CN116041539A - IL-2 mutant immunoconjugates - Google Patents

IL-2 mutant immunoconjugates Download PDF

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CN116041539A
CN116041539A CN202211346800.XA CN202211346800A CN116041539A CN 116041539 A CN116041539 A CN 116041539A CN 202211346800 A CN202211346800 A CN 202211346800A CN 116041539 A CN116041539 A CN 116041539A
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immunoconjugate
amino acid
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cancer
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CN116041539B (en
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饶木顶
宋德勇
窦昌林
王巧平
潘荣荣
邢平平
矫婕
黄蕊
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Shandong Boan Biotechnology Co Ltd
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Abstract

The present invention relates to an immunoconjugate of an anti-PD 1 antibody with an IL-2 mutant, said immunoconjugate comprising an interleukin-2 (IL-2) polypeptide mutant and an antibody or antigen-binding fragment that binds to PD 1. In particular, polynucleotides encoding the immunoconjugates, expression vectors, host cells, methods of expression, and pharmaceutical compositions are also provided. The use of the immunoconjugate for the preparation of a pharmaceutical composition for the treatment or prevention of cancer, preferably the cancer is an anti-PD 1 treatment low response cancer, is provided. The immunoconjugate of the anti-PD 1 antibody and the IL-2 mutant provided by the invention has reduced affinity to an IL-2 receptor, reduced activation capacity to cells expressing the IL-2 receptor, effective avoidance of specific activation of tumor T lymphocytes by IL-2 molecules, reduced toxic and side effects and high safety; through mutual synergy of the activation of the PD1 signal channel and the IL-2 molecule, the activation efficiency of T cells is improved, the killing of tumor cells is realized efficiently, and the method has wide application prospect.

Description

IL-2 mutant immunoconjugates
Technical Field
The invention relates to the technical field of biomedicine or biopharmaceuticals, in particular to an immunoconjugate of an anti-PD 1 antibody and an IL-2 mutant.
Background
Interleukin-2 (IL-2), also known as T Cell Growth Factor (TCGF), is a 15.5kDa globular glycoprotein that plays a central role in lymphopoiesis, survival and homeostasis. It has a length of 133 amino acids and consists of four antiparallel amphipathic alpha helices forming the quaternary structure essential for its function.
IL-2 mediates its actions by binding to the IL-2 receptor (IL-2R), which consists of up to three individual subunits, whose different combinations can produce receptor forms that differ in their affinity for IL-2. The combination of the α (CD 25), β (CD 122) and γ (CD 132) subunits results in trimeric high affinity IL-2 receptors. The dimer medium affinity IL-2 receptor consists of beta and gamma subunits. The alpha subunit forms the monomeric low affinity IL-2 receptor. The affinity of the trimeric high affinity receptor is 100 times that of the dimeric medium affinity receptor, and both dimeric and trimeric IL-2 receptor variants are capable of signaling upon IL-2 binding.
PD-1 (programmed death receptor 1), an important immunosuppressive molecule, is the immunoglobulin superfamily, a membrane protein of 288 amino acid residues. The immunoregulation with PD-1 as a target spot has important significance for resisting tumors, infections, autoimmune diseases, organ transplantation survival and the like. The ligand PD-L1 can also serve as a target point, and the corresponding antibody can also play the same role.
The ability of IL-2 to expand lymphocyte populations in vivo and to increase effector functions of these cells imparts anti-tumor effects to IL-2, making IL-2 immunotherapy an attractive treatment option for certain metastatic cancers. High dose IL-2 therapy has been approved for patients with metastatic renal cell carcinoma and malignant melanoma, but IL-2 alone has a short half-life, a narrow therapeutic window, a large toxic side effect, and at the same time stimulates Treg activation, limiting clinical use.
PD1/PDL1 inhibitor is a drug widely applied to tumor immunotherapy at present, but the response rate of patients to PD1/PDL1 inhibitor is found to be low in clinical application, and drug resistance is easy to occur.
Therefore, how to avoid the clinical drawbacks of individual IL-2 molecules and individual PD1 antibodies, and to prepare anticancer drugs, is a problem to be solved.
Disclosure of Invention
The invention provides an IL-2 mutant immunoconjugate, and a coding polynucleotide, an expression vector, a host cell, an expression method, a pharmaceutical composition and pharmaceutical application thereof.
In one aspect, the invention provides an IL-2 mutant immunoconjugate comprising an interleukin-2 (IL-2) polypeptide mutant and an antibody or antigen-binding fragment that binds to PD1, wherein the IL-2 polypeptide mutant has reduced IL-2rβ binding activity and/or abolished IL-2rα binding activity, the IL-2 polypeptide mutant comprising an amino acid substitution selected from one of the following a) -e):
a)R38G,P65R；
b)L19H,R38G,P65R；
c)R38E,P65R；
d)R38E,P65R，L19H；
e)L19H；
wherein the mutation number is the number relative to the amino acid sequence shown in the human IL-2 sequence SEQ ID NO. 1;
and wherein the PD1 antibody and antigen-binding fragment thereof comprises: (a) A heavy chain variable region (VH) comprising a heavy chain complementarity determining region HCDR1 of the amino acid sequence shown in SEQ ID No. 19, HCDR2 of the amino acid sequence shown in SEQ ID No. 20, and HCDR3 of the amino acid sequence shown in SEQ ID No. 21; and (b) a light chain variable region (VL) comprising a light chain complementarity determining region LCDR1 of the amino acid sequence set forth in SEQ ID NO. 16, LCDR2 of the amino acid sequence set forth in SEQ ID NO. 17 and LCDR3 of the amino acid sequence set forth in SEQ ID NO. 18.
Further, the PD1 antibody and antigen-binding fragment thereof comprise a heavy chain variable region (VH) as shown in SEQ ID NO. 22 and a light chain variable region (VL) as shown in SEQ ID NO. 23.
Further, the IL-2 polypeptide mutant further comprises an amino acid substitution T3A and/or an amino acid substitution C125A.
Further, the IL-2 mutant immunoconjugates of the invention, wherein the IL-2 polypeptide mutant is fused at its amino terminal amino acid to the carboxy terminal amino acid of the Fc domain of the antibody or antigen binding fragment via a linker peptide.
In a second aspect of the invention, there is provided an isolated polynucleotide or recombinant polynucleotide encoding said IL-2 mutant immunoconjugate.
In a third aspect, the invention provides one or more vectors, in particular expression vectors, comprising a polynucleotide encoding an IL-2 mutant immunoconjugate.
In a fourth aspect, the invention provides a host cell comprising a polynucleotide encoding an IL-2 mutant immunoconjugate or a vector comprising a polynucleotide encoding an IL-2 mutant immunoconjugate.
