WO2023116770A1 - BINDING MOLECULES FOR FCγRIIA AND USE THEREOF - Google Patents

Abstract

Provided are a binding molecule for FcγRIIa or an antigen-binding fragment thereof, a nucleic acid encoding the binding molecule or the antigen-binding fragment thereof, an expression vector and a host cell comprising the nucleic acid, and a related conjugate and composition. Further provided is the use of the above-mentioned substance in the aspects of treatment, detection and diagnosis.

Description

Binding molecules for FcγRIIA and applications thereof

This disclosure claims the priority of Chinese patent application 2021115815306 with the filing date of 2021/12/22, and this disclosure refers to the full text of the above-mentioned Chinese patent application.

technical field

The present disclosure belongs to the field of immunology, and more specifically, the present disclosure relates to FcγRIIA binding molecules or antigen-binding fragments thereof, nucleic acids encoding them, expression vectors and host cells, and related conjugates and compositions, as well as the above-mentioned substances in the treatment, Uses in detection and diagnosis.

Background technique

FcyRs are a family of cell surface receptors that bind the Fc portion of antibodies of the immunoglobulin (IgG) subclass. Human Fcγ receptors (FcγRs) have diverse functions, binding affinities and cellular distributions. FcγR is a protein receptor present on the cell surface, widely expressed on various immune cells, such as B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, monocytes, neutrophils, Eosinophils, basophils, human platelets and mast cells, etc. In humans, there are five FcγRs: the high-affinity receptor FcγRI (CD64), which can bind monomeric IgG; and the low-affinity receptors FcγRIIA (CD32a), FcγRIIB (CD32b), FcγRIIIA (CD16a) and FcγRIIIB (CD16b), It binds monomeric IgG weakly, but readily binds to immune complexes of IgG. FcyRI, FcyRIIA, FcyRIIIA and FcyRIIIB are believed to have activating properties, whereas FcyRIIB is primarily inhibitory. Fc[gamma]RIIA is an activating Fc receptor that, in its intracellular tail, contains an ITAM motif, an immunoreceptor tyrosine-based activation motif (ITAM). According to the 131st amino acid sequence of the extracellular domain, there are mainly two FcγRIIA alleles in humans, namely FcγRIIA131H (also known as FcγRIIAH or CD32a-H) and FcγRIIA131R (also known as FcγRIIAR or CD32a-R).

FcyRIIA and FcyRIIB are the most closely related receptors, the extracellular regions of these receptors responsible for interaction with IgG share greater than 90% sequence identity. Sequence differences in the intracellular signaling regions of FcyRIIA and FcyRIIB mediate distinct cellular responses.

FcyRIIA is a low-affinity receptor for monomeric IgG that binds the Fc effector region of immunoglobulin G (IgG) as a ligand. Thus, IgG immobilized on the cell surface or aggregated into immune complexes (ICs) can bind to FcγRIIA to induce inflammatory responses in neutrophils, monocytes, platelets, and other immune cells, which is thought to lead to Heparin-induced thrombocytopenia (HIT), rheumatoid arthritis, systemic lupus erythematosus (SLE), immune thrombocytopenia (ITP) and autoimmune hemolytic anemia. The immune complex (IC) plays a very important role in the development of different autoimmune diseases. IC is present in circulation and deposited in affected tissues and organs. Binding of ICs to immune cells bearing Fc receptors can trigger cell recruitment and activation, local inflammation, adaptive immunity, and histopathology. Immune complexes bind to activating Fc receptors (FcRs) and inhibitory FcRs expressed by innate immune effector cells such as basophils, mast cells, neutrophils, monocytes, and macrophages, thereby activating the corresponding effect response.

FcγRIIA plays a critical role in IC-mediated FcγR-dependent immune cell activation. In lupus, IC binding to FcγRIIA on a type of dendritic cell called pDC drives IC-mediated type I interferon (IFN) production and is important for pathological development of SLE, psoriasis, type 1 diabetes . So recently, targeting type I interferons and other cytokines secreted by pDCs has become a new therapeutic approach in the treatment of autoimmune diseases.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a systemic autoimmune disease characterized by inflammation of small vessels. The pathogenesis of AAV is complex, including the pathogenic role of ANCA and neutrophils as mediators of injury, the dysregulation of the complement system, and the involvement of T cells. Neutrophils are recognized to play an important role in the occurrence and development of AAV. Through FcγRIIA, ANCA-immune complexes activate neutrophils, activate the downstream signaling pathway NADPH (reducing coenzyme), and release superoxide, thereby further activating neutrophils. So FcγRIIA plays an important role in the occurrence and development of antineutrophil cytoplasmic antibody-associated vasculitis.

Since FcγRIIA plays an important role in the development and pathogenesis of antibody-mediated autoimmune diseases, specifically blocking FcγRIIA function is a potential key strategy for the treatment of these diseases. Although antibodies against FcγRIIA have been reported in the prior art (such as CN109863174A), there is still a need in the art to develop antibodies with better performance, especially to develop antibodies with further improved specificity to FcγRIIA and further reduced binding to FcγRIIB. Specific antibodies to better meet the requirements of toxicity and safety.

Contents of the invention

The purpose of the present disclosure is to provide a binding molecule capable of specifically blocking FcγRIIA while hardly binding to FcγRIIB and an antigen-binding fragment thereof, the binding molecule or an antigen-binding fragment thereof capable of specifically binding FcγRIIA while hardly binding to FcγRIIB .

The present disclosure provides an FcγRIIA binding molecule or an antigen-binding fragment thereof, comprising:

i) heavy chain complementarity determining region 1 (CDRH1), the amino acid sequence of which is shown in SEQ ID NO: 3;

ii) heavy chain complementarity determining region 2 (CDRH2), the amino acid sequence of which is shown in SEQ ID NO: 4, or a CDRH2-H61 D-V mutation is introduced on the basis of SEQ ID NO: 4;

iii) Heavy chain complementarity determining region 3 (CDRH3), the amino acid sequence of which is shown in SEQ ID NO: 5, or a CDRH3-H101 D-A mutation is introduced on the basis of SEQ ID NO: 5;

iv) light chain complementarity determining region 1 (CDRL1), the amino acid sequence of which is shown in SEQ ID NO: 6, or on the basis of SEQ ID NO: 6, a mutation selected from the following groups is introduced:

a) CDRL1-L26 S-W and/or CDRL1-L27c L-P; or

b) CDRL1-L24 R-L and/or CDRL1-L27c L-P; or

c) CDRL1-L24 R-L and/or CDRL1-L32 Y-Q; or

d) CDRL1-L24 R-L and/or CDRL1-L26 S-W; or

e) CDRL1-L26 S-W and/or CDRL1-L32 Y-Q; or

f) CDRL1-L27c L-P and/or CDRL1-L32 Y-Q;

v) light chain complementarity determining region 2 (CDRL2), the amino acid sequence of which is shown in SEQ ID NO: 7; and

vi) Light chain complementarity determining region 3 (CDRL3), the amino acid sequence of which is shown in SEQ ID NO: 8.

The above numbering adopts the Kabat nomenclature, where CDRH2-H61 D-V means that the D at position 61 of the complementarity determining region 2 of the heavy chain is mutated to V, CDRL1-L24 R-L means that the R at position 24 of the light chain complementarity determining region 1 is mutated to L, and the rest are analogous .

Preferably, the amino acid sequence of the light chain complementarity determining region 1 (CDRL1) of the FcγRIIA binding molecule or antigen-binding fragment thereof is shown in SEQ ID NO: 11.

More preferably, the FcγRIIA-binding molecule or antigen-binding fragment thereof comprises:

I) The heavy chain variable region as shown in SEQ ID NO: 9, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, A heavy chain variable region of 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity; and/or;

II) The light chain variable region as shown in SEQ ID NO: 10, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, Light chain variable regions of 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity.

