Anti-CD16a nanobody, and preparation method therefor and use thereof

Abstract

Provided are an anti-CD16A nanobody, and a preparation method therefor and the use thereof. Specifically, the method comprises: immunizing alpaca with a CD16A recombinant protein to obtain a high-quality immune library, and screening the immune library by means of using a phage display technique, to obtain a nanobody with high specificity for binding to CD16A. Further provided are a corresponding anti-CD16A antibody, chimeric antigen receptor, fusion protein and recombinant protein, an encoding nucleic acid, an expression vector, a host cell and a CAR thereof, etc. The anti-CD16A nanobody has the advantages of high affinity, simple structure and easy preparation, and has application prospects in the fields of tumor treatment, immunological detection, etc.

Description

Anti-CD16A nanobody and its preparation method and application Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to anti-CD16A nanoantibodies and preparation methods and applications thereof. Background Art

[0002] CD16, also known as FcγRIII, is a single-pass transmembrane receptor widely expressed on the surface of natural killer cells, neutrophils, monocytes, macrophages, and some T cells. CD16 is divided into two highly similar variants, FcγRIIIa (CD16a) and FcγRIIIb (CD16b), which participate in signal transduction of immune cells, triggering NK cells to lyse target cells, and is a molecule of the immunoglobulin superfamily involved in antibody-dependent cellular cytotoxicity (ADCC).

[0003] CD16 is a type III Fcγ receptor that exists in two distinct forms in the human body: FcγRIIIa (CD16a) and FcγRIIIb (CD16b), which share 96% sequence similarity in the extracellular immunoglobulin-binding region. FcγRIIIa is a transmembrane receptor expressed on mast cells, macrophages, and natural killer cells, while FcγRIIIb is expressed exclusively on neutrophils. FcγRIIIb is the only Fc receptor anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) linker and plays a crucial role in triggering calcium flux and neutrophil degranulation. Together, FcγRIIIa and FcγRIIIb activate degranulation, phagocytosis, and the oxidative burst, enabling neutrophils to eliminate opsonized pathogens.

[0004] CD16 binds to the Fc portion of IgG antibodies, which then activates antibody-dependent cell-mediated cytotoxicity (ADCC) in NK cells. CD16 is an essential mediator of ADCC in human monocytes. Binding of CD16 on human NK cells to ligands, such as the conserved portion of IgG antibodies, induces gene transcription for surface activation molecules such as IL2R (CD25) and inflammatory cytokines such as IFN-γ and TNF.

[0005] CD16 plays an important role in the early activation of natural killer (NK) cells after vaccination. In normal healthy individuals, cross-linking of immune complexes to CD16 (FcγRIII) induces antibody-dependent cellular cytotoxicity (ADCC) in NK cells, a pathway through which cancer cells can also be targeted by immunotherapy. CD16-positive T cells are found in patients with chronic viral infections or after organ transplantation, as well as in patients with severe viral infections. CD16 expression enables antibody-mediated degranulation, thereby achieving T cell receptor-independent cytotoxicity. In patients with severe COVID-19, CD16-positive T cells may lead to increased cytotoxicity, promote microvascular endothelial cell damage and contribute to disease severity. Due to its expression on neutrophils, CD16 represents a possible target for cancer immunotherapy.

[0006] However, there is currently a lack of anti-CD16A nanobodies with high affinity, high specificity and high biological activity against CD16A. Therefore, there is an urgent need in the art to develop anti-CD16A nanobodies with high affinity, high specificity and high biological activity. Summary of the Invention

[0007] The purpose of the present invention is to provide an anti-CD16A nanobody with high affinity, high specificity and high biological activity.

[0008] In a first aspect of the present invention, an anti-CD16A nanobody is provided, wherein the anti-CD16A nanobody has one or more complementary determining regions (CDRs) selected from the group consisting of:

[0009] (1) CDR1 shown in SEQ ID NO: 7, CDR2 shown in SEQ ID NO: 8, and CDR3 shown in SEQ ID NO: 9;

[0010] (2) CDR1 shown in SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 11, and CDR3 shown in SEQ ID NO: 12;

[0011] (3) CDR1 shown in SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 13, and CDR3 shown in SEQ ID NO: 14;

[0012] (4) CDR1 shown in SEQ ID NO: 15, CDR2 shown in SEQ ID NO: 16, and CDR3 shown in SEQ ID NO: 17;

[0013] (5) CDR1 shown in SEQ ID NO: 18, CDR2 shown in SEQ ID NO: 19, and CDR3 shown in SEQ ID NO: 20;

[0014] (6) CDR1 shown in SEQ ID NO: 21, CDR2 shown in SEQ ID NO: 22, and CDR3 shown in SEQ ID NO: 23.