In a fifth aspect, the invention provides a method of producing the IL-2 mutant immunoconjugate, the method comprising (a) culturing a host cell comprising a polynucleotide encoding the IL-2 mutant immunoconjugate or a vector comprising a polynucleotide encoding the IL-2 mutant immunoconjugate under conditions suitable for expression of the immunoconjugate, and optionally (b) recovering the immunoconjugate.
In a sixth aspect, the invention provides a pharmaceutical composition comprising said immunoconjugate, a nucleotide encoding said immunoconjugate, an expression vector comprising a nucleotide encoding said immunoconjugate, a host cell comprising an expression vector encoding said immunoconjugate nucleotide, and a pharmaceutically acceptable carrier.
In a seventh aspect, the present invention provides a pharmaceutical use of the immunoconjugate or pharmaceutical composition comprising the immunoconjugate for the manufacture of a medicament for ameliorating or treating cancer; preferably, the cancer is a low response cancer to anti-PD 1 treatment; preferably, the cancer is selected from: colon cancer, breast cancer, liver cancer, melanoma, gastric cancer, etc., and further, the melanoma is refractory melanoma.
The immunoconjugates of the anti-PD 1 antibodies and IL-2 mutants provided herein have one or more of the following advantages:
(1) The unexpected finding of the invention that certain amino acid sites on the binding interface of IL-2 and IL-2Rα have the effects of eliminating IL-2Rα affinity and weakening IL-2Rβ affinity, and the combination of specific mutations by constructing IL-2 mutant immunoconjugates has the effects of eliminating IL-2Rα affinity and weakening IL-2Rβ affinity is verified from the aspects of binding kinetics and cell activation, and experiments show that the R38 and P65 sites of IL-2 are positioned on the binding interface of IL-2 and IL-2Rα, but the effects are not limited to eliminating the affinity with IL-2Rα subunits, but also weakening the affinity of IL-2 and IL-2Rβ receptors, and the mutations weaken the activation of IL-2 on cells, and exhibit improved antitumor effects in different animal models of transplanted tumors.
(2) The creative discovery of the invention can realize the great reduction of IL-2 Rbeta affinity by singly mutating the L19 position, shows the characteristics of low affinity and low activation on HH cells expressing IL-2 Rbeta, improves the mutation efficiency by single point mutation on one hand, also overcomes the problem of larger conformational change caused by multi-point mutation, and takes into account the principles of high efficiency and economy;
(3) The invention adjusts the structure of the immunoconjugate, and unexpectedly discovers that the construction form of the immunoconjugate of the anti-PD 1 antibody and the IL-2 mutant constructed by adopting the structures 2 and 3 has the advantages of minimum steric hindrance for the anti-PD 1 antibody and maximum affinity for PD1 expression cells, so that the conjugate constructed by the invention has the guidance of the PD1 antibody, leads the immunoconjugate to be biased to combine with tumor T lymphocytes, activates the tumor T lymphocytes specifically by IL-2 molecules, reduces toxic and side effects and expands a treatment window
(4) The IL-2 mutant immunoconjugate anti-PD 1 antibody of the invention can cooperate with the activation of IL-2 molecules by inhibiting the PD1 signal path, thereby improving the activation efficiency of T cells and effectively realizing the killing of tumor cells.
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FIG. 1 is a schematic diagram of IL-2 mutant immunoconjugate structure;
FIG. 2 is a graph showing the results of analysis of response values of IL-2Rα binding interface mutants of different IL-2 mutant immunoconjugates;
FIG. 3 is a graph showing the results of analysis of response values of IL-2Rβ binding interface mutants of different IL-2 mutant immunoconjugates;
FIG. 4 is a flow-through binding assay of different IL-2 mutant immunoconjugates SU-DHL-1 cells of example 4;
FIGS. 5-6 are results of flow-through binding assays of different IL-2 mutant immunoconjugates GS-J2-PD1 cells of example 4;
FIGS. 7-12 are results of flow-through binding assays of HH cells of different IL-2 mutant immunoconjugates of examples 4-5;
FIGS. 13-17 are results of assays for activation activity of HH cells of different IL-2 mutant immunoconjugates of example 6;
FIG. 18 is a graph showing the analysis of the low concentration group MC38 colon cancer cell mouse engraftment tumor model;
FIG. 19 is a graph showing the analysis of the high concentration group MC38 colon cancer cell mouse engraftment tumor model;
FIG. 20 shows analysis results of a model of A20 lymphoma cell mice engraftment tumor;
FIG. 21 is an EMT6 breast cancer cell mouse engraftment tumor model analysis;
FIG. 22 shows the results of B16-F10 melanocyte mouse engraftment tumor model analysis.
Definition of the definition
Unless otherwise defined below, the terms used herein are generally as used in the art.
The terms "interleukin 2", "IL-2" as used herein refer to natural, wild-type IL-2 from any known source. The term encompasses unprocessed IL-2 as well as any form of IL-2 derived from processing in a cell, as well as naturally occurring variants of IL-2, such as splice variants or allelic variants. An exemplary IL-2 herein is uniprot: P60568, the amino acid sequence aa21-153 of the removed signal peptide, abbreviated as wild-type IL-2 (wtIL-2).
The term mutation as used herein refers to a template constructed with the wild type as a mutant, and may be generated using genetic or chemical methods well known in the art, including, but not limited to, site-directed mutagenesis, PCR, gene synthesis, and the like; in the invention, amino acids from 21 st to 153 th of IL-2 removed signal peptide shown in uniprot P60568 are taken as wild type IL-2 to construct IL-2 mutant, aa21 is taken as first amino acid to be numbered, and the mutation site number refers to the amino acid sequence shown in SEQ ID NO. 1.
The terms "PD1", "PD-1", and Programmed death molecule (PD-1) are also known as CD279, which refers to a further important negative costimulatory molecule following CTLA4 discovery, the ligand PD-L1, also known as CD274, belonging to the CD28/B7 superfamily. After PD-1 is combined with the ligand, the conducted negative co-stimulatory signal can inhibit T lymphocyte proliferation, plays a key role in regulating T cell activation and immune tolerance, and plays an important role in preventing the occurrence and development of autoimmune diseases.
The term "affinity" as used herein refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., receptor) and its binding partner (e.g., ligand). Unless otherwise indicated, as used herein, "binding affinity" refers to an intrinsic binding affinity that reflects interactions between members of a binding pair (e.g., a receptor and a ligand). The affinity of a molecule X for its partner Y can be generally expressed in terms of dissociation constant (KD), which is the ratio of dissociation to binding rate constants (K dissociation and K binding, respectively). As such, equal affinities may contain different rate constants, as long as the ratio of rate constants remains the same. Affinity can be measured by established methods known in the art, including the methods described herein.