The FcγRIIA binding molecule of the present disclosure or an antigen-binding fragment thereof further comprises a heavy chain constant region and/or a light chain constant region; preferably, the heavy chain constant region comprises Fc; more preferably, the Fc is derived from a mouse or a human; more preferably Preferably, the sequence of the Fc is native or modified.

In a specific embodiment, the heavy chain constant region is a human IgG1 heavy chain constant region, and the sequence is from the Uniprot database, and the number is P01857 (https://www.uniprot.org/uniprot/P01857); the light chain constant region is human kappa light The chain constant region, the sequence is from the Uniprot database, the number is P01834 (https://www.uniprot.org/uniprot/P01834).

The FcγRIIA binding molecules of the present disclosure or antigen-binding fragments thereof can be monoclonal antibodies, bispecific binding molecules, multispecific binding molecules, humanized antibodies, chimeric antibodies, modified antibodies, fully human antibodies, full-length antibodies , heavy chain antibody, Nanobody, Fab, Fv, scFv, F(ab') ₂ , linear antibody or single domain antibody.

The present disclosure also provides a conjugate, which is formed by conjugating the FcγRIIA binding molecule of the present disclosure or an antigen-binding fragment thereof with a capture label or a detection label or a biologically active molecule;

Preferably, the detection markers include radionuclides, luminescent substances, colored substances or enzymes;

Preferably, the bioactive molecule is a small molecule drug; more preferably, the FcγRIIA binding molecule or an antigen-binding fragment thereof is connected to the bioactive molecule through a linker.

The present disclosure also provides a nucleic acid encoding an FcγRIIA binding molecule of the present disclosure or an antigen-binding fragment thereof.

The present disclosure also provides an expression vector comprising the aforementioned nucleic acid.

The present disclosure also provides a host cell comprising the aforementioned nucleic acid or vector;

Preferably, the host cell is a prokaryotic cell or a eukaryotic cell; the prokaryotic cell is preferably Escherichia coli; the eukaryotic cell is preferably a mammalian cell or yeast; more preferably, the mammalian cell is a CHO cell, an Expi293 cell or HEK293 cells.

The present disclosure also provides a composition comprising the aforementioned FcγRIIA binding molecules or antigen-binding fragments thereof, conjugates, nucleic acids, expression vectors and/or host cells;

Preferably, the composition is a pharmaceutical composition, which further comprises a pharmaceutically acceptable carrier; more preferably, the pharmaceutical composition further comprises one or more additional therapeutic agents;

Preferably, the composition is a diagnostic reagent.

The present disclosure also provides a method for preparing the aforementioned FcγRIIA-binding molecules or antigen-binding fragments thereof, the method comprising: culturing the aforementioned host cells under suitable conditions.

The present disclosure also provides the aforementioned FcγRIIA binding molecules or antigen-binding fragments thereof, conjugates, nucleic acids, expression vectors, host cells and/or compositions in the preparation of treatment, alleviation and/or prevention of inflammatory diseases or autoimmune diseases or other FcγRIIA Use in medicines for mediated diseases;

Preferably, the inflammatory disease or autoimmune disease or other FcγRIIA-mediated disease is selected from: primary immune thrombocytopenia (ITP) or antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV ).

The present disclosure also provides the use of the aforementioned FcγRIIA binding molecules or antigen-binding fragments thereof, conjugates, nucleic acids, expression vectors, host cells and/or compositions in the preparation of detection reagents or diagnostic reagents, which are used for Detection or diagnosis of inflammatory diseases or autoimmune diseases or other FcγRIIA mediated diseases.

The present disclosure also provides a method for treating, alleviating and/or preventing inflammatory diseases or autoimmune diseases or other FcγRIIA-mediated diseases in a subject in need thereof, which comprises administering the aforementioned FcγRIIA binding molecules to the subject or an antigen-binding fragment thereof, a conjugate, a nucleic acid, an expression vector, a host cell and/or a composition.

The present disclosure also provides a method for detecting FcγRIIA in a sample, comprising:

(1) contacting the sample with the aforementioned FcγRIIA binding molecule or antigen-binding fragment thereof;

(2) Detecting the formation of a complex of the FcγRIIA-binding molecule or antigen-binding fragment thereof with FcγRIIA; optionally, the FcγRIIA-binding molecule or antigen-binding fragment thereof is detectably labeled.

The technical solution of the present disclosure has the following beneficial effects: (1) the antibody of the present disclosure has higher selectivity, and can specifically recognize CD32a but not CD32b; (2) Cell level tests show that the antibody of the present disclosure has stronger CD32a binding ability.

Description of drawings

The accompanying drawings further illustrate the novel features disclosed in this specification. The features and advantages disclosed in this specification will be better understood with reference to these drawings, it being understood that these drawings are only illustrative of specific embodiments of the principles disclosed herein and are not intended to limit the scope of the appended claims. The scope is limited.

Figure 1A and Figure 1B show the distribution of mutation sites of positive clones after single-site mutation ELISA screening, wherein Figure 1A is the heavy chain CDR, and Figure 1B is the light chain CDR.

Figure 2A and Figure 2B show the ELISA experiment results of QLS0309-A, QLS0309-D, and QLS0309-G, and the antigen concentrations in Figure 2A and Figure 2B are 2 μg/ml and 0.4 μg/ml, respectively.

Figure 3A and Figure 3B show the dose-response curves of the binding activity of QLS0309-E to FcγRIIA-CHO-K1 stably transfected cell lines of different species of non-human primates, wherein Figure 3A is for cynomolgus monkeys and Figure 3B is for rhesus monkeys.

Figure 4A and Figure 4B respectively show the secretion of human cell TNF-α and IL-6 caused by QLS0309-E blocking immune complexes, and Figure 4C and Figure 4D respectively show the food-eating effects caused by QLS0309-E blocking immune complexes. Secretion of TNF-α and IL-6 in cynomolgus monkey cells.

Figure 5A, Figure 5B, and Figure 5C show the results of the affinity between QLS0309-E and human monocytes, and Figure 5A, Figure 5B, and Figure 5C correspond to the results of donors Y376, 804F, and 405F, respectively.

Figure 6A, Figure 6B, Figure 6C, and Figure 6D show the results of the affinity between QLS0309-E and human B cells, and Figure 6A, Figure 6B, Figure 6C, and Figure 6D correspond to the results of donors Y376, 804F, 405F, and 304F, respectively.

Figure 7A, Figure 7B, Figure 7C, Figure 7D, and Figure 7E show the binding of QLS0309-E to different FcγR subtypes, and the subtypes in Figure 7A, Figure 7B, Figure 7C, Figure 7D, and Figure 7E are FcγRI, FcγRIIA-131H, FcγRIIB, FcγRIII-158F, FcγRIII-158V.

Figure 8A, Figure 8B, Figure 8C, and Figure 8D show the results of QLS0309-E-mediated FcγRIIA endocytosis, and Figure 8A, Figure 8B, Figure 8C, and Figure 8D correspond to the results of donors Y376, 804F, 405F, and 304F, respectively.

Figure 9A, Figure 9B, and Figure 9C are the dose-response curves for the detection of FcγRIIB endocytosis on the surface of B cells in human whole blood induced by QLS0309-E. In the detection experiment at the cellular level, H2B is an FcγRIIIB-recognizing antibody. Figure 9A, Figure 9B, and Figure 9C correspond to the results of donors Y376, 804F, and 304F, respectively.

Figure 10A, Figure 10B, and Figure 10C show the ability of QLS0309-E to block ANCA-induced neutrophil superoxide production, and Figure 10A, Figure 10B, and Figure 10C correspond to donors Z0231, Z0285, and Z0299, respectively.

Detailed ways

the term

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference and into this article.

Before the present disclosure is described in detail below, it is to be understood that this disclosure is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present disclosure. Unless defined otherwise, 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 disclosure belongs.

