IL-21 fusion protein targeting pd-1 and preparation method and application thereof

Abstract

The application relates to the technical field of biological medicine, in particular to an IL-21 fusion protein targeting PD-1 and a preparation method and application thereof. The IL-21 fusion protein targeting PD-1 is a fusion protein, which comprises an anti-PD-1 antibody and IL-21 in operable linkage; the IL-21 has an amino acid sequence shown in Seq ID No. 18 or 19. The application fuses the PD-1 antibody and the reformed IL-21, thereby obtaining a fusion protein which is stable in the body and has a targeted selection effect on PD-1 positive cells. It is verified that the fusion protein shows a significant anti-tumor effect, has good drug properties and exhibits great clinical application potential.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to an IL-21 fusion protein targeting PD-1 and its preparation method, nucleic acid molecules, vectors, cells and their construction methods and applications, and drugs containing the same. Background Technology

[0002] Anti-PD-1 or PD-L1 antibodies block the binding of PD-1 to its ligand PD-L1 on T cells, thereby relieving the inhibitory signal of PD-1 in T cells and restoring tumor-reactive CD8+. + The effector function of T cells has enabled complete and sustained tumor remission in patients with advanced or conventionally resistant cancers. Currently, over 20 anti-PD-1 and PD-L1 antibody drugs are approved for marketing in China and the United States, used to treat more than 20 types of cancer and nearly 100 indications, becoming a major means of clinical cancer treatment. However, even in clinically approved malignant tumor indications, the efficacy rate of anti-PD-1 or PD-L1 antibody therapy is low, only about 20%, and the proportion achieving sustained remission is even lower, less than 10%, meaning it is ineffective for the vast majority of cancer patients. To improve the efficacy of anti-PD-1 or PD-L1 antibody therapy for cancer, numerous combination therapies have been developed, including those with chemotherapy, targeted therapy, and radiotherapy. While some combination therapies have indeed shown significant improvement in efficacy compared to single anti-PD-1 or PD-L1 antibody therapy, the overall improvement in the efficacy of malignant tumor treatment is very limited.

[0003] Numerous studies have confirmed that the tumor therapeutic effect of anti-PD-1 or PD-L1 antibodies is mainly mediated by CD8. + T cell-mediated, tumor-reactive CD8 cells dependent on the functional exhaustion of the tumor microenvironment + T cell reactivation, proliferation, and effector functional cell differentiation. T cell activation, proliferation, differentiation, and effector responses are regulated by numerous signals, leading to the development of various dual-target fusion protein drugs, such as anti-PD-1 / VEGF bispecific antibodies.

[0004] Cytokines play a crucial regulatory role in the activation, proliferation, differentiation, and effector function of T cells, and hold great potential in immunotherapy. However, their systemic application faces many challenges, primarily including systemic toxicity and pharmacokinetic (PK) limitations. Cytokines target numerous cell types, and systemic application often leads to systemic toxicity. Furthermore, the short half-life of most cytokines limits their exposure and therapeutic efficacy. Therefore, this application aims to develop an in vivo targeted application strategy for cytokines. Summary of the Invention

[0005] Based on this, one or more embodiments of this application provide an IL-21 fusion protein targeting PD-1, its preparation method, and its application, particularly relating to an IL-21 fusion protein targeting PD-1, its preparation method, nucleic acid molecules, vectors, cells, their construction methods, and applications, and drugs containing the thereof. The technical solutions include the following:

[0006] One or more embodiments of this application provide an IL-21 fusion protein targeting PD-1, comprising an anti-PD-1 antibody and IL-21 operably linked together; said IL-21 has the amino acid sequence shown from position 1 to position 127 in Seq ID No. 18 or 19;

[0007] Optionally, the heavy chain variable region of the anti-PD-1 antibody has the amino acid sequences CDR1 to CDR3 as shown in Seq ID No. 48 to 50, and the light chain variable region has the amino acid sequences CDR1 to CDR3 as shown in Seq ID No. 51 to 53.

[0008] Optionally, the anti-PD-1 antibody may be a humanized anti-PD-1 antibody or a mouse-derived anti-PD-1 antibody.

[0009] In some embodiments of this application, the IL-21 is operatively linked to one or two heavy chains of the anti-PD-1 antibody; or / and, the IL-21 is operatively linked to the C-terminus of the heavy chain of the anti-PD-1 antibody.

[0010] In some embodiments of this application, the anti-PD-1 antibody has a heavy chain variable region with an amino acid sequence as described in any one of Seq ID Nos. 1 to 5; or / and, the anti-PD-1 antibody has a light chain variable region with an amino acid sequence as described in any one of Seq ID Nos. 6 to 10.

[0011] In some embodiments of this application, the anti-PD-1 antibody has a heavy chain variable region as shown in Seq ID No. 5 and a light chain variable region as shown in Seq ID No. 10.

[0012] In some embodiments of this application, the anti-PD-1 antibody has a constant region of amino acid sequence as shown in Seq ID No. 55 and / or Seq ID No. 56.

[0013] In some embodiments of this application, the operable connection uses a connector including (G4S)n, where n is an integer in the range of 2-4.

[0014] One or more embodiments of this application provide a PD-1 specific binding protein, wherein the specific binding protein is a humanized anti-PD-1 antibody as defined above.

[0015] One or more embodiments of this application provide a nucleic acid molecule that encodes the IL-21 fusion protein targeting PD-1.

[0016] In some embodiments of this application, the nucleotide sequence of the nucleic acid molecule satisfies one or more of the conditions shown in (1) and (2) below:

[0017] (1) Includes any of the nucleotide sequences shown in Seq ID No. 30 to 33; or / and includes any of the nucleotide sequences shown in Seq ID No. 35 to 38;

[0018] (2) Includes the nucleotide sequence shown in positions 1 to 381 of Seq ID No. 43 or 44;

[0019] Optionally, the nucleotide sequence of the nucleic acid molecule includes any of the nucleotide sequences shown in Seq ID No. 41 to 42.

[0020] One or more embodiments of this application provide a vector, wherein the nucleotide sequence of the vector comprises the nucleotide sequence of the nucleic acid molecule.

[0021] One or more embodiments of this application provide a cell that expresses the IL-21 fusion protein targeting PD-1, the nucleic acid molecule described herein, or the vector described herein.

[0022] One or more embodiments of this application provide a method for constructing the cell described above, the method comprising the step of introducing the nucleic acid molecule or the vector into the cell to be modified.

[0023] One or more embodiments of this application provide a method for preparing the IL-21 fusion protein targeting PD-1, the preparation method including the step of culturing the cells.

[0024] One or more embodiments of this application provide the use of the PD-1-targeting IL-21 fusion protein, the nucleic acid molecule, the vector, or the cell in the preparation of an antitumor drug.

[0025] In some embodiments of this application, the antitumor drug is used to treat solid tumors; optionally, the solid tumor is lung cancer, melanoma, head and neck malignant tumor, liver cancer, pancreatic cancer, renal cell carcinoma, urothelial carcinoma, bladder cancer, prostate cancer, gastric cancer, esophageal cancer, intestinal cancer, cervical cancer, ovarian cancer, breast cancer, thyroid cancer, or malignant tumors of the nervous system.

[0026] In some embodiments of this application, the antitumor drug is used to prevent and treat hematologic malignancies; optionally, the hematologic malignancies are lymphoma or leukemia.

[0027] One or more embodiments of this application provide a medicament comprising the PD-1-targeting IL-21 fusion protein and a pharmaceutically acceptable carrier.

[0028] Compared with traditional technologies, this application has the following advantages:

[0029] This application fuses a PD-1 antibody with a modified IL-21 to obtain a stable fusion protein in vivo that has targeted selectivity for PD-1 positive cells. The fusion protein has been verified to have significant anti-tumor effects, good drug-like properties, and great potential for clinical application. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This involves the binding of humanized antibodies to the PD-1 protein.

[0032] Figure 2 It has the blocking function of humanized antibodies.

[0033] Figure 3 Schematic diagram of the construction of different fusion proteins.

[0034] Figure 4 Flowchart for constructing the antibody heavy chain-IL-21 fusion protein.

[0035] Figure 5 This is a schematic diagram of the antibody-IL-21 fusion protein structure.

[0036] Figure 6 Comparison of blood drug stability of different fusion proteins in mice; where: left figure, blood drug concentration of the antibody portion of the fusion protein; right figure, blood drug concentration of the full-length fusion protein.

[0037] Figure 7 This refers to the binding of PD-1AbIL-21 with PD-1.

[0038] Figure 8 This represents the blocking effect of PD-1AbIL-21 on the PD-1 / PD-L1 interaction.

[0039] Figure 9 It is a combination of PD-1AbIL-21 and IL-21R.

[0040] Figure 10 PD-1AbIL-21 fusion protein activates STAT3 phosphorylation.

[0041] Figure 11 For PD-1AbIL-21 fusion protein against PD-1 + Selective targeting of cells.

[0042] Figure 12 The PD-1AbIL-21 fusion protein showed significant anti-tumor therapeutic effects in a mouse melanoma model; where: a is the mouse melanoma model construction and experimental design diagram; b is the tumor growth curve of the mouse melanoma model; c is the mouse survival curve.

[0043] Figure 13 The PD-1AbIL-21 fusion protein showed significant anti-tumor therapeutic effects in a mouse pancreatic cancer model; where: a) is the construction and experimental design diagram of the mouse pancreatic cancer model; b) is the tumor growth curve of the mouse pancreatic cancer model; and c) is the survival curve of the mouse.

[0044] Figure 14 The PD-1AbIL-21 fusion protein showed significant anti-tumor therapeutic effects in a mouse Lewis lung cancer model; where: a) is the construction and experimental design diagram of the mouse Lewis lung cancer model; b) is the tumor growth curve of the mouse Lewis lung cancer model; c) is the mouse survival curve.

[0045] Figure 15 The PD-1AbIL-21 fusion protein showed significant anti-tumor therapeutic effects in a humanized mouse breast cancer model; where: a) diagram of the construction and experimental design of the humanized mouse breast cancer model; b) tumor growth curve of the humanized mouse breast cancer model; c) survival curve of the humanized mouse.

[0046] Figure 16 The PD-1AbIL-21 fusion protein has significant anti-tumor therapeutic effects in a humanized mouse leukemia model; where: a) is the diagram of the construction and experimental design of the humanized mouse leukemia model; b) is the tumor growth curve of the humanized mouse leukemia model; c) is the survival curve of the humanized mouse.

[0047] Figure 17 The PD-1AbIL-21 fusion protein showed significant anti-tumor therapeutic effects in a humanized mouse lymphoma model. Among them: a) diagram of the construction and experimental design of the humanized mouse lymphoma model; b) tumor growth curve of the humanized mouse lymphoma model; c) survival curve of the humanized mouse; d) changes in the proportion of hCD3+hCD45+ T cells in the peripheral blood of mice at different time points after treatment. Figure 18 A comparison of the in vivo antitumor effects of IL-21M2 fusion protein and natural IL-21 fusion protein. Detailed Implementation

[0048] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0050] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0051] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0052] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0053] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0054] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0055] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0056] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0057] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0058] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0059] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0060] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0061] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0062] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass-volume percentage.

[0063] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0064] Despite the immense potential of cytokines in immunotherapy, systemic application presents numerous challenges, including pharmacokinetic (PK) limitations and side effects. The short half-lives of most cytokines restrict their exposure and efficacy. More critically, the numerous target cells for cytokines mean that systemic application can lead to severe side effects, and some cytokines can simultaneously activate immunosuppressive signaling pathways, inhibiting anti-tumor immunity. Therefore, targeting cytokines to tumor-specific T cells can significantly enhance their anti-tumor efficacy and reduce side effects.

[0065] In cancer patients, tumor-specific CD8 + T cells express high levels of PD-1. The presence of PD-1 can differentiate tumor-reactive CD8+ cells. + T lymphocyte pool. Within tumor-infiltrating lymphocytes (TILs), only the PD-1 positive cell population contains T lymphocytes specific to tumor neoantigens or related antigens (e.g., Melan-A). This is in contrast to PD-1 cells from TILs. - Compared to T cells, only PD-1 + Adoptive T-cell transfer therapy has shown promising therapeutic effects in mouse tumor models. Furthermore, the presence of CD8+ with high PD-1 expression in the tumor further influences the outcome. + T cells can predict remission and survival in lung cancer patients receiving PD-1 blockade therapy. Therefore, PD-1 is considered a molecular marker of activated tumor-reactive T cells. Thus, by fusing IL-21 with an anti-PD-1 antibody, IL-21 can be specifically targeted and delivered to PD-1. + T cells significantly enhance their anti-tumor effects while reducing their side effects.

[0066] However, in practical applications, modifications to IL-21 primarily involve fusion with IgFc or with anti-albumin antibodies to prolong its half-life, but these strategies still fail to address the issue of systemic toxicity. Furthermore, while reducing the affinity of IL-21 for its receptor can extend its half-life, it also significantly reduces its biological activity (by 100-1000 times), making it difficult to achieve ideal anti-tumor efficacy even after forming fusion proteins with antibodies. On the other hand, the IL-21 protein exhibits poor structural stability; the C-helix and D-loop regions in its molecule can undergo conformational changes at different temperatures, leading to protein instability.