The term "antibody" or "antigen-binding fragment" as used herein refers to antibodies of various structures exhibiting binding activity to an antigen, i.e., binding activity to PD-1, antibodies of substantially similar structure to the native antibody structure, a portion of antibodies, fragments having binding ability to an antigen to which the antibodies bind, antibodies, and antigen-binding fragments including, but not limited to FV, fab ' Fab ' -sh, F (ab ') 2 Diabodies, linear antibodies, single chain antibodies scFV and single domain antibodies sdabs.
The term "CDR" as used herein is a discrete amino acid sequence present within the variable region of an antibody as known in the art, as it is sterically structured to form a precise complementarity determining region with an epitope. The prior art provides a variety of antibody numbering systems (e.g., kabat/Chothia/IMGT/Gelfand/Aho/Martin) for labeling and numbering antibody variable regions. In one example of the invention, the IMGT antibody numbering system used defines the CDR regions of an anti-PD 1 antibody or antigen-binding fragment thereof in an immunoconjugate as: LCDRS shown as SEQ ID NO 16-18 and HCDRS shown as SEQ ID NO 19-21; a preferred antibody VL sequence is shown in SEQ ID NO. 22 and VH is shown in SEQ ID NO. 23.
The term "immunoconjugate" refers to a construct covalently bonded to an antibody moiety through a linker, and the present invention exemplifies immunoconjugates comprising an IL-2 mutant bonded to an anti-PD 1 antibody.
Detailed Description
The invention will be further illustrated with reference to specific examples. The described embodiments are some, but not all, embodiments of the invention. It is to be understood that the following examples are set forth to provide those of ordinary skill in the art with a complete disclosure and description of how the methods and compositions of the present invention may be utilized and are not intended to limit the scope of what the present invention may be used. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 design of IL-2 mutant
The experimental method comprises the following steps:
utilizing Analyze Protein Interface function of Analyze Protein Complexes module of Discovery Studio software to analyze IL-2 and IL-2 receptor binding interface amino acid in a three-dimensional conformation, wherein the three-dimensional conformation is a protein tertiary structure (PDB: 2B 5I) numbered as 2B5I in a PDB database, and screening and analyzing key amino acid of IL-2 and IL-2 receptor alpha and beta binding interface; the Calculate Mutation Energy function in the Design protein module of Discovery Studio software is used for carrying out mutation simulation screening on the IL-2 binding interface amino acid, and an IL-2 mutant form which influences the IL-2 receptor binding after mutation is selected to construct an IL-2 mutant for subsequent IL-2 activity analysis.
Experimental results:
a) The results of the interface analysis are shown in Table 1, and the calculated interface amino acid between IL-2 and IL-2Rα is K35R 38T 41F 42K 43F 44Y 45E 61E 62K 64P 65E 68V 69L 72Q 74Y 107, and the calculated interface amino acid between IL-2 and IL-2Rβ is L12Q 13E 15H 16L 19M 23R 81D 83S 87N 88V 91I 92E 95.
TABLE 1 IL-2 interface amino acid sites for IL-2 receptor binding
Figure SMS_1
b) Based on the experimental results of IL-2 and IL-2Rα, beta binding interface sites, further carrying out amino acid mutation simulation screening on the IL-2Rα, L2Rβ binding interface amino acid sites, wherein the experimental results are recorded in tables 2-3, and the more the mutation energy increases, the more unstable the protein binding after mutation; the more reduced the mutation energy, representing more stable protein binding after mutation, the candidate mutation sites for constructing IL-2 mutant immunoconjugates were selected based on the different mutant forms shown in tables 2-3, the energy change after mutation, and the effect of the mutation on IL-2 binding to IL-2R.
Table 2 IL-2 and IL-2Rα binding interface amino acid mutation mimetic screening
Figure SMS_2
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Figure SMS_3
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Figure SMS_4
TABLE 3 simulation screening of IL2 and IL2 Rbeta binding interface amino acid mutations
Figure SMS_5
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Figure SMS_6
EXAMPLE 2 construction of IL-2 mutant conjugates
The experimental method comprises the following steps:
construction and expression of 4 structural IL-2 mutant conjugates A schematic of the IL-2 mutant conjugate structure is shown in FIG. 1.
1) Structure 1 (IL-2 mutant Fc fusion protein):
wild type IL-2 (uniprot: P60568, aa21-153, abbreviated wtIL-2) was used as template for mutant construction. IL-2 gene and IL-2 mutant gene sequences were amplified by molecular biology PCR (2X Phanta Max Master Mix Vazyme cat# P515-P1-AA lot # 7E512E 1) and then ligated into vector pCDNA3.4 with antibody heavy chain constant region sequences by homologous recombination (ClonExpress II One Step Cloning Kit Vazyme cat# C112-02-AB lot # 7E550B 1).
The positive clone after sequencing is subjected to plasmid extraction and then is co-transfected into HEK293 cells to be cultured in a shaking table at 37 ℃ and 8% CO2 and 125rpm, supernatant is subjected to Protein A affinity chromatography after 7 days of transient expression, antibody is obtained by purification, and the concentration of the antibody is determined by combining UV280 with a theoretical extinction coefficient.
The amino acid sequence of wild-type IL-2 is shown below:
Figure SMS_7
the amino acid sequence of structure 1 is the sequence shown by connecting IL2 and mutants to SEQ ID NO. 2, wherein the italic underlined part is the linker sequence:
Figure SMS_8
2) Structure 2:
construction of heavy chain: the heavy chain variable region gene of the human or murine PD1 antibody was amplified by molecular biology PCR (2X Phanta Max Master Mix Vazyme cat# P515-P1-AA lot # 7E512E 1) and then ligated into vector pCDNA3.4 with the antibody heavy chain constant region sequence by homologous recombination (ClonExpress II One Step Cloning Kit Vazyme cat# C112-02-AB lot # 7E550B 1).
Construction of a light chain: the light chain gene (comprising variable region VL and constant region CL), IL-2 gene and its mutant gene sequence gene of human or mouse PD1 antibody are amplified by conventional molecular biological PCR, then the light chain gene and different IL-2 mutant genes are linked together by linker by overlay PCR technique, finally by homologous recombination into vector pCDNA3.4.
The positive clone after sequencing is subjected to plasmid extraction and then is co-transfected into HEK293 cells to be cultured in a shaking table at 37 ℃ and 8% CO2 and 125rpm, supernatant is subjected to Protein A affinity chromatography after 7 days of transient expression, antibody is obtained by purification, and the concentration of the antibody is determined by combining UV280 with a theoretical extinction coefficient.