Certain embodiments disclosed herein include numerical ranges, and certain aspects of the disclosure can be described in terms of ranges. Unless otherwise stated, it should be understood that the numerical range or the way described in the range is only for the purpose of brevity and convenience, and should not be regarded as a strict limitation on the scope of the present disclosure. Accordingly, description in range form should be considered to specifically disclose all possible subranges and all possible specific numerical points within that range, as if such subranges and numerical points had been expressly written herein. The above principles apply equally regardless of the breadth or narrowness of the numerical values stated. When a range is used, that range includes the range endpoints.

When referring to measurable values such as amounts, temporal durations, etc., the term "about" is meant to include ±20%, or in some cases ±10%, or in some cases ±5%, or within ±1% in some cases, or ±0.1% in some cases.

As used herein, the three-letter and one-letter codes for amino acids are as described in J. Biol. Chem, 243, p3558 (1968).

The term "antibody" herein may include whole antibodies (e.g., full-length monoclonal antibodies) and any antigen-binding fragments thereof (i.e., antigen-binding portions) or single chains thereof, and may also include whole antibodies or antigen-binding fragments thereof or single chains thereof. A product with antigen-specific binding ability formed on the basis of chain modification (such as linking other peptides, rearrangement of functional units, etc.).

In one embodiment, an antibody typically refers to comprising two heavy (H) polypeptide chains (HC) and two light (L) polypeptide chains (LC) held together by covalent disulfide bonds and non-covalent interactions Y-tetrameric protein. Natural IgG antibodies have such a structure. Each light chain consists of a variable domain (VL) and a constant domain (CL). Each heavy chain comprises a variable domain (VH) and constant region (CH).

Five major classes of antibodies are known in the art: IgA, IgD, IgE, IgG, and IgM, and the corresponding heavy chain constant domains are called α, δ, ε, γ, and μ, respectively. IgG and IgA can be further divided into different For example, IgG can be divided into IgG1, IgG2, IgG3, IgG4, and IgA can be divided into IgA1 and IgA2. The light chains of antibodies from any vertebrate species can be assigned to one of two distinct classes, called kappa and lambda, based on the amino acid sequence of their constant domains.

In the case of IgG, IgA and IgD antibodies, this constant region comprises three domains called CH1, CH2 and CH3 (IgM and IgE have a fourth domain, CH4). In the IgG, IgA and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, which is a proline- and cysteine-rich segment of variable length. Each class of antibodies further comprises interchain and intrachain disulfide bonds formed by paired cysteine residues.

The term "variable region" or "variable domain" exhibits significant variation in amino acid composition from one antibody to another and is primarily responsible for antigen recognition and binding. The variable regions of each light chain/heavy chain pair form the antibody combining site such that an intact IgG antibody has two binding sites (ie it is bivalent). The variable region (VH) of the heavy chain and the variable region (VL) of the light chain each contain three regions of extreme variability known as hypervariable regions (HVR) or, more commonly, Complementarity determining region (CDR), VH and VL each have four framework regions FR, denoted by FR1, FR2, FR3, FR4 respectively. Thus, the CDR and FR sequences typically appear in the following sequence of the heavy chain variable domain (or light chain variable domain): FR1-CDRH1(CDRL1)-FR2-CDRH2(CDRL2)-FR3-CDRH3(CDRL3)- FR4.

The term "Fc" is used to define the C-terminal region of an immunoglobulin heavy chain, which region comprises at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions.

"Antibody" may be used in the broadest sense herein and may include, for example, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primatized antibodies, CDR-grafted antibodies (CDR- grafted antibody), human antibody (including recombinantly produced human antibody), recombinantly produced antibody, intrabody, multispecific antibody, bispecific antibody, monovalent antibody, multivalent antibody, anti-idiotype antibody, synthetic antibody ( Including muteins and variants thereof) and the like.

The term "monoclonal antibody" (or "monoclonal antibody") refers to a substantially homogeneous antibody produced by a single cell clone that only targets a specific epitope. Monoclonal antibodies can be prepared using various techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals, synthetic technology or a combination of the above technologies, etc.

It should be noted that the division of CDR and FR of the variable region of the monoclonal antibody of the present disclosure is determined according to the definition of Kabat. However, other naming and numbering systems, such as Chothia, IMGT or AHo etc., are also known to those skilled in the art. Thus, humanized antibodies comprising one or more CDRs derived from any nomenclature system based on the mAb sequences of the disclosure expressly remain within the scope of the disclosure.

The substitutions referred to in the present disclosure are preferably represented by the kabat numbering system, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

The term "chimeric antibody" refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species, eg, antibodies in which the variable regions are derived from a mouse antibody and the constant regions are derived from a human antibody.

The term "humanized antibody" refers to an antibody in which all or part of the amino acids other than the CDRs of a non-human antibody (such as a mouse antibody) are replaced with corresponding amino acids derived from human immunoglobulins. Minor additions, deletions, insertions, substitutions or modifications of amino acids are permissible so long as they do not eliminate the ability of the antibody to bind a particular antigen. A "humanized" antibody retains similar antigen specificity to the original antibody.

The term "antibody fragment" includes at least a portion of an intact antibody. As used herein, a "fragment" of an antibody molecule includes an "antigen-binding fragment" of an antibody, and the term "antigen-binding fragment" refers to a fragment of an immunoglobulin or antibody that specifically binds to a selected antigen or an immunogenic determining portion thereof. Or reacted polypeptide fragments, or fusion protein products further derived from such fragments, such as single-chain antibodies, extracellular binding regions in chimeric antigen receptors, etc. Exemplary antibody fragments or antigen-binding fragments thereof include, but are not limited to: variable light chain fragments, variable heavy chain fragments, Fab fragments, F(ab') ₂ fragments, Fd fragments, Fv fragments, single domain antibodies, linear Antibodies, single-chain antibodies (scFv), bispecific antibodies or multispecific antibodies formed from antibody fragments, etc.

The term "antigen" refers to a substance that is recognized and specifically bound by an antibody or antibody-binding fragment. In a broad sense, an antigen can include any immunogenic fragment or determinant of a selected target, including single-epitope, multi-epitope, single-structure domains, multiple domains, complete extracellular domains (ECDs), or proteins. Peptides, proteins, glycoproteins, polysaccharides and lipids, parts thereof and combinations thereof can constitute antigens. Non-limiting exemplary antigens include tumor antigens or pathogen antigens, among others. "Antigen" can also refer to a molecule that elicits an immune response. Any form of antigen or cells or preparations containing the antigen can be used to generate antibodies specific for an antigenic determinant. The antigen can be an isolated full-length protein, a cell surface protein (e.g., immunized with a cell expressing at least a portion of the antigen on its surface), or a soluble protein (e.g., immunized with only the ECD portion of the protein) or protein Constructs (eg, Fc antigens). The antigen can be produced in genetically modified cells. Any of the foregoing antigens may be used alone or in combination with one or more immunogenicity enhancing adjuvants known in the art. The DNA encoding the antigen may be genomic or non-genomic (eg, cDNA), and may encode at least a portion of the ECD sufficient to elicit an immunogenic response. Any vector may be used to transform the cells in which the antigen is expressed, including but not limited to adenoviral vectors, lentiviral vectors, plasmids, and non-viral vectors such as cationic lipids.

The term "epitope" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed from contiguous amino acids, or non-contiguous amino acids that are juxtaposed by the tertiary folding of the protein. Epitopes formed from adjacent amino acids are generally maintained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are generally lost upon denaturing solvent treatment. Epitopes generally exist in a unique spatial conformation and comprise at least 3-15 amino acids. Methods for determining the epitope bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays, among others. Methods for determining the spatial conformation of epitopes include techniques in the art, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, among others.

