-2V on PD-1, we replaced the anti-PD-1 antibody portion of part of PD-1-IL-2V with an antibody directed against an unrelated antigen, while retaining the mutation of the IL-2 portion, resulting in IgG-PC, and corresponding IgG-V91T containing V91T on IL-2, and corresponding IgG-D20A containing D20A on IL-2, and corresponding IgG-N88D containing N88D on IL-2. IgG-PC and IgG-V91T, igG-D20A, igG-N88D differ only in part by IL-2. The sequence is as follows:

Knob chain of IgG-PC

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNSWIGWFRQAPGQGLEWIGDIYPGGGYTNYNEIFKGKATMTADTSTNTAYMELSSLRSEDTAVYYCSRGIPGYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSAPASSSTKKTQLQLEHLLLDLQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:44)

Knob chain of IgG-V91T

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNSWIGWFRQAPGQGLEWIGDIYPGGGYTNYNEIFKGKATMTADTSTNTAYMELSSLRSEDTAVYYCSRGIPGYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSAPASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINTIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:45)

Knob chain of IgG-N88D

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNSWIGWFRQAPGQGLEWIGDIYPGGGYTNYNEIFKGKATMTADTSTNTAYMELSSLRSEDTAVYYCSRGIPGYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSAPASSSTKKTQLQLEHLLLDLQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:46)

Knob chain of IgG-D20A

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNSWIGWFRQAPGQGLEWIGDIYPGGGYTNYNEIFKGKATMTADTSTNTAYMELSSLRSEDTAVYYCSRGIPGYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSAPASSSTKKTQLQLEHLLLALQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT

(SEQ ID NO:47)

Hole chain of IgG-PC, igG-V91T, igG-D20A or IgG-N88D QVQLVQSGAEVKKPGASVKVSCKASGYTFTNSWIGWFRQAPGQGLEWIGDIYPGGGYTNYNEIFKGKATMTADTSTNTAYMELSSLRSEDTAVYYCSRGIPGYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSP(SEQ ID NO:48)

Light chain of IgG-PC, igG-V91T, igG-D20A or IgG-N88D DIQMTQSPSSLSASVGDRVTMSCKSSQSLLNSGDQKNYLTWYQQKPGKAPKLLIYWASTGESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQNDYSYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:49)

The fusion Protein is expressed in HEK293 cells, and purified by Protein A, and the molecular weight of the purified product is confirmed to meet the expectations by SDS-PAGE, thus proving that the target Protein is obtained.

In addition, the wild-type IL-2 sequences of the present disclosure are:

>wt IL-2

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT(SEQ ID NO:50)

EXAMPLE 5 detection of PD-1-IL-2v pro-M07 e cell proliferation Activity

To examine the activity of the above-mentioned PD-1-IL-2v fusion proteins with different IL-2 mutants and the dependence on PD-1 expression, we examined the effect of the fusion protein of example 4 on cell proliferation in M07e and M07e-hPD-1 cell lines. Experimental methods see example 3. The relative cell proliferation activity of PD-1-IL-2v was calculated as follows:

wherein, Represents the activity of PD-1-IL-2v at the concentration of conc,Represents the luminescence intensity of the cell lysate upon stimulation with PD-1-IL-2v at the concentration of conc,Represents the luminescence intensity of the cell lysate after stimulation of cells with the positive control molecule A-PC at a concentration of 1000nM in the M07e or M07e-hPD-1 cell line,Represents the background luminescence intensity without any PD-1-IL2v or PEG-IL 2v stimulation in the M07e or M07e-hPD-1 cell line with medium alone.