Structure 2 heavy chain amino acid sequence (human PD 1) is shown below, wherein the bolded underlined parts are the HCDR1-HCDR3 sequences numbered SEQ ID NOs: 19-21 in sequence:
Figure SMS_9
the light chain amino acid sequence (human PD 1) of the structure 2 is a sequence shown as SEQ ID NO. 4, IL-2 and mutants thereof are connected through a terminal linker, wherein the bolded underlined parts are LCDR1-LCDR3 sequences with the numbers of SEQ ID NO. 16-18 in sequence, and the italic underlined parts are the linkers:
Figure SMS_10
the heavy chain amino acid sequence of structure 2 (murine PD 1) is shown below:
Figure SMS_11
Figure SMS_12
the light chain sequence of structure 2 (murine PD 1) is characterized in that IL-2 and mutants thereof are linked after the sequence shown in SEQ ID NO:6, the underlined in italics is linker: .
Figure SMS_13
3) Structure 3:
construction of heavy chain 1 (knob chain): the heavy chain variable region gene and heavy chain (knob chain) constant region sequence of the antibody, IL-2 and its mutant gene sequence were amplified by molecular biology PCR (2X Phanta Max Master Mix Vazyme, lot: P515-P1-AA, lot: 7E512E 1), respectively, then the heavy chain variable region gene and heavy chain (knob chain) constant region sequence and the different IL-2 mutant genes were ligated together by overlay PCR technique (IL-2 was ligated to the FC end of knob chain by linker), and finally ligated into vector pCDNA3.4 by homologous recombination (ClonExpress II One Step Cloning Kit Vazyme, lot: C112-02-AB, lot: 7E550B 1);
construction of heavy chain 2 (hole chain): amplifying heavy chain variable region genes and heavy chain (hole chain) constant region sequences of antibodies by conventional molecular biology PCR, then connecting the heavy chain variable region genes and the heavy chain (hole chain) constant region sequences together by an overlap PCR technology, and finally connecting the heavy chain variable region genes and the heavy chain (hole chain) constant region sequences into a vector pCDNA3.4 by homologous recombination;
construction of a light chain: the antibody light chain variable region gene was ligated into vector pcdna3.4 with the antibody light chain constant region sequence.
The positive clone after sequencing is subjected to plasmid extraction and then is co-transfected into HEK293 cells to be cultured in a shaking table at 37 ℃ and 8% CO2 and 125rpm, supernatant is subjected to Protein A affinity chromatography after 7 days of transient expression, antibody is obtained by purification, and the concentration of the antibody is determined by combining UV280 with a theoretical extinction coefficient.
The heavy chain 1 sequence (human PD 1) of the structure 3 is shown as SEQ ID NO:7, and IL2 and mutants thereof are connected to the rear of the heavy chain 1 sequence, wherein the bolded underlined parts are HCDR1-HCDR3 sequences with the numbers of SEQ ID NO:19-21 in sequence, and the italic underlined parts are linker:
Figure SMS_14
structure 3 heavy chain 2 amino acid sequence (human PD 1) is shown below, wherein the bolded underlined parts are the HCDR1-HCDR3 sequences numbered SEQ ID NOs: 19-21 in sequence:
Figure SMS_15
Figure SMS_16
structure 3 the light chain amino acid sequence (human PD 1) is shown below, wherein the bolded underlined sections are the LCDR1-LCDR3 sequences numbered SEQ ID NOs: 16-18 in sequence:
Figure SMS_17
the amino acid sequence of the heavy chain 1 (murine PD 1) of the structure 3 is shown in SEQ ID NO:10, and IL2 and mutants thereof are connected at the back, and the underlined part in italics is linker:
Figure SMS_18
structure 3 heavy chain 2 amino acid sequence (murine PD 1) is shown below:
Figure SMS_19
structure 3 the light chain amino acid sequence (murine PD 1) is shown below:
Figure SMS_20
4) Structure 4:
construction of heavy chain 1 (knob chain): amplifying heavy chain (knob chain) constant region sequences (CH 2CH 3), IL-2 and mutant gene sequences thereof respectively by molecular biology PCR (2X Phanta Max Master Mix Vazyme, cat# P515-P1-AA, cat# 7E512E 1), then linking the heavy chain (knob chain) constant region sequences (CH 2CH 3) and different IL-2 mutant genes together by overlap PCR (IL-2 is linked to the N-terminal of knob chain by linker), and finally linking into vector pCDNA3.4 by homologous recombination (ClonExpress II One Step Cloning Kit Vazyme, cat# C112-02-AB, cat# 7E550B 1);
construction of heavy chain 2 (hole chain): amplifying heavy chain variable region genes and heavy chain (hole chain) constant region sequences of antibodies by a molecular biology technology PCR, then connecting the heavy chain variable region genes and the heavy chain (hole chain) constant region sequences together by an overlap PCR technology, and finally connecting the heavy chain variable region genes and the heavy chain (hole chain) constant region sequences into a vector pCDNA3.4 by homologous recombination;
construction of a light chain: the antibody light chain variable region gene was ligated into vector pcdna3.4 with the antibody light chain constant region sequence.
The positive clone after sequencing is subjected to plasmid extraction and then is co-transfected into HEK293 cells to be cultured in a shaking table at 37 ℃ and 8% CO2 and 125rpm, supernatant is subjected to Protein A affinity chromatography after 7 days of transient expression, antibody is obtained by purification, and the concentration of the antibody is determined by combining UV280 with a theoretical extinction coefficient.
The heavy chain 1 sequence of structure 4 (murine PD 1) is shown in SEQ ID NO:13, preceded by an IL-2 or IL-2 mutant, underlined in italics as linker:
Figure SMS_21
the heavy chain 2 sequence (murine PD 1) of structure 4 is shown in SEQ ID NO. 14:
Figure SMS_22
the light chain sequence (murine PD 1) of structure 4 is shown in SEQ ID NO. 15:
Figure SMS_23
IL-2 mutant immunoconjugate expression:
experimental results:
the IL-2 mutant immunoconjugates prepared are described in tables 4-1 to 4-3.
TABLE 4-1 IL-2 mutant immunoconjugates
Figure SMS_24
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Figure SMS_25
TABLE 4-2 IL-2 mutant conjugates
Figure SMS_26
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Figure SMS_27
TABLE 4-3 IL-2 mutant conjugates
Mutant numbering IL-2 mutation site Antibodies to Construction of structural forms
3001 P65K Murine PD1 antibodies Structure 3
3006 R38E P65R Murine PD1 antibodies Structure 3
3011 NA Human PD1 antibodies Structure 3
3013 R38E P65R Human PD1 antibodies Structure 3
3014 T3A F42A Y45A L72G C125A Human PD1 antibodies Structure 3
3015 R38E Y45R Human PD1 antibodies Structure 3
3017 R38A Y45R Human PD1 antibodies Structure 3
3019 R38D P65R Human PD1 antibodies Structure 3
3020 R38G P65R Human PD1 antibodies Structure 3
3025 F42A E62A E15L Human PD1 antibodies Structure 3
3026 F42A E62A E15V Human PD1 antibodies Structure 3
3027 F42A E62A E15Y Human PD1 antibodies Structure 3
3028 F42A E62A L19H Human PD1 antibodies Structure 3
3029 F42A E62A S87F Human PD1 antibodies Structure 3
3030 F42A E62A S87L Human PD1 antibodies Structure 3
3031 F42A E62A S87P Human PD1 antibodies Structure 3
3032 F42A E62A I92T Human PD1 antibodies Structure 3
3033 R38E P65R L19H Human PD1 antibodies Structure 3
3034 R38G P65R L19H Human PD1 antibodies Structure 3
3042 R38E P65R T3A Human PD1Antibodies to Structure 3
3043 R38E P65R C125A Human PD1 antibodies Structure 3
3044 T3A R38G P65R Human PD1 antibodies Structure 3
3047 L19H Human PD1 antibodies Structure 3
3048 T3A L19H Human PD1 antibodies Structure 3
4002 R38E P65R Murine PD1 antibodies Structure 4
EXAMPLE 3 IL-2R-IL-2 mutant conjugate binding kinetics Studies
The equilibrium dissociation constants (KD) of the IL-2 mutant conjugates of the invention (mutant numbers see tables 4-1 to 4-3) and their receptors were determined using a biological membrane interferometry (forteBio) assay.