The terms "bispecific binding molecule" and "multispecific binding molecule" respectively refer to binding molecules (such as antibodies or molecules comprising antibody fragments) specific for two or more different antigens (or epitopes), preferably bispecific antibody.

When using the variable regions in the present disclosure to produce antibodies, binding molecules, bispecific binding molecules or multispecific binding molecules, the constant regions are not particularly limited, and constant regions known to those skilled in the art or obtained by themselves can be used. Amino acid mutations (for example, mutations that increase or decrease binding to Fcγ receptors or FcRn) can also be introduced into the constant region.

The method for obtaining the binding molecule, antigen-binding fragment, antibody, bispecific binding molecule or multispecific binding molecule of the present disclosure is not particularly limited, and can be obtained by any method, such as Cold Spring Harbor's Antibody Experimental Technical Guide, chapters 5-8 and 15 chapters. The binding molecules, antigen-binding fragments, antibodies, bispecific binding molecules or multispecific binding molecules of the present disclosure can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in serum-free medium in bioreactors for antibody production. The culture fluid that secretes the antibody can be purified and collected using conventional techniques. Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and aggregates can also be removed by conventional methods such as molecular sieves and ion exchange.

The term "affinity" or "binding affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). The term "KD" refers to the dissociation constant for a particular antibody-antigen interaction. Binding affinity can be determined using various techniques known in the art, such as surface plasmon resonance, biolayer interferometry, dual polarization interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultrafast centrifugation and flow cytometry, etc.

The term "biological activity" refers to the ability of an antibody to bind antigen and cause a measurable biological response, which can be measured in vitro or in vivo.

The pharmaceutical composition of the present disclosure can be prepared by mixing the binding molecules of the present disclosure with appropriate pharmaceutically acceptable carriers, media, etc. as needed.

The binding molecules or antigen-binding fragments of the present disclosure can be used in combination with other drugs, and the active ingredients can be mixed together to form a single administration unit, or can be used independently as administration units.

The term "effective amount" refers to the dose of a pharmaceutical formulation of an antibody or fragment of the present disclosure which, when administered to a patient in single or multiple doses, produces the desired effect in a treated patient. An effective amount can be readily determined by the attending physician, who is skilled in the art, by considering various factors such as ethnic differences; body weight, age and health; the particular disease involved; the severity of the disease; the response of the individual patient; The specific antibody administered; the mode of administration; the bioavailability characteristics of the formulation administered; the chosen dosing regimen; and the use of any concomitant therapy.

The term "kit" or "kit" includes an effective amount of one or more pharmaceutical compositions of the present disclosure in unit dosage form. In some embodiments, a kit may contain sterile containers; such containers may be in the form of boxes, ampoules, bottles, vials, tubes, bags, blister packs, or other suitable container forms known in the art. Such containers may be made of plastic, glass, laminated paper, foil, or other materials suitable for holding medications. In addition, the kit includes instructions for administering a pharmaceutical composition of the present disclosure to an individual. The instructions generally include methods of using the disclosed pharmaceutical compositions to treat diseases.

The term "individual" or "subject" as used herein refers to any animal, such as a mammal or a marsupial. Subjects of the present disclosure include, but are not limited to, humans, non-human primates (such as cynomolgus monkeys or rhesus monkeys or other types of cynomolgus monkeys), mice, pigs, horses, donkeys, cows, sheep, rats, and Any kind of poultry.

As used herein, the term "disease" or "condition" or "disorder" and the like refers to any change or disorder that damages or interferes with the normal function of a cell, tissue or organ. For example, the "disease" includes but is not limited to: tumor, pathogenic infection, autoimmune disease, T cell dysfunction disease, or immune tolerance defect (such as transplant rejection).

The term "treatment" as used herein refers to clinical intervention in an attempt to alter the course of a disease caused by an individual or a cell, either for prevention or for intervention in the course of clinical pathology. Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, relieving symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing down the progression of the disease, improving or relieving the disease, remission or improving the prognosis, etc.

Example

The present disclosure will be further elaborated below in conjunction with specific embodiments. It should be understood that these examples are only for illustrating the present disclosure and are not intended to limit the scope of the present disclosure. The experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions (such as the conditions described in "J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, 2002"), or as recommended by the manufacturer.

Example 1. Design and screening of single point mutations

Based on the amino acid sequence information of VIB9600 recorded in the patent application CN109863174A (see Table 1), 65 mutation sites were designed for the CDR region, and primers were designed for each mutation site to replace the original coding nucleic acid sequence with the NNK form (N stands for A/T/G/C, and K stands for T/G), using overlapping PCR to build libraries and synthesize single-chain antibodies (scFv), a total of 65 libraries were obtained, and the libraries were numbered 1-65 according to the mutated amino acid positions. Based on counts, the size of each library was approximately 5×10 ⁶ . The diversity of each library is good enough for the next step of screening. In each library, 100 single clones were selected for subsequent screening.

Table 1 The sequence of VIB9600

The mutant library was divided into two parts and screened by ELISA method. FcγRIIAR, FcγRIIB, and FcγRIIAH antigens were used in sequence in libraries 1-23 for screening; FcγRIIB, FcγRIIAR, and FcγRIIAH antigens were used in sequence for screening in libraries 24-65. For FcγRIIAH/FcγRIIAR antigen screening, OD450>2 were identified as positive clones; while for FcγRIIB antigen screening, OD450<1 were identified as negative clones. The statistical results of all batch screening are shown in Table 2 and Table 3:

Table 2 Screening results of libraries 1-23

Table 3 Screening results of library 24-65

The mutated amino acids of all unique sequences were counted, and the results are shown in Figure 1A and Figure 1B. The mutations marked underlined were used in the next screening. In the mutation screening, it was found that the amino acids at these positions could only retain the binding effect when mutated into an additional amino acid, which proved that the site could bind to the antigen. It is relatively conservative, so it is hoped that more excellent candidate antibodies can be obtained by screening mutations of these key amino acids.

After ELISA screening, 20 positive clones with the underlined marks above were selected for the next step of off-rate screening. Antigens were first coupled to SA biosensors and then immersed in antibody samples. Antigens were diluted to a working concentration of 5 μg/ml in 1× Kinetics buffer, and all scFv samples were used undiluted for testing. The signal of conjugated antibody was subtracted from the signal without conjugated antibody calculated with Octet data analysis software (version 10.0). Dissociation constant data were obtained for analysis and positive screening. The dissociation constant data are shown in Table 4:

Table 4 Dissociation rate screening results

From the off-rate screen, samples with reads kd<kd VIB9600 were selected for the next mutational engineering. Six samples (CDRL1-L26/CDRH3-H101/CDRL1-L32/CDRL1-L27c/CDRH2-H61/CDRL1-L24) were identified for further screening.

Example 2. Design and construction of double-site mutations

According to the screening results in Example 1, 7 double-site mutant antibody molecules were constructed by PCR site-directed mutagenesis. Using the PCR method, the VH and VL sequences of the seven double-site mutations were respectively amplified, and then subcloned into the transient expression vector pCP-hCg1/ pCP-hCk (Shanghai Ruizhi Chemical Research Co., Ltd.). After confirming that the bacterial clone contains a complete and correct heavy chain or light chain sequence by DNA sequencing, the plasmid DNA is then extracted. Matched plasmid DNA containing heavy and light chains was transiently co-transfected into Expi293T cells, the supernatant was collected, and the expressed antibody was purified by protein A affinity chromatography.

The construction scheme of the double-site mutation antibody molecule is shown in Table 5. The heavy chain constant region is derived from human IgG1, and the light chain constant region is derived from the human kappa light chain constant region.