The experimental results are shown in table 11 and fig. 3A and 3B. The results showed that 1) in M07e cells, A-PC was comparable to PEG-IL-2V, demonstrating that the activity of IL-2 bound to the antibody and PEG-conjugated IL-2 was consistent in the absence of PD-1 overexpression, that A-V91T and IgG-V91T were comparable and weaker than A-PC, demonstrating that V91T did decrease IL-2 activity but still stimulated M07e proliferation, and that other mutants, including D20A, D20N, N88R, N88D and Q126D, did not substantially activate M07e cells, and that A-N88D was only weakly active at 1000 nM. 2) In M07e-hPD-1 cells, since PD-1 is overexpressed 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 improved the dependence of PD-1-IL-2V pro-cell proliferation activity on PD-1 expression, and that other PD-1-IL-2V had slightly lower maximum activity on M07e-hPD-1 cell proliferation than A-PC and A-V91T. 3) Comparing the activity of each PD-1-IL-2V in the M07e and M07e-hPD-1 cell proliferation experiments, the PD-1-IL2V with the D20A, D20N, N88R, N88D or Q126D mutation does not activate M07e cells basically, but can strongly stimulate proliferation of M07e-hPD-1 cells, which shows that PD-1-IL-2V reduces activation of PD-1 negative cells while maintaining selective activation of PD-1 positive cells, while A-PC and A-V91T have selective activation of PD-1 positive cells but can activate also of PD-1 negative cells.

TABLE 11 proliferation-promoting Activity of PD-1-IL-2v with different IL-2 mutations on M07e and M07e-hPD-1 cells

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 with IgG-N88D, A-D20A with IgG-D20 on cell proliferation in the M07e-hPD-1 cell line. The experimental results are shown in Table 12 and FIG. 4. The results showed that at higher concentrations (1000 nM) IgG-N88D and IgG-D20A still failed to activate M07e-hPD-1 cell proliferation, but A-N88D and A-D20A activated M07e-hPD-1 cell proliferation at lower concentrations.

TABLE 12 proliferation-promoting Activity of PD-1-IL-2v or IgG-IL-2v on M07e-hPD-1 cells

Example 6 detection of PD-1-IL-2v T-cell proliferation-promoting Activity

Non-stimulated T cells do not express or underexpress PD-1, and CD3 antibody stimulated T cells highly express PD-1. To examine whether the above-described PD-1-IL-2v fusion proteins with different IL-2 mutants were able to activate T cells in human peripheral blood cells (PBMC) that underexpress PD-1 and that highly express PD-1, we isolated T cells in PBMC and stimulated the isolated T cells with CD3 antibodies, and examined the effect of the fusion proteins on proliferation of unstimulated and stimulated T cells.

T cell proliferation experiments stimulated with CD3 antibodies T cells were isolated after resuscitating cryopreserved human PBMC using Pan T Cell Isolation Kit (Miltenyi, 130-096-535). The CD3 antibody was diluted to 0.1. Mu.g/mL with PBS, added to a10 cm dish, and incubated at 37℃for 2 hours for coating, and washed with PBS for use. T cells were plated at 2 x 10 ⁶ cells/mL in 10cm dishes pre-coated with CD3 antibody and stimulated for 24h at 37 ℃ under 5% CO ₂. Collecting stimulated T cells, washing and counting, regulating the cell density to 5X 10 ⁵ cells/mL, spreading 200 mu L of each hole in a 96-well plate, gradient diluting a sample to be tested with a culture medium to prepare 5X working solution, adding 50 mu L of each concentration sample to be tested into the stimulated T cells, uniformly mixing, and culturing for 4 days. Cells after drug addition treatment were washed twice with FACS buffer (PBS+2% FBS), labeled with Fixable Viability Dye (eBioscience, 65-0865-14) for cell death, stained with CD3 (BD, 565491), CD4 (BD, 558116), CD8 (BD, 565310), ki-67 (eBioscience, 12-5698-82)) for flow antibody, and FACS-on-line for detection of cell proliferation.

And (3) performing unstimulated T cell proliferation experiments, namely counting the separated T cells, paving the counted T cells in 96-well plates according to 5X 10 ⁵ cells/mL, diluting 200 mu L of each well of the T cells with a culture medium gradient to prepare 5X working solution, adding 50 mu L of each concentration of the sample to be tested into the stimulated T cells, uniformly mixing, and culturing for 4 days. Cells treated with the drug were washed twice with FACS buffer, labeled with FVD780, stained with flow antibody, and examined for proliferation on FACS.

