CN113583127A - Bispecific antibody targeting NKG2A and PD-L1 and application thereof - Google Patents

Abstract

According to the invention, based on the light and heavy chain amino acid sequence of an anti-NKG2A antibody reported in the prior art, a mutant antibody with improved affinity and/or improved dissociation constant is obtained by constructing a light and heavy chain mutant antibody library for affinity maturation, CDRs (complementary deoxyribonucleic acid) regions of the mutant antibody are constructed into a human Fab heavy chain gene expression vector and a mammalian cell expression vector containing human kappa subclass light chain constant region genes, the heavy chain vector and the light chain vector of the affinity matured antibody are in cross pairing, and a mutant Fab antibody of anti-NKG2A is obtained by screening and is connected with a human antibody Fc segment. A second antigen-binding molecule (e.g., an anti-PD-L1 antibody) is linked to the C-terminus of the Fc fragment via a linker to obtain a bispecific antibody.

Description

Bispecific antibody targeting NKG2A and PD-L1 and application thereof

This patent application claims priority to chinese patent application No. CN202010369911.7 filed on 30/4/2020, which is incorporated by reference in its entirety.

Technical Field

The invention relates to the field of antibody medicines, in particular to the field of tumor therapeutic antibodies. Specifically, the invention relates to a high-affinity humanized antibody targeting human NKG2A, and preparation and application of a bispecific antibody composed of the antibody and an anti-human PD-L1 nano antibody.

Background

In recent years, tumor immunotherapy has been unsuccessful, and only a few patients have shown long-lasting efficacy. Improving clinical response and overcoming resistance mechanisms are ongoing challenges in the field of tumor immunotherapy, and blocking other inhibitory immune receptors may be a viable strategy.

NKG2A (killer cell receptor C1), also known as KLRC1 or CD159A, is a type II transmembrane protein belonging to the NKG2/CD94 natural killer cell lectin receptor family. NKG2A has a Carbohydrate-recognizing domain CRD (Carbohydrate-recognition domain, usually consisting of 115-130 amino acids, containing 2-3 disulfide bonds, having 2-3N-linked glycosylation sites, the process of ligand recognition is often Ca2+ -dependent) in the extracellular domain. NKG2A is mainly expressed in NK cells, NKT cells and T cells; the relative molecular mass is 43000, which is composed of 233 amino acids, and the extracellular region has 135 amino acids. NKG2A inhibits immune cell activation through interaction with its ligand HLA-E. HLA-E is widely expressed on the surfaces of various tumor cells such as head and neck cancer, lung cancer, prostate cancer, colorectal cancer and the like, and IFN-gamma released by immune cells further up-regulates the expression of HLA-E (J Clin invest.2019; 129(5): 2094-2106). Like other receptor-ligands (such as PD-1/PD-L1), NKG2A/HLA-E is also an important signal path for tumor immune escape for immune checkpoints, and blocking the interaction between NKG2A/HLA-E becomes a very potential target in the field of tumor immunotherapy. Monoclonal antibodies against NKG2A are currently developed by several companies (e.g., Innate Pharma/Novo Nordisk/AstraZeneca and ChemPartner) to kill tumor cells by blocking the NKG2A/HLA-E interaction to increase the immunocompetence of NK cells as well as T cells.

Recent research shows that NKG2A and PD-1 are co-expressed on CD8+ T cells infiltrating head and neck cancer and melanoma, and have strong synergistic antitumor effect on blocking two signal pathways of NKG2A/HLA-E and PD-1/PD-L1 (cell.2018; 175:1-13, cell.2018; 175: 1744-1755). Meanwhile, the treatment aiming at various targets has positive effects of improving the response rate of tumor immunotherapy and reducing immune tolerance. At present, the affinity of a therapeutic antibody aiming at NKG2A to antigen is insufficient, the therapeutic effect of the single target point binding to NKG2A to tumors is poor, and no medicine aiming at NKG2A is on the market in the world. The French Paoli-Calmettes institute is advancing a phase I clinical safety test (NCT02921685) for treating hematological malignancies using humanized anti-NKG2A monoclonal antibody IPH2201 in combination with allogeneic stem cell transplantation.

Disclosure of Invention

In order to solve the problems, the invention constructs a mutant antibody with improved affinity and/or improved dissociation constant by constructing a light and heavy chain mutant antibody library based on the light and heavy chain amino acid sequence of an anti-NKG2A antibody reported in the prior art and performing affinity maturation, constructs a CDRs region of the mutant antibody into a human Fab heavy chain gene expression vector and a mammalian cell expression vector containing a human kappa subclass light chain constant region gene, cross-matches the heavy chain vector and the light chain vector of the affinity matured antibody, screens and obtains a mutant Fab antibody of the anti-NKG2A, and connects with an Fc segment of a human antibody. An anti-PD-L1 antibody was linked to the C-terminus of the Fc fragment by a linker to obtain an antibody bispecific to NKG2A and PD-L1. Specifically, the method comprises the following steps:

in one aspect, the invention provides an anti-NGK 2A antibody or an antigen-binding fragment thereof, which is an antibody with improved affinity and/or improved dissociation constant obtained by constructing a mutant antibody library on the basis of hum270 for affinity maturation, wherein the amino acid sequence of the light chain variable region of hum270 is shown as SEQ ID No. 4, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 6.

Further, the anti-NGK 2A antibody or antigen-binding fragment thereof according to the present invention is characterized in that the CDRs regions of the antibody are distinguished by the heavy chain CDRs regions compared to the CDRs regions of hum270, wherein the sequence of the antibody HCDR1-3 is as set forth in any one of H0-H6 in table 3.

Further, the anti-NGK 2A antibody or antigen-binding fragment thereof according to the present invention is characterized in that the CDRs regions of the antibody are distinguished by the light chain CDRs regions compared to the CDRs regions of hum270, wherein the sequence of the antibody LCDR1-3 is as shown in LCDR1-3 of any one of K0-270KX in table 4.

Further, the anti-NGK 2A antibody or antigen-binding fragment thereof of the invention is characterized in that the CDRs in the heavy chain variable region of the antibody are selected from HCDR1-3 of any one of H0-H3 in Table 3, and/or the CDRs in the light chain variable region of the antibody are selected from LCDR1-3 of any one of K0-K2 in Table 4.

Further, the anti-NGK 2A antibody or antigen-binding fragment thereof according to the present invention is characterized in that the heavy chain variable region of the antibody is selected from the group consisting of SEQ ID nos. 6, 12, and 13, and the light chain variable region is selected from the group consisting of SEQ ID nos. 4, 14, and 15;

also, the antibody does not include the case where the heavy chain variable region is SEQ ID NO. 4, while the light chain variable region is SEQ ID NO. 6.