[0067] One or more embodiments of this application provide an IL-21 fusion protein targeting PD-1, comprising an anti-PD-1 antibody and IL-21 operably linked; said IL-21 has an amino acid sequence represented by positions 1 to 127 in Seq ID No. 18 or 19 or has a sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with said sequence;

[0068] Optionally, the heavy chain variable region of the anti-PD-1 antibody has the amino acid sequences CDR1 to CDR3 as shown in Seq ID No. 48 to 50, and the light chain variable region has the amino acid sequences CDR1 to CDR3 as shown in Seq ID No. 51 to 53.

[0069] Optionally, the anti-PD-1 antibody may be a humanized anti-PD-1 antibody or a mouse-derived anti-PD-1 antibody.

[0070] The antibody of this application may have the aforementioned CDRs, or a derivative fragment having the aforementioned CDRs. The derivative fragment is formed by replacing amino acids at no more than six sites relative to its corresponding CDR (“conservative modification” or “conservative substitution”), retaining the biological activity consistent with its corresponding complementarity-determining region. For example, the derivative fragment may replace one amino acid with another, or one amino acid with multiple amino acids (e.g., two), at sites 1, 2, 3, 4, 5, or 6 of its corresponding complementarity-determining region.

[0071] In the CDRs provided in this application, the derived fragments (conserved variants) refer to polypeptides formed by replacing one, two, or three amino acids with amino acids of similar or related properties compared to the amino acid sequence of the antibody in this application. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0072] Table A

[0073]

[0074] "Conservative modification" or "conservative substitution" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity), allowing for frequent alterations without changing the protein's biological activity. Those skilled in the art will recognize that, in general, the substitution of a single amino acid in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. (1987), Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to disrupt biological activity.

[0075] The three-letter and single-letter codes for amino acids used in this application are as described in J.biol.chem, 243, p3558 (1968).

[0076] The "antibody" mentioned in this application refers to immunoglobulins. A complete antibody is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of its hinge region and the number and position of disulfide bonds in its heavy chain, it can be further divided into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ chains or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ chain or a λ chain.

[0077] The sequence of approximately 110 amino acids near the N-terminus of both the antibody heavy and light chains varies considerably and is known as the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are called the constant region. The variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FR). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0078] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by grafting a non-human species' CDR sequence into the variable region framework of a human antibody, i.e., a human antibody framework sequence of different types. This overcomes the heterologous response induced by chimeric antibodies carrying a large number of heterologous protein components. Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences or from publicly available references. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available at www.mrccpe.com.ac.uk / vbase) and in Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse or reversion mutations can be performed on the aforementioned human antibody variable region framework sequence to maintain activity. The humanized antibodies in this application also include humanized antibodies further derived from yeast exhibiting affinity maturation mutations of CDR.

[0079] The "variants" of the heavy chain constant region and light chain constant region of human antibodies described in this application refer to prior art variants of the heavy chain constant region or light chain constant region derived from humans that do not alter the structure and function of the antibody variable region. Exemplary variants include IgG1, IgG2, IgG3, or IgG4 heavy chain constant region variants that involve site-specific modifications and amino acid substitutions in the heavy chain constant region; specific substitutions include prior art known YTE mutations, L234A and / or L235A mutations, S228P mutations, and / or mutations that obtain a knock-in-hole structure (giving the antibody heavy chain a knock-Fc and hole-Fc combination), which have been shown to give antibodies new properties without altering the function of the antibody variable region.

[0080] The terms "human antibody," "human-derived antibody," "fully human antibody," and "completely human antibody" are used interchangeably. An antibody can be derived from a human being or obtained from a transgenic organism "modified" to produce specific human antibodies in response to antigenic stimulation, and can be produced by any method known in the art. In some techniques, human heavy and light chain locus elements are introduced into cell lines derived from embryonic stem cell lines, where endogenous heavy and light chain loci are targeted and disrupted. The transgenic organism can synthesize human antibodies specific to human antigens, and can be used to produce hybridomas that secrete human antibodies. A human antibody can also be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources. Completely human antibodies can also be constructed using gene or chromosome transfection methods and phage display technology, or from in vitro activated B cells, all of which are known in the art.

[0081] The terms “full-length antibody,” “intact antibody,” “complete antibody,” and “all antibody” are used interchangeably herein to refer to an antibody in substantially its complete form, as distinguished from the antigen-binding fragment as defined below. This term specifically refers to antibodies containing constant regions in both the light and heavy chains. The term “antibody” in this application includes “full-length antibodies” and their antigen-binding fragments.

[0082] The full-length antibody of this application includes the full-length antibody formed by linking the light chain variable region with the light chain constant region and the heavy chain variable region with the heavy chain constant region in the light and heavy chain variable region combination in the following embodiments. Those skilled in the art can select different antibody sources of light chain constant regions and heavy chain constant regions according to actual needs, such as light chain constant regions and heavy chain constant regions derived from human antibodies.

[0083] The term "antigen-binding fragment" or "functional fragment" of an antibody refers to one or more fragments that retain the ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used for antigen-binding function. Examples of binding fragments included in the term "antigen-binding fragment" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments connected by disulfide bridges on the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VH and VL domains on a single arm of the antibody; (v) dsFv, an antigen-binding fragment formed by interchain disulfide bonds between VH and VL; and (vi) bispecific, bispecific, and multispecific antibodies containing fragments such as scFv, dsFv, and Fab. Furthermore, although the two domains VL and VH of the Fv fragment are linked by a synthetic linker, enabling it to produce a single protein chain (referred to as a single-chain Fv (scFv) where the VL and VH regions pair to form a monovalent molecule; see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also included in the term "antigen-binding fragment" of antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding moieties can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0084] Fab is an antibody fragment with antigen-binding activity obtained by treating IgG antibody molecules with an enzyme of the same activity as papain.

[0085] F(ab')2 is an antibody fragment with antigen-binding activity obtained by digesting IgG with an enzyme of the same activity as pepsin.

[0086] Fab' is an antibody fragment with antigen-binding activity obtained by cleaving the above-mentioned F(ab')2.

[0087] In addition, the Fab' can be produced by inserting DNA encoding the Fab' fragment into an expression vector and then introducing the vector into a host.

[0088] The terms "single-chain antibody," "single-chain Fv," or "scFv" refer to molecules that contain a variable domain (or region; VH) of the antibody heavy chain and a variable domain (or region; VL) of the antibody light chain linked by a linker. Such scFv molecules can have a generic structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Other connectors that may be used in this application are described in the following literature, for example, but not limited to: Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Alfthan et al. (1995), Protein Eng. 8:725-731; Choi et al. (2001), Eur. J. Immunol. 31:94-106; Hu et al. (1996), Cancer Res. 56:3055-3061; Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

[0089] dsFv is obtained by linking polypeptides in which one amino acid residue in each VH and VL is replaced by a cysteine ​​residue via disulfide bonds between cysteine ​​residues. The amino acid residues to be replaced by cysteine ​​residues can be selected based on the prediction of the antibody's three-dimensional structure using known methods (e.g., Protein Engineering, 7, 697 (1994)).

[0090] The term "amino acid difference" or "amino acid mutation" refers to an alteration or mutation of amino acids in a variant protein or polypeptide compared to the original protein or polypeptide. This includes the insertion, deletion, or substitution of one, two, three, or more amino acids in the original protein or polypeptide.

[0091] The term "antibody framework" or "FR region" refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain. Essentially, it is a variable domain without a CDR.

[0092] The terms "complementarity-determining region," "CDR," or "hypervariant region" refer to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. Typically, each heavy chain variable region contains three CDRs (HCDR1, HCDR2, HCDR3), and each light chain variable region contains three CDRs (LCDR1, LCDR2, LCDR3). The amino acid sequence boundaries of CDRs can be determined using any of a variety of well-known schemes, including the “Kabat” numbering rule (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the “Chothia” numbering rule (see B Al-Lazikani et al., JMol Biol. 1997 Nov 7;273(4):927-48.), and the ImMunoGeneTics (IMGT) numbering rule (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.P. et al., Dev Comp Immunol. 2003 Jan;27(1):55-77., etc.). For example, in the classic format, following Kabat rules, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Following Chothia rules, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions of Kabat and Chothia, the CDR is composed of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL.Following the IMGT rules, the CDR amino acid residues in VH are approximately numbered 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), while those in VL are approximately numbered 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3). Following the IMGT rules, the CDR regions of antibodies can be determined using the IMGT / DomainGap Align procedure.

[0093] The term "epitope" or "antigenic determinant" refers to a site on an antigen that is bound by immunoglobulins or antibodies. Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a distinctive spatial conformation. See, for example, Epitope Mapping Protocols: Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

[0094] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to a pre-defined epitope on an antigen. Typically, antibodies bind at a concentration of approximately less than 10... -8 M, for example, approximately less than 10 -9 M, 10 - 10 M, 10 -11 M, 10 -12 M or a smaller equilibrium dissociation constant (KD) are combined.

[0095] The term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. Typically, the antibodies in this application have a dissociation equilibrium constant of less than approximately 10. -7 M, for example, less than approximately 10 -8 M or 10 -9 The dissociation equilibrium constant (KD) of M binds to the antigen or its epitope. For example, in this application, the affinity of the antibody for the cell surface antigen is determined by the FACS method to determine the KD value.

[0096] In some embodiments of this application, the IL-21 is operatively linked to one or two heavy chains of the anti-PD-1 antibody; or / and, the IL-21 is operatively linked to the C-terminus of the heavy chain of the anti-PD-1 antibody.

[0097] "Identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit—for example, if every position in two DNA molecules is occupied by adenine—then the molecules are homologous at that position. The percentage of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared multiplied by 100. For example, in optimal sequence alignment, if 6 out of 10 positions in two sequences match or are homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in two sequences match or are homologous, then the two sequences are 95% homologous. Typically, comparisons are made when aligning two sequences to give the maximum percentage of identity. For example, comparisons can be performed using the BLAST algorithm, where the algorithm's parameters are chosen to give the maximum match between the sequences over the entire length of each reference sequence. The following references relate to the BLAST algorithm commonly used in sequence analysis: BLAST ALGORITHMS: Altschul, S.F. et al., (1990) J. Mol. Biol. 215:403-410; Gish, W. et al., (1993) Nature Genet. 3:266-272; Madden, T.L. et al., (1996) Meth. Enzymol. 266:131-141; Altschul, S.F. et al., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J. et al., (1997) Genome Res. 7:649-656. Other common BLAST algorithms, such as those provided by NCBI BLAST, are also well-known to those skilled in the art.

[0098] In some embodiments of this application, the anti-PD-1 antibody has a heavy chain variable region with an amino acid sequence as described in any one of Seq ID Nos. 1 to 5, or has an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of Seq ID Nos. 1 to 5.

[0099] In some embodiments of this application, the anti-PD-1 antibody has a light chain variable region with an amino acid sequence as described in any one of Seq ID Nos. 6 to 10, or has an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of Seq ID Nos. 6 to 10.

[0100] In some embodiments of this application, the anti-PD-1 antibody has a heavy chain variable region as shown in Seq ID No. 5 and a light chain variable region as shown in Seq ID No. 10.

[0101] In some embodiments of this application, the anti-PD-1 antibody has a constant region of amino acid sequence as shown in Seq ID No. 55 and / or Seq ID No. 56, or has a sequence of amino acid sequence identity with any one of Seq ID No. 55 and / or Seq ID No. 56 of not less than 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).

[0102] In some embodiments of this application, the operable connection uses a connector including (G4S)n, where n is an integer in the range of 2-4, for example, n is 2, 3, or 4.

[0103] One or more embodiments of this application provide a PD-1 specific binding protein, wherein the specific binding protein is a humanized anti-PD-1 antibody as defined above.

[0104] One or more embodiments of this application provide a nucleic acid molecule that encodes the IL-21 fusion protein targeting PD-1.

[0105] As used herein, the term "nucleic acid molecule" refers to DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, preferably double-stranded DNA or single-stranded mRNA or modified mRNA. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0106] The nucleic acid molecules in this application primarily refer to isolated nucleic acid molecules. "Isolated" means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth media. Generally, the term "isolated" is not intended to mean the complete absence of these materials or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the compounds described herein.

[0107] In some embodiments of this application, the nucleotide sequence of the nucleic acid molecule satisfies one or more of the conditions shown in (1) and (2) below:

[0108] (1) Includes any of the nucleotide sequences shown in Seq ID No. 30 to 33 or includes a sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity with such sequence; or / and includes any of the nucleotide sequences shown in Seq ID No. 35 to 38 or includes a sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity with such sequence;

[0109] (2) Includes the nucleotide sequence shown in positions 1 to 381 of Seq ID No. 43 or 44, or includes a sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity with that sequence.