The experimental method comprises the following steps:
ForteBio affinity assay the affinity of candidate IL-2 mutants to IL-2Rα, IL-2Rβ or IL-2Rβγ, respectively, was measured as follows: step 1, the sensor balances for 20 minutes under the line in an analysis buffer solution, and then a baseline is established after 60 seconds of on-line detection; loading Fc fusion expressed IL-2 mutants onto a ProA sensor (Sartorius, 18-5010) for ForteBio affinity; samples of IL-2 mutants loaded with Fc fusion expression were placed in a solution containing 10ug/ml IL-2Rα, IL-2Rβ or IL-2Rβγ until the plateau phase, after which the sensor was transferred to assay buffer for dissociation 300s for binding and dissociation rates. Analysis of kinetics and response values was performed using a 1:1 binding model.
Step 2, the sensor balances for 20 minutes under the line in the analysis buffer solution, and then a baseline is established after 60 seconds of on-line detection; the amount of ForteBio affinity performed on IL-2rα, IL-2rβ or IL-2rβγ loaded with human His tag to His1K sensor (Sartorius, 18-5120); the His-tagged IL-2Rα, IL-2Rβ or IL-2Rβγ -loaded sensor was placed in a solution containing 10ug/ml IL-2 mutant until the plateau phase, after which the sensor was transferred to assay buffer for dissociation 300s for binding and dissociation rate. Analysis of kinetics and response values was performed using a 1:1 binding model.
Experimental results:
a) Analysis of the mutant response values of structure 1-form immunoconjugates with IL-2 mutations located at the IL-2rα and/or β binding interfaces are shown in figures 2-3, respectively, and it can be seen from the histogram of the results of figure 2 that IL-2 mutant immunoconjugates made by mutating IL-2 IL-2rα binding interface amino acids exhibit a general decrease or even disappearance of IL-2rα affinity, with only 2 control immunoconjugates: conjugate numbered 1001 (wild-type IL-2, uniprot: P60568, aa21-153), conjugate numbered 1002 (Medicenna Co., MDNA 109) showed higher affinity for IL-2Rα; whereas IL-2rα and β binding interface amino acid mutations did not bring about a general decrease in affinity for IL-2rβ, only IL-2rα with numbers 1003, 1018, 1081, 1082, 1091, 1095, 1097, 1098, 1101, 1121, showed low or even no affinity for IL-2rα, as seen in fig. 3; whereas mutations to the IL-2Rα and β binding interface amino acids did not result in the disappearance of IL-2Rβγ binding capacity.
b) Structure of theIL-2Rα, β binding KD values for form 1 IL-2 mutant conjugates, and IL-2 cell MFI values are reported in Table 5, from which it can be seen that the IL-2 mutant conjugates exhibit a general decrease or even disappearance of IL-2Rα affinity by mutation of one or more of the IL-2Rα binding interface amino acids (see Table 1) of IL-2 in the conjugates, but at the same time the amino acids of the mutant conjugates in which IL-2 is located in the IL-2Rα and β binding interface, are not able to bind the IL-2Rβ to eliminate the binding capacity of the conjugates, and that the IL-2 mutant immunoconjugates shown in Table 5 have affinity to IL-2Rβ and affinity KD values for IL-2 wild type conjugates of 10 -9 There is no defined mutation site or combination that can achieve the effect of attenuation of IL-2. Beta. Affinity. IL-2 mutant conjugates with IL-2R elimination, as well as sets of mutation sites, were obtained based on the above experiments.
TABLE 5 analysis of IL-2 mutant antibody conjugate binding Activity
Figure SMS_28
EXAMPLE 4 in vitro evaluation of the biological Activity of IL-2 mutant conjugates 1
The binding activity of the IL-2 mutant conjugate to IL-2Rα and PD1 on the cell level is determined by flow cytometry, and IL-2 mutant conjugate which binds to the cell surface PD1 and has IL-2Rα receptor binding elimination type and IL-2 mutant set are screened out. FACS examined binding activity of different constructs of the mutant IL-2 conjugates with SU-DHL-1 cells (ATCC, CRL-2955), GS-J2/PD-1 cells (Gensgript, M00612). An experimental group and a control group were set, wherein the control group was a isotype-independent antibody (isotype). The experimental method comprises the following steps:
step 1, lightly blowing the cells in the logarithmic phase into single cells by a liquid transfer device, transferring the single cells into a 50mL centrifuge tube, centrifuging for 300g and 5min to remove the supernatant, re-suspending and washing the cells once by using DPBS, and re-suspending and counting 10mLDPBS after centrifuging to remove the supernatant, and diluting the cells to 2E6/mL by using DPBS;
step 2, diluting the IL-2 mutant antibody by DPBS to initial concentrations of 1200nM, 534nM and 534nM respectively, and diluting the IL-2 mutant antibody by 3 times, 5 times and 5 times concentration gradient, wherein the total concentration is 8;
step 3, mixing the antibody and 50 mu L of cells uniformly to make the cells in a 100 mu L system be 1E5, wherein the final concentration of the antibody is 600nM, 267nM and 267nM at the highest; blowing and mixing antibody cells uniformly, and incubating at 4 ℃ for 1h;
after washing with DPBS and centrifuging to remove unbound primary antibody, adding fluorescent secondary antibody anti-human IgG Fcgamma (Jackson, 109-545-008) diluted with DPBS 1:600, and incubating at 4deg.C in the absence of light for 0.5h; after washing and centrifugation of DPBS to remove unbound secondary fluorescent antibody, cells were resuspended with 100. Mu.L/well DPBS, the average fluorescence intensity values of the cells were measured using a flow cytometer (Essen organism, novoCyte 2060R), and the EC50 of IL-2 mutant antibody binding to cells was calculated using GraphPad software, and the binding activity of the antibodies to cells was analyzed.