Further, the anti-NGK 2A antibody or an antigen-binding fragment thereof according to the present invention is characterized in that the antibody or the antigen-binding fragment thereof is derived from a mouse, a rat, a rabbit, a goat, a sheep, a camel, an alpaca, a human, or a chimeric antibody derived from the above species.

Further, the anti-NGK 2A antibody or antigen binding fragment thereof of the present invention is characterized in that the antibody or antigen binding fragment thereof includes but is not limited to Fab, Fab ', F (ab')₂Fv fragments, scFv and single domain fragments.

In a second aspect, the present invention provides a multispecific antibody or antigen-binding fragment thereof, comprising at least one first antigen-binding portion that binds a first antigen, and at least one second antigen-binding portion that binds a second antigen; wherein the first antigen-binding portion that binds a first antigen is an anti-NGK 2A antibody or antigen-binding fragment thereof according to the first aspect of the invention.

Further, the multispecific antibody or antigen-binding fragment thereof of the present invention, wherein the second antigen is an antibody therapy target, including but not limited to CD19, CD3, CD80, CD86, CD25, OX40, PD-L1, PD-1, VEGF, CTLA4, RANKL, EGFR, HER2, TNF-a.

Further, the multispecific antibody or antigen-binding fragment thereof of the present invention, wherein the second antigen is a tumor therapy-associated antigen, preferably PD-L1, PD-1.

Further, the multispecific antibody or antigen-binding fragment thereof of the present invention, wherein the first antigen-binding portion is selected from a human antibody, a humanized antibody, a human chimeric antibody; the second antigen-binding portion is selected from the group consisting of a single chain antibody, a single domain antibody.

Further, the multispecific antibody or antigen-binding fragment thereof of the present invention, wherein the first antigen-binding portion comprises an intact antibody Fc fragment; and, the second antigen-binding portion is covalently coupled to the Fc region of the first antigen-binding portion.

Further, the multispecific antibody or antigen-binding fragment thereof of the present invention, wherein the second antigen-binding portion is fused to the C-terminus of the heavy chain of the first antigen-binding portion by a Linker.

Further, the multispecific antibody or antigen-binding fragment thereof provided by the invention, wherein the Linker is (GnS) m, wherein n and m are 1-6.

In a third aspect, the present invention provides a composition comprising an anti-NGK 2A antibody or antigen-binding fragment thereof according to the first aspect of the invention, and/or a multispecific antibody or antigen-binding fragment thereof according to the second aspect of the invention.

Furthermore, the composition is characterized by further comprising a pharmaceutically acceptable carrier, and is used as a pharmaceutical composition, preferably the pharmaceutical composition is a water agent, an injection and a powder injection.

In a fourth aspect, the present invention also provides the use of the anti-NGK 2A antibody or antigen binding fragment thereof of the first aspect, the multispecific antibody or antigen binding fragment thereof of the second aspect, or the composition of the third aspect in the preparation of a medicament for treating a tumor and/or enhancing an immune response in a body.

In a fifth aspect, the present invention provides a polynucleotide encoding the anti-NGK 2A antibody or antigen-binding fragment thereof according to the first aspect of the invention, or the multispecific antibody or antigen-binding fragment thereof according to the second aspect of the invention.

In a sixth aspect, the present invention provides a vector comprising a polynucleotide according to the fifth aspect of the invention.

In a seventh aspect, the present invention provides a host cell comprising a polynucleotide according to the fifth aspect of the present invention or a vector according to the sixth aspect of the present invention.

In an eighth aspect, the present invention provides a method of making an anti-NGK 2A antibody or antigen binding fragment thereof, or making a multispecific antibody or fragment thereof comprising an anti-NGK 2A antibody or antigen binding fragment thereof, comprising the steps of:

(1) culturing a host cell according to the seventh aspect of the invention under conditions suitable for expression of an anti-NGK 2A antibody or antigen-binding fragment thereof, or a multispecific antibody or fragment thereof comprising an anti-NGK 2A antibody or antigen-binding fragment thereof;

(2) isolating and purifying the anti-NGK 2A antibody or antigen-binding fragment thereof, or a multispecific antibody or fragment thereof comprising the anti-NGK 2A antibody or antigen-binding fragment thereof from the cell culture.

For a better understanding of the present invention, certain terms are first defined. Other definitions are listed throughout the detailed description section.

The term "NKG 2A" is an inhibitory receptor present in NK, NKT and T cell subsets, NKG2A (OMIM 161555, the entire disclosure of which is incorporated herein by reference) is a member of the NKG2 group of transcripts (Houchins, et al (1991) J.exp.Med.173: 1017-. NKG2A is encoded by 7 exons spanning 25kb showing some differential splicing. NKG2A forms together with CD94 the heterodimeric inhibitory receptor CD94/NKG2A found on the surface of NK cells, α/β T cells, γ/δ T cells and subsets of NKT cells. Like the inhibitory KIR receptor, it has an ITIM in its cytoplasmic domain. As used herein, "NKG 2A" refers to any variant, derivative or isoform (isoform) of the NKG2A gene or encoded protein. Also included are any nucleic acid or protein sequences that share one or more biological properties or functions with wild-type full-length NKG2A and share at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more nucleotide or amino acid identity. Human NKG2A comprises 233 amino acids in 3 domains, wherein the cytoplasmic domain comprises residues 1-70, the transmembrane region comprises residues 71-93, and the extracellular region comprises residues 94-233.

The term "specific" refers to the determination of the presence or absence of a protein in a heterogeneous population of proteins and/or other organisms, e.g., the binding reaction of a monoclonal antibody of the invention to NKG2A antigen. Thus, under the conditions specified, a particular ligand/antigen binds to a particular receptor/antibody and does not bind in significant amounts to other proteins present in the sample.

The term "antibody" herein is intended to include full-length antibodies and any antigen-binding fragment (i.e., antigen-binding portion) or single chain thereof. Full-length antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains, the heavy and light chains being linked by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH 3. Each light chain is composed of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region is composed of one domain CL. The VH and VL regions can also be divided into hypervariable regions, called Complementarity Determining Regions (CDRs), which are separated by more conserved Framework Regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. The variable regions of the heavy and light chains comprise binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component of the classical complement system (C1 q).

The term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

The term "antigen-binding fragment" of an antibody (or simply antibody portion), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind antigen. It has been demonstrated that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments comprised in the "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH 1; (ii) a F (ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a hinge region disulfide bridge; (iii) an Fd fragment consisting of VH and CH 1; (iv) an Fv fragment consisting of VL and VH antibody single arms; (v) dAb fragments consisting of VH (Ward et al, (1989) Nature 341: 544-546); (vi) an isolated Complementarity Determining Region (CDR); and (vii) a nanobody, a heavy chain variable region comprising a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by different genes, they can be joined by recombinant methods via a synthetic linker that makes the two single protein chains, in which the VL and VH regions pair to form monovalent molecules (known as single chain Fc (scFv); see, e.g., Bird et al., (1988) Science 242: 423-. These single chain antibodies are also intended to be included within the term meaning. These antibody fragments can be obtained by conventional techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as intact antibodies.