Table 5 Two-site mutation design

Combined mutant name	Mutation site-01	Mutation site-02
QLS0309-E	CDRL1-L26 S-W	CDRL1-L27c L-P
QLS0309-A	CDRH2-H61 D-V	CDRH3-H101 D-A
QLS0309-B	CDRL1-L24 R-L	CDRL1-L26 S-W
QLS0309-C	CDRL1-L24 R-L	CDRL1-L27c L-P
QLS0309-D	CDRL1-L24 R-L	CDRL1-L32 Y-Q
QLS0309-F	CDRL1-L26 S-W	CDRL1-L32 Y-Q
QLS0309-G	CDRL1-L27c L-P	CDRL1-L32 Y-Q

Note: Position numbers in the table above are expressed in the kabat numbering system.

Example 3. Affinity Assessment of Candidate Molecules to CD32 Antigen

The 7 purified antibodies were tested by ELISA to identify their binding activity to CD32. Figure 2A and Figure 2B show the results of ELISA experiments for QLS0309-A, QLS0309-D, and QLS0309-G. It can be seen that QLS0309-D and QLS0309-G have basically lost their binding properties.

According to the above results, the screening scope was narrowed down to 5 antibody molecules, QLS0309-A, QLS0309-B, QLS0309-C, QLS0309-E, QLS0309-F. The ELISA data results of these five IgG molecules are shown in Table 6, among which the QLS0309-F molecule is more obvious, and the binding ability decreases the fastest after the antigen concentration decreases.

Table 6 ELISA experiment results of QLS0309-A, QLS0309-B, QLS0309-C, QLS0309-E, QLS0309-F

Example 4. Evaluation of the binding ability of candidate molecules to FcγRIIA on the surface of human stably-transformed CD32a cells and to FcγRIIB on the surface of human stably-transformed CD32b cells Binding evaluation

The binding of candidate molecules to FcγRIIA on the surface of stably transfected CD32a(131R/H) cells was verified using FACS, and the basic steps were as follows: Cell preparation: Spread CHOK1-CD32aH/CHOK1-CD32aR/CHOK1-CD32b/CHOK1 cells on a FACS plate. Add purified IgG samples (1:3 serial dilution); IgG1-VIB9600 as a positive control, and isotype IgG as a negative control. Incubate at 4°C for 1 hour. After the cells were washed twice with FACS buffer, Alexa Fluor 633 goat anti-human IgG (H+L) Cross-Adsorbed fluorescent secondary antibody (dissolved in FACS buffer at 1:500) was added. Incubate at 4°C for 1 hour. The cells were washed twice with FACS buffer and resuspended, and the plate was read on a BD FACSVerse ^™ II instrument.

Table 7A is the FACS experimental data of QLS0309-A, QLS0309-B, QLS0309-C, QLS0309-D and QLS0309-G. It can be seen that the binding ability of QLS0309-D and QLS0309-G to CD32a is significantly weaker than that of other molecules. Among the remaining 3 molecules, QLS0309-B and QLS0309-C have certain binding ability to CD32b, and QLS0309-A has the weakest binding ability to CD32b.

Table 7A FACS experiment results of QLS0309-A, QLS0309-B, QLS0309-C, QLS0309-D and QLS0309-G

The FACS experiment result of table 7B QLS0309-E

Table 7B is the FACS experimental data of QLS0309-E (Note: Table 7A and Table 7B are two separate experiments), showing that QLS0309-E and VIB9600 have better binding to CD32a (decreased EC ₅₀ ). In terms of binding ability to CD32b, among the candidate antibody molecules, QLS0309-E has significantly weaker binding ability than VIB9600. Therefore, in the next experiment, QLS0309-E was selected to continue testing. QLS0309-E comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:9 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:10, wherein the amino acid sequence of CDRL1 is as shown in SEQ ID NO:11 Show.

Embodiment 5. species selectivity

amino acid sequence homology

In order to determine the relevant animal species for QLS0309-E pharmacokinetics and toxicology studies, according to literature research, FcγRIIA only exists in primates, so this study limited the selection of relevant animal species to non-human primates ( In this study, cynomolgus monkeys and rhesus monkeys were selected as alternative species). The FcγRIIA amino acid sequences of humans and different animal species (cynomolgus monkeys, rhesus monkeys) were checked on the uniprot website, and the homology of the amino acid sequences was compared by Clustal W. The results are shown in Table 8.

Table 8 Homology analysis results of different species and human FcγRIIA amino acid sequence

Comparative analysis of the affinity of QLS0309-E to cynomolgus FcγRIIA and rhesus FcγRIIA proteins

In this experiment, the Biacore 8K molecular interaction instrument was used to detect the affinity of QLS0309-E to recombinant FcγRIIA in cynomolgus monkeys and rhesus monkeys. The results showed that the affinity KD values of QLS0309-E to cynomolgus monkey and rhesus monkey FcγRIIA were 1.43E-07M and 2.93E-07M.

Rhesus monkey FcγRIIA reported on human For the binding characteristics of source IgG, we selected the most common variant H9BMP0 (named Rhesus monkey FcγRIIA), and according to the detection method of the patent CN109863174A, select the cynomolgus FcγRIIA-V3 sequence (see CN109863174A, which is the most common variant in cynomolgus monkeys), gene synthesis and in vitro expression of these two macaque proteins to detect FcγRIIA antibodies The strength of its affinity was compared with two gene polymorphism variants (R131, H131) of human FcγRIIA.

The affinity of QLS0309-E to FcγRIIA protein of cynomolgus monkey and rhesus monkey was determined by SPR technology (Biacore 8K). As shown in Table 9, the KD value difference between QLS0309-E and human and cynomolgus monkey is within 100 times, and the KD value difference between human and rhesus monkey is more than 100 times. Therefore, cynomolgus monkeys are closer to humans than rhesus monkeys.

Table 9 The affinity of QLS0309-E to cynomolgus monkey, rhesus monkey and human FcγRIIA

Immobilized Ligand (IgG)	antigen	KD(M)	Cynomolgus monkey KD/human KD
QLS0309-E	Cynomolgus FcγRIIA_v3	1.43E-07	the
QLS0309-E	Rhesus FcγRIIA_v1	2.93E-07	the
QLS0309-E	Human FcγRIIA 131H	1.94E-09	73.71134
QLS0309-E	Human FcγRIIA 131R	2.98E-09	47.98658

Binding activity of QLS0309-E to engineered cells in cynomolgus monkeys and rhesus monkeys

In the species-selective study, the binding of QLS0309-E to engineered cells of FcγRIIA of different species (cynomolgus monkey, rhesus monkey) of non-human primates was evaluated respectively.

In this study, cynomolgus monkeys and rhesus monkey FcγRIIA-CHO-K1 stably transfected cell lines (engineered cell lines expressing FcγRIIA) were incubated with different concentrations of QLS0309-E (1.3×10 ^-3 -66.7nM) at 4°C for 60 minutes After the incubation, goat anti-human IgG Fc fluorescent secondary antibody (Goat anti human IgG Fc Alexa Fluor 647) was added, and incubated at 4°C in the dark for 60 minutes. After the incubation, the average fluorescence intensity of the cell surface was detected by flow cytometry . The study found that QLS0309-E can bind to FcγRIIA on the cell surface of cynomolgus monkey-FcγRIIA CHO-K1 and rhesus monkey-FcγRIIA CHO-K1 in a dose-dependent manner, with EC ₅₀ of 1.099 and 142.7nM, respectively. The test results are shown in Figure 3A and Figure 3B, indicating that QLS0309-E has a strong binding ability to cynomolgus monkey FcγRIIA.

Through FcγRIIA, the immune complex can activate monocytes to produce TNF-α and IL-6, which are key cytokines involved in immune inflammation. By blocking the signaling pathway of FcγRIIA, it can inhibit the growth of macrophages or monocytes. Production of pro-inflammatory factors. In vitro cell experiments, we used biotin-labeled IgG-avidin immune complex (IgIC) to activate and induce human and cynomolgus peripheral blood mononuclear cells (PBMC) to secrete the expression of IL6 and TNFα. QLS0309-E can effectively block the secretion of TNF-α and IL-6 caused by immune complexes. As shown in Figures 4A-4D and Table 10, the _EC50 of QLS0309-E in inhibiting immune complex-mediated cytokine release in cynomolgus monkey and human PBMCs was within 10-fold. The related animal species of QLS0309-E is cynomolgus monkey.