The experimental results are shown in Table 13, table 14 and FIGS. 5A, 5B, 6A and 6B. The results showed that 1) in unstimulated T cells, A-PC showed activity in activating T proliferation at concentrations above 1nM (CD 8 ⁺ T cell proliferation EC ₅₀＝21.46nM,CD4⁺ T cell proliferation EC ₅₀ =8.61 nM), A-V91T activity was weaker than A-PC, indicating that V91T did decrease IL-2 activity but could still promote proliferation of unstimulated T, and other mutants, including D20A, D20N, N88R, N88D, and Q126D, did not show activation of T cells at concentrations up to 100 nM. 2) In CD3 antibody stimulated T cells, each PD-1-IL-2v showed significant activity in promoting T cell proliferation at a lower concentration (0.1 nM). PD-1-IL-2V carrying other mutations had slightly lower maximal activity on pan T cell proliferation stimulated with CD3 antibodies than A-PC and A-V91T. 3) Comparing the activity of each fusion protein in the unstimulated and CD3 antibody stimulated T cell proliferation experiments, the PD-1-IL-2V with the D20A, D20N, N88R, N88D, or Q126D mutations, respectively, did not activate unstimulated T cell proliferation, but could strongly stimulate CD3 antibody stimulated T cell proliferation, while A-PC and A-V91T, while able to strongly activate CD3 antibody stimulated T cells, could also strongly activate unstimulated CD3 antibody stimulated T cells.

TABLE 13 proliferation Activity of PD-1-IL-2v carrying different IL-2 mutations on unstimulated T cells

TABLE 14 proliferation Activity of different IL-2 mutations PD-1-IL-2v on CD3 antibody stimulated T cells

EXAMPLE 7 identification of binding affinity of PD-1-IL2v to the antigens PD-1 and IL-2Rα or IL-2Rβ/γ

The binding affinities of PD-1 and human IL-2Rα or IL-2Rβ/γ of the PD-1-IL-2v fusion proteins with different IL-2 mutants described above were examined using surface plasmon resonance (surface plasmon resonance, SPR). The experiment was performed using a Biacore 8K (GE Healthcare) instrument. CM5 sensor chip is selected, and HBS-EP+ buffer solution (10mM HEPES,150mM NaCl,3mM EDTA,0.05%surfactant P20) is adopted as mobile phase. Anti-human IgG (Fc) antibodies were prepared as 25 μg/mL solutions in 10mM sodium acetate buffer (pH 5.0) and the immobization procedure was selected to automatically perform amino-coupled Immobilization of the anti-human IgG (Fc) antibody channels. The PD-1-IL2v to be detected is respectively prepared by using HBS-EP+ buffer solution as a ligand, and the anti-human IgG (Fc) antibody on a chip channel is used for capturing. 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, which were formulated with HBS-EP+ buffer, 2-fold gradient diluted from 200nM, 7 concentration points total, and flowed through the experimental and reference channels at a flow rate of 30. Mu.L/min, binding time 140s, dissociation time 600s. Regeneration buffer 10mM Glycine pH 1.5 (GE HEALTHCARE, 29238268-AA) was run at a flow rate of 10. Mu.L/min for 30s. The binding rate Ka and dissociation rate Kd, as well as the dissociation constant (i.e., the affinity K _D value) are calculated. The results are as follows.

TABLE 15 SPR affinity of PD-1-IL2v binding to human PD-1 antigen

Antibody numbering	ka(1/Ms)	Kd(1/s)	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

TABLE 16 SPR binding avidity of anti-PD-1 antibodies for binding to human IL-2Rα with fusion proteins of IL-2 mutants

Antibody numbering	ka(1/Ms)	Kd(1/s)	K_D(M)
				A-PC	NB	NB	NB
A-N88D	NB	NB	NB
				A-D20A	NB	NB	NB

(NB: not combined)

TABLE 17 SPR binding avidity of anti-PD-1 antibodies for binding to human IL-2Rβ/γ with fusion proteins of IL-2 mutants

Antibody numbering	ka(1/Ms)	Kd(1/s)	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

Binding to human PD-1 antigen results showed that A-PC, A-N88D and A-D20A have similar binding to human PD-1 and to human PD-1 as A17m 0902-hIgG 4 (Table 8). The results (Table 16) for binding to human IL-2Rα show that neither A-PC, A-N88D nor A-D20A bind to human IL-2Rα. The results (Table 17) for binding human IL-2Rβ/γ show that A-PC has the strongest affinity for human IL-2Rβ/γ, and that A-N88D and A-D20A have about 13-fold and about 63-fold reduced affinities for human IL-2Rβ/γ, respectively.