[0110] Optionally, the nucleotide sequence of the nucleic acid molecule includes any of the nucleotide sequences shown in Seq ID No. 41 to 42 or includes a sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with that sequence.

[0111] One or more embodiments of this application provide a vector, wherein the nucleotide sequence of the vector comprises the nucleotide sequence of the nucleic acid molecule.

[0112] The term "vector," also known as "expression vector," refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop to which an additional DNA segment can be linked. In another embodiment, the vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. The vectors disclosed herein are capable of autonomous replication in host cells upon introduction (e.g., bacterial vectors with bacterial origins of replication and augmented mammalian vectors) or can be integrated into the host cell's genome after introduction, thereby replicating along with the host genome (e.g., non-augmented mammalian vectors). In general, vectors may be selected from, but are not limited to, mammalian cell viruses, bacterial plasmids, bacteriophages, yeast plasmids, or combinations thereof.

[0113] One or more embodiments of this application provide a cell that expresses the IL-21 fusion protein targeting PD-1, the nucleic acid molecule, or the vector.

[0114] The term "cell," also known as "host cell," refers to a cell into which an expression vector has been introduced. Host cells can include bacteria, microorganisms, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; members of the Bacillaceae family, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0115] The terms “cell,” “cell line,” and “cell culture” used herein are used interchangeably, and all such names include progeny. Therefore, “transformant” and “transformed cell” include primary test cells and cultures derived from them, regardless of passage number. It should also be understood that, due to intentional or unintentional mutations, all progeny cannot be exactly identical in DNA content. This includes mutant progeny with the same function or biological activity as those screened from the original transformed cells. Where different names are intended, the context will be clear.

[0116] One or more embodiments of this application provide a method for constructing the cell described above, the method comprising the step of introducing the nucleic acid molecule or the vector into the cell to be modified.

[0117] One or more embodiments of this application provide a method for preparing the IL-21 fusion protein targeting PD-1, the preparation method including the step of culturing the cells.

[0118] The proteins described in this application can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect host cells. As a more preferred prior art, mammalian expression systems lead to antibody glycosylation, particularly at the highly conserved N-terminal site in the Fc region. Stable clones are obtained by expressing the specific binding protein, and positive clones are scaled up in a bioreactor to produce antibodies. The culture medium secreting the antibody can be purified using conventional techniques, such as using an A or GSepharose FF column with adjusted buffer. Non-specifically bound components are washed away. The bound antibody is then eluted using a pH gradient, and antibody fragments are detected by SDS-PAGE and collected. The antibody can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange. The resulting product should be immediately frozen, e.g., at -70°C, or lyophilized.

[0119] One or more embodiments of this application provide the use of the PD-1-targeting IL-21 fusion protein, the nucleic acid molecule, the vector, or the cell in the preparation of an antitumor drug.

[0120] In some embodiments of this application, the antitumor drug is used to treat solid tumors; optionally, the solid tumor is lung cancer, melanoma, head and neck malignant tumor, liver cancer, pancreatic cancer, renal cell carcinoma, urothelial carcinoma, bladder cancer, prostate cancer, gastric cancer, esophageal cancer, intestinal cancer, cervical cancer, ovarian cancer, breast cancer, thyroid cancer, or malignant tumors of the nervous system.

[0121] In some embodiments of this application, the antitumor drug is used to prevent and treat hematologic malignancies; optionally, the hematologic malignancies are lymphoma or leukemia.

[0122] One or more embodiments of this application provide a medicament comprising the PD-1-targeting IL-21 fusion protein and a pharmaceutically acceptable carrier.

[0123] "Drug" or "pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0124] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for delivering antibody or antigen-binding fragments. Carriers can be anti-adhesion agents, adhesives, coatings, disintegrants, fillers or diluents, preservatives (such as antioxidants, antibacterial agents, or antifungal agents), sweeteners, absorption delay agents, wetting agents, emulsifiers, buffers, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (such as olive oil), saline, buffer solutions, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.

[0125] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0126] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0127] Example 1: Humanization of anti-PD-1 monoclonal antibody

[0128] A murine anti-PD-1 antibody with blocking activity was previously screened using hybridoma technology. Its heavy chain variable region (VH) and light chain variable region (VL) amino acid sequences are: Seq ID No. 1 (119 aa) and Seq ID No. 6 (107 aa), respectively; the corresponding nucleotide sequences are: Seq ID No. 29 (357 bp) and Seq ID No. 34 (321 bp), respectively. Humanization of this antibody was performed using the following experimental procedure:

[0129] Based on the obtained murine anti-PD-1 antibody gene coding sequence, the heavy chain and light chain CDR sequences of the antibody were compared with the fully human antibody Germline database to obtain highly homologous human Germline templates. The human Germline light chain framework region was derived from the human K light chain gene, with human germline light chain templates IGKV4-1*01 and IGKJ4*01 being preferred; the human germline heavy chain framework region was derived from the human heavy chain gene, with human germline heavy chain templates IGHV3-23*04 and IGHJ4*01 being preferred. The murine antibody CDR was then transplanted onto the selected humanized templates, replacing the humanized framework region, to obtain the CDR-transplanted antibody (cPD-1Ab).

[0130] Then, based on the three-dimensional structure of the murine antibody, reverse mutations were performed on embedded residues, residues that directly interact with the CDR region, and residues that significantly affect the conformation of VL and VH, resulting in a series of humanized light and heavy chain molecules. The amino acid sequences of the humanized heavy chain variable region molecules are: Seq ID No. 2-5; the amino acid sequences of the humanized light chain variable region molecules are: Seq ID No. 7-10. The heavy and light chain amino acid sequences of the designed CDR transplantation antibody, along with the signal peptide sequence Seq ID No. 54 (encoding nucleic acid as shown in Seq ID No. 57), were codon-optimized and synthesized (Beijing Qingke Biotechnology Co., Ltd.). After PCR amplification, the obtained DNA amplified fragments were seamlessly cloned and ligated into pCDNA3.1 vectors containing constant regions (Seq ID No. 55 / Seq ID No. 58, Seq ID No. 56 / Seq ID No. 59), transformed into Top10 competent bacteria, and positive clones were screened and identified, verified by sequencing. All other humanized sequences were obtained by introducing corresponding mutations into the CDR transplantation antibody sequence via QuickChange PCR. The molecular pairings of the heavy chain variable region and the light chain variable region are shown in Table 1.

[0131] Table 1. Heavy / light chain pairing of chimeric and humanized PD-1 antibodies

[0132]

[0133] Example 2: Preparation and functional identification of humanized anti-PD-1 antibody

[0134] 1. Preparation of anti-PD-1 antibody

[0135] Next, the constructed antibody vectors were transfected into 293F cells (Thermo Fisher Scientific) according to the heavy and light chain combinations in Table 1 to complete the preparation of chimeric and humanized PD-1 antibodies. The specific methods are as follows:

[0136] (1) Plasmid transfection and protein expression

[0137] 1) Add 30 μg of target plasmid DNA (heavy chain to light chain ratio of 1:1) to 1.8 mL of Opit-MEM serum-depleted medium, and add 120 μL of PEI to 1.68 mL of Opit-MEM serum-depleted medium. Gently vortex to mix and incubate at room temperature for 5 min. Add the diluted PEI dropwise to the diluted plasmid DNA, gently vortex to mix, and incubate at room temperature for 15-20 min.

[0138] 2) Slowly add the PEI / DNA complex dropwise to 30 mL of 293F cells (1 mL of cells corresponds to 1 μg of DNA transfection). Gently shake the culture flask during the addition process, then place it on a track shaker and culture the cells at 37°C and 8% CO2. 18-22 hours after transfection, add Profeed (Optimum Nutrition) feed to the culture flask to a final concentration of 5% of the total volume.

[0139] 3) On day 4 after transfection, the density and viability of live cells were measured to identify the protein expression in the cell supernatant.

[0140] 4) When the cell viability is around 60% (around day 6), collect the cell supernatant and purify the protein.

[0141] (2) Protein purification

[0142] According to the instructions for AmMag ProteinA Magmetic Beads (GenScript, L00695-80), after mixing the beads with the supernatant and incubating at room temperature for 4 hours, the magnetic beads were adsorbed and aggregated using a magnetic rack. The supernatant was discarded, and the magnetic beads were washed with PBS. 50 mM pH 3.0 citric acid solution was added and eluted for 10 min. After removing the magnetic beads with a magnetic rack, 1 M pH 9.0 Tris-HCl was added to the elution buffer to adjust the pH, which is generally ±1 of the protein's pI. After filtration through a 0.22 μm filter membrane, the concentration was measured, and the product was aliquoted and stored at -80℃.

[0143] 2. Identification of the biological functions of anti-PD-1 antibodies

[0144] (1) Compare the binding of different anti-PD-1 antibodies to PD-1 molecules.

[0145] The binding interaction between anti-PD-1 antibody and PD-1 protein was detected using ELISA. The specific experimental procedure is as follows:

[0146] Add 1 μg / mL of PD-1-His protein (a fusion protein of the extracellular region of the human PD-1 receptor molecule and the His molecule tag, purchased from Sinocare) to the wells of an ELISA plate and incubate overnight at 4°C. Wash away unbound protein. Serially dilute the chimeric antibody or humanized antibody and add it to the wells coated with PD-1-His protein, incubating at room temperature for 1 hour. Wash away unbound antibody, add HRP-labeled anti-human IgG, and incubate at room temperature for 1 hour. Wash away unbound labeled antibody, add substrate reaction solution, and terminate the reaction by adding stop solution based on color change. Detect the absorbance of the reaction system using a Varioskan LUX multi-functional microplate reader. The obtained OD values ​​are fitted using a 4-factor formula, combined with the experimental formula: Y = Bottom + (Top-Bottom) / (1 + 10^(LogEC)). 50 -X)*HillSlope)).

[0147] The results are as follows Figure 1 As shown, the binding affinity of humanized anti-PD-1 antibodies to PD-1 molecules is similar to that of chimeric antibodies, while the EC50 of chimeric antibodies... 50 The EC50 of the humanized antibody was 0.02487 nM. 50 Between 0.010 and 0.022 nM, the affinity of the antibody increased slightly after humanization.

[0148] (2) Determination of the affinity of anti-PD-1 antibody for binding to PD-1 molecules

[0149] The affinity of different anti-PD-1 antibodies for PD-1 molecules was determined using the surface plasmon resonance (SPR) method. The specific experimental procedure is as follows:

[0150] The instrument used was a Biacore 8K with the Protein A S-series sensor chip. Antibodies were injected into the Protein A chip and captured by the chip. Binding phase: The antigen was serially diluted two-fold from 400 nM to 12.5 nM using running buffer (1×HBS-EP+ buffer pH 7.4) and injected into the flow cell on the chip surface. The binding time was 120 seconds. Dissociation phase: Immediately after binding, running buffer was injected, and the dissociation time was set to 600 seconds. Analysis was performed on the Biacore 8K using Biacore Insight Evaluation.

[0151] The results are shown in Table 2. The binding of the humanized anti-PD-1 antibody to the PD-1 molecule was similar to that of the chimeric antibody, and the affinity of the chimeric antibody to the PD-1 molecule was approximately 1.75 × 10⁻⁶. -8 M, after humanization, the affinity of the antibodies all increased slightly, ranging from 3.83 to 9.22 × 10. -9 Between M.

[0152] Table 2. Affinity test of humanized PD-1 antibody

[0153]

[0154] (3) Compare the blocking effects of different anti-PD-1 antibodies on PD-1 binding to PD-L1.

[0155] The blocking effect of humanized antibodies on PD-1 / PD-L1 interaction was detected using ELISA. The specific experimental procedure is as follows:

[0156] Add 1 μg / mL of PD-L1-hFc protein (a fusion protein of the extracellular region of PD-L1 protein and the human IgG Fc fragment, self-produced) to the wells of an ELISA plate, coat overnight at 4°C, and wash away unbound protein. Serially dilute the chimeric antibody or humanized antibody, then mix each with 100 nM of PD-1-His protein, pre-incubate at room temperature for 1 hour, add the premix to the PD-L1 protein-coated wells, incubate at room temperature for 1 hour, and wash away unbound protein. Add Anti-His Tag (HRP) antibody, incubate at room temperature for 1 hour. Wash away unbound antibody, add substrate reaction solution, and terminate the reaction by adding stop solution based on color change. Detect the absorbance of the reaction system using a Varioskan LUX multi-functional microplate reader. The obtained OD values ​​are fitted using a 4-factor formula. The blocking experiment formula is: Y = Bottom + (Top-Bottom) / (1 + 10^(LogIC)). 50 -X)*HillSlope)).

[0157] The results are as follows Figure 2 As shown, the blocking effect of humanized anti-PD-1 antibody on PD-1 / PD-L1 interaction is similar to that of chimeric antibody, while the IC50 of chimeric antibody is lower. 50 The EC50 of the humanized antibody was 72.54 nM. 50 The values ​​ranged from 97.81 to 128.4 nM. These results indicate that the germline selection and reversion mutation design of the anti-PD-1 antibody were reasonable and effective, and had little impact on the antibody's binding and blocking functions.