Table 10 QLS0309-E blocks the secretion of TNF-α and IL-6 caused by IgIC

Example 6. Evaluation of the binding ability of QLS0309-E to FcγRIIA on the surface of human immune cells

In order to further study the affinity of candidate antibody molecules, QLS0309-E (1:3 gradient dilution) and human peripheral blood mononuclear cells (Peripheral blood mononuclear cell, PBMC) were incubated at 4°C for 1 hour, then centrifuged and washed twice with phosphate buffered saline, Add fluorescently labeled anti-human IgG Fc secondary antibody (Jackson Imm.109-606-170, Goat anti-human IgG Secondary Antibody, Alexa Fluor 647), 100 μL per well, incubate at 4°C for 1 hour, then centrifuge and wash with phosphate buffer Twice, add CD14-APC CY7 (Biolegend, 301820) fluorescent antibody, incubate at 4°C for 30 min, centrifuge and wash twice with phosphate buffer saline, add 7AAD (BD, 559925) for staining and washing, and then put the prepared samples in Detection was carried out on a flow cytometer (BD LSRFortessa), the average fluorescence intensity of each concentration was calculated by software, and then the half maximal binding concentration (hereinafter referred to as EC ₅₀ ) and the highest average fluorescence intensity (Top MFI) were calculated by GraphPad software. The results are shown in the table 11.

Table 11 Binding of CD32a antibody to human monocyte CD32a from 3 donors

the	ID: Y376	ID: 804F	ID: 405F
the	EC50 (nM)	EC50 (nM)	EC50 (nM)
VIB9600	0.1036	0.1657	0.1055
QLS0309-E	0.0313	0.052	0.04542

Note: The number shown after the ID is the donor number, Y376 corresponds to Y1376 in the figure, 804F corresponds to P121051804F in the figure, and 405F corresponds to P121051405F in the figure, the same below.

Figures 5A-5C and Table 11 show the affinity results of QLS0309-E and VIB9600 to monocytes. The results showed that: the EC ₅₀ binding to FcγRIIA on the surface of monocytes in PBMCs was 0.0429±0.0106nM, N=3; the EC ₅₀ of VIB9600 binding to monocytes in PBMCs under the same reaction conditions was 0.125±0.0353nM, N= 3. Compared with VIB9600, the binding ability of QLS0309-E to FcγRIIA antigen on the surface of monocytes is enhanced.

Example 7. Selective evaluation of the binding ability of QLS0309-E to FcγRIIB on the surface of PBMC-derived B cells

To further study the affinity of candidate antibody molecules to FcγRIIB, in this study, flow cytometry was used to determine the binding ability of QLS0309-E to FcγRIIB on the surface of B cells in human PBMCs, so as to evaluate the binding activity of QLS0309-E to human B cells.

Human B cells were co-incubated with different concentrations of QLS0309-E, then added fluorescently labeled anti-human IgG Fc secondary antibody, incubated at 4°C for 1 hour, centrifuged and washed with phosphate buffer, added CD20-BV510 fluorescent antibody, 4°C After incubation for 30 min, centrifugation and washing, adding 7-AAD staining and washing, the prepared samples were then detected on a flow cytometer (BD LSRFortessa), and the average fluorescence intensity of each concentration was calculated by software, and then analyzed by GraphPad software. The half maximal binding concentration (hereinafter referred to as EC ₅₀ ) and the highest mean fluorescence intensity (Top MFI) were calculated, and the results are shown in FIGS. 6A-6D and Table 12. The mean fluorescence intensity (MFI) of the cell surface was detected by flow cytometry.

Table 12 Binding of CD32a antibody to human B cell CD32b

the	ID: Y376	ID: 804F	ID: 405F	ID: 304F
Antibody	EC50 (nM)	EC50 (nM)	EC50 (nM)	EC50 (nM)
VIB9600	3.96	6.16	5.27	2.71
QLS0309-E	NA	NA	NA	NA
H2B	0.02	0.02	0.02	0.03

Note: The number shown after the ID is the donor number, Y376 corresponds to Y1376 in the figure, 804F corresponds to P121051804F in the figure, 405F corresponds to P121051405F in the figure, 304F corresponds to P121051304F in the figure, the same below; H2B is the hFcγRIIB control antibody, as a negative control, only Fc[gamma]RIIB is identified; NA fits a straight line and no _EC50 can be obtained.

The results of QLS0309-E binding to B cells in PBMC showed that the half maximal binding concentration (EC ₅₀ ) of VIB9600 binding to FcγRIIB on the surface of B cells in PBMC was 4.525±1.51nM, N=4, QLS0309-E did not under the same reaction conditions Binding to B cell FcyRIIB (N=4).

Experiments show that: Compared with hIgG1-TM (isotype control), QLS0309-E does not bind to FcγRIIB on the surface of human B cells. B cells express FcγRIIB but no FcγRIIA expression, indicating that QLS0309-E has significantly lower affinity for human B cell FcγRIIB than VIB9600. QLS0309-E has better binding specificity to FcγRIIA than VIB9600.

Example 8. Comparative analysis of the affinity of QLS0309-E to FcγR subtypes

Binding of antibodies to recombinant FcyRI, FcyRIIA-131H, FcyRIIB, FcyRIII-158F, or FcyRIII-158V was assessed by ELISA. 0.125 μg/ml FcγRs (FcγRI, FcγRIIA-131H, FcγRIIB, FcγRIII-158F or FcγRIII-158V) were added to a 96-well plate and incubated overnight at 4°C (100 μl per well). Wash the plate 5 times with a PBS solution (PBST solution) with 0.1% Tween 20 added, then block with a blocking solution (Thermofisher, Superblock) for 3 hours at room temperature (200 microliters per well), then wash the plate 5 times with PBST solution. Prepare a 4-fold dilution of the antibody to be tested (the highest concentration is 12.5 μg/ml) and add it to a 96-well plate (100 μl per well), incubate at room temperature for 2 hours, and wash 5 times with PBST. Finally, add detection secondary antibody (0.2 μg/ml, 50 μl per well, Peroxidase AffiniPure Donkey Anti-Human IgG, Fcγfragment specific/Jackson ImmunoResearch/709-035-098 or Peroxidase AffiniPure Goat Anti-Mouse IgG ( subclasses 1+2a+2b+3), FcγFragment Specific/Jackson ImmunoResearch/115-035-164, select according to whether the primary antibody is human or mouse), incubate for 1 hour, wash with PBST 10 times, add 100 microliters of TMB bottom After reacting for a certain period of time, 100 microliters of 0.2M sulfuric acid solution was added to terminate the reaction (reaction times were 20 minutes, 2.5 minutes, 10 minutes, 15 minutes, and 15 minutes), and the OD450 absorbance was detected by a microplate reader (TECAN, SPARK). Anti-human IgG1 antibody was used as QLS0309-E isotype control, while antibodies H2B, VIB9600, 16-115, 3G8 were used as positive controls for FcγRII B, FcγRIIA, FcγRI and FcγRIII, respectively. Among them, 16-115 is a commercial anti-FCγRIA antibody (purchased from https://www.antibodies-online.com/antibody/515560/anti-Fc+Fragment+of+IgG,+High+Affinity+Ia,+Receptor+ CD64+FCGR1A+AA+16-115+antibody/); 3G8 is a commercially available anti-FCγRIIIA antibody (purchased from https://www.abcam.cn/cd16-antibody-3g8-low-endotoxin-azide-free-ab176528. html).

