CN112724263B

CN112724263B - Method for improving drug efficacy of anti-CD 20 monoclonal antibody by modifying anti-CD 20 monoclonal antibody and application thereof

Info

Publication number: CN112724263B
Application number: CN202110360712.4A
Authority: CN
Inventors: 何虹霖; 严宇媛; 徐阳华; 杨敏; 夏爱坤; 岳翠华; 钟子洋
Original assignee: Shanghai Luotuo Biotechnology Co ltd
Current assignee: Shanghai Luotuo Biotechnology Co ltd
Priority date: 2021-04-02
Filing date: 2021-04-02
Publication date: 2021-08-03
Anticipated expiration: 2041-04-02
Also published as: CN112724263A

Abstract

The invention discloses a method for improving the curative effect of a medicament by modifying an anti-CD 20 monoclonal antibody and application thereof. The method comprises the steps of connecting the gene of the anti-CD 20 monoclonal antibody with the gene of the IL-2 mutant, and obtaining the fusion protein through expression, wherein the fusion protein is applied to inhibiting tumors. The fusion protein obtained by the method provided by the invention has an excellent inhibition effect on tumor inhibition, and the toxic and side effects are controllable.

Description

Method for improving drug efficacy of anti-CD 20 monoclonal antibody by modifying anti-CD 20 monoclonal antibody and application thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a method for improving the curative effect of a CD 20-resistant monoclonal antibody by modifying the monoclonal antibody and application of the monoclonal antibody.

Background

CD20 is a transmembrane protein expressed on B cells and functions to regulate B cell activation and proliferation. CD20 is expressed in most B cell malignancies and thus CD20 is a hot target for the treatment of lymphomas, leukemias, and certain autoimmune diseases. An anti-CD 20 antibody drug, rituximab, was approved by the FDA for marketing on 26/11/1997 for the treatment of non-hodgkin's lymphoma, chronic lymphocytic leukemia, and other non-neoplastic diseases. Although some patients have well-controlled disease after rituximab injection, there are a significant number of patients who have failed or recurred after administration, and therefore there is a need to develop more effective drugs, which will benefit more patients.

Interleukin-2 (IL-2) is a cytokine produced by activated T cells, and enhances the immunocompetence of a human body by promoting the proliferation of helper T lymphocytes, cytotoxic T lymphocytes and NK cells, thereby achieving the purpose of killing tumor cells by self immune cells, because it is also applied to the treatment of malignant tumors. However, due to the super-strong immune response brought by IL-2, immune cells are excessively activated, and when tumor cells are killed, serious toxicity is caused to normal organs, such as vascular leakage syndrome, so that the clinical application of the IL-2 is limited.

At present, an anti-CD 20 antibody and IL-2 are also made into a fusion protein, on one hand, the targeting property of the anti-CD 20 antibody is utilized to specifically act on tumor cells with high expression of CD20, and on the other hand, the effect that the IL-2 can activate immune cells is utilized to attract the immune cells to the vicinity of the tumor cells, so that the tumor cells are killed more effectively. Therefore, the effect of the fusion protein on tumor treatment may be better than that of the anti-CD 20 antibody drug alone.

However, the preparation of a fusion protein of anti-CD 20 antibody and IL-2 also suffers from the problems of toxicity of IL-2 to the body, damage to normal tissues of the patient, and limited dosage, which further affects the efficacy of the anti-CD 20 antibody moiety. Therefore, how to reduce the toxicity of the IL-2 part and better exert the anti-tumor effect becomes a difficult problem to be solved by making the anti-CD 20 antibody and the IL-2 into a fusion protein and better applying the fusion protein to clinic.

Disclosure of Invention

The invention aims to provide a method for modifying an anti-CD 20 monoclonal antibody to improve the curative effect of a medicament.

In a first aspect of the present invention, there is provided a method of modifying an anti-CD 20 monoclonal antibody to improve its pharmaceutical efficacy, comprising: linking an anti-CD 20 monoclonal antibody to the IL-2 mutant; wherein the IL-2 mutant has a mutation in the IL-2 receptor binding region such that binding to its receptor is inhibited; the receptor binding region comprises: the binding domain of IL-2 to IL-2R α (CD25), and/or the binding domain of IL-2 to IL-2R β (CD 122).

In one alternative, the inhibition comprises total inhibition or partial inhibition; the partial suppression includes: inhibiting more than 20%, preferably inhibiting more than 40%, more preferably inhibiting more than 60%; such as 70%, 80% or 90% inhibition.

In another preferred embodiment, the IL-2 mutant is obtained by replacing amino acid NNYKNPKLTRML from position 29 to position 40 of IL-2 with an amino acid sequence with a different amino acid sequence, wherein the sequence length is 5-10 (such as 6, 7, 8, 9).

In another preferred embodiment, the IL-2 mutant is characterized in that the amino acid NNYKNPKLTRML from the 29 th position to the 40 th position of IL-2 is replaced by QSMEIDAT; wherein each amino acid in the QSMEIDAT sequence can be replaced by amino acids with the same or similar properties.

In another preferred embodiment, the IL-2 mutant also includes a mutation (including 1, 2 or more mutations) selected from the group consisting of: (1) mutating the 16 th H; (2) mutating D at position 84; (3) mutating N at position 88; (4) mutation of I at position 92.

In another preferred embodiment, (1) the 16 th H is mutated to E; (2) in (2), mutating the 84 th D to T; (3) in the method, the 88 th N is mutated into A; (4) in (1), the 92 th I is mutated to A.

In another preferred embodiment, the mutation is 1, 2 or more mutations selected from (1) to (4).

In another preferred embodiment, the IL-2 mutant is linked to the end of one or both heavy chains of the anti-CD 20 monoclonal antibody.

In another preferred embodiment, a linker (linker peptide) of 4-20 amino acids is included between the amino acid sequence of the anti-CD 20 monoclonal antibody and the amino acid sequence of the IL-2 mutant.