Experimental results:
a) Binding activity of IL-2 mutant conjugates to IL-2Rα receptor was demonstrated at cellular level using SU-DHL-1 cells, FIG. 4
It can be seen that the constructed IL-2 mutant conjugates successfully eliminate alpha receptor binding, resulting in IL-2 mutant conjugates and IL-2 mutant sets.
b) The function of cell-level validated IL-2 ra receptor binding-abrogating IL-2 mutant conjugates to bind PD1 was analyzed using flow-through binding of GS-J2-PD1 cells, and it can be seen from fig. 5 that the ability of IL-2 mutant conjugates to bind PD1 was unaffected, consistent with the control Keytruda binding ability, indicating that the mutant conjugates did not affect antibody moiety function.
c) Two sets of samples with identical mutations but different structures were compared for binding to PD1 using a flow-through binding assay curve with GS-J2/PD-1 cells
The differences in capacity are shown in FIG. 6 for IL-2 mutation to R38E P65R, but two sets of samples 3006 and 4002 of different structure were analyzed by GS-J2/PD-1 cell flow binding assay, table 5 for EC50 values calculated from the curves, and both the EC50 values shown in FIG. 6 and Table 6 show: under the same conditions as the IL-2 mutation site, the construct of structure 3 has a higher PD1 binding sensitivity than the construct of structure 4.
TABLE 6
Sample numbering EC50(nM)
3006 0.36
4002 4.08
EXAMPLE 5 IL-2 mutant conjugate biological Activity in vitro evaluation 2
The experimental method comprises the following steps:
and (3) measuring the binding activity of the IL-2 mutant immunoconjugate and IL-2 Rbeta on the cellular level by adopting a flow cytometry, and screening out IL-2 mutant conjugate with strong attenuation type IL Rbeta and IL-2 mutation set. The binding activity of IL-2 mutant conjugates of different structures to HH cells expressing IL-2Rβ (ATCC, CRL-2105) was examined by FACS in the same manner as in example 4.
Experimental results:
a) FIG. 7 shows a flow-through binding assay of IL-2 mutant conjugates with interface mutations to IL-2Rα to HH cells, table 7 shows EC50 values calculated from the curves, and the results indicate that IL-2 mutant conjugates with interface mutations to IL-2Rα, numbered 3011, 3013, 3015, 3017, 3019, 3020, 3014, can still bind to IL-2Rβ expressing cells without affecting their binding capacity to IL-2Rβ expressing cells after mutation of the interface amino acids to IL-2Rα.
TABLE 7
Sample numbering EC50(nM)
3011 40.48
3013 63.53
3015 54.07
3017 43.04
3019 36.11
3020 32.26
3014 26.28
b) FIG. 8 shows a flow-through binding analysis of IL-2 mutant conjugates with IL-2Rβ binding interface mutations to HH cells for constructs of structure 3 as well, and Table 8 shows EC50 values obtained from curve calculations, wherein IL-2 mutant conjugates numbered 3028 exhibit reduced IL-2Rβ affinity and EC50 values 241.40nM, which are obtained by increasing the L19H mutation based on IL-2Rα binding interface mutation F42A, E A.
TABLE 8
Figure SMS_29
Figure SMS_30
c) FIG. 9 shows the effect on IL-2Rβ binding activity of IL-2 mutant conjugates with interface mutations (R38E and P65R) associated with increased physicochemical properties at mutation point T3A/C125A, table 9 shows the EC50 values: the results show that the IL-2Rα binding interface mutation (R38E and P65R) in combination with the T3A mutation does not alter the affinity activity of the IL-2 mutant conjugate for IL-2Rβ; IL-2Rα binding interface mutations (R38E and P65R) in combination with the C125A mutation significantly reduced the affinity activity of IL-2Rβ in the IL-2 mutant conjugates. The results show that the amino acid mutation T3A, which eliminates the O-glycosylation site of IL-2, does not affect mutant binding activity.
TABLE 9
Sample numbering EC50(nM)
3042 24.34
3043 41.04
3013 29.22
d) FIG. 10 shows the results of IL-2 mutant conjugate combination mutants having both IL-2Rα, β binding interface mutations for HH cell surface IL-2Rβ binding activity analysis, and Table 10 shows the EC50 values thereof: the results indicate that samples 3044, 3034 have strongly attenuated IL-2Rβ binding activity, wherein 3033 has an interface mutation for binding to IL-2Rα (R38E, P R) and an interface mutation for binding to IL-2Rβ (L19H), and 3034 has an interface mutation for binding to IL-2Rα (R38G, P R) and an interface mutation for binding to IL-2Rβ (L19H)
Table 10
Sample numbering EC50(nM)
3020 14.24
3034 258.60
3013 15.87
3033 257.60
e) Amino acid mutations T3A that eliminate the O-glycosylation site of IL-2, combined IL-2Rα binding interface mutations (R38G and P65R), or combined IL-2Rβ binding interface mutation (L19H) were studied for their ability to bind to the IL-2Rβ on the surface of HH cells, and the results of flow-binding assays for HH cells are shown in FIGS. 11-12, where 3020 is the R38G+P65R mutation, 3044 is the R38G+P65R+T3A, where 3047 is the L19H mutation, 3048 is the L1H+T3A mutation, and the EC50 values for the mutations listed in FIGS. 11-12 are shown in Table 11. The results indicate that increasing the T3A mutation in the mutant conjugate does not affect its binding activity.
TABLE 11
Sample numbering EC50(nM)
3020 22.29
3044 21.76
3047 398.90
3048 225.00
EXAMPLE 6 analysis of activation Activity of IL-2 mutants on HH cells
The activation activity of IL-2 mutants on HH cells was examined using flow cytometry.
The experimental method comprises the following steps:
step 1, preparing a reaction Buffer: RPMI1640+10% FBS (gibco);
step 2, preparing a 2X antibody to be tested: preparing the antibody to be detected into a solution with an initial concentration of 400nM by using a reaction Buffer, and diluting the solution by 3 times for 11 concentrations;
step 3. Preparing 2X cells: suspension cells HH (ATCC, CRL-2105) were washed 2 times with PBS and the cell density was adjusted to 4X10 with reaction Buffer 6 individual/mL;
step 4. 50ul of the antibody from step 2 and the cells from step 3, respectively, were added to 96 well U-bottom cell culture plates (NEST, 701111) at an initial antibody concentration of 200nM, 2X10 cells per well 5 And each. Incubating the reaction system at 37 ℃ for 15min;
step 5, centrifuging the reaction system in the step 4 to remove the supernatant, adding 200uLPBS to wash for 1 time, adding 100uLFix Buffer I (BD, 557870) into each hole, and standing for 15 minutes at room temperature;
step 6, centrifuging the reaction system in the step 5 to remove the supernatant, adding 200uLPBS to wash for 1 time, adding 100uL of Perm Buffer III (BD, 558050) into each hole, and standing on ice for 30min;
step 7, centrifuging the reaction system in the step 6 to remove the supernatant, adding 200uLPBS to wash for 1 time, adding 100uL PE Mouse Anti-Stat5 (pY 694) (BD, 562077) into each hole, and standing at 4 ℃ for 30min;8. the reaction system of step 7 was centrifuged to remove the supernatant, and after washing with 200uLPBS for 1 time, 100uL of PBS was added to each well to resuspend the cells, and the MFI value of the PE channel was measured by a flow cytometer (ACEA, novoCyte).