Results As shown in Figures 7A-7E, QLS0309-E exhibited high binding affinity to human FcγRIIA, but not to other FcγRs. These data demonstrate that QLS0309-E only specifically binds CD32a in FcγR.

Example 9. QLS0309-E mediates FcγRIIA endocytosis at the surface of monocytes in PBMCs

After FcγRIIA antibody binds to FcγRIIA, it can mediate the endocytosis of FcγRIIA. Therefore, after antigen-antibody binding, we evaluate the efficacy of candidate antibody endocytosis by detecting the expression of FcγRIIA on the surface of human immune cells. We evaluated the efficacy of FcγRIIA antibodies using a human PBMC endocytosis assay.

After the antibody and PBMC were incubated, the mononuclear cells (anti-CD14) were stained with immune cell biomarker antibody and anti-FcγRIIA antibody, IV.3-FITC, and then the prepared samples were detected on the flow cytometer, The mean fluorescence intensity (MFI for short) of each concentration was calculated by software, and the half effective concentration (hereinafter referred to as EC ₅₀ ) was calculated by GraphPad software. IV.3 is an existing mouse monoclonal antibody in the prior art. In the FcγR family, cloneIV.3 (IgG, stemcell, 60012FI) has the strongest binding to the FcγRII subtype, and can simultaneously recognize FcγRII R131 and FcγRII H131 ( Boruchov A M, Heller G, Veri M C, et al. Activating and inhibiting IgG Fc receptors on human DCs mediate opposing functions [J]. The Journal of clinical investigation, 2005, 115(10): 2914-2923.). H2B is hFcγRIIB control antibody, as a negative control, it only recognizes FcγRIIB.

The test results are shown in Figure 8A-8D and Table 13, QLS0309-E is the same as VIB9600, both can promote the endocytosis of FCγRIIA on the surface of monocytes, QLS0309-E is the same as VIB9600, both can promote the endocytosis of FcγRIIA on the surface of monocytes, QLS0309 The half effective concentration (EC ₅₀ ) of -E half promoting FcγRIIA endocytosis is 0.02±0.034nM, N=4, and the EC ₅₀ of VIB9600 is 0.0472±0.009nM, N=4. Therefore, QLS0309-E can promote the endocytosis of FcγRIIA on the surface of monocytes, and exhibits a better endocytosis effect on FcγRIIA receptors than VIB9600.

Table 13 Endocytosis of monocyte FcγRIIA

the	ID: Y376	ID: 804F	ID: 405F	ID: 304F
Antibody	EC50 (nM)	EC50 (nM)	EC50 (nM)	EC50 (nM)
VIB9600	0.0458	0.0599	0.039	0.044
QLS0309-E	0.016	0.024	0.019	0.0209

Example 10. QLS0309-E mediates the endocytosis ability of FcγRIIB on the surface of human PBMC-derived B cells

The specific binding of QLS0309-E antibody to FcγRIIA is further different from that of VIB9600, which is also reflected in the endocytosis ability of FcγRIIB on the surface of immune cells. Therefore, we tested the ability of candidate antibodies to internalize FcγRIIB on the surface of B cells. In this study, QLS0309-E induced endocytosis of FcγRIIB on the surface of B cells.

The main research process is to extract peripheral blood mononuclear cells from healthy donors, and after incubation with antibodies and human PBMCs, use immune cell biomarker antibodies and anti-FcγRIIA antibodies to stain B cells (anti-CD20), and then prepare samples in Detection was performed on a flow cytometer, and the mean fluorescence intensity (MFI for short) of each concentration was calculated by software, and the half effective concentration (EC ₅₀ hereinafter) was calculated by GraphPad software.

The results of antibody-mediated endocytosis of B cells in PBMC are shown in Figures 9A-9C and Table 14: EC ₅₀ of VIB9600-mediated endocytosis of FcγRIIB on the surface of B cells is 7.999±1.302nM, N=3, QLS0309-E under the same conditions Down, does not mediate endocytosis of B cell surface FcγRIIB, N=3.

Therefore, QLS0309-E has a lower affinity for FcγRIIB than VIB9600 (does not bind to FcγRIIB), and does not mediate endocytosis of FcγRIIB receptors. QLS0309-E can specifically recognize FcγRIIA antigen and does not bind to FcγRIIB, so compared with VIB9600, it will not affect the endocytosis of FcγRIIB on the surface of B cells, while VIB9600 can recognize FcγRIIB antigen at high concentrations and promote the endocytosis of FcγRIIB on the surface of B cells . Therefore, compared with VIB9600, QLS0309-E has similar binding specificity to FcγRIIA, and reduces the binding to FcγRIIB, which enhances the specificity of the antibody. This makes the selectivity and functionality of QLS0309-E significantly enhanced compared with VIB9600.

Table 14 QLS0309-E causes endocytosis of FcγRIIB on the surface of human B cells

the	ID: Y376	ID: 804F	ID: 304F
Antibody	EC50 (nM)	EC50 (nM)	EC50 (nM)
VIB9600	7.994	9.303	6.699
QLS0309-E	NA	NA	NA
H2B	0.0055	0.0056	0.0093

(NA: curve fitting, EC ₅₀ not available).

Example 11.QLS0309-E Detection of neutrophil superoxide production induced by antineutrophil cytoplasmic antibody (ANCA)

In the pathogenesis of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), autoantibodies against neutrophil surface antigens rely on FcγRIIA to activate neutrophils and induce tissue damage. Antineutrophil cytoplasmic antibodies (ANCA) activate signal transduction pathways in neutrophils. Proinflammatory cytokines and chemokines (eg, tumor necrosis factor) Tumor necrosis factor (TNF) leads to neutrophil activation and, at low doses, induces synthetic expression of myeloperoxidase (MPO) in neutrophils And released from the cytoplasm to the cell membrane, where it binds to autoantibodies, activates neutrophils through FcγRIIA, ANCA, activates the downstream signaling pathway NADPH (reducing coenzyme), releases superoxide, and plays a role in the development of AAV diseases play an important role.

In order to detect the effect of QLS0309-E on neutrophil superoxide production induced by antineutrophil cytoplasmic antibody (ANCA), we used flow cytometry to detect the substrate of superoxide, Dihydrorhodamine 123 (Dihydrorhodamine 123 , DH123), so as to verify whether the FcγRIIA antibody can inhibit neutrophil antineutrophil cytoplasmic antibody (ANCA)-induced superoxide production. DH123 is an uncharged and non-fluorescent reactive oxygen species (ROS) indicator that passively diffuses across membranes, where it is oxidized to the cationic rhodamine 123, which localizes to mitochondria and displays green fluorescence.

The neutrophils were resuspended in RPMI 1640 buffer and the cell density was adjusted, and seeded into a 96-well plate, 45 μL per well. Prepare antibody sample solution with 2 times concentration, transfer the antibody sample solution or isotype control antibody (Baiying Biotechnology, B485801) to the corresponding well of 96-well cell plate, 45 μL per well, and place in a cell culture incubator (37°C/5% CO ₂ ) preincubation for 45 minutes. Then 5 μL of TNF-α (R&D, 210-TA-020) and 1 μg/mL of anti-myeloperoxidase (MPO) antibody (Abcam, ab134132) with a final concentration of 1 ng/mL were added to the cell culture incubator (37 ℃/5% CO ₂ ) for 30 minutes, add dihydrohodamine (dihydrohodamine, DHR) 123 (Thermofisher Scientific, D632) at a final concentration of 1 μg/mL and incubate for 20 minutes in a cell culture incubator (37 ℃/5% CO ₂ ) , take it out and place it on ice for 10 minutes, collect the cell pellet by centrifugation, and resuspend in ice-cold HBSS at a density of 2x10 ⁶ cells/mL. Mean fluorescence intensity (MFI) was measured by flow cytometry (BD Biosciences, LSR Fortessa), and statistical differences were calculated by GraphPad software.