Antigen-binding fragments of the invention include those capable of specifically binding coronavirus RBD. Examples of antibody binding fragments include, for example, but are not limited to, Fab ', F (ab')₂Fv fragments, single chain Fv (scFv) fragments and single domain fragments.

The Fab fragment contains the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab 'fragments are generated by cleavage of the disulfide bond at the hinge cysteine of the F (ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art. Fab and F (ab')2 fragments lack the fragment crystallizable (Fc) region of intact antibodies, clear more rapidly from the circulation of animals, and may have less non-specific tissue binding than intact antibodies (see, e.g., Wahl et al, 1983, J.Nucl. Med.24: 316).

As is generally understood in the art, an "Fc" region is a fragment crystallizable constant region of an antibody that does not comprise an antigen-specific binding region. In IgG, IgA and IgD antibody isotypes, the Fc region consists of two identical protein fragments derived from the second and third constant domains of the two heavy chains of an antibody (CH2 and CH3 domains, respectively). The IgM and IgE Fc regions contain three heavy chain constant domains (CH2, CH3, and CH4 domains) in each polypeptide chain.

The "Fv" fragment is the smallest fragment of an antibody that contains the entire target recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain (VH-VL dimer) in tight non-covalent association. In this configuration, the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer. Typically, six CDRs confer target binding specificity on an antibody. However, in some cases, even a single variable domain (or half of an Fv comprising only three CDRs specific for a target) may have the ability to recognize and bind to a target, although at a lower affinity than the entire binding site.

"Single chain Fv" or "scFv" antibody binding fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form a structure that facilitates target binding.

A "single domain fragment" consists of a single VH or VL domain that exhibits sufficient affinity for a coronavirus RBD. In a particular embodiment, the single domain fragments are camelized (see, e.g., Riechmann, 1999, Journal of immunological Methods 231: 25-38).

The anti-NKG2A antibodies of the invention include derivatized antibodies. For example, derivatized antibodies are typically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins. Any of a number of chemical modifications can be made by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the derivative may contain one or more unnatural amino acid, e.g., using ambrx technology (see, e.g., Wolfson, 2006, chem. biol.13(10): 1011-2).

"human antibodies" include antibodies having the amino acid sequence of a human immunoglobulin, and include antibodies isolated from a human immunoglobulin library or an animal that is transgenic for one or more human immunoglobulins and does not express endogenous immunoglobulins. Human antibodies can be made by various methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Pat. nos. 4,444,887 and 4,716,111; and PCT publication WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO 96/34096; WO 96/33735; and WO 91/10741. Human antibodies can also be produced using transgenic mice that do not express functional endogenous immunoglobulins, but can express human immunoglobulin genes. See, for example, PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; U.S. patent nos. 5,413,923; 5,625,126, respectively; 5,633,425, respectively; 5,569,825; 5,661,016, respectively; 5,545,806; 5,814, 318; 5,885,793, respectively; 5,916,771, respectively; and 5,939,598. Alternatively, using techniques similar to those described above, companies such as LakePharma, Inc (Belmont, CA) or Creative BioLabs (Shirley, NY) may be engaged in providing human antibodies to selected antigens. Fully human antibodies that recognize selected epitopes can be generated using a technique known as "guided selection". In this method, a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of fully human antibodies that recognize the same epitope (see Jespers et al, 1988, Biotechnology 12: 899-903).

The term "chimeric antibody" refers to an antibody that: its light and heavy chain genes have been constructed, usually by genetic engineering, from immunoglobulin variable and constant region genes derived from different species. For example, variable segments of genes from mouse monoclonal antibodies can be linked to human constant segments.

The terms "antibody recognizing an antigen" and "antibody specific for an antigen" are used herein interchangeably with the term "antibody specifically binding to an antigen".

The term "binding affinity" is used herein as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or fragment thereof, and an antigen). The term "binding affinity" is used to describe monovalent interactions (intrinsic activity). The binding affinity between two molecules (e.g., an antibody or fragment thereof, and an antigen) via a monovalent interaction can be quantitatively determined by determining the dissociation constant (KD). KD can then be determined by measurement of complex formation and dissociation kinetics, for example by SPR methods. The rate constants corresponding to the association and dissociation of monovalent complexes are referred to as the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. KD is related to ka and KD by the equation KD ═ KD/ka. According to the above definitions, the binding affinities associated with different molecular interactions, e.g. the binding affinities of different antibodies for a given antigen, can be compared by comparing the KD values of the individual antibody/antigen complexes. Similarly, the specificity of an interaction can be evaluated by determining and comparing the KD value for the interaction of interest (e.g., a specific interaction between an antibody and an antigen) to the KD value for an interaction not of interest. The value of the dissociation constant can be determined directly by well-known methods, e.g., by standard assays that assess the binding ability of a ligand (e.g., an antibody) to a target are known in the art and include, e.g., ELISA, western blot, RIA, and flow cytometry analysis. The binding kinetics and binding affinity of the antibody can also be assessed by standard assays known in the art, such as SPR. A competitive binding assay may be performed in which the binding of an antibody to a target is compared to the binding of another ligand (e.g., another antibody) to the target.

The term "high affinity" for IgG antibodies means a KD for the antigen of 1.0x10^-6M or less, preferably 5.0x10^-8M or less, more preferably 1.0x10^-8M below, 5.0x10^-9M or less, more preferably 1.0x10^-9M is less than or equal to M. For other antibody subtypes, "high affinity" binding may vary. For example, "high affinity" binding of an IgM subtype means a KD of 10^-6M is less, preferably 10^-7M is less, more preferably 10^-8M is less than or equal to M.

The term "Kassoc" or "Ka" refers to the association rate of a particular antibody-antigen interaction, while the term "Kdis" or "Kd" refers to the dissociation rate of a particular antibody-antigen interaction. The term "KD" refers to the dissociation constant, derived from the KD to Ka ratio (KD/Ka), and expressed in molar concentration (M). The KD value of an antibody can be determined by methods known in the art. A preferred way of determining the KD of an antibody is by measurement using a Surface Plasmon Resonance (SPR), preferably a biosensing system such as the Biacore (TM) system.

The term "EC 50," also called half maximal effect concentration, refers to the concentration of antibody that causes 50% of the maximal effect.