In another preferred embodiment, when the IL-2 mutant is linked to the end of one heavy chain of the anti-CD 20 monoclonal antibody, the two heavy chains of the anti-CD 20 monoclonal antibody are provided with a Knob and a Hole respectively, so as to form a Knob-into-Hole structure.

In another preferred embodiment, the IL-2 mutant is linked to a non "antigen binding site" of the anti-CD 20 monoclonal antibody.

In another preferred example, the amino acid sequence of the linker is (GGGS) n, wherein n is 1-5. More preferably, n is 1 to 4, and still more preferably, n is 2 to 3. More preferably, the linker has the amino acid sequence of GGGSGGGSGGGS.

In another preferred embodiment, the anti-CD 20 monoclonal antibody comprises an IgG1, IgG2 or IgG4 type antibody.

In another preferred embodiment, the anti-CD 20 monoclonal antibody comprises: a monovalent antibody, a single chain antibody, a diabody, a chimeric antibody, or a derivative, functional equivalent or homolog thereof.

In another preferred embodiment, the anti-CD 20 monoclonal antibody comprises an antibody fragment or a polypeptide comprising an antigen binding domain.

In another preferred embodiment, the anti-CD 20 monoclonal antibody is rituximab.

In another preferred embodiment, the anti-CD 20 monoclonal antibody has a heavy chain variable region in which the CDR1 amino acid sequence is shown in SEQ ID NO. 4, the CDR2 amino acid sequence is shown in SEQ ID NO. 5, and the CDR3 amino acid sequence is shown in SEQ ID NO. 6; the amino acid sequence of CDR1 in the variable region of the light chain is shown as SEQ ID NO. 7, the amino acid sequence of CDR2 is shown as SEQ ID NO. 8, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 9.

In another preferred embodiment, the amino acid sequence of the variable region of the heavy chain of the anti-CD 20 monoclonal antibody is shown in SEQ ID NO. 2, and the amino acid sequence of the variable region of the light chain thereof is shown in SEQ ID NO. 3.

In another aspect of the present invention, there is provided a fusion protein comprising: anti-CD 20 monoclonal antibodies and IL-2 mutants; wherein the IL-2 mutant has a mutation in the IL-2 receptor binding region such that binding to its receptor is inhibited; the receptor binding region comprises: the binding domain of IL-2 to IL-2R α (CD25), and/or the binding domain of IL-2 to IL-2R β (CD 122).

In a preferred embodiment, the IL-2 mutant is obtained by replacing amino acid NNYKNPKLTRML from position 29 to position 40 of IL-2 with an amino acid sequence with a different amino acid sequence, wherein the sequence length is 5-10 (such as 6, 7, 8, 9).

In another preferred embodiment, the fusion protein further comprises a mutation (including 1, 2 or more mutations) selected from the group consisting of: (1) a 16 th H mutation; (2) a D mutation at position 84; (3) an 88 th N mutation; (4) mutation at position 92.

In another preferred embodiment, (1) the 16 th H mutation is E; (2) in, the 84 th D mutation is T; (3) in the middle, the 88 th N is mutated into A; (4) in the second place, the 92 nd I mutation is A.

In another aspect of the invention, a nucleic acid molecule is provided, which encodes the fusion protein.

In another aspect of the present invention, there is provided a vector comprising said nucleic acid molecule.

In another aspect of the invention, there is provided a genetically engineered cell comprising said vector; or said nucleic acid molecule is integrated into the genome of said cell.

In another aspect of the present invention, there is provided a method for preparing the fusion protein, the method comprising: culturing said cell such that said cell produces said fusion protein.

In a preferred embodiment, the method further comprises: recovering said fusion protein; more preferably, it comprises isolating and purifying said fusion protein.

In another aspect of the invention, the fusion protein is provided for use in preparing a pharmaceutical composition for inhibiting tumor.

In a preferred embodiment, the tumor is a tumor expressing the CD20 antigen.

In another preferred embodiment, the CD20 antigen-expressing tumor includes (but is not limited to): b cell lymphoma (including diffuse large B cell lymphoma), follicular lymphoma, chronic lymphocytic leukemia.

In another aspect of the present invention, there is provided a pharmaceutical composition for inhibiting tumor, the pharmaceutical composition comprising: (i) the fusion protein; and (ii) a biologically acceptable carrier.

In another aspect of the present invention, there is provided a kit for inhibiting tumor, comprising the fusion protein; or, a pharmaceutical composition containing the same.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1 sequence alignment of IL-2 mutants of the invention that have removed binding to the receptor IL-2R α (CD25) with wild-type IL-2.

FIG. 2 is a schematic structural diagram of a fusion protein of anti-CD 20 and IL-2 constructed by the present invention.

FIG. 3, the affinity of the IL-2 mutant of the invention (RT001-10) and the wild type IL-2(wt IL-2) for IL-2R α (CD 25).

FIG. 4 shows the affinity of the IL-2 mutant of the present invention for IL-2R β (CD 122).

FIG. 5 shows the affinity of the fusion protein of anti-CD 20 and IL-2 mutant of the present invention for CD20 on cell membrane.

FIG. 6A shows the activation of intracellular pSTAT5 signaling by RT001-10, a fusion protein of the invention anti-CD 20 with IL-2 mutant, as compared to wild type.

FIG. 6B shows the activation function of the fusion proteins RT001-10, RT001-24, RT001-27, RT001-30 and RT001-33 of the anti-CD 20 and IL-2 mutant of the present invention on intracellular pSTAT5 signal.

FIG. 7 shows the killing function of the fusion protein of anti-CD 20 and IL-2 mutant on tumor cells.

FIG. 8 is a diagram of the structure of a fusion protein of anti-CD 20 and IL-2 mutants of different IL-2 mutants of the present invention.

FIG. 9 shows the affinity of the fusion protein of anti-CD 20 of various IL-2 mutants of the present invention and IL-2 mutants for CD20 on cell membranes.