The PD-1-IL2v (A-N88D, knob strand shown as SEQ ID NO:42, hole strand shown as SEQ ID NO: 43) was purified as follows.

Example 8 affinity chromatography and protein detection

Subjecting the cell supernatant comprising the PD-1-IL2v fusion protein to affinity chromatography, comprising the following specific steps:

step 1, loading the cell clarified liquid onto an affinity chromatography column which is balanced, wherein the affinity chromatography filler is N Mab;

Step 2, flushing the chromatographic column subjected to sample loading by using an equilibrium buffer solution until UV ₂₈₀, pH and conductivity are stable, wherein the equilibrium buffer solution is 0.02mol/L phosphate, 1.0mol/L sodium chloride and pH is 7.4;

Step 3, washing 5-8 column volumes by using a first eluting buffer solution, wherein the types of the solutions are shown in a table 18;

Step 4, flushing 2-3 column volumes by using a second eluting buffer solution to increase the pH value of the affinity chromatography column to more than 4.5, wherein the types of the solutions are shown in a table 18;

step 5, flushing 2-3 column volumes by using a third leaching buffer, wherein the types of the solutions are shown in a table 18, and the conductivity of the third leaching buffer is less than 5mS/cm;

step 6, eluting with 0.05mol/L sodium acetate-acetic acid and pH 3.6 solution, and collecting eluent;

and 7, calculating the chromatography yield, and detecting the fusion protein in the eluent obtained by the obtained chromatography.

TABLE 18 affinity chromatography and protein detection

Example 9 affinity chromatography and protein detection

Affinity chromatography of PD-1-IL2v was performed as described under number 1 in example 8, with the first rinse volume taking the upper limit (8 volumes). And continuing anion exchange chromatography and cation exchange chromatography after obtaining the fusion protein of the affinity chromatography. The SEC-HPLC purity, RP-HPLC purity and NRCE purity of the fusion protein obtained in each step were checked. The results are shown in Table 19.

Table 19 preparation of Fine sample and detection of fusion proteins

Example 10 affinity chromatography and protein detection

Subjecting the cell supernatant containing PD-1-IL2v to affinity chromatography, wherein the method comprises the following specific steps:

step 3, using 0.05mol/L M acetic acid-sodium acetate solution with pH of 5.0 to wash until UV280, pH and conductivity are stable;

Step 4, eluting by using 0.05mol/L sodium acetate-acetic acid with pH of 3.6, and collecting eluent;

The fusion protein SEC-HPLC purity in the obtained affinity chromatography eluent is 82.07%, and the RP-HPLC purity is 81.14%.

EXAMPLE 11 affinity chromatography and protein detection

The fusion protein was prepared according to the affinity chromatography method described in example 10, and anion exchange chromatography and cation exchange chromatography were continued after the fusion protein of the affinity chromatography was obtained. The SEC-HPLC purity, RP-HPLC purity and NRCE purity of the chromatographic fusion protein were examined in each step. The results are shown in Table 20.

TABLE 20 preparation of precision samples and detection of fusion proteins

Example 12 affinity chromatography and protein detection

Step 3, washing 5-8 column volumes by using a first eluting buffer solution, wherein the types of the solutions are shown in a table 21;

step 4, flushing 2-3 column volumes by using a second eluting buffer solution, wherein the types of the solutions are shown in a table 21;

step 5, flushing 2-3 column volumes by using a third eluting buffer solution, wherein the types of the solutions are shown in a table 21;

Step 6, eluting with 0.05mol/L sodium acetate-acetic acid, and collecting eluent, wherein the pH value of the eluent is 3.6;

Step 7, calculating the chromatographic yield, and detecting the fusion protein in the obtained eluent;

TABLE 21 affinity chromatography and intermediate detection

Claims

1. A method of purifying a fusion protein, the method comprising the steps of:

(c) Leaching the affinity chromatography packing by using leaching buffer;

wherein the fusion protein comprises a PD-1 binding domain and a cytokine;

Preferably, the fusion protein has an isoelectric Point (PI) of 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 points between the first polypeptide chain and the second polypeptide chain differ by no more than about 3.0 units.