Compared with the prior art, the technical scheme of the invention has the following advantages:

firstly, the invention is modified on the basis of the anti-NKG2A antibody with therapeutic activity known in the prior art, so that the key properties such as affinity, specificity and the like are further improved. And (3) respectively carrying out mutation library construction on CDRs (complementary deoxyribonucleic acid) regions of the light and heavy chain antibodies, screening mutants with single-chain CDRs which are mutated and have improved specific binding capacity, and further carrying out cross-pairing screening to obtain the mutant antibodies with stronger binding capacity and improved specificity compared with the original antibodies.

Secondly, the anti-NKG2A antibody with optimized structure and improved performance is fused with the PD-L1 targeting fragment to construct the NKG2A/PD-L1 bispecific antibody, and the NKG2A/PD-L1 bispecific antibody has high affinity and strong specificity for two targets, namely NKG2A and PD-L1 through FACs and ELISA detection.

Third, the therapeutic effect of the NKG2A/PD-L1 bispecific antibody of the present invention on animal models of tumors is beyond the reasonable expectation of those skilled in the art. In a tumor-bearing mouse model, the treatment effect of the NKG2A/PD-L1 bispecific antibody is not only remarkably superior to that of two monospecific antibodies used alone, but also can reach a treatment effect even superior to that of two antibodies used together at a higher total dose, namely, two targeting parts of the NKG2A/PD-L1 bispecific antibody generate a synergistic effect.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. Also, like reference numerals are used to refer to like parts throughout the drawings. In the drawings:

FIG. 1: hum270 was analyzed for affinity to human NKG2A-CD94 recombinant protein.

FIG. 2: primers used to construct the CDR mutation library.

FIG. 3: mutant clones with improved dissociation constants.

FIG. 4: mutant antibody light and heavy chain variable region gene.

FIG. 5: the light and heavy chains of each mutant antibody were cross-paired to generate Fab numbering designs.

FIG. 6: NKG2A and PD-1/PD-L1 pathway inhibitors have inhibitory effects on PBMC immunization for reconstitution of subcutaneous tumor-bearing A375 tumor growth.

FIG. 7: effect of NKG2A and PD-1/PD-L1 pathway inhibitors on PBMC immunization to reconstitute subcutaneous tumor-bearing a375 tumor weights.

FIG. 8: hum270-F2 immune reconstruction of tumor growth inhibition effect of A375 subcutaneous transplantation tumor on PBMC

FIG. 9: hum270-F2 and AM10-F2 had an inhibitory effect on the growth of A375 subcutaneous transplantation tumor by immune reconstitution of PBMC.

FIG. 10: FACS analysis of hum270-F2/AM10-F2 binding activity to NK92 cell surface NKG 2A.

FIG. 11: FACS detection of hum270-F2/AM10-F2 binding Activity assay with MDA-MB-231 cell surface PD-L1.

FIG. 12: ELISA was used to detect that hum270-F2/AM10-F2 blocked the binding activity of recombinant PD-L1 and PD-1.

Detailed Description

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

Example 1 analysis of the expression of anti-NKG 2A-specific antibody Z270 antibody

The coding genes of NKG2A (genebank: AF461812.1, 157aa-233aa) and CD94 (genebank: AB009597.1, 4aa-148aa) are connected through GGSGGS coding genes to synthesize a coding gene (SEQ ID NO.1) of NKG2A-CD94, and the coding gene is further cloned into a eukaryotic transient expression vector with an mFc tag at the N end by an enzyme digestion method and is transferred into escherichia coli for amplification, an mFc-NKG2A-CD94 expression plasmid is obtained through separation, and the plasmid is transferred into HEK293 cells for recombinant expression according to the operation instruction of a transfection reagent 293fectin (Cat:12347019, Gibco). And 5-6 days after cell transfection, taking culture supernatant, and purifying the expression supernatant by using a ProA affinity chromatography column to obtain a pure mFc-NKG2A-CD94 recombinant protein.

The light-heavy chain amino acid sequence of the antibody hum270 is obtained from WO2012/172102A1/CN 103635487B patent, after further mutation of the heavy chain amino acid in Q1E, carrying out whole-gene synthesis on optimized light chain variable region hum270VL (SEQ ID NO.3) and heavy chain variable region hum270VH (SEQ ID NO.5), carrying out enzyme digestion cloning on hum270VH into the upstream of a heavy chain constant region (SEQ ID NO.9) coding gene of human IgG4 of a eukaryotic expression vector pKN034, carrying out enzyme digestion cloning on hum270VL into the upstream of a coding gene of a human light chain Ck (SEQ ID NO.7) of the eukaryotic expression vector pKN019, constructing a hum270 light chain and heavy chain expression vector, transferring the mixture into escherichia coli for amplification, separating to obtain hum270 light and heavy chain expression plasmid hum270-L, hum270-H, and performing transient expression by using HEK293 cells, and purifying an expression supernatant by using a MabSelect affinity chromatography column to obtain a recombinant protein pure product of the recombinant hum270 antibody.

The Fortebio protein interaction system is used for determining the binding affinity of the hum270 recombinant antibody and the mFc-NKG2A-CD94 recombinant antigen. The method mainly comprises the following steps:

the binding of the antibody to the bivalent antigen mFc-NKG2A-CD94 was determined by capturing the Fc fragment of the antibody with an anti-human antibody Fc fragment capture Antibody (AHC) bioprobe using an Octet QKe system instrument from Fortebio. Hum270 recombinant antibody (4ug/mL) was flowed over the surface of an AHC probe (Cat:18-5060, PALL) for 240 s. The mobile phase was composed of mFc-NKG2A-CD94 at concentrations of 60nM, 30nM, 7.5nM, and 5 nM. The binding time was 300s and the dissociation time was 300 s. After the experiment, blank control response values were deducted, and the software was run for 1: 1Langmuir binding pattern was fitted and kinetic constants for antigen-antibody binding were calculated. The results show that the response curves of hum270 to human NKG2A-CD94 recombinant protein are shown in FIG. 1, the curves are fitted and the affinities are calculated, the affinity (KD) of hum270 is 4.08E-10M, the affinity (KD) of kon is 4.81E + 051/Ms, and the kdis is 1.96E-041/s.

Example 2 affinity maturation of anti-NKG2a antibody hum270

And (3) carrying out random mutation library construction on 6 CDR (complementary deoxyribonucleic acid) region amino acids of the heavy chain variable region and the light chain variable region of the hum270 of the anti-NKG2a antibody by adopting a yeast display technology, and screening candidate antibody molecules with higher affinity.

1. Mutant antibody library design and construction

The humanized antibody hum270 is selected as a template for affinity maturation modification, and a heavy chain variable region and a light chain variable region of the humanized antibody are connected by a GS linker to construct a single-chain variable fragment (scFv). The CDR region of the single-chain antibody is the target of affinity maturation modification, while the framework region will remain unchanged in the affinity maturation modification, and the partial amino acids (table 1) of the light and heavy chains in 6 CDR regions are subjected to mutation design to respectively construct a mutation library.