FIG. 10 shows the activation function of anti-CD 20 fusion proteins with IL-2 mutants of different IL-2 mutants of the present invention on intracellular pSTAT5 signaling.

FIG. 11 shows the tumor cell killing function of the fusion protein of anti-CD 20 and IL-2 mutant of different IL-2 mutants of the present invention.

FIG. 12 shows the affinity of the fusion protein of the IL-2 mutant of the present invention grafted to the ends of the two heavy chains of the anti-CD 20 antibody to CD20 on the cell membrane.

FIG. 13 shows the fusion of the IL-2 mutant of the present invention to the ends of the two heavy chains of an anti-CD 20 antibody and the activation of intracellular pSTAT5 signaling.

FIG. 14 shows the tumor cell killing function of the fusion protein of the IL-2 mutant of the present invention connected to the two heavy chain ends of the anti-CD 20 antibody.

FIG. 15 toxicity test of anti-CD 20 fusion proteins with different IL-2 mutants in mice.

Detailed Description

The present inventors have conducted extensive studies and experiments to disclose a method for improving the therapeutic effect of an anti-CD 20 monoclonal antibody, which comprises fusing an anti-CD 20 monoclonal antibody with an IL-2 mutant. The fusion protein obtained by the method of the invention has excellent effect on inhibiting tumor, which is greatly higher than the tumor inhibiting effect of CD20 alone.

As used herein, "operably linked" or "operably linked" refers to a condition in which certain portions of a linear DNA sequence are capable of affecting the activity of other portions of the same linear DNA sequence. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the coding sequence.

As used herein, the terms "comprising," having, "or" including "include" comprising, "" consisting essentially of … …, "" consisting essentially of … …, "and" consisting of … …; "consisting essentially of … …", "consisting essentially of … …" and "consisting of … …" are subordinate concepts of "comprising", "having" or "including".

As used herein, a "biologically acceptable carrier" is a solvent, suspending agent or excipient or the like that is used to deliver the fusion protein of the present invention to a subject in need of treatment (including food, feed), that is controllable in toxicity, side effects, environmentally friendly or harmless to humans and animals. The carrier may be a liquid or a solid, and is preferably a carrier capable of maintaining the biological activity of the fusion protein of the present invention to a high degree.

Protein engineering

The invention provides a novel method for modifying an anti-CD 20 monoclonal antibody to improve the curative effect of a medicament, which comprises the step of fusing an anti-CD 20 monoclonal antibody and an IL-2 mutant, wherein the fusion protein obtained by the method comprises a CD20 monoclonal antibody (comprising a bioactive fragment thereof) and an IL-2 mutant (comprising a bioactive fragment thereof). The fusion protein of the invention may be a separate/isolated protein, a purified product of recombinant host cell culture or as a purified extract.

IL-2 mutants

The inventor obtains some IL-2 mutants which can be well compatible and matched with the anti-CD 20 monoclonal antibody through deep analysis; the ability of the obtained fusion protein to inhibit tumor cells is obviously enhanced, and the amino acid number in the mutant protein can be based on the number of the amino acid sequence shown in SEQ ID NO. 1.

The mutants (muteins) of the present invention are synthetic or recombinant proteins, i.e., can be the product of chemical synthesis, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants). According to the host used in the recombinant production protocol. The mutant proteins of the present invention may or may not also include an initial methionine residue.

The invention also includes fragments, derivatives and analogs of the IL-2 muteins. As used herein, the terms "fragment," "derivative," and "analog" refer to a protein that retains substantially the same biological function or activity as the muteins listed in the examples of the present invention.

A fragment, derivative or analogue of an IL-2 mutein of the invention may be (i) a mutein wherein one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a mutein having a substituent group in one or more amino acid residues, or (iii) a mutein formed by fusing a mature mutein with another compound, such as a compound that extends the half-life of the mutein, e.g.polyethylene glycol, or (iv) a mutein formed by fusion of an additional amino acid sequence to the mutein sequence (e.g.a leader or secretory sequence or a sequence used to purify the mutein or a proprotein sequence, or a fusion protein formed with an antigenic IgG fragment). However, the amino acid sequences of the fragments, derivatives and analogs described herein are such that mutations in the key mutation sites/regions, including the IL-2 receptor binding region, claimed by the present invention, corresponding to wild-type IL-2, are conserved; that is, the sequence changes covered by the fragments, derivatives and analogs, which are present at noncritical sites on IL-2, can still be the same or substantially the same as the functions/activities of the mutants listed in the examples of the present invention.

In addition, the mutant protein can be modified. Modified (generally without altering primary structure) forms include: chemically derivatized forms of the mutein such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during synthesis and processing of the mutein or during further processing steps. Such modification may be accomplished by exposing the mutein to an enzyme that performs glycosylation, such as mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are muteins which have been modified to increase their resistance to proteolysis or to optimize solubility. Similarly, the amino acid sequence of the modified IL-2 mutant protein is such that mutations in key mutation sites/regions claimed herein, including the IL-2 receptor binding region, corresponding to wild-type IL-2, are conserved; that is, the modified IL-2 mutant protein covers the sequence modification changes in the non-critical location of IL-2, and the modified IL-2 mutant protein can still have the same or substantially the same function/activity as the mutants listed in the examples of the present invention.

The term "mutant protein encoding polynucleotide" can be included encoding the IL-2 mutant protein polynucleotides, but also can also include additional coding and/or non-coding sequence of the polynucleotide.

anti-CD 20 monoclonal antibody

As used herein, the term "anti-CD 20 monoclonal antibody" refers to an antibody that is capable of specifically binding to the cell surface CD20 antigen. In the present invention, the "antibody" may encompass an anti-CD 20 specific binding member having a binding domain with the desired specificity. Thus, this term encompasses antibody fragments, derivatives, and functional equivalents and homologues of antibodies homologous thereto, and also encompasses any polypeptide, whether natural or synthetically produced, that comprises an antigen-binding domain.