3. The method of any one of claims 1-2, wherein:

The leaching step comprises the steps of sequentially using a first leaching buffer solution, a second leaching buffer solution and a third leaching buffer solution for washing;

The pH of the first wash buffer is from about 3.8 to about 5.0, preferably from about 4.0 to about 4.7, more preferably from about 4.1 to about 4.3;

The pH of the second leaching 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 leaching 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 equilibration buffer has a pH of 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 from about 3.0 to about 5.0, preferably from about 3.4 to about 4.0, more preferably from about 3.4 to about 3.8.

4. A method according to claim 3, wherein the buffer substances in the first, second and third elution buffers are each independently selected from one or more of sodium acetate, sodium citrate, tris, sodium dihydrogen phosphate, disodium hydrogen phosphate, acetic acid, citric acid, phosphoric acid;

Preferably, the buffer substances in the first, second and third elution buffers 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 buffer substance in the first wash buffer is from about 0.03 to about 0.1mol/L, preferably from about 0.04mol/L to about 0.06mol/L;

The concentration of buffer material in the second wash buffer is from about 0.03 to about 0.1mol/L, preferably from about 0.04mol/L to about 0.06mol/L, and/or,

The concentration of buffer substance in the third leaching buffer is about 0.03 to about 0.1mol/L, preferably about 0.04mol/L to about 0.06mol/L, preferably the conductivity of the third leaching buffer is <5.0mS/cm;

Preferably, the method comprises the steps of,

The first leaching buffer further comprises an inorganic salt selected from one or more of sodium chloride, sodium sulfate, ammonium sulfate, preferably sodium chloride, or sodium chloride and sodium sulfate, more preferably sodium chloride, preferably the concentration of the inorganic salt in the first leaching buffer is about 0.1mol/L to about 1.0mol/L, preferably about 0.1mol/L to about 0.6mol/L, preferably about 0.4mol/L to about 0.6mol/L;

the second wash buffer further comprises an inorganic salt selected from one or more of sodium chloride, sodium sulfate, ammonium sulfate, preferably sodium chloride, or sodium chloride and sodium sulfate, more preferably sodium chloride, preferably the concentration of the inorganic salt in the second wash buffer is from about 0.2 to about 1.0mol/L, preferably from about 0.3mol/L to about 0.7mol/L, preferably from about 0.4mol/L to about 0.6mol/L, and/or,

The buffer material in the equilibration buffer is a phosphate buffer having a concentration of from about 0.01mol/L to about 0.1mol/L, preferably from about 0.01mol/L to about 0.05mol/L, more preferably from about 0.01mol/L to about 0.03mol/L, preferably the equilibration buffer further comprises an inorganic salt, preferably sodium chloride, more preferably from about 0.1mol/L to about 2.0mol/L sodium chloride, more preferably from about 0.5mol/L to about 1.5mol/L sodium chloride.

5. The method of any one of claims 3-4, wherein the (c) rinsing step comprises:

(c-1) eluting the affinity chromatography packing with a first eluting buffer having a pH of about 3.8 to about 5.0, more preferably about 4.0 to about 4.7, more preferably about 4.1 to about 4.3;

(c-2) eluting the affinity chromatography packing with a second eluting buffer to increase the pH of the affinity chromatography packing to above 4.5;

(c-3) eluting the affinity chromatography packing with a third eluting buffer to provide an affinity chromatography packing conductivity of less than 5.0 mS/cm;

Preferably, the first eluting buffer, the second eluting buffer and the third eluting buffer are used for flushing 1-10 column volumes, more preferably, the first eluting buffer is used for flushing 2-8 column volumes, the second eluting buffer is used for flushing 1-5 column volumes and the third eluting buffer is used for flushing 1-5 column volumes;

Preferably, the affinity chromatography packing is a Protein A affinity chromatography packing, more preferably, the Protein A affinity chromatography packing is selected from the group consisting of MabSelectSure, mabselect Sure LX, mabselect PrismA, N Mab, N Mab Pro or Unimab HC;

preferably, the method reduces or removes homodimers in the sample.