[0015] In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0016] In another preferred example, the amino acid sequence of the VHH chain of the anti-CD16A Nanobody is as shown in any one of SEQ ID NOs: 1-6.

[0017] In another preferred example, the VHH chain of the anti-CD16A Nanobody comprises an amino acid sequence having a sequence similarity of at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% to any one of the amino acid sequences of SEQ ID NO: 1-6.

[0018] In another preferred example, the amino acid sequence of the VHH chain of the anti-CD16A Nanobody comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, compared with any one of the amino acid sequences in SEQ ID NO: 1-6.

[0019] In another preferred embodiment, any one of the above amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one (such as 1-3, preferably 1-2, more preferably 1) amino acid and can retain the ability to specifically bind to CD16A.

[0020] In another preferred embodiment, the anti-CD16A nanobody includes humanized antibodies, animal-derived antibodies, and chimeric antibodies.

[0021] In the second aspect of the present invention, an anti-CD16A multivalent antibody or an antibody against multiple epitopes is provided, wherein the multivalent antibody or the antibody against multiple epitopes comprises at least one antibody element against a CD16A epitope, and the antibody element is the anti-CD16A nanobody described in the first aspect of the present invention.

[0022] In another preferred embodiment, the anti-CD16A multivalent antibody or the antibody targeting multiple epitopes comprises one or more anti-CD16A nanobodies.

[0023] In another preferred embodiment, the anti-CD16A multivalent antibody includes a monomer, a bivalent body (divalent antibody), a tetravalent body (tetravalent antibody), and / or a multivalent body (multivalent antibody).

[0024] In another preferred embodiment, the anti-CD16A multivalent antibody comprises one or more VHH chains having an amino acid sequence as shown in any one of SEQ ID NOs: 1-6.

[0025] In another preferred embodiment, the anti-CD16A multivalent antibody comprises two VHH chains having the amino acid sequences shown in SEQ ID NOs: 1-6.

[0026] In another preferred embodiment, the anti-CD16A nanobody includes humanized antibodies, animal-derived antibodies, and chimeric antibodies.

[0027] In a third aspect of the present invention, a recombinant protein is provided, wherein the recombinant protein has:

[0028] (i) the anti-CD16A Nanobody of the first aspect of the invention, or the anti-CD16A multivalent antibody or antibody against multiple epitopes of the second aspect of the invention; and

[0029] (ii) optionally a tag sequence to facilitate expression and / or purification.

[0030] In another preferred embodiment, the tag sequence includes a Flag tag, an Fc tag, an HA tag and a His tag.

[0031] In another preferred embodiment, the tag sequence is a His tag.

[0032] In another preferred embodiment, the recombinant protein specifically binds to CD16A protein.

[0033] In the fourth aspect of the present invention, an antibody-drug conjugate is provided, wherein the antibody-drug conjugate comprises:

[0034] (a) the Nanobody as described in the first aspect of the invention, the multivalent antibody or antibody against multiple epitopes as described in the second aspect of the invention, or the recombinant protein as described in the third aspect of the invention; and

[0035] (b) a conjugated moiety conjugated to the antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0036] In another preferred embodiment, the antibody portion and the coupling portion are coupled via a chemical bond or a linker.

[0037] In the fifth aspect of the present invention, a chimeric antigen receptor (CAR) is provided, wherein the CAR comprises an extracellular domain, wherein the extracellular domain comprises the anti-CD16A nanobody as described in the first aspect of the present invention, or the anti-CD16A multivalent antibody as described in the second aspect of the present invention.

[0038] In another preferred embodiment, the extracellular domain further includes a signal peptide.

[0039] In another preferred embodiment, the extracellular domain also includes other exogenous proteins.

[0040] In another preferred embodiment, the CAR has a structure shown in formula (I): L-Nb-H-TM-C-CD3ζ (I)

[0041] Where,

[0042] The "-" is a connecting peptide or peptide bond;

[0043] L is none or a signal peptide sequence;

[0044] Nb is the antigen-binding domain that specifically binds to CD16A;

[0045] H is none or hinge region;

[0046] TM is the transmembrane domain;

[0047] C is the costimulatory signaling domain;

[0048] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type, or mutants / modified forms thereof).