The anti-CD 20 monoclonal antibodies of the invention may be monovalent or single chain antibodies, diabodies, chimeric antibodies, and derivatives, functional equivalents and homologues of the foregoing, including antibody fragments and any polypeptides comprising an antigen binding domain. Antibodies can be modified in a number of ways and recombinant DNA techniques can be used to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable regions or Complementarity Determining Regions (CDRs) of an antibody into the constant regions or constant region plus framework regions of different immunoglobulins. Genetic mutations or other changes may also be made to the hybridoma cells or other cells that produce the antibody, which may or may not alter the binding specificity of the produced antibody.

The anti-CD 20 monoclonal antibody of the present invention is a framework region except for the hypervariable regions CDR1, CDR2 and CDR3 and the linking sequences in the heavy and light chains. The framework regions can be replaced by other sequences under conditions where the three-dimensional structure required for binding is not affected, and the molecular basis for antibody specificity arises primarily from its highly variable regions CDR1, CDR2, and CDR3, which are key sites for antigen binding. To maintain preferred binding properties, the sequence of the CDRs should be preserved as much as possible, however, some amino acid changes may be required to optimize binding properties.

In a preferred mode of the invention, the anti-CD 20 monoclonal antibody is rituximab or an engineered or modified monoclonal antibody which has the same function with rituximab.

Connection of

In the novel fusion protein, the anti-CD 20 monoclonal antibody and the IL-2 mutant are operably connected, and have ideal compatibility and high biological activity.

In a preferred embodiment of the present invention, the IL-2 mutant and the CD20 monoclonal antibody are linked by a polypeptide linker to form a fusion protein. The connection of an amino acid linker with a proper length between the amino acid sequences of the IL-2 mutant and the CD20 monoclonal antibody is beneficial to the high biological activity of the two.

In a preferred embodiment of the invention, the linker comprises 4 to 20 amino acids. In a more preferred embodiment of the present invention, the linker has an amino acid sequence of (GGGS) n (wherein n is 1 to 5); more preferably, n is 1 to 4; most preferably, n-3, i.e., the linker is GGGSGGGSGGGS.

Preferably, the IL-2 mutant is linked to the end of one amino acid sequence of the heavy chain of the anti-CD 20 monoclonal antibody when expressed to form a Y-shaped structure of the antibody. When expressed, the Y-shaped structure of the antibody is formed, which constitutes a linkage as in the left or middle panel of FIG. 2 in the examples of the invention.

As another preferred mode, the IL-2 mutant is linked to the ends of the two amino acid sequences of the heavy chain of the anti-CD 20 monoclonal antibody when the Y-shaped structure of the antibody is formed after expression. Which constitutes the connection means as the right drawing of fig. 2 in the embodiment of the present invention.

Alternatively, the IL-2 mutant and the CD20 monoclonal antibody are directly linked, for example, the anti-CD 20 monoclonal antibody is directly linked with the gene coding for the IL-2 mutant, and fusion expression is carried out without adding an amino acid linker between the two.

The fusion protein of the invention can be further connected or coupled with other heterologous functional molecules to form an immunoconjugate; the heterologous functional molecule may include, but is not limited to: cytokines, detectable markers, toxins (e.g., tumor suppressor toxins), transcriptional activation domains, transcriptional repression domains, nucleases, deaminases, methylases, demethylases, transcriptional release factors, and the like. The detectable labels may include, but are not limited to: fluorescent markers, chromogenic markers, reporter genes, localization signals, and the like. The heterologous functional domain may be linked, coupled or conjugated to the N-terminus, C-terminus or inside the fusion protein of the invention.

Such detectable labels may include, for example, but are not limited to: fluorescent markers, chromogenic markers; such as: enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and nonradioactive paramagnetic metal ions. More than one label may also be included. The label used to label the antibody for detection and/or analysis and/or diagnostic purposes depends on the particular detection/analysis/diagnostic technique and/or method used, e.g., immunohistochemical staining of (tissue) samples, flow cytometry, etc. Suitable labels are well known to those skilled in the art for detection/analysis/diagnostic techniques and/or methods known in the art.

Nucleic acid molecules and constructs or cells comprising same

In another aspect, the invention provides an isolated nucleic acid encoding the fusion protein, and optionally the complementary strand thereof.

Furthermore, the present invention provides a vector comprising a nucleic acid molecule encoding the fusion protein. The vector may further comprise an expression control sequence operably linked to the sequence of the nucleic acid molecule to facilitate expression of the fusion protein.

Any suitable nucleic acid molecule encoding an IL-2 mutant is suitable for use in the present invention. Any suitable nucleic acid molecule encoding an anti-CD 20 monoclonal antibody is also suitable for use in the present invention. The sequences mentioned in the examples below are all suitable for use in the method of the invention. It will be appreciated that when the amino acid sequence of a protein is provided, the person skilled in the art will be able to readily determine the nucleic acid molecule encoding it.

Any suitable vector may be used, such as some vectors used for cloning and expression of mammals, bacteria, fungi, yeast, e.g., Pouwels, et al, cloning vectors: described in a laboratory manual (latest edition of Elsevier). In a preferred embodiment of the present invention, the vector is a vector suitable for mammalian cells.

In some embodiments, the vector may be a viral vector, such as, but not limited to, a retroviral vector, a phage vector, an adenoviral vector, a Herpes Simplex Virus (HSV) vector, an AAV vector, or a lentiviral vector.

Expression vectors include fusion protein DNA sequences linked to appropriate transcriptional and translational regulatory sequences, such as mammalian, microbial, viral, or insect genes. Regulatory sequences include transcriptional promoters, operators, enhancers, ribosome binding sites, or suitable sequences that control transcription and translation initiation and termination. Where the fusion protein sequence requires the function of a regulatory sequence, an appropriate regulatory sequence is ligated. Thus, the promoter sequence is ligated in front of the DNA sequence encoding the fusion protein. The ability to replicate in a host cell is generally controlled by the origin of replication. A selection gene for transformant identification may also be added to the expression vector.