6. A method of purifying a fusion protein comprising the steps of:

(c) Leaching the affinity chromatography packing by using leaching buffer;

(c-1) eluting the affinity chromatography packing with a first eluting buffer, preferably having a pH of about 3.8 to about 5.0, more preferably about 4.0 to about 4.7, more preferably about 4.1 to about 4.3;

(c-2) eluting the affinity chromatography packing with a second eluting buffer, preferably having a pH of about 4.5 to about 8.0, more preferably about 4.5 to about 6.5, more preferably about 4.5 to about 5.5;

(c-3) eluting the affinity chromatography packing with a third eluting buffer, preferably having a pH of about 4.5 to about 8.0, more preferably about 4.5 to about 6.5, more preferably about 4.5 to about 5.5;

(d) An elution step of eluting the lower fusion protein from the affinity chromatography packing with an elution buffer, preferably having a pH of about 3.0 to about 5.0, more preferably about 3.4 to about 4.0, and even more preferably about 3.4 to about 3.8, and collecting the eluate;

Preferably, the first wash buffer contains from about 0.03 to about 0.1mol/L of sodium acetate-acetate, and from about 0.1 to about 0.6mol/L of sodium chloride, preferably from about 0.04mol/L to about 0.06mol/L of sodium acetate-acetate, and from about 0.4mol/L to about 0.6mol/L of sodium chloride;

The second wash buffer contains from about 0.03 to about 0.1mol/L of sodium acetate-acetate, and from about 0.3 to about 0.7mol/L of sodium chloride, preferably from about 0.04mol/L to about 0.06mol/L of sodium acetate-acetate, and from about 0.4mol/L to about 0.5mol/L of sodium chloride;

The third wash buffer contains from about 0.03 to about 0.1mol/L of acetic acid-sodium acetate, preferably from about 0.04mol/L to about 0.06mol/L of acetic acid-sodium acetate, preferably no sodium chloride;

The elution buffer contains from about 0.01mol/L to about 0.5mol/L of sodium acetate-acetate, preferably from about 0.05mol/L to about 0.1mol/L of sodium acetate-acetate;

The equilibration buffer contains from about 0.01mol/L to about 0.1mol/L phosphate and from about 0.1mol/L to about 2.0mol/L sodium chloride, preferably from about 0.01mol/L to about 0.05mol/L phosphate and from about 0.5mol/L to about 1.5mol/L sodium chloride;

Preferably, the affinity chromatography packing is a Protein A affinity chromatography packing, preferably selected from the group consisting of MabSelect Sure, mabselect Sure LX, mabselect PrismA, N Mab, N Mab Pro or Unimab HC;

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 fusion protein comprises asymmetric first and second polypeptide chains, wherein the PI of the fusion protein differs by no more than about 3.0 units between any two of the PI of the first polypeptide chain and the PI of the second polypeptide chain;

Or:

(c) Leaching the affinity chromatography packing by using leaching buffer;

(c-1) eluting the affinity chromatography packing with 2-8 column volumes of a first eluting buffer, preferably having a pH of about 3.8 to about 5.0, more preferably about 4.0 to about 4.7, more preferably about 4.1 to about 4.3;

(c-2) eluting the affinity chromatography packing with 1-5 column volumes of a second elution buffer, preferably having a pH of about 4.5 to about 8.0, more preferably about 4.5 to about 6.5, and even more preferably about 4.5 to about 5.5, to raise the pH of the affinity chromatography packing above 4.5;

(c-3) eluting the affinity chromatography packing with a third eluting buffer of 1-5 column volumes to reduce the conductivity of the affinity chromatography packing to below 5.0mS/cm, the pH of the third eluting buffer preferably being about 4.5 to about 8.0, more preferably about 4.5 to about 6.5, more preferably about 4.5 to about 5.5;