[0049] In another preferred embodiment, the L is selected from the signal peptides of the following histones: CD28, 4-1BB, GM-CSF, CD3, CD8a or a combination thereof.

[0050] In another preferred embodiment, the sequence of Nb is shown in any one of SEQ ID NOs: 1-6.

[0051] In another preferred embodiment, the H is selected from the hinge region of the following histones: CD8, CD28, CD137, IgG, or a combination thereof.

[0052] In another preferred embodiment, the C is selected from the costimulatory domains of the following histones: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD-1, Dap10, LIGHT, NKG2C, B7-H3, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, OX40L, 2B4, TLR, or mutants / modified forms thereof, or combinations thereof.

[0053] In the sixth aspect of the present invention, a polynucleotide is provided, which encodes a protein selected from the following group: the nanobody described in the first aspect of the invention, or the multivalent antibody or antibody against multiple epitopes described in the second aspect of the invention, the recombinant protein described in the third aspect of the invention, or the CAR described in the fifth aspect of the invention.

[0054] In the seventh aspect of the present invention, an expression vector is provided, wherein the expression vector contains the polynucleotide described in the sixth aspect of the present invention.

[0055] In another preferred embodiment, the expression vector is selected from the following group: DNA, RNA, viral vector, plasmid, transposon, other gene transfer systems, or a combination thereof.

[0056] In another preferred embodiment, the expression vector is a lentiviral vector.

[0057] In the eighth aspect of the present invention, a host cell is provided, which contains the expression vector described in the seventh aspect of the present invention, or the polynucleotide described in the sixth aspect of the present invention is integrated into its genome, or expresses the nanoantibody described in the first aspect of the present invention, the multivalent antibody or antibody against multiple epitopes described in the second aspect of the present invention, the recombinant protein described in the third aspect of the present invention, or the CAR described in the fifth aspect of the present invention.

[0058] In another preferred embodiment, the cell is an isolated cell, and / or the cell is a genetically engineered cell.

[0059] In another preferred embodiment, the cells are mammalian cells.

[0060] In another preferred embodiment, the cells are T cells.

[0061] In another preferred embodiment, the host cell is an engineered immune cell.

[0062] In another preferred embodiment, the engineered immune cells are selected from the following group:

[0063] (i) Chimeric antigen receptor αβ T cells (CAR-T cells);

[0064] (ii) chimeric antigen receptor γδ T cells (CAR-T cells);

[0065] (iii) chimeric antigen receptor NKT cells (CAR-NKT cells);

[0066] (iv) chimeric antigen receptor NK cells (CAR-NK cells);

[0067] (v) Chimeric antigen receptor macrophages.

[0068] In another preferred embodiment, the engineered immune cells include autologous or allogeneic αβT cells, γδT cells, NKT cells, NK cells, or a combination thereof.

[0069] In another preferred embodiment, the engineered immune cells are CAR-T cells.

[0070] In the ninth aspect of the present invention, a preparation is provided, which contains the nanobody described in the first aspect of the present invention, the multivalent antibody or the antibody against multiple epitopes described in the second aspect of the present invention, the recombinant protein described in the third aspect of the present invention, the antibody-drug conjugate described in the fourth aspect of the present invention, the CAR described in the fifth aspect of the present invention, or the vector described in the seventh aspect of the present invention, or an immune cell expressing the CAR described in the fifth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0071] In a tenth aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0072] (i) the Nanobody of the first aspect of the invention, the multivalent antibody or the antibody against multiple epitopes of the second aspect of the invention, the recombinant protein of the third aspect of the invention, the antibody-drug conjugate of the fourth aspect of the invention, or the host cell of the eighth aspect of the invention; and

[0073] (ii) a pharmaceutically acceptable carrier.

[0074] In another preferred embodiment, the pharmaceutical composition further contains other drugs for treating diseases.

[0075] In another preferred embodiment, the pharmaceutical composition is used to prepare a drug for preventing and / or treating diseases or conditions associated with CD16A.

[0076] In another preferred embodiment, the disease or condition associated with CD16A is selected from the following group: hematological tumors, solid tumors, or a combination thereof.

[0077] In another preferred embodiment, the blood tumor is selected from the group consisting of T cell tumors, B cell tumors, or a combination thereof.

[0078] In another preferred embodiment, the solid tumor is selected from the group consisting of pancreatic cancer, colorectal cancer, lung cancer, gastric cancer, breast cancer, ovarian cancer, or a combination thereof.