In addition, the coding sequence for the signal peptide of the non-native IL-2 mutant or CD20 monoclonal antibody may be introduced into an expression vector. For example: the signal peptide (secretion guide) sequence may be fused to the fusion protein coding sequence so that the translated fusion protein may be secreted extracellularly. The signal peptide may enhance secretion of the chimeric polypeptide extracellularly by the host cell. The signal peptide may be cleaved off during secretion of the polypeptide from the cell.

In addition, recombinant cells containing nucleic acid sequences encoding the fusion proteins are also encompassed by the invention. In one form of the invention, the cell may be a eukaryotic cell. Preferably, the cells may include, but are not limited to: a CHO cell or a cell engineered based thereon, a NS2/0 cell or a cell engineered based thereon, a 293 cell or a cell engineered based thereon; more preferably, the cell is a CHO cell or a cell engineered based thereon.

Methods of producing fusion proteins are also encompassed by the present invention. The method comprises culturing a recombinant cell comprising a nucleic acid encoding a fusion protein. The fusion protein comprises an IL-2 mutant and a CD20 monoclonal antibody. The method can include allowing the cell to express the encoded fusion protein, and allowing renaturation of the expressed fusion protein. In one example, the method may further comprise isolation and/or purification of the renatured fusion protein. The product of the process is also protected.

The fusion protein prepared as described above can be purified to substantially uniform properties, for example, as a single band on SDS-PAGE electrophoresis. For example, when the recombinant protein is expressed for secretion, the expression supernatant may be first concentrated using a commercially available ultrafiltration membrane, such as Millipore, Amicon, Pellicon, or the like. The concentrated solution can be further purified by gel chromatography or ion exchange chromatography. Such as anion exchange chromatography (DEAE etc.) or cation exchange chromatography. The gel matrix can be agarose, dextran, polyamide, etc. commonly used for protein purification. The SP group is preferably an ion exchange group. Finally, the purified product can be further purified by reversed phase high performance liquid chromatography (RP-HPLC). All of the above purification steps can be combined in different ways to achieve a substantially uniform protein purity.

The expressed fusion polypeptide can be purified using an affinity column containing an antibody, receptor or ligand to the IL-2 mutant or CD20 monoclonal antibody. The fusion polypeptide bound to the affinity column can be eluted by conventional methods, such as high salt buffer, pH change, etc., depending on the characteristics of the affinity column used.

The recombinant protein and the nucleic acid encoding the recombinant protein can be prepared by any suitable method, such as chemical synthesis, recombinant expression, or a combination thereof.

Use of fusion proteins

When the anti-CD 20 antibody and wild type IL-2 form a fusion protein, the inventor finds that IL-2 has high toxicity, causes harm to human bodies, causes poor drug effect, does not have high drug dosage, and further influences the dosage of the anti-CD 20 antibody part. The invention aims to overcome the defect, IL-2 is modified in two aspects, one aspect is that the combination of IL-2 and IL-2R alpha (CD25) is removed, the other aspect is that the affinity of IL-2 and IL-2R beta (CD122) is reduced, and IL-2 with toxicity which is properly regulated and greatly reduced is prepared into a fusion protein with a CD20 antibody.

The affinity of IL-2 and IL-2R beta (CD122) is further reduced while the combination of IL-2 and a receptor IL-2R alpha (CD25) is reduced or even eliminated, so that the toxic and side effects of the fusion protein on organisms can be further reduced, but the killing effect on tumors is not influenced.

One important reason for the toxicity of IL-2 in vivo is its strong affinity for IL-2R α, and the mutants of the present invention achieve substantially complete prevention of IL-2 binding to IL-2R α, thus completely eliminating the toxicity associated with IL-2R α binding. In contrast, the combination of IL-2 and IL-2R beta is related to the efficacy of IL-2, and too low combination can affect the effect, but too high combination can cause toxicity.

The fusion protein of the IL-2 mutant and the CD20 monoclonal antibody can be used for inhibiting tumors. The surface molecule on the cell surface of some tumor cells, such as cell lymphoma (including diffuse large B cell lymphoma) is CD20, when the fusion protein of the invention enters the body, the fusion protein has the characteristic of specifically binding CD20 to target the tumor, and can also play a role by binding phagocytes or killing cells, so that the tumor cells are phagocytized by the phagocytes or killed by the killing cells; alternatively, the antibody can be bound to CD20 to activate complement, which can "tunnel" through the lymphoma cells and cause them to die. Due to the specific targeting of the antibody, normal cells are less affected (normal B lymphocytes also have CD20 and are cleared, but most normal cells are not affected). Therefore, the fusion protein can realize targeted therapy aiming at tumor cells.

In some embodiments, the use/method may be an in vitro method, an in vivo method, or an ex vivo method.

The fusion protein of the anti-CD 20 antibody and the IL-2 mutant not only utilizes the fact that the anti-CD 20 antibody can effectively recognize and kill tumor cells, but also utilizes the fact that IL-2 can activate immune cells of an organism, and the fusion protein of the anti-CD 20 antibody and the IL-2 mutant can play a synergistic role, can attract the immune cells to the periphery of the tumor cells, and plays a better role in killing the tumor cells. By reducing or even eliminating the combination of IL-2 and a receptor IL-2R alpha (CD25), the toxic and side effects of the fusion protein on the body can be reduced while the function of the fusion protein for killing tumor cells is maintained.

The research result of the inventor shows that the fusion protein of the invention not only has very high curative effect, but also has low toxicity, and can greatly improve the dosage under the condition of controllable toxicity in the clinical treatment of tumors.

Pharmaceutical composition or kit

The present invention also provides a pharmaceutical composition for inhibiting tumor, comprising: the fusion protein or the polynucleotide for coding the fusion protein, or an expression vector containing the polynucleotide or a recombinant cell for expressing the fusion protein; and a pharmaceutically or physiologically acceptable carrier.