[0158] Example 3: Construction of a humanized anti-PD-1 antibody and IL-21 fusion protein

[0159] The screened humanized anti-PD-1 antibody (uPD1Ab6) was fused with natural or modified IL-21 with different amino acid mutations to construct a fusion protein. Simultaneously, a fusion protein of IL-21 with His or IgG Fc was constructed. Figure 3 ).

[0160] 1. Construction of antibody-IL-21 fusion protein

[0161] Primers IL-21Fusion-F (Seq ID No. 27) and IL-21Fusion-R (Seq ID No. 28) were designed and synthesized based on the IL-21 gene coding sequence, with a (G4S)3 linker sequence added to primer IL-21Fusion-F. Using native IL-21 and two mutant IL-21s (IL-21M1 and IL-21M2) as templates, the corresponding IL-21 DNA fragments were amplified by PCR. Similarly, primers PD1Ab-F (Seq ID No. 25) and PD1Ab-R (Seq ID No. 26) were designed and synthesized based on the heavy chain sequence of the humanized anti-PD-1 antibody (uPD1Ab6), and the antibody heavy chain fragment was amplified by PCR. The PCR-amplified antibody heavy chain fragments were then fused with different IL-21 gene fragments via overlap PCR to obtain the gene coding fragments of the antibody heavy chain-IL-21 fusion protein. After double digestion with HindIII and XbaI, the protein was ligated into the expression vector pCDNA3.1 to obtain an expression vector for the humanized anti-PD-1 antibody (uPD1Ab6) heavy chain and IL-21 fusion protein (construction process see [link to documentation]). Figure 4 ).

[0162] The gene encoding fragment of the humanized anti-PD-1 antibody (uPD1Ab6) light chain was amplified using the same method, and then ligated into the expression vector pCDNA3.1 by enzyme digestion to obtain the humanized PD-1 antibody (uPD1Ab6) light chain expression vector.

[0163] The amino acid / DNA sequence of the constructed antibody heavy chain and IL-21 fusion protein is as follows:

[0164] uPD1Ab_VH4-IL-21, Seq ID No.11 (593aa), Seq ID No.39 (1782bp);

[0165] uPD1Ab VH4-IL-21M1, Seq ID No.12 (587aa), Seq ID No.40 (1764bp);

[0166] uPD1Ab_VH4-IL-21M2, Seq ID No.13 (587aa), Seq ID No.41 (1764bp);

[0167] The amino acid / DNA sequence of the constructed antibody light chain is as follows: variable region uPD1Ab_VL4, Seq ID No. 10 linked to constant region kappa, Seq ID No. 56.

[0168] Figure 5 This is a schematic diagram of the final complete fusion protein structure.

[0169] 2. Construction of IL-21-His fusion protein

[0170] Primers were designed and synthesized based on the IL-21 gene coding sequence: IL-21-F (Seq ID No. 20) and IL-21His-R (Seq ID No. 21). The gene coding sequence with a His tag was added to primer IL-21His-R. Using native IL-21 and two mutant IL-21s (IL-21M1 and IL-21M2) as templates, the corresponding IL-21 DNA fragments were amplified by PCR. The amplified fragments were double-digested with HindIII and XbaI, and then ligated into the vector pCDNA3.1. Figure 3 The amino acid / DNA sequence of the constructed IL-21His fusion protein:

[0171] IL-21-His, Seq ID No.14 (143aa), Seq ID No.42 (432bp),

[0172] IL-21M1-His, Seq ID No.15 (137aa), Seq ID No.43 (414bp),

[0173] IL-21M2-His, Seq ID No. 16 (137aa), Seq ID No. 44 (414bp).

[0174] 3. Construction of Fc-tagged protein

[0175] Primers IL-21Fc-R (Seq ID No. 22) were designed and synthesized based on the IL-21 gene coding sequence. Using native IL-21 and two mutant IL-21s (IL-21M1 and IL-21M2) as templates, PCR amplification was performed using primers IL-21-F and IL-21Fc-R to obtain the corresponding IL-21 DNA fragments. Primers Fc-F (Seq ID No. 23) and Fc-R (Seq ID No. 24) were designed and synthesized based on the IgGFc gene coding sequence, with the (G4S)2 linker coding sequence added to primer Fc-F.

[0176] Using an IgGFc expression plasmid as a template, the IgGFc gene fragment was amplified. Different IL-21 gene fragments amplified by PCR were fused with the IgGFc gene fragment via overlap PCR to obtain the gene encoding the IL-21-Fc fusion protein. After double digestion with HindIII and XbaI, the fragment was ligated into the expression vector pCDNA3.1. Figure 3 An expression vector for the IL-21-Fc fusion protein was obtained. The amino acid / DNA sequence of the constructed protein is as follows:

[0177] IL-21-Fc, Seq ID No.17 (366aa), Seq ID No.45 (1101bp),

[0178] IL-21M1-Fc (Seq ID No.18 (360aa), Seq ID No.46 (1083bp),

[0179] IL-21M2-Fc (Seq ID No. 19 (360aa), Seq ID No. 47 (1083bp).

[0180] Example 4: Comparison of expression levels of different IL-21 fusion proteins

[0181] In this application, the constructed expression vectors of uPD1Ab6-IL-21, uPD1Ab6-IL-21M1, uPD1Ab6-IL-21M2, IL-21-Fc, IL-21M1-Fc, and IL-21M2-Fc were prepared according to the scheme in Example 2. The expression of IL-21-His, IL-21M1-His, and IL-21M2-His proteins was the same as in Example 2, and the purification scheme is as follows:

[0182] Purification using Ni magnetic beads: Taking 100 μL of pure Ni magnetic beads as an example, resuspend the beads in the cell supernatant and incubate at room temperature for 2 hours or overnight at 4°C. Wash three times with PBS. Add 500 μL of 10 mM imidazole, mix gently at room temperature for 5 min, collect the eluent and measure the protein concentration. If the protein concentration is low, replace the eluent with 200 mM imidazole and then 500 mM imidazole until the protein concentration in the eluent is 0 mg / mL, then stop elution. After PAGE analysis of the imidazole eluent, immediately replace it with PBS, replacing approximately 5 times the volume of the eluent. Measure the conductivity of the filtered solution; if it matches that of the PBS, the replacement was successful. Measure the protein concentration.

[0183] After protein preparation, the purity of the protein was determined by SEC using liquid chromatography. The method is as follows: After rinsing the liquid chromatograph with ultrapure water, the mobile phase was replaced (0.15M PB, pH 7.0). An SEC-300 (Saifen) detection column was installed. After the baseline on the detection page stabilized, the sample was loaded. 10 μg of the protein to be tested (maximum loading volume is 50 μL) was placed in the sample vial and placed in the detection rack. After the protein sample was processed, the protein purity was analyzed using the software.

[0184] The expression levels of IL-21-His, IL-21-Fc, and antibodies were compared using a 30 mL transfection system with IL-21 fusion protein and the corresponding IL-21 mutant fusion protein.

[0185] The results are shown in Table 3. Compared with wild-type IL-21, the expression levels of all IL-21 mutants increased, especially the IL-21M2 mutant, whose expression levels were significantly higher than those of the wild-type and M1 mutants when fused with His tag, Fc tag, and anti-PD-1 antibody, respectively. Furthermore, we also tested the purity of the Fc-tagged fusion protein and the anti-PD-1 antibody fusion protein. The results showed that no effective purity data could be obtained for the wild-type IL-21 group under the same experimental conditions, while the purity values ​​of the two mutant fusion proteins were in the range of 90%-97%, which was in line with expectations. These expression and purification results indicate that IL-21 protein expression was significantly improved through mutant design, especially after fusion expression with anti-PD-1 antibody, which significantly increased the expression level of the fusion protein.

[0186] Table 3. Expression levels of IL-21-related proteins

[0187]

[0188] Example 5: In vivo stability evaluation of antibody-IL-21 fusion protein

[0189] C57BL / 6 mice were injected via tail vein with three different types of anti-PD-1 antibody-IL-21 fusion protein and PD-1 antibody (uPD1Ab6, as a control), 200 μg per mouse, with three mice per group. One hour after administration, and on days 1, 3, 5, 7, 9, 11, and 14, approximately 100 μL of blood was collected via orbital sampling. The blood was incubated at 37°C for half an hour, centrifuged at 4500 rpm for 3 minutes, and the supernatant was collected to obtain serum. The concentrations of the PD-1 antibody fragment and the complete fusion protein in the serum were detected using ELISA. The specific detection method is as follows:

[0190] 1. Partial detection of antibody fragments

[0191] The concentration of the PD-1 fusion protein antibody fragment in mouse serum was quantitatively determined using ELISA. First, 1 μg / mL of PD-1His protein was coated onto a 96-well plate and incubated overnight. Then, using the corresponding fusion protein as a standard, serial dilutions were performed starting at 1000 ng / mL. Serum samples or standards were added to the coated 96-well plates and incubated at room temperature for 1 hour. Unbound material was washed away, and 10000-fold diluted HRP-labeled anti-human IgG (Siuthern Biotech) was added and incubated at room temperature for 1 hour. Substrate reaction solution was then added, and unbound labeled antibody was washed away. Substrate reaction solution was added again, and the reaction was stopped by adding stop solution based on color change. The absorbance of the reaction system was measured using a Varioskan LUX multi-functional microplate reader, and a standard curve was plotted. The PD-1 protein concentration in the sample was calculated based on the standard curve. The standard curve used four parameters with a weighting factor of 1 / Y. 2 Regression was performed, and the concentration of the PD-1 antibody fragment in serum was calculated based on the standard curve.

[0192] 2. Detection of full-length fusion proteins

[0193] 1 μg / mL of PD-1 His protein was coated onto a 96-well ELISA plate. The corresponding fusion protein was then used as a standard, serially diluted starting at 1000 ng / mL. Serum samples or standards were added, and the plate was incubated at room temperature for 1 hour. Unbound material was washed away, and 500 ng / mL of biotin-labeled anti-IL-21 antibody was added. The plate was incubated at room temperature for 1 hour, and unbound labeled antibody was washed away. Avidin-labeled HRP (SA-HRP, Jackon Immuno Research) diluted 90,000 times was added, and the plate was incubated at room temperature for 1 hour. Unbound HRP was washed away, and substrate reaction solution was added. Based on the color change, the reaction was terminated by adding stop solution. The absorbance of the reaction system was measured using a Varioskan LUX multi-well microplate reader. The OD450nm / 620nm values ​​were recorded using an ELISA reader. A standard curve was constructed using four parameters with a weighting factor of 1 / Y. 2 Regression was performed. The concentration of the intact fusion protein in serum was calculated based on the standard curve. Concentration-time curves of the partial PD-1 antibody fragment and the intact fusion protein in serum were plotted separately.

[0194] The results are as follows Figure 6As shown, the left figure represents the concentration-time trend of the antibody fraction in the fusion protein, while the right figure represents the concentration-time trend of the complete fusion protein. It can be seen that the antibody fraction concentration of the uPD1Ab6-IL-21 fusion protein rapidly decreased to one-tenth of that of the single antibody one hour after injection, and its metabolic rate thereafter was essentially the same as that of the single antibody. The metabolic kinetics of the antibody fractions of the uPD1Ab6-IL-21M1 and uPD1Ab6-IL-21M2 fusion proteins showed no significant difference from those of the single antibodies. Simultaneously, the clearance rate of the complete uPD1Ab6-IL-21 fusion protein in serum was significantly faster than that of its antibody fraction; the situation for uPD1Ab6-IL-21M1 improved slightly, but remained significantly faster than that of its antibody fraction, indicating that only the antibody fraction of the fusion protein remained in the serum, and the IL-21 fraction had been degraded; while the metabolic kinetics of the complete uPD1Ab6-IL-21M2 fusion protein were essentially consistent with those of its antibody fraction, demonstrating optimal in vivo stability. The above results indicate that the IL-21M2 mutation exhibits optimal in vivo protein stability after fusion with the anti-PD-1 antibody, effectively addressing the drug-likeness issue. Based on these findings, this application selected the uPD1Ab6-IL-21M2 fusion protein (abbreviated as: PD-1AbIL-21 fusion protein) for subsequent functional studies.

[0195] Example 6: Identification of the biological function of anti-PD-1 antibody and IL-21 fusion protein (PD-1AbIL-21)

[0196] 1. Functional study of PD-1AbIL-21 fusion protein binding to PD-1 molecules

[0197] (1) Compare the binding of PD-1AbIL-21 fusion protein and PD-1 antibody to PD-1 molecules.