The equilibration buffer contains from about 0.01mol/L to about 0.1mol/L phosphate, from about 0.1mol/L to about 2.0mol/L sodium chloride, preferably from about 0.01mol/L to about 0.05mol/L Phosphate (PB), from about 0.5mol/L to about 1.5mol/L sodium chloride;

Preferably, the affinity chromatography packing is a Protein A affinity chromatography packing, preferably selected from MabSelect Sure, mabselect Sure LX, mabselect PrismA, N Mab, N Mab Pro, or Unimab HC;

Preferably, the fusion protein comprises asymmetric first and second polypeptide chains, wherein the PI of the fusion protein differs by no more than about 3.0 units between any two of the PI of the first polypeptide chain and the PI of the second polypeptide chain.

7. The method of any one of claims 1-6, wherein the fusion protein comprises a PD-1 binding domain and a cytokine;

The PD-1 binding domain comprises an immunoglobulin single variable domain comprising CDR1, CDR2 and CDR3 of any one of the amino acid sequences shown in SEQ ID NO. 2, 8-15, or the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NO. 3, 16, 17, respectively, the CDRs being defined according to the Kabat, IMGT, chothia, abM or Contact numbering system;

preferably, the CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are each

As shown in SEQ ID NO. 3, 18 and 19,

As shown in SEQ ID NO 3, 4, 5, or

As shown in SEQ ID NO 3, 4 and 20;

Preferably, the PD-1 binding domain comprises an amino acid sequence as set forth 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 set forth 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 mutations:

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 comprises the following mutations:

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 comprises a mutation at position 3 and/or 125, preferably a mutation at position 3 is T3A, preferably a mutation at position 125 is C125A or C125S;

More preferably, the IL-2 variant comprises or has an amino acid sequence as set forth in any one of SEQ ID NOS.23-31 or a sequence having at least 90% identity thereto.

9. The method of any one of claims 1-8, wherein the fusion protein further comprises an immunoglobulin Fc region, preferably the Fc region is a human IgG1, igG2, or IgG4 Fc region;

Preferably, the Fc region of human IgG1 contains one or more amino acid mutations that reduce binding to fcγ receptors (fcγr) and/or reduce ADCC effects;

More preferably, the Fc region of human IgG1 comprises a mutation of L234, L235, P329 or any combination thereof;

most preferably, the Fc region of human IgG1 contains the L234A/L235A, L234A/L235A/P329G mutation;

the mutations of 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 the association of the first and second subunits of the Fc region;

Preferably, 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 create a bulge in the CH3 region of the first subunit that can be placed in a cavity within 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 create a cavity within the CH3 region of the second subunit that can be placed in a bulge within the CH3 region of the first subunit;

Preferably, the first subunit of the Fc region comprises a T366W mutation, the second subunit comprises a mutation selected from the group consisting of T366S, L368 53407V or any combination thereof, the first subunit of the Fc region comprises a S354C or E356C mutation, the second subunit comprises a Y349C mutation, or the first subunit of the Fc region comprises a S354C/T366W mutation, the second subunit comprises a Y349C/T366S/L368A/Y407V mutation;

Preferably, the second subunit of the Fc region further comprises the 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 the C-terminus of the Fc region, more preferably the cytokine is located in the polypeptide chain in which the first subunit of the Fc region is located;

Preferably, the fusion protein comprises the structure shown below:

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 variant thereof ],

2) The second polypeptide chain comprises [ PD-1 binding domain 1] - [ Fc region second subunit ] from N-terminal to C-terminal;

Wherein a is selected from 0 or 1 and the linker has the structure (G _xS)_y, wherein x is selected from an integer from 1 to 5 and y is selected from an integer from 1 to 6, preferably x is 4 and y is 1,2 or 3;

More preferably, the fusion protein comprises a combination of polypeptide chains of the amino acid sequences shown below:

SEQ ID NOS 42 and 43;

SEQ ID NOS.21 and 22;

SEQ ID NOS 36 and 37;

SEQ ID NOS 38 and 39, or,

SEQ ID NOS.40 and 41.