Suitable pharmaceutically acceptable carriers are well known to those of ordinary skill in the art. Sufficient information about pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences. Pharmaceutically acceptable carriers in the compositions may comprise liquids such as water, phosphate buffered saline, ringer's solution, physiological saline, balanced salt solution, lyoprotectants such as glycerol or sorbitol, and the like. In addition, auxiliary substances, such as lubricants, glidants, wetting or emulsifying agents, pH buffering substances and stabilizers, such as albumin and the like, may also be present in these carriers.

In use, a safe and effective amount of the fusion protein of the invention or a polynucleotide encoding it, or an expression vector containing the polynucleotide or recombinant cell expressing the fusion protein, is administered to a mammal (e.g., a human), wherein the safe and effective amount is typically at least about 0.001 micrograms/kg of body weight, and in most cases no more than about 10 milligrams/kg of body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The precise effective amount for a subject will depend upon the size and health of the subject, the nature and extent of the disorder, and the therapeutic agent and/or combination of therapeutic agents selected for administration. The effective amount can be determined by routine experimentation for a given condition, as will be appreciated by a clinician.

The invention also provides a kit or kit comprising: the fusion protein or the polynucleotide for coding the fusion protein, or an expression vector containing the polynucleotide or a recombinant cell for expressing the fusion protein; or the pharmaceutical composition.

For convenience of clinical application, the pharmaceutical composition of the present invention may be contained in an administration device for injection (e.g., a needle for injection), in which the pharmaceutical composition may be contained in an amount administered at one time. The administration device for injection may be contained in a cartridge for convenient storage and use.

The kit or kit of the present invention may further comprise instructions for use, which will facilitate the use of the kit or kit in a proper manner by those skilled in the art.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Sequence information

The respective CDR regions are as follows:

HCDR1(SEQ ID NO: 4)：SYNMH；

HCDR2(SEQ ID NO: 5)：AIYPGNGDTSYNQKFKG；

HCDR3(SEQ ID NO: 6)：STYYGGDWYFNV；

LCDR1(SEQ ID NO: 7)：RASSSVSYIH；

LCDR2(SEQ ID NO: 8)：ATSNLAS；

LCDR3(SEQ ID NO: 9)：QQWTSNPPT。

example 1 mutation of IL-2 and fusion thereof with Rituximab

1. Establishment of mutants

In order to achieve the goal of reducing IL-2 toxicity, the present inventors have conducted repeated studies and experiments to analyze many sites of IL-2 sequence to determine a means for reducing toxicity while maintaining its biological activity well. The inventors expect to remove the ability of IL-2 to bind to its receptor IL-2R α (CD25) but retain its good biological activity and to have a good effect after fusion with CD 20.

First, the present inventors replaced a part of the sequence derived from IL-15 in the IL-2 binding site with IL-2R α by replacing amino acid NNYKNPKLTRML (SEQ ID NO: 10) from position 29 to position 40 of IL-2 with QSMEIDAT (SEQ ID NO: 11) of IL-15.

Second, the inventors also mutated C at 125 to S. After mutation, the sequence is shown as RT001-10 in FIG. 1.

2. Establishment of fusion protein and research on effect and affinity

The structure of the fusion protein is shown in FIG. 2, the mutant IL-2 mutant is respectively connected to the end of one or two heavy chains of rituximab, and a section of polypeptide with the amino acid sequence of GGGSGGGSGGGS (SEQ ID NO: 12) is used as a linker in the middle. Taking the example of connecting the IL-2 mutant to the end of one of the heavy chains, the specific method is to adopt the knob-into-hole technology (oncotarget, 2017 Aug 1; 8(31): 51037 and 51049), connect the genes of the IL-2 mutant to the end of one of the heavy chain genes of rituximab respectively, the Fc segment of the heavy chain is transformed into a hole chain through gene mutation, construct the connected genes into the BstBI/PacI enzyme cutting site of the pCGS3(Merck) expression vector of the light chain gene with the rituximab already, and name the expression vector as pCGS3-RTX-LC-RTX-hole-HC-IL2 mutant 1. In addition, the heavy chain gene of the rituximab is transformed into a knob chain through gene mutation, the knob chain is constructed on a pCGS3 expression vector which already carries the light chain gene of the rituximab, and the expression vector is named as pCGS 3-RTX-LC-RTX-knob-HC. The above two plasmids, pCGS3-RTX-LC-RTX-hole-HC-IL2 mutant1 and pCGS3-RTX-LC-RTX-knob-HC, were transfected into ExpCHO cells together and expressed, and the fusion protein (FIG. 2, panel) was obtained by purification, which the present inventors named RT 001-10. The same is true for the ligation of the IL-2 mutant to the end of the other heavy chain (FIG. 2 left panel); the same is true for the ligation of the IL-2 mutant to the ends of both heavy chains (FIG. 2, right panel).

The affinity of the fusion protein to CD25 was determined using a conventional Octet affinity meter. The results are shown in FIG. 3, and compared with the wild type IL-2 with obvious binding signal, the fusion protein RT001-10 of the mutant IL-2 has no binding signal at all, which shows that after mutation, the binding ability to CD25 is completely removed.

Example 2 further mutation of IL-2 and fusion thereof with Rituximab

After obtaining a fusion protein of an IL-2 mutant without the binding ability of CD25, the inventors further performed mutagenesis.

Through intensive research, the inventors selected 4 mutation modes, which are as follows:

the first method comprises the following steps: mutating the 16 th H to E;

and the second method comprises the following steps: mutating D at position 84 to T;

and the third is that: mutating the 88 th N to A;

and fourthly: the 92 nd I was mutated to A.

In the same manner as in example 1, the present inventors constructed the genes of these human IL-2 mutants at the end of one of the genes of the heavy chain of rituximab, respectively, constructed the ligated genes on the pCGS3 expression vector that already carries the gene of the light chain of rituximab, and named pCGS3-RTX-LC-RTX-hole-HC-IL2 reduced. The pCGS3-RTX-LC-RTX-hole-HC-IL2 reduced and pCGS3-RTX-LC-RTX-knob-HC were transfected into ExpicHO cells together for expression, and the fusion protein was obtained by purification.