Experimental results:
a) Comparing the activation activities of HH cells of the samples of structure 2 and structure 3, respectively, but with the same mutation, the results are shown in FIG. 13 and Table 12. As can be seen from the graph of FIG. 13, the sample of sample number 3020 has a poor ability to activate HH cells relative to 2067, and has a large EC50 value, so that the activation activity of structure 2 is significantly higher than that of structure 3 under the condition that the mutation is located at R38 and P65 at the interface between IL-2 and IL-2Rα, indicating that structure 2 has a bivalent effect
Table 12
Sample numbering EC50(nM)
3020 2.57
2067 0.88
b) Comparing the ability of the conjugate constructed by combining different mutations on the combination surface of IL-2 and IL-2Rα to activate HH cells, and screening mutants with weak activation of HH cells by IL-2 mutant immunoconjugate; the results are shown in fig. 14 and table 13, and table 13 shows that the EC50 of activation of HH cells by selected candidate IL-2 mutant immunoconjugates was about 2-fold that of the wild-type IL-2-PD1 conjugate with sample number 3011.
TABLE 13
Sample numbering EC50(nM)
3011 0.45
3014 0.45
3013 0.93
3015 0.89
3017 0.72
3019 1.08
3020 0.83
c) Structure 3 was selected and analyzed for the ability to previously identify HH cell activation by the β -attenuating mutant using the wild-type IL-2-PD1 conjugate with sample number 3011 as a control, and the results are set forth in fig. 15 and table 14, which show that sample number 3028 showed significant activation attenuation relative to 3011, 3029, 3030, 3032, and that the L19H mutation was identified as a key site for β -attenuating mutant activation by comparison of IL-2 mutations.
TABLE 14
Sample numbering EC50(nM)
3028 8.60
3011 2.47
3014 1.67
3029 6.06
3030 4.35
3031 1.77
3032 5.11
d) Structure 3, an analysis of the ability of IL-2 mutant immunoconjugates with surface mutations binding to IL-2rβ and IL-2rα, showed significant activation attenuation as shown in fig. 16 and table 15, numbered 3020, 3034, further verifying that r38g+p65r alone, or in combination with L19H, showed strong IL-2rβ attenuation.
TABLE 15
Figure SMS_31
Figure SMS_32
e) Structure 3 was selected and analyzed for the effect of composition modifications, e.g., amino acid mutations at the O-glycosylation site of IL-2, on HH cell activation activity, the results are shown in fig. 17A-17B and table 16, and the conclusion shows that increasing the r38g+p65r mutant IL-2 conjugate after T3A modification does not show a difference in activation activity, whereas the L19H mutation combination T3A has a significantly reduced activation activity, which combination exhibits a strong IL-2rβ -reducing effect. The results indicate that increasing the T3A mutation in the mutant conjugate does not affect its activation activity.
Table 16
Sample numbering EC50(nM)
3020 1.66
3044 1.48
3047 40.99
3048 28.38
EXAMPLE 7 evaluation of drug efficacy of PD1 antibody-IL-2 mutant conjugate on tumor cell/mouse transplantation tumor model
The experimental method comprises the following steps:
MC38-hPDL1 mouse colon cancer cell PD1 humanized mouse transplantation tumor model
MC38-hPDL1 mouse colon cancer cells were purchased from Beijing Bai Sago Gene biotechnology Co., ltd, and the cells were cultured in an incubator at 37℃with 5% CO2, the medium composition being DMEM medium containing 10% FBS. PD1 humanized mice (C57 BL/6JSMoc-Pdcd1em1 (hPDCD 1)/Smoc), females, 6-8 weeks old, were purchased from Shanghai's model biotechnology Co., ltd. MC38-hPDL1 cell concentration was adjusted to 5.0X10 by PBS 6 The right side of the PD1 humanized mice was inoculated subcutaneously at 0.1 mL/volume per mL. When the average tumor volume reaches 99mm 3 At this time, mice were grouped according to tumor volume of the mice, 5 mice per group were administered starting on the day of grouping at a dose of 2.5mg/kg or 5mg/kg, and were given once weekly by intraperitoneal injection.
A20 mouse B cell lymphoma cell PD1 humanized mouse transplantation tumor model
A20 mouse B cell lymphoma cells were purchased from ATCC, and the cells were cultured at 37℃in 5% CO 2 The medium composition was RPMI-1640 medium containing 10% FBS. PD1 humanized mice (BALB/cAnSmoc-Pdcd 1em1 (hPDCD 1) Smoc), females, 7-8 weeks old, were purchased from Jiangsu Jiuyaokang Biotech Co. A20 cell concentration was adjusted to 8.0x10 with PBS 6 The right side of the PD1 humanized mice was inoculated subcutaneously at 0.1 mL/volume per mL. When the average tumor volume reached 92mm 3 At this time, mice were grouped according to tumor volume of the mice, 5 mice per group were administered starting on the day of grouping at a dose of 5.0mg/kg, by intraperitoneal injection, once weekly.
EMT6 mouse breast cancer cell PD1 humanized mouse transplantation tumor model
EMT6 mouse breast cancer cells were purchased from ATCC, and the cells were cultured at 37℃with 5% CO 2 The culture medium composition is RPMI-1640 culture medium containing 10% FBS. PD1 humanized mice (BALB/cAnSmoc-Pdcd 1em1 (hPDCD 1) Smoc), females, 7-8 weeks old, were purchased from Jiangsu Jiuyaokang Biotech Co. EMT6 cell concentration was adjusted to 2.0X10 with PBS 6 The right side of the PD1 humanized mice was inoculated subcutaneously at 0.1 mL/volume per mL. When the average tumor volume reached 83mm 3 At this time, mice were grouped according to tumor volume of the mice, 5 mice per group were administered starting on the day of grouping at a dose of 4.0mg/kg by intraperitoneal injection, once weekly.