TABLE 1 amino acid sequences for mutation design

CDR region mutation primers were designed and synthesized to introduce only 30% of the nucleotide mutations at each mutation position, with approximately 20 base pairs of unmutated bases flanking the primer (as shown in FIG. 2). The mutations are integrated into the final single-chain antibody gene by two-step PCR of the mutant primers with other conventional primers. The single-chain antibody gene has an overhang of 148bp at the 5 'end and an overhang of 222bp at the 3' end, which are identical to the sequences on the vector, so-called homologous regions. The antibody fragment and the linearized vector gene are transformed into a yeast strain EBY100 together by an electroporation method, and the homologous recombination mechanism of the yeast is utilized to realize the in vivo recombination of the single-chain antibody gene and the display vector, so that a mutant yeast display library is constructed. The library capacity is shown in Table 2.

TABLE 2 library Capacity statistics for each mutant

CDR-H3 amino acids were abundant, and thus two separate antibody libraries were constructed

2. Flow sorting and identification of affinity matured antibody libraries

The antibody repertoires in Table 2 above were classified and pooled into 3 combinatorial antibody repertoires, the first of which is a heavy chain repertoire consisting of H1-H4; the second combinatorial library was a light chain library comprising L1-L3 and the third was a full sequence library, i.e., EP.

The 3 combined antibody libraries were flow sorted, each round of sorting was combined with 10nM antigen for 30min at room temperature, unbound antigen was washed off, and a final concentration of 1uM hz270 IgG was added for competitive screening, followed by flow analysis and sorting after shaking incubation at room temperature. Collecting libraries by FACS sorting in each round

The clones of (2) are cultured, then the next round of sorting is carried out, and finally, the mutant clones with improved affinity are separated through 4-5 rounds of sorting in total. The CDR region amino acid sequences of the light and heavy chain mutants are shown in tables 3 and 4.

TABLE 3 CDRs sequences of heavy chain mutants

	HCDR1	HCDR2	HCDR3
				H0	GYTFTNYWMN	RIDPYDSETHYAQKLQGR	GGYDFDVGTLYWFFDV
H1	SYDFSWYWIN	RIDPYDSETHYAQKLQGR	GGYDFDVGTLYWFFDV
				H2	GYTFTNYWLN	RIDPYDSETHYAQKLQGR	GGYDFDVGTLYWFFDV
H3	GYTFDSYWMN	RIDPYDSETHYAQKLQGR	GGYDFDVGTLYWFFDV
				H4	SYDFSWYWIN	RIDPYDSETHYAQKLQGR	GGYDWDVGTLYWFFDV
H5	SYDFSWYWIN	RIDPYDSETHYAQKLQGR	GGYDFDVGTLYWFPDI
				H6	SYDFSWYWIN	RIDPYDSETHYAQKLQGR	GGYDWDVGTLYWFPDI

TABLE 4 CDRs sequences of light chain mutants

	LCDR1	LCDR2	LCDR3
				K0	RASENIYSYLA	NAKTLAE	QHHYGTPRT
K1	RASENIYSYLA	NSTALAK	QHHYGTPRT
				K2	RASENIYSYLA	NAKTLAE	QQHYGTPRT
K3	RASENIYSYLA	NSTALAK	QQHYGKPRT
				K4	RASENIYSYLA	NSTALAK	LQHYSKPRT
K5	RASENIYSYLA	NTTAFVE	QQHYGKPRT
				K6	RASENIYSYLA	NTTAFVE	LQHYSKPRT
K7	RASENIYSYLA	NGTTSTD	QQHYGKPRT
				K8	RASENIYSYLA	NGTTSTD	LQHYSKPRT
K9	RASENIYSYLA	NAKTLAE	QQHYGKPRT
				270-KX	RASENIYSYLA	NAKTLAE	LQHYSKPRT

The yeast cloned by the mutant is inoculated in a yeast culture medium to induce the expression of the related single-chain antibody. Yeast cells were harvested by centrifugation and washed 1 time with 1% BSA in PBS. Resuspend yeast cells to a density of 5000,000 cells/ml and add 100ul cells per well in a 96U-shaped well plate. 100ul of antigen solution with a final concentration of 10nM was added, incubated with shaking at room temperature for 30 minutes, and then placed on ice for 10 minutes. On the one hand, the bound EC50 values of the mutants were analyzed by FACS using fluorescence after washing, and the results are shown in table 5.

Table 5: EC50 value for yeast cell surface display mutant clones

On the other hand, the Koff improvement of the mutant antibody is evaluated by a competition method. That is, 1uM hz270-IgG is added to the washed yeast resuspension solution, the mixture is shaken at room temperature and placed, 20ul solutions are taken out at different time points (such as10 minutes, 20 minutes, 30 minutes, 60 minutes, 90 minutes to 240 minutes, respectively recorded as t 1-t 8), the solution is centrifuged at 14000rpm for 1 minute at 4 ℃ to collect cells, the cells are washed with precooled PBS containing 1% BSA for 2-3 times and the precipitate is collected by centrifugation, and after the last sampling, 100 microliters of 1:500 dilutions of fluorescent secondary antibody were incubated on ice for 30 min. A T0 control with no Hz270-IgG added was also set. The cells were harvested by centrifugation at 14000rpm for 1 minute at 4 ℃ and washed 2-3 times with pre-cooled PBS containing 1% BSA. The mean fluorescence readings of the cell populations were analyzed by flow cytometry, the data plotted, and the Koff values calculated using GraphPad PRISM software to obtain Koff improved mutant antibodies as shown in figure 3.

3. Recombinant expression and affinity analysis of affinity matured antibody Fab

The mutation sites of the above affinity-improving clones were analyzed in combination, and the relevant mutation sites of the respective CDR regions were combined. Synthesizing an antibody variable region heavy chain gene as shown in FIG. 4, and constructing it into a mammalian cell expression vector containing the heavy chain gene in the form of Fab of a human monoclonal antibody; the light chain gene shown in FIG. 4 was synthesized and constructed into a mammalian cell expression vector containing the light chain constant region gene of the kappa subclass of the human monoclonal antibody. The heavy chain vector and the light chain vector of the constructed affinity matured antibody are cross-paired, a HEK293 cell is transfected by using Polyethyleneimine (PEI), and after about 7 days, cell supernatant is collected and purified to obtain the Fab protein of the affinity matured antibody (FIG. 5).