[0198] The binding of the fusion protein to human PD-1 molecules was assessed using ELISA. The specific experimental procedure is as follows:

[0199] Add 1 μg / mL of PD-1-His protein to the wells of an ELISA plate and incubate overnight at 4°C. Wash away unbound protein. Add the fusion protein, PD-1 antibody (Keytruda®), or irrelevant negative control antibody (NC is anti-HEL antibody, purchased from Thermo, catalog number MA5-55107) in 3-fold serial dilutions (from 100,000 nM to 0.002 nM) to the wells coated with PD-1-His protein and incubate at room temperature for 1 hour. Wash away unbound protein or antibody, add HRP-labeled anti-human IgG, and incubate at room temperature for 1 hour. Wash away unbound labeled antibody, add substrate reaction solution, and terminate the reaction by adding stop solution based on color change. Measure the absorbance of the reaction system using a Varioskan LUX multi-functional microplate reader and calculate the half-maximal effective concentration (EC5). 50 The calculation formula is as follows: Y = Bottom + (Top - Bottom) / (1 + 10^(LogEC)) 50 -X)*HillSlope)).

[0200] The results are as follows Figure 7 As shown, the half-maximum effective concentration (EC50) of the PD-1AbIL-21 fusion protein and the PD-1 antibody (Keytruda®) binding to the PD-1 molecule is... 50 The values ​​were 0.2559 nM and 0.5568 nM, respectively, meaning that the interaction between the PD-1AbIL-21 fusion protein and the PD-1 molecule was almost exactly the same as that between the PD-1 antibody and the PD-1 molecule. This indicates that the fusion of the PD-1 antibody and IL-21 does not affect the interaction between the PD-1 antibody and the PD-1 molecule.

[0201] (2) Determine the affinity of the PD-1AbIL-21 fusion protein for binding to PD-1 molecules.

[0202] The affinity between the PD-1AbIL-21 fusion protein and the PD-1 molecule was determined using surface plasmon resonance (SPR). The specific experimental procedure is as follows: The instrument used was a Biacore 8K with the Protein A S-series sensor chip. The fusion protein was injected into the Protein A chip and captured by the chip. Binding phase: The hPD-1-His protein was serially diluted two-fold from 100 nM to 3.125 nM using running buffer (1×HBS-EP+ buffer pH 7.4) and injected into the flow cell on the chip surface. The binding time was 180 seconds. Dissociation phase: After binding, running buffer was injected immediately, and the dissociation time was set to 1200 seconds. After detection, the chip was regenerated using glycine-HCl (pH=1.5). Experimental data were analyzed on a Biacore 8K using Biacore Insight Evaluation. The equilibrium dissociation constant (KD) between the PD-1AbIL-21 fusion protein and human PD-1 protein was 3.07E-10 M (Table 4).

[0203] (3) The blocking effect of PD-1AbIL-21 fusion protein on PD-1 binding to PD-L1

[0204] The flow cytometry technique was used to detect the blocking effect of the PD-1AbIL-21 fusion protein on the binding of PD-1 molecules expressed on the cell surface to PD-L1. The specific experimental procedure is as follows:

[0205] Previously, stable CHO-K1 (CHO-K1-PD-1-GFP) and PD1+HuT78 cell lines expressing PD-1 and GFP were constructed, and PD-L1-mFc protein was prepared. Specifically, the CHO-K1 cell line was purchased from ATCC (CCL-61), and the full-length PD-1 gene and the extracellular sequence of PD-L1 were synthesized by Beijing Qingke Biotechnology Co., Ltd. The PD-1 gene was constructed into a transposon stable transfection plasmid (with a GFP fluorescent tag), which was transfected into the CHO-K1 or HuT78 cell lines. After antibiotic-treated pressure selection culture, stable single clones were screened by limiting dilution. The PD-L1 extracellular sequence was constructed into a pCDNA3.4 vector containing an mFc tag by enzyme digestion and ligation, and then expressed and purified.

[0206] Add 2×10 to the flow cytometer 5CHO-K1-PD-1-GFP cells. PD-1AbIL-21 fusion protein or PD-1 antibody (Keytruda®) was serially diluted 3-fold from 600.0 nM to 0.01 nM. 100 μL of each was added to flow cytometry tubes to suspend CHO-K1-PD-1-GFP cells, and incubated at 4°C for 20 min. Then, PD-L1-mFc protein was added, and the cells were incubated at 4°C for 20 min. Unbound protein was washed away by centrifugation. Next, APC-labeled goat anti-mouse IgG antibody was added, and the cells were incubated at 4°C for 20 min. Unbound antibody was washed away by centrifugation. The cells were then resuspended in FACS buffer for flow cytometry analysis. A curve showing the binding of the inhibitory agent to PD-L1 protein was plotted, and the half-maximal inhibitory concentration (IC50) was calculated. 50 The calculation formula is as follows: Y = Bottom + (Top - Bottom) / (1 + 10^(LogIC) 50 -X)*HillSlope)).

[0207] The results are as follows Figure 8 As shown: The PD-1AbIL-21 fusion protein, like the PD-1 antibody, can effectively block the binding of PD-L1 to PD-1 on the cell surface. Moreover, the PD-1AbIL-21 fusion protein exhibits a stronger blocking effect. The IC50 of the PD-1AbIL-21 fusion protein is [not specified in the original text]. 50 The concentration was 4.629 nM, while the IC50 of the PD-1 antibody was... 50 It is 13.12 nM.

[0208] 2. Assay of the biological activity of IL-21 in the PD-1AbIL-21 fusion protein

[0209] (1) Compare the interactions between different IL-21 fusion proteins and IL-21R.

[0210] The interaction between the IL-21 fusion protein and the IL-21Rα molecule was detected using ELISA. The specific experimental procedure is as follows:

[0211] Add 1 μg / mL of IL-21R-His protein (a fusion protein of the extracellular region of the human IL-21 receptor α molecule and the His molecule tag, purchased from Sinocare) to the wells of an ELISA plate and incubate overnight at 4°C. Wash away unbound protein. Perform 3-fold serial dilutions (from 150,000 nM to 0.003 nM) of PD-1AbIL-21 fusion protein, IL-21-Fc fusion protein, or negative control anti-OX40 antibody (NC) and add them to the wells coated with IL-21R-His protein. Incubate at room temperature for 1 hour. Wash away unbound protein, add HRP-labeled anti-human IgG, and incubate at room temperature for 1 hour. Wash away unbound labeled antibody, add substrate reaction solution, and terminate the reaction by adding stop solution based on color change. Detect the absorbance of the reaction system using a Varioskan LUX multi-functional microplate reader and calculate the half-maximal effective concentration (EC50). 50 The calculation formula is as follows: Y = Bottom + (Top - Bottom) / (1 + 10^(LogEC)) 50 -X)*HillSlope)).

[0212] The results are as follows Figure 9 As shown, the EC2 binding of the PD-1AbIL-21 and IL-21-Fc fusion proteins to the IL-21R molecule... 50 The values ​​were 0.5341 nM and 0.2077 nM, respectively, indicating that the interaction between IL-21M2 and native IL-21R in the PD-1AbIL-21 fusion protein was weaker than that between native IL-21 and native IL-21.

[0213] (2) Compare the affinity of different IL-21 fusion proteins for binding to IL-21Ra.

[0214] The affinity of different IL-21 fusion proteins for Human IL-21R was determined using the surface plasmon resonance (SPR) method. The specific experimental procedure is as follows:

[0215] The instrument used was a Biacore 8K with the Protein A S-series sensor chip. The PD-1AbIL-21, IL-21-Fc, and IL-21M2-Fc fusion proteins were injected into the Protein A chip and captured by the chip. Binding phase: hIL-21R-His protein was serially diluted two-fold from 400 nM to 12.5 nM using running buffer (1×HBS-EP+ buffer, pH 7.4) and injected into the flow cell on the chip surface. The binding time was 120 seconds. Dissociation phase: Immediately after binding, running buffer was injected, and the dissociation time was set to 600 seconds. After detection, the chip was regenerated using 10 mM glycine-HCl (pH=1.5). Experimental data were analyzed using Biacore Insight Evaluation on the Biacore 8K.

[0216] The results showed that the PD-1AbIL-21 fusion protein, IL-21-Fc and IL-21M2-Fc had comparable affinity for hIL-21R, with KD values ​​of 8.58E-09, 8.18E-09 and 1.13E-08M, respectively (Table 4).

[0217] Table 4. Affinity test of fusion proteins

[0218]

[0219] (3) Compare the biological activity of PD-1AbIL-21 and IL-21-Fc fusion proteins in activating cellular IL-21R signaling.

[0220] To compare the activation effect of the IL-21 fusion protein on IL-21R in PD-1-expressing and PD-1-non-expressing cells, this application constructed HuT78 cells stably expressing PD-1 and GFP via expression plasmid transfection, as well as control HuT78 cells transfected with mock plasmids. Flow cytometry was then used to detect the activation of intracellular STAT3 phosphorylation in these two cell types by IL-21 fusion protein stimulation. The specific experimental procedure is as follows:

[0221] Take HuT78 (ATCC, TIB-161) and PD-1 respectively + HuT78 cells (construction method described in Section 1 of this embodiment) (5×10 5 Cells were placed in flow cytometry tubes, and the PD-1AbIL-21 and IL-21-Fc fusion proteins were diluted 3-fold from 75.00 nM to 0.01 nM. HuT78 and PD-1 were added to each concentration sample, respectively. +HuT78 cells were mixed in flow cytometry tubes and incubated at 37°C for 30 min. Paraformaldehyde was added to a final concentration of 2%, and the cells were incubated at room temperature for 30 min. After centrifugation, the cells were resuspended in pre-chilled ice-cold methanol and incubated at 4°C for 30 min. After centrifugation to wash away the methanol, Phospho-STAT3 (Tyr705) (Thermo Fisher Scientific) antibody was added, and the cells were incubated at room temperature in the dark for 30 min. After centrifugation to wash away unbound antibody, the cells were resuspended in FACS buffer for flow cytometry analysis. A curve was plotted on the protein dose versus the proportion of pSTAT3-positive cells, and EC50 was calculated. 50 EC 50 Calculation formula: Y = Bottom + (Top - Bottom) / (1 + 10^(LogEC)) 50 -X)*HillSlope)).

[0222] The results are as follows Figure 10 As shown: IL-21-Fc fusion protein stimulates HuT78 and PD-1 + HuT78 cells exhibit similar activity in activating STAT3 phosphorylation; IL-21-Fc stimulation of the EC50 cells in both cell lines... 50 The concentrations were 1.354 nM and 0.7463 nM, respectively. However, the PD-1AbIL-21 fusion protein in PD-1... + The activity of activating STAT3 phosphorylation in HuT78 cells was significantly higher than its activity in HuT78 cells, EC 50 The values ​​were 0.0737 nM and 4.724 nM, respectively, indicating that the PD-1AbIL-21 fusion protein activates PD-1. + The phosphorylation activity of STAT3 in HuT78 cells was 10 times that of the IL-21-Fc fusion protein. These results indicate that the PD-1AbIL-21 fusion protein can target IL-21 to PD-1 positive cells and exert its biological effects.

[0223] Example 7: Selective targeting effect of PD-1AbIL-21 fusion protein on PD-1 positive cells

[0224] To further demonstrate the targeting effect of the fusion protein on PD-1 positive cells, this application directly compared the effects of the PD-1AbIL-21 and IL-21-Fc fusion proteins on PD-1 in the same environment. - and PD-1 + The cell binding process is illustrated in the following experimental procedure:

[0225] The PD-1 constructed in the previous stage (see item 1 of this embodiment) +HuT78 cells (expressing both PD-1 and IL-21R) and HuT78 cells (expressing only IL-21R) were mixed at a 1:1 ratio. The PD1AbIL-21 and IL-21M2-Fc fusion proteins were then serially diluted 3-fold from 600.00 nM to 0.003 nM. Each diluted sample was added to the mixed cells and incubated on ice for 30 min. The cells were then washed three times with PBS, incubated with APC-labeled anti-human IgG antibody for 30 min, washed three more times with PBS, and then resuspended in FACS buffer for flow cytometry analysis to detect the fusion protein bound to the cell surface. During analysis, GFP-positive and GFP-negative cells represented cells expressing and not expressing PD-1, respectively.

[0226] The results are as follows Figure 11 As shown, PD1AbIL-21 preferentially binds to GFP at low concentrations. + PD-1 + Cells that were near saturation only began to bind GFP-PD-1 to cells that were in contact with EC2 cells in both cell types. 50 The difference was greater than 60-fold. However, the binding of IL-21M2-Fc protein to PD-1-expressing and non-PD-1-expressing cells was almost identical, with no difference. These results fully demonstrate that PD1AbIL-21, through the targeting action of anti-PD-1 antibodies, preferentially binds to the surface of PD-1-positive cells, thereby achieving its selective targeting function.