The names of the fusion proteins and the corresponding IL-2 mutation patterns are shown in Table 1.

TABLE 1

The affinity of the fusion protein to CD122 was tested by ELISA. The results are shown in FIG. 4, and the fusion proteins with four human IL-2 mutants, H16E, D84T, N88A and I92A respectively, have significantly reduced maximum signal values of A450 compared with RT001-10, which indicates that the four mutations all can significantly reduce the affinity of the fusion protein to CD 122.

Example 3 affinity of the fusion protein of the invention to CD20 on cell membranes

By adopting flow cytometry, the fusion proteins of different IL-2 mutants and Raji cells of human lymphoma cells expressing CD20 are incubated firstly, then unbound antibodies are washed away, secondary antibodies of anti-human Fc antibodies with PE labels are added, and after incubation, the unbound secondary antibodies are washed away and detected by a flow cytometer.

The results are shown in fig. 5, after the mutated IL-2 and anti-CD 20 antibody are constructed into a fusion protein, the affinity of the fusion protein to CD20 on the cell membrane is similar to that of rituximab, i.e., the fusion protein is mutated for the IL-2 part, and the affinity of the anti-CD 20 antibody part to CD20 is completely retained.

Example 4 activation of the T cell pSTAT5 Signaling pathway by fusion proteins of the invention

The fusion proteins were incubated with T cells isolated from human PBMC and pSTAT signals were detected using the pSTAT5 kit (Cisbio).

As shown in FIG. 6A, compared with wild type IL2, the fusion protein RT001-10 formed by fusing the IL-2 mutant obtained by the method of the invention and the anti-CD 20 antibody has a significant reduction in the activation function of the T cell pSTAT5 signal channel, which is about 10 times lower. The toxicity of the fusion protein RT001-10 obtained by the method of the invention in vivo is greatly reduced.

As shown in FIG. 6B, the fusion proteins RT001-24, RT001-27, RT001-30 and RT001-33 formed by fusing the IL-2 mutant obtained by the method of the present invention with the anti-CD 20 antibody significantly reduce the activation function of the T cell pSTAT5 signal channel by about 2-30 times compared with the fusion protein RT001-10, wherein RT001-33 is reduced by the most times and RT001-30 times.

This result demonstrates that the fusion proteins RT001-24, RT001-27, RT001-30, RT001-33 obtained by the method of the invention are less toxic in vivo than RT 001-10.

Example 5 killing function of the fusion protein of the present invention against tumor cells

Mixing the fusion protein with Raji cells (human Burkitt lymphoma cells), adding human PBMC, putting the mixture into a CO2 incubator to incubate for 20-24 hours, and finally detecting the killing capacity of the fusion protein on the Raji cells by using an LDH kit (Invitrogen).

The results are shown in fig. 7, and the killing function of the fusion protein on tumor cells is obviously superior to that of rituximab. However, the killing effect on tumor cells is good whether only CD25 binding (RT001-10) is removed or the fusion proteins (RT001-24, RT001-27, RT001-30 and RT001-33) which are combined with CD25 and reduce IL-2 mutant combined with CD122 are removed at the same time, which shows that the fusion protein with IL-2 mutant has the capability of activating immune cells although the affinity with IL-2 receptor is reduced, so the killing effect on tumor cells is obviously better than that of rituximab alone.

Example 6 function of anti-CD 20 fusion protein with IL-2 of different Structure

Based on the IL-2 mutant in RT001-10, the IL-2 mutant was ligated to the end of either the knob heavy chain or the hole heavy chain of rituximab, and the function of the fusion protein was verified in the presence or absence of a linker (linker) (see FIG. 8). The names and corresponding structures of the fusion proteins are shown in table 2.

TABLE 2

The affinity of the fusion protein with different structures to CD20 on the cell membrane was verified by flow cytometry, and the result is shown in FIG. 9, and the affinity of the fusion protein to CD20 on the cell membrane was high regardless of whether IL-2 was attached to the end of the heavy chain of knob or hole, or a linker was present.

By examining the activation function of the fusion protein on the T cell pSTAT5 signal path, the result is shown in FIG. 10, and whether IL-2 is connected to the end of the heavy chain of knob or hole, or whether a linker exists, the activation function of the fusion protein on the T cell pSTAT5 signal path is obviously inhibited.

By detecting the killing function of the fusion protein to the tumor cells, the result is shown in FIG. 11, and no matter IL-2 is connected to the end of the heavy chain of knob or hole, or a linker exists or not, the killing function of the fusion protein to the tumor cells is ideal and superior to that of rituximab.

Example 7 functional Change of IL-2 mutants grafted at different termini of anti-CD 20 antibody

In this example, the function of a fusion protein in which an IL-2 mutant was ligated to the ends of both heavy chains of an anti-CD 20 antibody was verified.

Based on the IL-2 mutant in RT001-10, the IL-2 mutant was ligated to the ends of both heavy chains of rituximab, the structure is shown in FIG. 2, and the names and corresponding structures of the fusion proteins are shown in Table 3.

TABLE 3

The affinity of the fusion protein of different IL-2 mutants with CD20 on cell membrane was verified by flow cytometry, and the results are shown in FIG. 12, where the affinity of the fusion protein with CD20 on cell membrane is high.

By examining the activation function of the fusion protein on the signal path of T cell pSTAT5, the results are shown in FIG. 13, wherein the signal of RT001-13 is stronger than that of RT001-10, which indicates that the activation function of the fusion protein with two IL-2 on the signal path of T cell pSTAT5 is stronger than that of the fusion protein with only one IL-2.

RT001-14 and RT001-15 signal less strongly than RT001-10, indicating that fusion proteins with reduced affinity for CD122 will also have reduced T cell activation.