The binding kinetic parameters of the affinity matured antibody Fab to the antigen mFC-huNKG2A/CD94 were analyzed using a Fortebio (BLITZ pro1.1.0.28) instrument. Before measurement, AMC biological probe is soaked in PBS for 10 minutes; the probe was then captured in PBS containing 100nM mFC-huNKG2A/CD94 for 300 seconds; further carrying out a binding reaction on the probe and 100nM antibody Fab for 400 seconds; the probe was then transferred to PBS and the dissociation reaction was carried out for 600 seconds. After the experiment, deducting the blank parent response value, and performing 1: 1Langmuir binding pattern was fitted and kinetic constants for antigen-antibody binding were calculated, and as a result, as shown in Table 6, it was seen that the affinity of the mutants was improved to various degrees.

TABLE 6 analysis of kinetic parameters of partial affinity matured antibody Fab

4. Analysis of the specific binding Activity of mutant Fab on cell-surface NKG2A

Flp-in CHO cells were co-stably transfected with plasmids encoding human NKG2A (huNKGA) and human CD94 (huncd 94), respectively, and expression-identified using anti-NKG2A and anti-CD94 antibodies, followed by sorting of double-positive cells using a flow cytometer, constructing NKG2A/CD94 stably expressing cell lines, and further performing FACS binding activity analysis of the above mutants.

A corresponding number of human NKG2A/CD94 stable cell lines were collected and plated at 0.06X 10 in 96-well clear bottom cell culture plates⁶Each cell/well was seeded in 100ul DMEM + 10% FBS overnight at 37 deg.C with 5% CO 2. The following morning, the plates were inverted in a water bath to gently remove the culture supernatant, and then the cells were gently washed 1-2 times with 1xPBS, gently tapped for drying, and collected for future use.

Affinity matured antibody, template antibody Fab and negative parent antibody Fab were formulated to the required concentrations by triple dilution with DMEM + 10% FBS, added immediately to wells (50 ul/well) and incubated at room temperature for 30 minutes. Wash 1-2 times with pre-cooled PBS containing 1% BSA, add 100 μ l 1:500 dilutions of fluorescent secondary antibody were incubated on ice for 30 min. The cell population was then washed 2 times with pre-chilled PBS containing 1% BSA, the mean fluorescence readings were analyzed by flow cytometry, the data were plotted, and EC50 values were calculated using GraphPad PRISM software (table 7). The results suggest that the following mutants all have different degrees of improvement in affinity compared to the parent antibody AM 00.

TABLE 7 EC50 values for the binding of mutant antibody Fab and cell-surface human NKG2A/CD94

Fab	KD[nM]	R square	KD/KD-AM00		Fab	KD[nM]	R square	KD/KD-AM00
									AM00	188.80	0.994	1.0		AM00	188.8	0.994	1.0
AM10	1.4310	0.946	131.9		AM11	1.127	0.946	167.5
									AM20	130.90	0.989	1.4		AM12	0.339	0.941	556.6
AM30	56.610	0.995	3.3		AM13	0.636	0.949	297.1
														AM14	0.466	0.843	405.2
AM01	117.50	0.991	1.6
									AM02	28.960	0.995	6.5		AM15	0.768	0.970	246.0
					AM16	0.597	0.943	316.2
														AM17	0.741	0.963	254.9
					AM18	0.614	0.947	307.7

Example 3 expression and affinity analysis of anti-NKG2a and PD-L1 bispecific antibodies

1. Bispecific antibody construction and affinity assays

The N end of a nucleotide sequence of a blocking humanized nano antibody F2 for coding anti-PD-L1 is connected into the C end of a hum270 antibody heavy chain nucleotide sequence through a connecting peptide by a PCR method to obtain a hum270-H-F2 coding sequence containing hum270 antibody heavy chain-F2, a coding sequence (hum270-H-F2) of the hum270 antibody heavy chain-F2 and a hum270 antibody light chain coding sequence hum270-L are respectively cloned into a eukaryotic transient expression vector by an enzyme digestion method, the obtained expression plasmid is transferred into escherichia coli for amplification, the hum270-H-F2 and the hum270-L expression plasmid are obtained by separation, and the plasmids are transferred into HEK293 cells for recombinant expression according to the operation instructions of a transfection reagent 293fectin (Cat:12347019, Gibco). 5-6 days after cell transfection, culture supernatant is taken, and expression supernatant is purified by utilizing a ProA affinity chromatography column to obtain the anti-NKG2A and anti-PD-L1 bispecific antibody hum270-F2 recombinant protein. And further carrying out the amino acid mutation obtained in example 2 on the light chain and the heavy chain of hum270 on the basis of the amino acid mutation, and constructing bispecific antibodies of corresponding mutant antibodies, such as AM10-F2, AM30-F2 and AM 12-F2.

The affinity of the antibody to NKG2A and PD-L1 was determined by capturing the Fc region of the antibody with an anti-human antibody Fc region capture Antibody (AHC) bio-probe using an Octet QKe system instrument from Fortebio, Inc. Bispecific antibodies hum270-F2, AM10-F2, AM30-F2 were diluted to 4ug/mL in PBS buffer and passed over the surface of an AHC probe (Cat:18-5060, PALL) for 240 s. Human PD-L1-His recombinant protein with the concentrations of 60nM, 30nM, 15nM and 7.5nM and human mFc-NKG2A-CD94 recombinant protein with the concentrations of 60nM, 30nM, 7.5nM and 5nM were used as mobile phases, respectively. The binding time was 300s and the dissociation time was 300 s. At the same time, similar affinity assays were performed for hum270 and F2-Fc monoclonal antibodies, respectively. After the experiment, blank control response values were deducted, and the software was run for 1: 1Langmuir binding pattern was fitted and kinetic constants for antigen-antibody binding were calculated. The results are shown in Table 8. The affinity of the bispecific antibody was not significantly changed compared to the monoclonal antibody.

TABLE 8 affinity assay results for hum270-F2 bispecific antibody

2. Bispecific antibody binding Activity assay for cell-surface NKG2A and PD-L1

The binding of bispecific antibodies to cell-surface NKG2A and PD-L1 was evaluated using NK92 cells and MBA-MD-231 cells, respectively.

Specifically, the natural cell NK92 suspension was incubated with the double anti-hum 270-F2 labeled AF488, AM10-F2 and control Antibody NC-IgG4(Mix-n-Stain CF 488A Antibody Labeling Kit: Cat. MX488AS100-1KT, sigma) at 4 ℃ for 40min at the cell concentration: 1 × 105 cells/sample, final antibody concentration: 55nM starts 3-fold serial dilution of 8 gradients. Cells were washed 3 times with ice-cold PBS (containing 0.05% Tween) and tested on the machine. The Mean Fluorescence Intensity (MFI) of the cells was measured by flow cytometry (model B49007AD, SNAW31211, BECKMAN COULTER) after PBST washing the cells 3 times to examine the binding ability of the humanized antibody to natural cell NK 92. The results are shown in figure 10, and the bispecific antibody can be specifically combined with the NKG2A antibody on the cell surface of NK 92. The binding activity of AM10-F2 is stronger than that of the parent antibody hum 270-F2.