[0227] Example 8: Preparation of large-scale expression of PD-1AbIL-21 fusion protein

[0228] To conduct in vivo experimental studies, a large quantity of protein was prepared for expression. The method is as follows:

[0229] (1) Cell transfection and protein expression

[0230] 1) Add 200 μg of target plasmid DNA (heavy chain to light chain ratio of 1:1) to 12 mL of Opit-MEM serum-depleted medium, and add 800 μL of PEI to 11.2 mL of Opit-MEM serum-depleted medium. Gently vortex to mix and incubate at room temperature for 5 min. Add the diluted PEI dropwise to the diluted plasmid DNA, gently vortex to mix, and incubate at room temperature for 15-20 min.

[0231] 2) Slowly add the PEI / DNA complex dropwise to 200 mL of cells (1 mL of cells corresponds to 1 μg of DNA transfection). Gently shake the culture flask during the addition process, then place it on a track shaker and culture the cells at 37°C and 8% CO2. 18-22 hours after transfection, add Profeed (Optimum Nutrition) feed to the culture flask to a final concentration of 5% of the total volume.

[0232] 3) On the fourth day after transfection, the density and viability of live cells were measured to identify the protein expression in the cell supernatant.

[0233] 4) When the cell viability is around 60% (around day 6), collect the cell supernatant and purify the protein.

[0234] (2) Purification

[0235] Purification was performed using an SCG purification system with NMab (nanomicro) packing material. The packing material was treated with 0.1M NaOH and then equilibrated with PBS. The cell culture supernatant was then loaded onto the sample. After loading, the sample was equilibrated with PBS and eluted with 50mM citrate. The UV peak at 280 nm was collected. Collection began when the signal value rose to 50 mAU and stopped when the signal value dropped to 50 mAU. The pH of the collected solution was adjusted to 6.0-7.5 by adding 1M Tris-HCl (pH 9.0). Sample affinity purification was then complete.

[0236] Example 9: Therapeutic effect of PD-1AbIL-21 fusion protein in human PD-1 and IL-21R transgenic mouse melanoma model

[0237] To investigate the in vivo antitumor effect of the PD-1AbIL-21 fusion protein, this application first conducted an antitumor therapeutic study in a human PD-1 and IL-21R double transgenic mouse melanoma model. The specific experimental procedure is as follows:

[0238] Transgenic mice B-hPD-1plus / hIL-21R were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd., and were subcutaneously injected with 5 × 10⁶ mice. 5 On day 4 of tumor-bearing, mice with melanoma cells (B16-F10) were randomly divided into 3 groups of 3 mice each. Each group received intraperitoneal injection of PBS (control), anti-PD-1 antibody (Keytruda®), or PD-1AbIL-21 fusion protein (all 10 mg / kg), respectively. Injections were administered every 3 days for a total of 6 treatments. Figure 12 (See Figure a). Starting from the day of grouping, tumor size was measured every 2 days using calipers, and the time of mouse death was recorded. Tumor growth curves and mouse survival curves were plotted. Tumor volume (mm) 3 = Length (mm) * Width (mm) 2 / 2.

[0239] The results are as follows Figure 12 Figure b in the middle and Figure 12As shown in Figure c, there was no significant difference in tumor growth and survival between the antibody-only treatment group and the PBS-treated control group; both groups of mice died within 24 days after tumor inoculation. However, treatment with the PD-1AbIL-21 fusion protein significantly inhibited tumor growth and greatly prolonged the survival of the mice. These results indicate that the PD-1AbIL-21 fusion protein still has a significant therapeutic effect on tumors resistant to PD-1 antibody therapy.

[0240] Example 10: Therapeutic effect of PD-1AbIL-21 in a human PD-1 and IL-21R transgenic mouse model of pancreatic cancer

[0241] Transgenic mice B-hPD-1plus / hIL-21R were subcutaneously injected with 5×10 5 Mice with pancreatic cholangiocarcinoma cells (KPC) were divided into three groups of three mice each on day 6 of tumor bearing, based on tumor size. Each group received an intraperitoneal injection of PBS (control), anti-PD-1 antibody (Keytruda®), and PD-1AbIL-21 fusion protein (all 10 mg / kg), respectively, every three days for a total of six treatments. Figure 13 (See Figure a). Tumor size was measured every three days starting from the grouping stage, mouse death time was recorded, and tumor growth curves and mouse survival curves were plotted.

[0242] The results are as follows Figure 13 Figure b in the middle and Figure 13 As shown in Figure c, there was no significant difference in tumor growth and survival between the antibody-only group and the control group, and all mice in both groups died within 27 days. However, treatment with the PD-1AbIL-21 fusion protein significantly reduced tumor size in mice, and all mice survived within the 30-day observation period. These results indicate that the PD-1AbIL-21 fusion protein still has a significant tumor treatment effect in pancreatic cancer models resistant to PD-1 antibody therapy.

[0243] Example 11: Therapeutic effect of PD-1AbIL-21 fusion protein in a subcutaneous lung cancer model of human PD-1 and IL-21R transgenic mice

[0244] Transgenic mice B-hPD-1plus / hIL-21R were subcutaneously injected with 5×10 5Mice with Lewis lung cancer (LLC) cells were divided into four groups of 10 mice each based on tumor size on day 11 of tumor bearing. Each group received intraperitoneal injection of PBS (control), anti-PD-1 antibody alone (Keytruda®, 10 mg / kg), anti-PD-1 antibody (Keytruda®, 8.2 mg / kg) combined with IL-21 (1.8 mg / kg), or PD-1AbIL-21 fusion protein (10 mg / kg), respectively. Injections were administered every three days for a total of seven treatments. Figure 14 (See Figure a). Tumor size was measured every three days starting from the grouping stage, mouse death time was recorded, and tumor growth curves and mouse survival curves were plotted.

[0245] The results are as follows Figure 14 As shown in Figures b and c of 14, both the antibody-only group and the combination therapy group showed significant therapeutic effects and significantly prolonged mouse survival compared to the PBS-treated control group. However, all mice in both groups died within 42 days of tumor inoculation. Compared to the antibody-only group and the combination therapy group, PD-1AbIL-21 fusion protein treatment further inhibited tumor growth. 40% of the mice survived 46 days after tumor inoculation, indicating that the PD-1AbIL-21 fusion protein has a more significant therapeutic effect on tumors responding to PD-1 antibody treatment. Furthermore, there was no significant difference in tumor growth and mouse survival between the antibody-only group and the combination therapy group, while the tumor therapeutic effect of the PD-1AbIL-21 fusion protein was greatly enhanced, suggesting that the anti-tumor effect of IL-21 depends on the targeting of PD-1.

[0246] Example 12: Therapeutic effect of PD-1AbIL-21 fusion protein in humanized breast cancer mouse model

[0247] To further investigate the anti-tumor effect of the PD-1AbIL-21 fusion protein in human tumor models, the applicant constructed a series of CAR-T cells and combined them with human tumor cell lines to establish humanized mouse tumor models in immunodeficient mice. The CAR-T cells involved are HER2CAR-T and CD19CAR-T cells, specifically constructed as follows: the heavy and light chain sequences of the anti-CD19 antibody FMC63 are derived from SEQ ID No:1 (Seq ID No. 60 in this application) and SEQ ID No:5 (Seq ID No. 61 in this application) in patent US20180265595A1, respectively; the sequences of the anti-HER2 antibody 4D5 are derived from SEQ ID No:41 (Seq ID No. 62 in this application) and SEQ ID No:42 (Seq ID No. 63 in this application) in patent US5821337A, respectively; the remaining expression elements of the CAR include the CD8a signal peptide (Uniprot: P01732-1, aa 1-21), the CD8a hinge transmembrane region (Uniprot: P01732-1, aa 138-206), and the 4-1BB intracellular domain (Uniprot: The CAR structure, from N-terminus to C-terminus, consists of a CD8a signal peptide, the antibody's scFv region, the CD8a hinge transmembrane region, the 4-1BB intracellular domain, and the CD3ζ signal transduction domain (P20963-1, aa 52-164). These sequences were synthesized by Beijing Qingke Biotechnology Co., Ltd. and cloned into the pCDH vector. Recombinant virus preparation: A certain proportion of the target plasmid, helper plasmids REV, RRE, and VSVG were transfected into well-grown 293T cells using the calcium phosphate transfection method. The cells were incubated for 8-10 hours, and the culture medium was replaced with fresh medium. Forty-eight hours after transfection, the supernatant was collected, concentrated, and purified. The virus was then aliquoted and stored at -80℃. CAR-T preparation: Peripheral blood was collected from healthy donors, and peripheral blood mononuclear cells (PBMCs) were separated using density gradient centrifugation. Subsequently, PBMCs were activated using anti-CD3 / CD28 Dynabeads (thermo, catalog number 11131D) in X-VIVO-15 medium supplemented with 100 IU / mL IL-2 at a magnetic bead to cell ratio of 3:1. After 24 hours, the activated T cells were placed in a system containing 200 IU / mL IL-2 and 8 µg / mL polybrene and lentivirally transduced by centrifugation (2000 g, 32°C, 90 min). After centrifugation for 6 hours, an equal volume of fresh medium containing IL-2 was added, and the cells were incubated at 37°C in a 5% CO2 incubator for 48 hours.After 48 hours, the culture medium was replaced to remove residual virus, and the cells were then cultured in a medium containing IL-2 (10 or 100 IU / mL) for another 3-5 days. The resulting HER2CAR-T was used in this example, and CD19CAR-T was used in Example 13.

[0248] A humanized tumor cell model was established in severely immunodeficient (NCG) mice. The specific experimental procedure is as follows:

[0249] Subcutaneous injection of 1×10⁻⁶ NCG-immune-deficient mice 7 Human breast cancer cells (BT474) were injected intravenously (iv) into mice via the tail vein 34 days after tumor bearing. 6 HER2CAR-T cells were used as tumor-specific T cells. Mice were then randomly divided into four groups of five mice each, based on tumor size. The mice received intraperitoneal injections of PBS (control), anti-PD-1 antibody alone (Keytruda®, 10 mg / kg), anti-PD-1 antibody (Keytruda®, 8.2 mg / kg) and IL-21M2 (1.8 mg / kg) in combination, and PD-1AbIL-21 fusion protein (10 mg / kg), respectively. Injections were administered every five days for a total of six treatments. Figure 15 (See Figure a). Tumor size was measured every three days starting from the grouping stage, mouse death time was recorded, and tumor growth curves and mouse survival curves were plotted.

[0250] The results are as follows Figure 15 As shown in Figures 15b and 15c, there was no significant difference in tumor growth between the antibody-only group and the combination therapy group and the control group. In the control group, tumors exceeded 2000 mm in all mice 66 days after tumor inoculation. 3 Mice in the antibody-only group and the combination therapy group were euthanized for the same reason. In the PD-1AbIL-21 fusion protein treatment group, except for one mouse that died unexpectedly, the tumors of the other four mice completely regressed, and these mice survived long-term. These results demonstrate that in tumor models resistant to PD-1 antibody therapy, the PD-1AbIL-21 fusion protein can still induce complete tumor regression and long-term survival in mice.

[0251] Example 13: Therapeutic effect of PD-1AbIL-21 fusion protein in a humanized B-cell leukemia mouse model

[0252] To investigate the therapeutic effect of the PD-1AbIL-21 fusion protein on hematological malignancies, this application also conducted an anti-tumor study in a humanized B-cell leukemia mouse model. The specific experimental procedure is as follows:

[0253] Immunodeficient NCG mice were injected via tail vein with 2×10 5Raji-Luc B-cell lymphoma cells were injected intravenously (iv) via the tail vein on day 11 of tumor bearing. 6 CD19CAR-T cells were used as tumor-specific T cells, and mice were treated with intraperitoneal injections of PBS (control) on days 11 and 17, or with anti-PD-1 antibody alone (Keytruda®, 10 mg / kg), anti-PD-1 antibody (Keytruda®, 8.2 mg / kg) and IL-21M2 (1.8 mg / kg) in combination, or PD-1AbIL-21 fusion protein (10 mg / kg). Five mice were in each group. Figure 16 (Figure a). Small animal in vivo imaging was performed every 3 days to detect tumor progression, and the time of mouse death was recorded to plot mouse survival curves.

[0254] The imaging methods for small animals are as follows:

[0255] 1) Each mouse was injected intraperitoneally with 3 mg of Luciferin substrate, and the timer was started for 3 minutes simultaneously;

[0256] 2) The mice were then transferred into an anesthesia chamber and anesthetized with isoflurane gas for about 7 minutes.

[0257] 3) Once the mouse is completely anesthetized, place it in the IVIS Lumina3 in vivo imaging system, set the exposure time to 1 minute, acquire images, and perform detection.

[0258] Experimental results are as follows Figure 16 Figures b and c show the results. The results indicated that, compared to the PBS-treated control group, the PD-1 antibody alone and the combination therapy group delayed tumor growth in some mice, and all mice died within 33 days after tumor inoculation. In contrast, the PD-1AbIL-21 fusion protein treatment rapidly cleared tumors in all mice, achieving a cure, and all mice achieved long-term survival. These results demonstrate that the PD-1AbIL-21 fusion protein has excellent therapeutic effects in combating PD-1 antibody-resistant leukemia models.