By detecting the killing function of the fusion protein on the tumor cells, the result is shown in fig. 14, the killing function of the fusion protein on the tumor cells is very ideal, and the result shows that the fusion protein with the IL-2 mutant has the capability of activating immune cells although the affinity with an IL-2 receptor is reduced, so that the killing effect on the tumor cells is obviously better than that of the rituximab alone.

Example 8 toxicity of the fusion protein of the invention in animals

The inventor adopts SCID mice and injects fusion proteins of different IL-2 mutants into the abdominal cavity to research the toxicity of the fusion proteins in animals. A total of 6 groups of 6 mice were prepared, in which RT001-10, RT001-24, RT001-33, RT001-14 and the fusion protein of wild type IL-2 to rituximab were injected at a dose of 3.3mg/kg once a week. The control group was injected with rituximab at a dose of 3mg/kg once a week. The results of the experiment are shown in FIG. 15:

the mice injected with rituximab in the control group were completely alive and normal in body weight, indicating that rituximab has little toxicity to the mice.

Mice injected with wild-type IL-2 all died within 5 days, indicating that wild-type IL-2 is very toxic to mice.

2 mice injected with RT001-10 died, and the body weight of other mice also decreased significantly, indicating that RT001-10 has relatively significant toxicity to mice, but lower toxicity than wild-type IL-2.

Mice injected with RT001-24, RT001-33 and RT001-14 all survived despite partial weight loss, indicating that RT001-24, RT001-33 and RT001-14 have minimal toxicity to the mice.

The above results demonstrate that the fusion protein with the IL-2 mutant bound to CD25 removed can reduce its toxicity in vivo, while the fusion protein with the IL-2 mutant bound to CD25 and reduced to CD122 can significantly reduce its toxicity in vivo.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

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Claims

1. A method of engineering an anti-CD 20 monoclonal antibody to improve its pharmaceutical efficacy, comprising: linking an anti-CD 20 monoclonal antibody to the IL-2 mutant; wherein the IL-2 mutant has a mutation in the IL-2 receptor binding region such that binding to its receptor is inhibited; the receptor binding region comprises: a binding region for IL-2 to IL-2R α, and/or a binding region for IL-2 to IL-2R β; wherein in the IL-2 mutant, the amino acid NNYKNPKLTRML from the 29 th position to the 40 th position of the IL-2 is replaced by QSMEIDAT, and the C at the 125 th position is replaced by S; the amino acid of the IL-2 is shown in SEQ ID NO. 1.

2. The method of claim 1, further comprising a mutation selected from the group consisting of:

(1) mutating the 16 th H to E;

(2) mutating D at position 84 to T;

(3) mutating the 88 th N to A;

(4) the 92 nd I was mutated to A.

3. The method of claim 1, wherein said IL-2 mutant is linked to the end of one or both heavy chains of said anti-CD 20 monoclonal antibody; and/or

A linker of 4-20 amino acids between the amino acid sequence of the anti-CD 20 monoclonal antibody and the amino acid sequence of the IL-2 mutant; and/or

When the IL-2 mutant is connected to the end of one heavy chain of the anti-CD 20 monoclonal antibody, two heavy chains of the anti-CD 20 monoclonal antibody are respectively provided with a Knob and a Hole to form a Knob-into-Hole structure.

4. The method of claim 1, wherein said anti-CD 20 monoclonal antibody comprises an IgG1, IgG2, or IgG4 type antibody; or

The anti-CD 20 monoclonal antibodies include: monovalent antibodies, single chain antibodies, diabodies, chimeric antibodies; or

The anti-CD 20 monoclonal antibodies include polypeptides comprising an antigen binding domain.

5. The method of claim 1, wherein said anti-CD 20 monoclonal antibody is rituximab.

6. A fusion protein, comprising: anti-CD 20 monoclonal antibodies and IL-2 mutants; wherein the IL-2 mutant has a mutation in the IL-2 receptor binding region such that binding to its receptor is inhibited; the receptor binding region comprises: a binding region for IL-2 to IL-2R α, and/or a binding region for IL-2 to IL-2R β; wherein in the IL-2 mutant, the amino acid NNYKNPKLTRML from the 29 th position to the 40 th position of the IL-2 is replaced by QSMEIDAT, and the C at the 125 th position is replaced by S; the amino acid of the IL-2 is shown in SEQ ID NO. 1.

7. The fusion protein of claim 6, further comprising a mutation selected from the group consisting of:

(1) the 16 th H mutation is E;

(2) the 84 th D mutation is T;

(3) the 88 th N is mutated into A;

(4) the 92 th I mutation is A.

8. The fusion protein of claim 6, wherein the IL-2 mutant is linked to the ends of one or both heavy chains of the anti-CD 20 monoclonal antibody; and/or

9. The fusion protein of claim 6, wherein the anti-CD 20 monoclonal antibody comprises an IgG1, IgG2, or IgG4 type antibody; or

10. The fusion protein of claim 6, wherein the anti-CD 20 monoclonal antibody is rituximab.

11. A nucleic acid molecule encoding the fusion protein of any one of claims 6 to 10.

12. A vector comprising the nucleic acid molecule of claim 11.

13. A genetically engineered cell comprising the vector of claim 12; or a cell having integrated into its genome the nucleic acid molecule of claim 11.

14. A method of making a fusion protein according to any one of claims 6 to 10, said method comprising: culturing the cell of claim 13, such that the cell produces the fusion protein.

15. Use of the fusion protein according to any one of claims 6 to 10 for the preparation of a pharmaceutical composition for inhibiting tumors.

16. A pharmaceutical composition for inhibiting a tumor, said pharmaceutical composition comprising:

(i) a fusion protein according to any one of claims 6 to 10; and

(ii) a biologically acceptable carrier.

17. A kit for inhibiting a tumor, comprising:

a fusion protein according to any one of claims 6 to 10; or

The pharmaceutical composition of claim 16.