On the other hand, MDA-MB-231 cell suspension was incubated with antibody-gradient diluted detection antibodies hum270-F2, AM10-F2 and negative control antibody NC-IgG4 at 4 ℃ for 60min at cell concentrations: 1X 10⁵cells/sample, final antibody concentration 10nM starting, 3 times serial dilution 10 gradient. After washing the cells 3 times with ice cold PBST (0.05% Tween), goat anti-human IgG-FITC (Cat.: F9512, Sigma) diluted 1:100 was added and incubated at 4 ℃ for 30 min. The Mean Fluorescence Intensity (MFI) of the cells was measured by flow cytometry after PBST washing the cells 3 times to examine the binding ability of the humanized antibody to human PD-L1 on the cell surface of MDA-MB-231. The results are shown in FIG. 11, where the bispecific antibodies all retained the binding activity to cell surface PD-L1.

3. Analysis of blocking Activity of bispecific antibodies on the binding of PD-1 and PD-L1

Human PD-1-hFc recombinant protein (SEQ ID NO: NP-005009.2, 21aa-167aa, C-terminal human Fc tag) was coated at 4 ℃ overnight at a coating concentration of 1. mu.g/mL; after washing the plate for 3 times with PBS, adding PBS containing 5% BSA, blocking for 120min at 37 ℃, and washing the plate for 3 times with PBST; adding hum270-F2, AM10-F2 and hzF2 (initial concentration is 50nM, 3-fold gradient is used for sequentially diluting 12 concentrations) with different dilution times and 3nM PD-L1-ECD-mFc (sequence number: NP-054862.1, 1aa-239aa, C-end mFc label) for co-incubation, arranging parallel holes at each concentration, incubating for 60min at 37 ℃, and washing the plate for 4 times by PBST; adding HRP-anti-mouse Fc (Cat: 115-; adding a TMB substrate for color development, incubating at 37 ℃ for 10min, and adding 2M HCl to stop the reaction; the absorbance A450nm-630nm of the well plate at a wavelength of 450nm was read and recorded using 630nm as the reference wavelength.

The experimental results (FIG. 12) show that hum270-F2, AM10-F2 and hzF2 can block the combination of PD-1 and PD-L1, and the half effective inhibitory concentration (IC50) values are 2.86nM, 2.90nM and 2.45nM respectively.

Example 4: research on anti-NKG2a and PD-1/PD-L1 dual-channel combined anti-tumor effect

Reconstitution of tumor-bearing mouse model by PBMC immunizationThe synergistic antitumor effect of the NKG2a pathway and the PD1/PD-L1 pathway is observed. Specifically, the mouse melanoma cell A375 was inoculated subcutaneously into the right anterior flank of a male NCG mouse, and the tumor was grown to 40-80mm³The administration was carried out in groups on the left and right, and the groups and the protocol were designed as shown in the following table. Tumor volumes were measured weekly. Blood is taken from orbital venous plexus of all mice at the end of the experiment, and the proportion of human CD45 positive cells in peripheral blood is detected by FACS; after euthanasia, the tumor-bearing mice were peeled off, weighed and photographed. The relative tumor volume ratio (T/C) and tumor growth inhibition ratio (1-T/C) of the treated group and the control group were calculated and statistically analyzed. Meanwhile, the expression conditions of NKG2A and PD-1 on tumor-infiltrating CD8 positive T cells of the control mice are detected at the end of the experiment.

TABLE 7. group of drug effect tests and dosing regimens for PBMC immune reconstitution tumor-bearing mouse model

# MW11 anti-PD-1 mAb, see invention patent application No. 201910022548.9

F2-hFc, humanized anti-PD-L1 Nanobody, see invention patent application No. 202010324761.8

The experimental results show (fig. 6 and 7) that the antibodies targeting PD-1, PD-L1 or NKG2A alone only showed slight antitumor effect, while the antibodies targeting NKG2A and PD-1 combined together showed significant antitumor effect; similarly, the bispecific antibody against NKG2A and PD-L1 under equal dosage has good antitumor effect. Suggesting that the NKG2A pathway and the PD-1/PD-L1 pathway have synergistic antitumor effect.

FACS analysis was further used to analyze co-expression of NKG2A and PD-1 on T cells in the control tumor microenvironment, with the results shown in table 8. The humanization rate of peripheral blood cells of mice mostly reaches more than 30%. The detection of huPD-L1 expression in the tumor microenvironment, as well as the presence of CD8+ T cells, cells that are double positive for CD8 and PD-1, positive for CD8 and NKG2A (hum270), and double positive for PD-1 and NKG2A, suggest a molecular mechanism for the combined action of the two pathways.

Table 8: analysis of expression of PD-1 and NKG2A in PBMC humanized mouse tumor microenvironment

Example 5: anti-NKG 2A/PD-L1 bispecific antibody anti-tumor dose and effect observation

Dose-effect relationship observation is carried out on the anti-tumor effect of hum270-F2 by using the efficacy model of example 4, and the experimental design is adjusted as follows: there are 4 groups of 8, each group is: a humG (20mg/kg, ip, biw x 6) group, a hum270-F2 high dose (20mg/kg, ip, biw x 6) group, a hum270-F2 medium dose (10mg/kg, ip, biw x 6) group, and a hum270-F2 low dose (4mg/kg, ip, biw x 6) group. Other scheme designs are the same as example 4.

The antitumor efficacy of hum270-F2 is shown in FIG. 8 and Table 9. hum270-F2 significantly inhibited tumor growth at doses of 10mg and 20mg/mL, with a terminal tumor inhibition rate of approximately 40% (P < 0.05).