[0259] Example 14: Therapeutic effect of PD-1AbIL-21 fusion protein in humanized lymphoma mouse model

[0260] Subcutaneous injection of 5×10 NCG immunodeficient mice 6 A humanized lymphoma model was constructed using Daudi B-cell tumor cells. On day 22 of tumor bearing, 2 × 10⁻⁶ cells were injected intravenously (iv) via the tail vein. 6CD19CAR-T cells (same as in Example 13) were used as tumor-specific T cells. Mice were divided into 4 groups of 5 mice each according to tumor size. Each group received intraperitoneal injection of PBS (control), anti-PD-1 antibody alone (Keytruda®, 10 mg / kg), anti-PD-1 antibody (Keytruda®, 8.2 mg / kg) and IL-21M2 (1.8 mg / kg) in combination, or PD-1AbIL-21 fusion protein (10 mg / kg). Injections were given every 5 days for a total of 5 treatments. Figure 17 (See Figure a). Tumor size was measured every three days, mouse death time was recorded, and tumor growth curves and mouse survival curves were plotted.

[0261] The results show Figure 17 As shown in Figures b and c, tumor growth in mice in the antibody-only group and the combination therapy group was not significantly different from that in the control group. All three groups of mice showed tumors exceeding 2000 mm² within 55 days after tumor inoculation. 3 Mice treated with the PD-1AbIL-21 fusion protein, however, had tumors that reached 1000 mm² at the time of treatment, and were euthanized. 3 Around 1000 mice underwent complete tumor regression, and the mice achieved long-term survival. These results demonstrate that, compared to PD-1 antibody therapy, the PD-1AbIL-21 fusion protein has a more significant therapeutic effect on lymphoma, even leading to complete regression of large tumors.

[0262] During treatment, the proportion of hCD3+hCD45+ T cells in the peripheral blood of mice was monitored using flow cytometry. Results showed that the proportion of hCD3+hCD45+ T cells in the peripheral blood of mice in the PBS treatment control group, the antibody-only group, and the combined treatment group increased transiently in the first two weeks, reaching a maximum of 2% in peripheral blood cells. After one month, CAR-T cells were undetectable in the peripheral blood of these mice. However, the proportion of CAR-T cells in the peripheral blood of mice treated with the PD-1AbIL-21 fusion protein significantly increased, peaking one month after the start of treatment, reaching a maximum of 60% in peripheral blood cells. Significant CAR-T cells were still detectable in the peripheral blood of mice two months later. Figure 17 (Figure d in the middle). Compared with the PD-1 antibody alone, the combination therapy of PD-1 antibody and IL-21 protein did not increase the proportion of CAR-T cells in the peripheral blood of mice, while the PD-1AbIL-21 fusion protein therapy significantly increased the proportion of CAR-T cells in the peripheral blood of mice. This result fully demonstrates that the PD-1AbIL-21 fusion protein can target and expand tumor reactive T cells in vivo.

[0263] Example 15: Comparison of in vivo antitumor effects of IL-21M2 fusion protein and natural IL-21 fusion protein

[0264] To further demonstrate the superior antitumor effect of the PD-1 antibody optimized and screened in this application, which is a fusion protein of stable mutant IL-21M2 (PD-1AbIL-21), this application used the aforementioned humanized B-cell lymphoma mouse model to compare the in vivo antitumor effects of uPD1Ab6-IL-21 (wild-type) and uPD1Ab6-IL-21M2 (mutant). The specific experimental procedure is as follows:

[0265] Subcutaneous injection of 5×10⁶ NCG mice 6 Daudi B-cell lymphoma cells were injected intravenously (iv) with 2 × 10⁻⁶ cells on day 23 of tumor bearing. 6 Mice were divided into four groups based on tumor size and treated with CD19CAR-T cells via intraperitoneal injection: control group (PBS), group receiving anti-PD-1 antibody alone (Keytruda®, 10 mg / kg), group receiving uPD1Ab6-IL-21 fusion protein (1 mg / kg), and group receiving uPD1Ab6-IL-21M2 fusion protein (1 mg / kg). Injections were administered every five days for a total of three treatments. Tumor size was measured every three days, and mouse mortality time was recorded. Tumor growth curves and mouse survival curves were plotted.

[0266] The results are as follows Figure 18 As shown, under low-dose fusion protein treatment conditions, compared with the PBS-treated control group, the uPD1Ab6-IL-21 fusion protein, like antibody treatment alone, failed to effectively inhibit tumor growth and significantly prolong mouse survival; that is, the uPD1Ab6-IL-21 fusion protein, like PD-1 antibody treatment alone, had no therapeutic effect. However, treatment with the uPD1Ab6-IL-21M2 fusion protein resulted in tumors exceeding 1000 mm² in size. 3 The tumors completely regressed, and all mice survived long-term except for one that died unexpectedly. These results strongly suggest that the stability of mutant IL-21 plays a crucial role in the tumor therapeutic effect of the PD-1AbIL-21 fusion protein.

[0267] Table 5

[0268]

[0269] >Seq ID No.1

[0270] EVQLVESGGGLVQPGGSLKLSCAASGFTFT SYTMS WVRQTPEKRLEWVA FISGGGGDTYYPDTVKG RFTISRDNAKNTLYLQMSSLKSEDTAMYYCAR HGYDGTWFAY WGQGTLVTVSA.

[0271] >Seq ID No.2

[0272] EVQLVESGGGLVQPGGSLRLSCAASGFTFT SYTMS WVRQAPGKGLEWVS FISGGGGDTYYPDTVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR HGYDGTWFAY WGQGTLVTVSS。

[0273] >Seq ID No.3

[0274] EVQLVESGGGLVQPGGSLRLSCAASGFTFT SYTMS WVRQAPGKGLEWVA FISGGGGDTYYPDTVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR HGYDGTWFAY WGQGTLVTVSS。

[0275] >Seq ID No.4

[0276] EVQLVESGGGLVQPGGSLRLSCAASGFTFTSYTMSWVRQTPGKGLEWVAFISGGGGDTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGYDGTWFAYWGQGTLVTVSS。

[0277] >Seq ID No.5

[0278] EVQLVESGGGLVQPGVSLRLSCAASGFTFTSYTMSWVRQTPGKGLEWVAFISGGGGDTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGYDGTWFAYWGQGTLVTVSS。

[0279] >Seq ID No.6

[0280] EIVLTQLPATLSLSVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVSNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYATPYTFGGGTKVEIK。

[0281] >Seq ID No.7

[0282] DIQLTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKSPKLLIYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYATPYTFGGGTKLEIK。

[0283] >Seq ID No.8

[0284] DIQLTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLISNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYATPYTFGGGTKLEIK。

[0285] >Seq ID No.9

[0286] DIQLTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKSPKLLVSNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYATPYTFGGGTKLEIK。

[0287] >Seq ID No.10

[0288] DVLTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKSPKLLVSNAKTLAEGVPSRFSGSGSGTQFSLTISSLQPEDFATYYCQQHYATPYTFGGGTKLEIK。

[0289] >Seq ID No.11

[0290] EVQLVESGGGLVQPGVSLRLSCAASGFTFTSYTMSWVRQTPGKGLEWVAFISGGGGDTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGYDGTWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS。

[0291] >Seq ID No.12

[0292] EVQLVESGGGLVQPGVSLRLSCAASGFTFTSYTMSWVRQTPGKGLEWVAFISGGGGDTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGYDGTWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRNLWGLAGLNSCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS。

[0293] >Seq ID No.13

[0294] EVQLVESGGGLVQPGVSLRLSCAASGFTFTSYTMSWVRQTPGKGLEWVAFISGGGGDTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGYDGTWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSQGQDRHMIRMRQLIDCVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVCIKKLKRNLWGLAGLNSCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS。

[0295] >Seq ID No.14

[0296] QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGSHHHHHH。

[0297] >Seq ID No.15

[0298] QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRNLWGLAGLNSCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGSHHHHHH。

[0299] >Seq ID No.16

[0300] QGQDRHMIRMRQLIDCVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVCIKKLKRNLWGLAGLNSCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGSHHHHHH。

[0301] >Seq ID No.17

[0302] QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGSGGGGSGGGGSCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

[0303] >Seq ID No.18

[0304] QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRNLWGLAGLNSCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGSGGGGSGGGGSCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

[0305] >Seq ID No.19

[0306] QGQDRHMIRMRQLIDCVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVCIKKLKRNLWGLAGLNSCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGSGGGGSGGGGSCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

[0307] >Seq ID No.20

[0308] AAACTTAAGCTTATGAGATCCAGTCCTGGC。

[0309] >Seq ID No.21

[0310] GGCCCTCTAGACTCGAGTCAATGATGGTGGTGATGGTGAGAGCCTCCACCAGAGTCCTCGGAGCCGTG。

[0311] >Seq ID No.22

[0312] ACCGCCACCGGATCCAGAGTCCTCGGAGCCGTG。

[0313] >Seq ID No.23

[0314] GGATCCGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGTGCCCACCATGTCCAGCA。

[0315] >Seq ID No.24

[0316] GCCCTCTAGACTCGAGTCAGCCCAGGCTCAGAGACAG。

[0317] >Seq ID No.25

[0318] AACTTAAGCTTGCCACCATGACCAGGCTGACAGTG。

[0319] >Seq ID No.26

[0320] AGAGCCGCCGCCGCCGCCCAGGCTCAGAGACAG。

[0321] >Seq ID No.27

[0322] GGCGGCGGCGGCTCTGGAGGAGGAGGATCCGGAGGAGGAGGATCCCAAGGTCAAGATCGCCAC。

[0323] >Seq ID No.28

[0324] GGCCCTCTAGACTCGAGTTATTAGGAATCTTCACTTCCGTG。

[0325] >Seq ID No.29

[0326] GAAGTGCAGCTGGTGGAGTCTGGGGGAGGTTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCACTAGCTATACCATGTCTTGGGTTCGCCAGACTCCCGAGAAGAGGCTGGAATGGGTCGCATTCATTAGTGGTGGTGGTGGTGACACCTACTATCCAGACACTGTAAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTATTTGCAAATGAGCAGTCTGAAGTCTGAGGACACGGCCATGTATTACTGTGCAAGGCATGGTTACGACGGGACCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA。

[0327] >Seq ID No.30

[0328] GAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGCAGCCTGGAGGCTCCCTGAGGCTGTCTTGCGCAGCAAGCGGCTTCACCTTTACATCCTACACCATGTCTTGGGTGAGACAGGCCCCAGGCAAGGGACTGGAGTGGGTGAGCTTCATCAGCGGCGGAGGAGGCGACACATACTATCCTGATACCGTGAAGGGCCGGTTTACCATCAGCAGAGACAACTCCAAGAATACACTGTATCTGCAGATGAACTCCCTGAGGGCAGAGGACACCGCCGTGTACTATTGCGCCAGACACGGCTACGATGGCACATGGTTCGCCTATTGGGGCCAGGGCACCCTGGTGACAGTGTCTAGC。

[0329] >Seq ID No.31

[0330] GAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGCAGCCTGGAGGCTCCCTGAGGCTGTCTTGCGCAGCAAGCGGCTTCACCTTTACATCCTACACCATGTCTTGGGTGAGACAGGCCCCAGGCAAGGGACTGGAGTGGGTGGCCTTCATCAGCGGCGGAGGAGGCGACACATACTATCCTGATACCGTGAAGGGCCGGTTTACCATCAGCAGAGACAACTCCAAGAATACACTGTATCTGCAGATGAACTCCCTGAGGGCAGAGGACACCGCCGTGTACTATTGCGCCAGACACGGCTACGATGGCACATGGTTCGCCTATTGGGGCCAGGGCACCCTGGTGACAGTGTCTAGC。

[0331] >Seq ID No.32

[0332] GAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGCAGCCTGGAGGCTCCCTGAGGCTGTCTTGCGCAGCAAGCGGCTTCACCTTTACATCCTACACCATGTCTTGGGTGAGACAGACCCCAGGCAAGGGACTGGAGTGGGTGGCCTTCATCAGCGGCGGAGGAGGCGACACATACTATCCTGATTCCGTGAAGGGCCGGTTTACCATCAGCAGAGACAACTCCAAGAATACACTGTATCTGCAGATGAACTCCCTGAGGGCAGAGGACACCGCCGTGTACTATTGCGCCAGACACGGCTACGATGGCACATGGTTCGCCTATTGGGGCCAGGGCACCCTGGTGACAGTGTCTAGC。

[0333] >Seq ID No.33

[0334] GAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGCAGCCTGGAGTATCCCTGAGGCTGTCTTGCGCAGCAAGCGGCTTCACCTTTACATCCTACACCATGTCTTGGGTGAGACAGACCCCAGGCAAGGGACTGGAGTGGGTGGCCTTCATCAGCGGCGGAGGAGGCGACACATACTATCCTGATTCCGTGAAGGGCCGGTTTACCATCAGCAGAGACAACTCCAAGAATACACTGTATCTGCAGATGAACTCCCTGAGGGCAGAGGACACCGCCGTGTACTATTGCGCCAGACACGGCTACGATGGCACATGGTTCGCCTATTGGGGCCAGGGCACCCTGGTGACAGTGTCTAGC。