Blank control well: there is only phosphate buffered saline (PBS) in the cell suspension. Positive control wells: Add TNF-α and MPO antibodies to the cell suspension. Test wells: TNF-α and anti-MPO antibodies and humanized antibodies were added to the cell suspension. In this test, the inhibitory rate of antibody to neutrophil superoxide production is calculated by the following formula: % inhibitory rate=100%×(positive control well average value-test well average value)/(positive control well well average value-blank control hole average). The inhibition rate results are marked in the figure.

Figures 10A-10C show that QLS0309-E can specifically block the ability of anti-neutrophil cytoplasmic antibody (ANCA)-induced neutrophil activation to produce superoxide. The experimental results show that: QLS0309-E has a good inhibitory ability on neutrophil superoxide production induced by anti-neutrophil cytoplasmic antibody (ANCA). At the concentration of 6.67μg/ml, there is an average inhibitory intensity of 62%, at the concentration of 2.22μg/ml, there is an average inhibitory intensity of 52.7%, and at the concentration of 0.74μg/ml, there is an average inhibitory intensity of 14.7%, while the isotype Controls had no inhibitory effect under the same conditions.

The above-described embodiments of the present disclosure are illustrative only, and any skilled in the art will recognize, or can ascertain, numerous equivalents to the specific compounds, materials, and procedures without undue undue experimentation. All such equivalents are within the scope of this disclosure and are encompassed by the claims.

Claims (15)

1. An FcγRIIA binding molecule or antigen-binding fragment thereof comprising:

   i) heavy chain complementarity determining region 1 (CDRH1), the amino acid sequence of which is shown in SEQ ID NO: 3;

   ii) heavy chain complementarity determining region 2 (CDRH2), the amino acid sequence of which is shown in SEQ ID NO: 4, or a CDRH2-H61 D-V mutation is introduced on the basis of SEQ ID NO: 4;

   iii) heavy chain complementarity determining region 3 (CDRH3), the amino acid sequence of which is shown in SEQ ID NO: 5, or a CDRH3-H101 D-A mutation is introduced on the basis of SEQ ID NO: 5;

   iv) light chain complementarity determining region 1 (CDRL1), the amino acid sequence of which is shown in SEQ ID NO: 6, or on the basis of SEQ ID NO: 6, a mutation selected from the following groups is introduced:

   a) CDRL1-L26 S-W and/or CDRL1-L27c L-P; or

   b) CDRL1-L24 R-L and/or CDRL1-L27c L-P; or

   c) CDRL1-L24 R-L and/or CDRL1-L32 Y-Q; or

   d) CDRL1-L24 R-L and/or CDRL1-L26 S-W; or

   e) CDRL1-L26 S-W and/or CDRL1-L32 Y-Q; or

   f) CDRL1-L27c L-P and/or CDRL1-L32 Y-Q;

   v) light chain complementarity determining region 2 (CDRL2), the amino acid sequence of which is shown in SEQ ID NO: 7; and

   vi) Light chain complementarity determining region 3 (CDRL3), the amino acid sequence of which is shown in SEQ ID NO: 8.
2. The FcγRIIA binding molecule or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the light chain complementarity determining region 1 (CDRL1) is shown in SEQ ID NO: 11.
3. The FcγRIIA binding molecule or antigen-binding fragment thereof according to claim 1 or 2, comprising:

   I) a heavy chain variable region as shown in SEQ ID NO: 9, or a heavy chain variable region having at least 80% amino acid sequence identity to SEQ ID NO: 9; and/or

   II) A light chain variable region as shown in SEQ ID NO: 10, or a light chain variable region having at least 80% amino acid sequence identity to SEQ ID NO: 10.
4. The FcγRIIA binding molecule or antigen-binding fragment thereof according to any preceding claim, further comprising a heavy chain constant region and/or a light chain constant region; preferably, the heavy chain constant region comprises Fc; more preferably, Fc Of murine or human origin; more preferably, the sequence of Fc is native or modified.
5. The FcγRIIA binding molecule or antigen-binding fragment thereof according to any one of the preceding claims, which is a monoclonal antibody, a bispecific binding molecule, a multispecific binding molecule, a humanized antibody, a chimeric antibody, a modified antibody, a fully Human antibody, full length antibody, heavy chain antibody, nanobody, Fab, Fv, scFv, F(ab') ₂ , linear antibody or single domain antibody.
6. A conjugate, which is formed by coupling the FcγRIIA binding molecule or antigen-binding fragment thereof according to any one of the preceding claims to a capture label or a detection label or a biologically active molecule;

   Preferably, the detection markers include radionuclides, luminescent substances, colored substances or enzymes;

   Preferably, the bioactive molecule is a small molecule drug; more preferably, the FcγRIIA binding molecule or an antigen-binding fragment thereof is connected to the bioactive molecule through a linker.
7. A nucleic acid encoding the Fc[gamma]RIIA binding molecule or antigen-binding fragment thereof of any preceding claim.
8. An expression vector comprising the nucleic acid of claim 7.
9. A host cell comprising the nucleic acid of claim 7 or the vector of claim 8;

   Preferably, the host cell is a prokaryotic cell or a eukaryotic cell; the prokaryotic cell is preferably Escherichia coli; the eukaryotic cell is preferably a mammalian cell or yeast; more preferably, the mammalian cell is a CHO cell, an Expi293 cell or HEK293 cells.
10. A composition comprising the FcγRIIA binding molecule or antigen-binding fragment thereof according to any one of claims 1-5, the conjugate according to claim 6, the nucleic acid according to claim 7, the expression expression according to claim 8 vector and/or the host cell of claim 9;

    Preferably, the composition is a pharmaceutical composition, which further comprises a pharmaceutically acceptable carrier; more preferably, the pharmaceutical composition further comprises one or more additional therapeutic agents;

    Preferably, the composition is a diagnostic reagent.
11. The method for preparing the FcγRIIA binding molecule or antigen-binding fragment thereof according to any one of claims 1-5, the method comprising: culturing the host cell according to claim 9 under suitable conditions.
12. The FcγRIIA binding molecule or antigen-binding fragment thereof according to any one of claims 1-5, the conjugate according to claim 6, the nucleic acid according to claim 7, the expression vector according to claim 8, or the expression vector according to claim 9 Use of the host cell and/or the composition according to claim 10 in the preparation of medicines for treating, alleviating and/or preventing inflammatory diseases or autoimmune diseases or other FcγRIIA-mediated diseases;

    Preferably, the inflammatory disease or autoimmune disease or other FcγRIIA-mediated disease is selected from: primary immune thrombocytopenia (ITP) or antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV ).
13. The FcγRIIA binding molecule or antigen-binding fragment thereof according to any one of claims 1-5, the conjugate according to claim 6, the nucleic acid according to claim 7, the expression vector according to claim 8, or the expression vector according to claim 9 Use of the host cell and/or the composition described in claim 10 in the preparation of detection reagents or diagnostic reagents for detection or diagnosis of inflammatory diseases or autoimmune diseases or other FcγRIIA mediated disease.
14. A method for treating, alleviating and/or preventing inflammatory diseases or autoimmune diseases or other FcγRIIA-mediated diseases in a subject in need thereof, comprising administering any one of claims 1-5 to the subject The FcγRIIA binding molecule or antigen-binding fragment thereof, the conjugate of claim 6, the nucleic acid of claim 7, the expression vector of claim 8, the host cell of claim 9, and/or The composition of claim 10.
15. A method for detecting FcγRIIA in a sample, comprising:

    (1) contacting the sample with the FcγRIIA binding molecule or antigen-binding fragment thereof according to any one of claims 1-5;

    (2) Detecting the formation of a complex of the FcγRIIA-binding molecule or antigen-binding fragment thereof with FcγRIIA; optionally, the FcγRIIA-binding molecule or antigen-binding fragment thereof is detectably labeled.

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