TABLE 9 tumor inhibiting action (tumor volume) of test substances on human melanoma A375 mouse graft tumor model

Example 6: in vivo efficacy comparison of different NKG2A mutant bispecific antibodies

The anti-tumor effects of hum270-F2 and the mutant AM10-F2 are compared and observed by using the drug effect model in example 4, and the experimental design is adjusted as follows: there are 5 groups of 8, each group is: a humG (10mg/kg, ip, biw x 6) group, a hum270-F2 high dose (10mg/kg, ip, biw x 6) group, a hum270-F2 low dose (5mg/kg, ip, biw x 6) group, an AM10-F2 high dose (10mg/kg, ip, biw x 6) group, and an AM10-F2 low dose (5mg/kg, ip, biw x 6). Other scheme designs are the same as example 4. The results are shown in FIG. 9. The AM10-F2 has slightly better drug effect than 270-F2 at high dose.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Sequence listing

<110> Miwei (Shanghai) Biotech Co., Ltd

<120> bispecific antibody targeting NKG2A and PD-L1 and application

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<212> PRT

<213> Hum270-HC

<400> 10

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 11

<211> 375

<212> DNA

<213> AM10-HV

<400> 11

gaggttcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60

tcctgcaagg cttcttccta cgacttttcc tggtactgga tcaactgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggaagg attgatcctt acgatagtga aactcactat 180

gcacagaagc tccagggcag agtcaccatg accacagaca catccacgag cacagcctac 240

atggagctga ggagcctgag atctgacgac acggccgtgt attactgtgc gagagggggc 300

tatgatttcg acgtaggaac tctctactgg ttcttcgatg tctggggcca agggacaacg 360

gtcaccgtct cttca 375

<210> 12

<211> 125

<212> PRT

<213> AM10-HV

<400> 12

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Ser Tyr Asp Phe Ser Trp Tyr

20 25 30

Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Ala Gln Lys Leu

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Asp Phe Asp Val Gly Thr Leu Tyr Trp Phe Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 13

<211> 125

<212> PRT

<213> AM30-HV

<400> 13

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Asp Ser Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Ala Gln Lys Leu

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Asp Phe Asp Val Gly Thr Leu Tyr Trp Phe Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 14

<211> 214

<212> PRT

<213> Hum270-L

<400> 14

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 15

<211> 214

<212> PRT

<213> K2

<400> 15

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Gly Thr Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

Claims (21)

1. An anti-NGK 2A antibody or antigen-binding fragment thereof, which is an antibody with improved affinity and/or improved dissociation constant obtained by affinity maturation by constructing a library of mutated antibodies on the basis of hum270, wherein the amino acid sequence of the light chain variable region of hum270 is as set forth in SEQ ID NO:4, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: and 6.

2. The anti-NGK 2A antibody or antigen-binding fragment thereof according to claim 1, wherein the CDRs regions of the antibody differ from the CDRs regions of hum270 by the heavy chain CDRs regions, wherein the sequence of the antibody HCDR1-3 is as set forth in any one of H0-H6 in table 3.

3. The anti-NGK 2A antibody or antigen-binding fragment thereof according to claim 1, wherein the CDRs regions of the antibody differ from the CDRs regions of hum270 by light chain CDRs regions, wherein the sequence of the antibody LCDR1-3 is as set forth in LCDR1-3 of any one of K0-270KX in table 4.

4. An anti-NGK 2A antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the heavy chain variable region CDRs are selected from HCDR1-3 of any one of H0-H3 in table 3 and/or the light chain variable region of the antibody is selected from LCDR1-3 of any one of K0-K2 in table 4.

5. The anti-NGK 2A antibody or antigen-binding fragment thereof of claim 4, wherein the heavy chain variable region of the antibody is selected from the group consisting of SEQ ID NO: 6. 12, 13, and the light chain variable region is SEQ ID NO:4 or SEQ ID NO: 15, a variable region; and, the antibody does not comprise a heavy chain variable region of SEQ ID NO:4, while the light chain variable region is SEQ ID NO: 6.

6. The anti-NGK 2A antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein said antibody or antigen-binding fragment thereof is derived from a mouse, rat, rabbit, goat, sheep, camel, alpaca, human, or is a chimeric antibody derived from the above species.

7. The anti-NGK 2A antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof includes but is not limited to Fab, Fab ', F (ab')2, Fv fragments, scFv, and single domain fragments.

8. A multispecific antibody or antigen-binding fragment thereof comprising at least one first antigen-binding portion that binds a first antigen, and at least one second antigen-binding portion that binds a second antigen; wherein the first antigen-binding portion that binds a first antigen is the anti-NGK 2A antibody or antigen-binding fragment thereof of any one of claims 1-7.

9. The multispecific antibody or antigen-binding fragment thereof of claim 8, wherein the second antigen is an antibody therapy target, including but not limited to CD19, CD3, CD80, CD86, CD25, OX40, PD-L1, PD-1, VEGF, CTLA4, RANKL, EGFR, HER2, TNF-a.

10. The multispecific antibody or antigen-binding fragment thereof of claims 8-9, wherein the second antigen is a tumor therapy-associated antigen, preferably PD-L1, PD-1.

11. The multispecific antibody or antigen-binding fragment thereof of claims 8-9, wherein the first antigen-binding portion is selected from a human antibody, a humanized antibody, a human chimeric antibody; the second antigen-binding portion is selected from the group consisting of a single chain antibody, a single domain antibody.

12. The multispecific antibody or antigen-binding fragment thereof of claims 8-9, wherein the first antigen-binding portion comprises an intact antibody Fc fragment; and, the second antigen-binding portion is covalently coupled to the Fc region of the first antigen-binding portion.

13. The multispecific antibody or antigen-binding fragment thereof of claim 12, wherein the second antigen-binding portion is fused to the C-terminus of the heavy chain of the first antigen-binding portion by a Linker.

14. The multispecific antibody or antigen-binding fragment thereof of claim 13, wherein the Linker is (CnS) m, wherein n and m are 1-6.

15. A composition comprising the anti-NGK 2A antibody or antigen-binding fragment thereof of claims 1-7, and/or the multispecific antibody or antigen-binding fragment thereof of claims 8-14.

16. The composition of claim 15, further comprising a pharmaceutically acceptable carrier, and being used as a pharmaceutical composition, preferably the pharmaceutical composition is a liquid, an injection, or a powder injection.

17. Use of the anti-NGK 2A antibody or antigen-binding fragment thereof of claims 1-7, the multispecific antibody or antigen-binding fragment thereof of claims 8-14, or the composition of claims 15-16 in the preparation of a medicament for treating a tumor and/or enhancing an immune response in a body.

18. A polynucleotide encoding the anti-NGK 2A antibody or antigen-binding fragment thereof of claims 1-7, the multispecific antibody or antigen-binding fragment thereof of claims 8-14.

19. A vector comprising the polynucleotide of claim 18.

20. A host cell comprising the polynucleotide of claim 18 or the vector of claim 19.

21. A method of making an anti-NGK 2A antibody or antigen-binding fragment thereof, or making a multispecific antibody or fragment thereof comprising an anti-NGK 2A antibody or antigen-binding fragment thereof, comprising the steps of:

(1) culturing the host cell of claim 20 under conditions suitable for expression of an anti-NGK 2A antibody or antigen-binding fragment thereof, or a multispecific antibody or fragment thereof comprising an anti-NGK 2A antibody or antigen-binding fragment thereof;

(2) isolating and purifying the anti-NGK 2A antibody or antigen-binding fragment thereof, or a multispecific antibody or fragment thereof comprising the anti-NGK 2A antibody or antigen-binding fragment thereof from the cell culture.

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