[0335] >Seq ID No.34

[0336] GAAATTGTGTTGACGCAGCTTCCAGCCACCCTGTCTTTGTCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAACTCCTGGTCTCTAATGCAAAAACCTTAGCAGAGGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATCATTATGCTACTCCGTACACGTTCGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTG。

[0337] >Seq ID No.35

[0338] GACATCCAGCTGACCCAGAGCCCATCCTCTCTGTCTGCCAGCGTGGGCGATAGGGTGACCATCACATGTCGCGCCTCTGAGAACATCTACAGCTATCTGGCCTGGTACCAGCAGAAGCCCGGCAAGTCCCCTAAGCTGCTGATCTACAATGCAAAGACCCTGGCAGAGGGAGTGCCATCTAGGTTCTCCGGCTCTGGCAGCGGCACCCAGTTTAGCCTGACAATCAGCTCCCTGCAGCCTGAGGATTTCGCCACATACTATTGTCAGCACCACTACGCCACCCCATATACATTTGGCGGCGGCACCAAGCTGGAGATCAAG。

[0339] >Seq ID No.36

[0340] GACATCCAGCTGACCCAGAGCCCATCCTCTCTGTCTGCCAGCGTGGGCGATAGGGTGACCATCACATGTCGCGCCTCTGAGAACATCTACAGCTATCTGGCCTGGTACCAGCAGAAGCCCGGCAAGTCCCCTAAGCTGCTGATCTCTAATGCAAAGACCCTGGCAGAGGGAGTGCCATCTAGGTTCTCCGGCTCTGGCAGCGGCACCCAGTTTAGCCTGACAATCAGCTCCCTGCAGCCTGAGGATTTCGCCACATACTATTGTCAGCACCACTACGCCACCCCATATACATTTGGCGGCGGCACCAAGCTGGAGATCAAG。

[0341] >Seq ID No.37

[0342] GACATCCAGCTGACCCAGAGCCCATCCTCTCTGTCTGCCAGCGTGGGCGATAGGGTGACCATCACATGTCGCGCCTCTGAGAACATCTACAGCTATCTGGCCTGGTACCAGCAGAAGCCCGGCAAGTCCCCTAAGCTGCTGGTGTCTAATGCAAAGACCCTGGCAGAGGGAGTGCCATCTAGGTTCTCCGGCTCTGGCAGCGGCACCCAGTTTAGCCTGACAATCAGCTCCCTGCAGCCTGAGGATTTCGCCACATACTATTGTCAGCAGCACTACGCCACCCCATATACATTTGGCGGCGGCACCAAGCTGGAGATCAAG。

[0343] >Seq ID No.38

[0344] GACGTGCTGACCCAGAGCCCATCCTCTCTGTCTGCCAGCGTGGGCGATAGGGTGACCATCACATGTCGCGCCTCTGAGAACATCTACAGCTATCTGGCCTGGTACCAGCAGAAGCCCGGCAAGTCCCCTAAGCTGCTGGTGTCTAATGCAAAGACCCTGGCAGAGGGAGTGCCATCTAGGTTCTCCGGCTCTGGCAGCGGCACCCAGTTTAGCCTGACAATCAGCTCCCTGCAGCCTGAGGATTTCGCCACATACTATTGTCAGCAGCACTACGCCACCCCATATACATTTGGCGGCGGCACCAAGCTGGAGATCAAG。

[0345] >Seq ID No.39

[0346]

[0347] >Seq ID No.40

[0348]

[0349] >Seq ID No.41

[0350]

[0351] >Seq ID No.42

[0352] CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGCTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCCGGTGGAGGCTCTCACCATCACCACCATCATTGA。

[0353] >Seq ID No.43

[0354] CAGGGCCAGGACAGACACATGATCAGGATGCGCCAGCTGATCGACATTGTGGATCAGCTGAAGAACTACGTGAATGACCTGGTGCCAGAGTTCCTGCCAGCACCTGAGGATGTGGAGACCAACTGCGAGTGGAGCGCCTTCTCCTGTTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACAGGCAACAATGAGCGGATCATCAACGTGTCTATCAAGAAGCTGAAGAGAAACCTGTGGGGACTGGCAGGCCTGAATTCTTGCCCATCTTGTGATAGCTATGAGAAGAAGCCACCCAAGGAGTTCCTGGAGCGGTTTAAGAGCCTGCTGCAGAAGATGATCCACCAGCACCTGTCTAGCAGAACCCACGGCTCCGAGGACTCTGGTGGAGGCTCTCACCATCACCACCATCATTGA。

[0355] >Seq ID No.44

[0356] CAGGGCCAGGACAGACACATGATCAGGATGCGCCAGCTGATCGACTGCGTGGATCAGCTGAAGAACTACGTGAATGACCTGGTGCCAGAGTTCCTGCCAGCACCTGAGGATGTGGAGACCAACTGCGAGTGGAGCGCCTTCTCCTGTTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACAGGCAACAATGAGCGGATCATCAACGTGTGCATCAAGAAGCTGAAGAGAAACCTGTGGGGACTGGCAGGCCTGAATTCTTGCCCATCTTGTGATAGCTATGAGAAGAAGCCACCCAAGGAGTTCCTGGAGCGGTTTAAGAGCCTGCTGCAGAAGATGATCCACCAGCACCTGTCTAGCAGAACCCACGGCTCCGAGGACTCTGGTGGAGGCTCTCACCATCACCACCATCATTGA。

[0357] >Seq ID No.45

[0358]

[0359] >Seq ID No.46

[0360]

[0361] >Seq ID No.47

[0362]

[0363] >Seq ID No.54

[0364] MTRLTVLALLAGLLASSRA。

[0365] >Seq ID No.55

[0366] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

[0367] >Seq ID No.56

[0368] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0369] >Seq ID No.57

[0370] ATGACCAGGCTGACAGTGCTGGCCCTGCTGGCAGGACTGCTGGCAAGCTCCAGGGCC。

[0371] >Seq ID No.58

[0372] GCCTCTACAAAGGGACCAAGCGTGTTTCCACTGGCACCATGCTCCAGATCTACCAGCGAGTCCACAGCCGCCCTGGGATGTCTGGTGAAGGACTATTTCCCTGAGCCAGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGAGTGCACACATTTCCTGCCGTGCTGCAGTCCTCTGGCCTGTACAGCCTGAGCTCCGTGGTGACCGTGCCATCTAGCTCCCTGGGCACAAAGACCTATACATGCAACGTGGATCACAAGCCCTCCAATACAAAGGTGGACAAGAGGGTGGAGTCTAAGTACGGACCACCTTGCCCACCATGTCCAGCACCTGAGGCTGCTGGAGGACCAAGCGTGTTCCTGTTTCCTCCAAAGCCTAAGGATACCCTGATGATCTCTCGCACCCCCGAGGTGACATGCGTGGTGGTGGATGTGAGCCAGGAGGACCCTGAGGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCTCGGGAGGAGCAGTTTAATTCCACCTACAGAGTGGTGTCTGTGCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCATCTAGCATCGAGAAGACCATCTCTAAGGCAAAGGGACAGCCTAGGGAGCCACAGGTGTACACACTGCCCCCTAGCCAGGAGGAGATGACCAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCTTCTGATATCGCCGTGGAGTGGGAGAGCAATGGCCAGCCAGAGAACAATTACAAGACAACCCCACCCGTGCTGGACAGCGATGGCTCCTTCTTTCTGTATAGCAAGCTGACCGTGGACAAGTCCCGCTGGCAGGAGGGCAACGTGTTTTCTTGTAGCGTGATGCACGAGGCCCTGCACAATCACTACACACAGAAGTCCCTGTCTCTGAGCCTGGGC。

[0373] >Seq ID No.59

[0374] AGGACAGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG。

[0375] >Seq ID No.60

[0376] LKPREVKLVESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINLDSSTINYTPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCARRYDAMDYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKASQ。

[0377] >Seq ID No.61

[0378] ASDIVLTQSPASLAVSLGQRATISCRASESVDDYGISFMNWFQQKPGQPPKLLIYAAPNQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKDVRWRHQAGDQTG。

[0379] >Seq ID No.62

[0380] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK。

[0381] >Seq ID No.63

[0382] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS.

[0383] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0384] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. An IL-21 fusion protein targeting PD-1, characterized in that, Including an operable link between an anti-PD-1 antibody and IL-21; The IL-21 has the amino acid sequence shown in positions 1 to 127 of Seq ID No. 18 or 19; Optionally, the heavy chain variable region of the anti-PD-1 antibody has the amino acid sequences CDR1 to CDR3 as shown in Seq ID No. 48 to 50, and the light chain variable region has the amino acid sequences CDR1 to CDR3 as shown in Seq ID No. 51 to 53. Optionally, the anti-PD-1 antibody may be a humanized anti-PD-1 antibody or a mouse-derived anti-PD-1 antibody.

2. The PD-1-targeting IL-21 fusion protein according to claim 1, characterized in that, The IL-21 is operatively linked to one or two heavy chains of the anti-PD-1 antibody; or / and the IL-21 is operatively linked to the C-terminus of the heavy chain of the anti-PD-1 antibody.

3. The IL-21 fusion protein targeting PD-1 according to claim 1, characterized in that, The anti-PD-1 antibody has a heavy chain variable region with an amino acid sequence as described in any one of Seq ID No. 1 to 5; or / and, the anti-PD-1 antibody has a light chain variable region with an amino acid sequence as described in any one of Seq ID No. 6 to 10.

4. The IL-21 fusion protein targeting PD-1 according to any one of claims 1 to 3, characterized in that, The anti-PD-1 antibody has a heavy chain variable region as shown in Seq ID No. 5 and a light chain variable region as shown in Seq ID No.

10.

5. The IL-21 fusion protein targeting PD-1 according to claim 4, characterized in that, The anti-PD-1 antibody has a constant region of amino acid sequence as shown in Seq ID No. 55 and / or Seq ID No.

56.

6. The IL-21 fusion protein targeting PD-1 according to any one of claims 1 to 3 and 5, characterized in that, The operable connections use connectors including (G4S)n, where n is an integer in the range of 2-4.

7. A PD-1 specific binding protein, characterized in that, The specific binding protein is a humanized anti-PD-1 antibody as defined in any one of claims 1 to 6.

8. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the IL-21 fusion protein targeting PD-1 as described in any one of claims 1 to 6.

9. The nucleic acid molecule according to claim 8, characterized in that, The nucleotide sequence of the nucleic acid molecule satisfies one or more of the conditions shown in (1) and (2) below: (1) Includes any of the nucleotide sequences shown in Seq ID Nos. 30 to 33; or / and, Includes any of the nucleotide sequences shown in Seq ID Nos. 35 to 38; (2) Includes the nucleotide sequence shown in positions 1 to 381 of Seq ID No. 43 or 44; Optionally, the nucleotide sequence of the nucleic acid molecule includes any of the nucleotide sequences shown in Seq ID No. 41 to 42.

10. A carrier, characterized in that, The nucleotide sequence of the vector comprises the nucleotide sequence of the nucleic acid molecule according to any one of claims 8 to 9.

11. A cell, characterized in that, The cells express the IL-21 fusion protein targeting PD-1 as described in any one of claims 1 to 6, the nucleic acid molecule as described in any one of claims 8 to 9, or the vector as described in claim 10.

12. The method for constructing cells according to claim 11, characterized in that, The construction method includes the step of introducing the nucleic acid molecule of any one of claims 8 to 9 or the vector of claim 10 into the cell to be modified.

13. The method for preparing the IL-21 fusion protein targeting PD-1 according to any one of claims 1 to 6, characterized in that, The preparation method includes the step of culturing the cells according to claim 11.

14. The use of the IL-21 fusion protein targeting PD-1 according to any one of claims 1 to 6, the nucleic acid molecule according to any one of claims 8 to 9, the vector according to claim 10, or the cell according to claim 11 in the preparation of an antitumor drug.

15. The application according to claim 14, characterized in that, The antitumor drug is used to treat solid tumors; optionally, the solid tumor is lung cancer, melanoma, head and neck malignant tumor, liver cancer, pancreatic cancer, renal cell carcinoma, urothelial carcinoma, bladder cancer, prostate cancer, gastric cancer, esophageal cancer, intestinal cancer, cervical cancer, ovarian cancer, breast cancer, thyroid cancer, or malignant tumor of the nervous system.

16. The application according to claim 14, characterized in that, The antitumor drug is used to prevent and treat hematologic malignancies; optionally, the hematologic malignancies are lymphoma or leukemia.

17. A drug, characterized in that, The drug comprises the IL-21 fusion protein targeting PD-1 as described in any one of claims 1 to 6, and a pharmaceutically acceptable carrier.

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