B16-F10 mouse melanoma cell PD1 humanized mouse transplantation tumor model
B16-F10 mouse melanoma cells were purchased from BNCC North Naorganism, and the cells were cultured in an incubator at 37℃with 5% CO2, the medium composition being DMEM medium containing 10% FBS. PD1 humanized mice (C57 BL/6JSMoc-Pdcd1em1 (hPDCD 1)/Smoc), females, 7-8 weeks old, were purchased from Shanghai's model biotechnology Co., ltd. MC38-hPDL1 cell concentration was adjusted to 1.0X106 cells/mL with PBS and inoculated subcutaneously on the right side of PD1 humanized mice at 0.1 mL/volume. When the average tumor volume reached 84mm3, mice were grouped according to their tumor volume, 5 mice per group were given starting on the day of grouping at a dose of 5.0mg/kg by intraperitoneal injection, once weekly.
Experimental results:
a) IL-2 mutant immunoconjugates with sample numbers 3015, 3017, 3020, 3028, 3030, 3029, 3032 were administered at 2.5mg/kg
(QW, i.p.) administration to MC38 colon cancer cell mice engraftment tumor model, anti-PD-1 antibody panel: keytruda, blank
Control group: PBS; the results are shown in FIG. 18, wherein the IL-2 mutant immunoconjugate of sample number 3020 is shown
Unexpected higher than the Keystuda control and higher than the numbers 3015, 3017, 3028, 3030, 3029, 3032 mutation avoidance
Anti-tumor efficacy of epidemic conjugate samples.
b) IL-2 mutant immunoconjugates of sample numbers 3020 and 3047 were administered at 5mg/kg (QW, I.P.) to the MC38 colon
Cancer cell mice engrafted tumor model, set up anti-PD-1 antibody group: keytruda, blank: PBS; results are shown in
In FIG. 19, IL-2 mutant conjugation, where sample numbers 3020, 3047, exhibited significantly better anti-than the Keystuda control
Tumor effect, based on this conclusion, IL-2 mutant immunoconjugates of 3020, 3047 were dosed at high concentrations (5 mg/kg)
The effect is significantly better than that of the Keystuda control compared with the low concentration (2.5 mg/kg) group; IL-2 with sample number 3020
The mutant immunoconjugate exhibited unexpectedly higher anti-tumor efficacy compared to 3047.
c) IL-2 mutant immunoconjugate with sample number 3044 was administered to A20 lymphoma cells at 5mg/kg (QW, I.P.)
Mice engrafted tumor model, set up anti-PD-1 antibody group: keytruda, blank: PBS; the application can be seen from FIG. 20
The anti-PD 1 group tumor volumes with Keytruda did not show any therapeutic effect, whereas the conjugate with sample number 3044 was anti-tumor
The tumor efficacy is significantly better than that of the Keystuda control, almost completely inhibits tumor growth, and low response against PD1 is obtained based on the tumor efficacy
The IL-2 conjugate containing R38G, P R and T3A mutations showed unexpected anti-tumor effects.
d) IL-2 mutant immunoconjugate with sample number 3020 was administered to EMT6 breast cancer cells at 4mg/kg (QW, I.P.)
Mouse engraftment tumor model, 5mg/kg (QW, I.P.) of administration to B16-F10 melanocyte mouse engraftment tumor model, respectively
Setting anti-PD-1 antibody group: keytruda, blank: PBS; from FIGS. 21-22, it can be seen that Keystuda is administered
The corresponding tumor volume in the anti-PD 1 group of (a) does not show any therapeutic effect, whether it is refractory melanoma or breast cancer model
If the anti-tumor efficacy of the conjugate with the sample number 3020 in two mouse cancer models is obviously better than that of Keystuda, the conjugate is shown
Based on the excellent antitumor effect, IL-2 containing R38G, P R mutation against PD1 low-response tumor was obtained
The conjugates showed unexpected anti-tumor effects.
The above disclosure is only a few specific embodiments of the present invention, but the present invention is not limited thereto, and any changes can be considered by those skilled in the art, which fall within the protection scope of the present invention.

Claims (13)

1. An IL-2 mutant immunoconjugate, characterized in that the immunoconjugate comprises an interleukin 2 (IL-2) polypeptide mutant and an antibody or antigen binding fragment that binds to PD1, wherein the IL-2 polypeptide mutant has reduced IL-2rβ binding activity and/or eliminated IL-2rα binding activity, the IL-2 polypeptide mutant comprises an amino acid substitution selected from a) or b), wherein the mutation number is the number relative to the amino acid sequence set forth in human IL-2 sequence SEQ ID No. 1:
a)R38G,P65R；
b)L19H；
the PD1 antibodies and antigen-binding fragments thereof comprise: (a) A heavy chain variable region (VH) comprising HCDR1 of the amino acid sequence shown in SEQ ID No. 19, HCDR2 of the amino acid sequence shown in SEQ ID No. 20 and HCDR3 of the amino acid sequence shown in SEQ ID No. 21; and (b) a light chain variable region (VL) comprising LCDR1 of the amino acid sequence shown in SEQ ID NO. 16, LCDR2 of the amino acid sequence shown in SEQ ID NO. 17 and LCDR3 of the amino acid sequence shown in SEQ ID NO. 18.
2. The immunoconjugate of claim 1, wherein the IL-2 polypeptide mutant further comprises an amino acid substitution T3A and/or an amino acid substitution C125A.
3. The immunoconjugate according to any one of claims 1 to 2, wherein the IL-2 polypeptide mutant is fused at its amino terminus to the carboxy terminus of the Fc domain of the antibody or antigen binding fragment by a linker peptide.
4. The immunoconjugate according to any one of claims 1 to 2, wherein the IL-2 polypeptide mutant is fused at its amino terminus to the carboxy terminus of the light chain of the antibody or antigen binding fragment by a linker peptide, the immunoconjugate comprising 1-2 IL-2 mutants.
5. One or more isolated polynucleotides encoding the immunoconjugate of any one of claims 1 to 4.
6. One or more vectors, characterized in that the vector is an expression vector, which vector comprises a polynucleotide according to claim 5.
7. A host cell comprising the polynucleotide of claim 5 or the vector of claim 6.
8. A method of producing an IL-2 mutant immunoconjugate according to claim 1, the method comprising (a) culturing the host cell according to claim 7 under conditions suitable for expression of the immunoconjugate, and optionally (b) recovering the immunoconjugate.
9. A pharmaceutical composition comprising the immunoconjugate of any one of claims 1 to 4 and a pharmaceutically acceptable carrier.
10. Use of an immunoconjugate according to any one of claims 1 to 4 or a pharmaceutical composition according to any one of claims 9 for the preparation of a medicament for ameliorating or treating cancer.
11. The use according to claim 10, characterized in that the cancer is a cancer that is resistant to low response to PD1 treatment.
12. Use according to any one of claims 10-11, characterized in that the cancer is selected from: colon cancer, breast cancer, liver cancer, melanoma and gastric cancer.
13. Use according to claim 12, characterized in that the melanoma is refractory melanoma.
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