[0079] In the eleventh aspect of the present invention, a kit is provided, wherein the kit contains the polynucleotide described in the sixth aspect of the present invention, the CAR described in the fifth aspect of the present invention, or the vector described in the seventh aspect of the present invention.

[0080] In another preferred embodiment, the kit is used to prepare immune cells expressing the CAR described in the fifth aspect of the present invention.

[0081] In the twelfth aspect of the present invention, there is provided the use of the nanobody described in the first aspect of the present invention, the multivalent antibody or the antibody against multiple epitopes described in the second aspect of the present invention, the recombinant protein described in the third aspect of the present invention, the antibody-drug conjugate described in the fourth aspect of the present invention, or the immune cell expressing the CAR described in the fifth aspect of the present invention for the preparation of a medicament for preventing and / or treating cancers or diseases associated with CD16A.

[0082] In the thirteenth aspect of the present invention, a method for treating a disease is provided, which comprises administering to a subject in need of treatment: the nanoantibody described in the first aspect of the invention, the multivalent antibody or antibody against multiple epitopes described in the second aspect of the invention, the recombinant protein described in the third aspect of the invention, the antibody-drug conjugate described in the fourth aspect of the invention, or an immune cell expressing the CAR described in the fifth aspect of the invention.

[0083] In another preferred embodiment, the disease is a tumor that is positive for CD16A expression.

[0084] In another preferred embodiment, the subject includes mammals, such as humans.

[0085] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] FIG1 shows the SDS-PAGE detection results of CD16A recombinant protein.

[0087] FIG2 shows the amplification results of the VHH fragment.

[0088] Figure 3 shows the diversity analysis of the phage display library.

[0089] FIG4 shows the results of phage ELISA detection of the fourth round of enrichment products.

[0090] FIG5 shows the FACS detection results of the target antibody expression supernatant.

[0091] Figure 6 shows the FACS binding validation results of the candidate antibodies; PC is a positive control and NC is a negative control.

[0092] FIG7 shows the affinity test results of 9-F05, 9-C05 and 9-G05 antibodies. DETAILED DESCRIPTION

[0093] After extensive and intensive research, the present inventors, through extensive screening, unexpectedly obtained, for the first time, anti-CD16A nanobodies with high affinity and specificity. Specifically, the present invention used recombinant CD16A protein to immunize alpacas, thereby obtaining a high-quality immune library. Phage display technology was used to screen the immune library, resulting in a class of specific nanobodies against CD16A. The anti-CD16A nanobodies of the present invention have high affinity, a simple structure, and are easy to prepare, with broad application prospects in fields such as tumor treatment and immunoassays. This is the basis for the completion of the present invention.

[0094] the term

[0095] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0096] The term "about" can refer to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0097] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0098] Sequence identity is determined by comparing two aligned sequences along a predetermined comparison window (which can be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein) and determining the number of positions at which identical residues occur. Typically, this is expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a method well known to those skilled in the art.

[0099] Antibody

[0100] As used herein, the term "antibody" refers to immunoglobulins, which are tetrapeptide chains composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.

[0101] As used herein, the terms "single-domain antibody (VHH)" and "nanobody" have the same meaning and refer to the cloning of the variable region of an antibody heavy chain to construct a single-domain antibody (VHH) consisting of only a single heavy chain variable region. This is the smallest antigen-binding fragment with complete function. Typically, an antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting of only a single heavy chain variable region.

[0102] The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CD16A). It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of an antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment of an antibody" include:

[0103] (i) Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains;

[0104] (ii) F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region;

[0105] (iii) an Fd fragment consisting of the VH and CH1 domains;

[0106] (iv) An Fv fragment consisting of the VH and VL domains of a single arm of an antibody.

[0107] Fv antibodies contain the variable regions of the antibody heavy chain and light chain, but no constant region, and are the smallest antibody fragments with all antigen-binding sites. Generally, Fv antibodies also include a polypeptide linker between the VH and VL domains and are able to form the structure required for antigen binding.

[0108] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al. (1991) Sequences of proteins of immunological interest. NIH Publication, No. 91-3242).

[0109] The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., a specific site on a CD16A molecule). An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation.

[0110] The terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. -7 M, for example, less than approximately 10 -8 M, 10 -9 M or 10 -10 Binds with an affinity (KD) of M or less.

[0111] As used herein, the term "antigenic determinant" refers to a discrete three-dimensional site on an antigen that is recognized by the antibodies or antigen-binding fragments of the present invention.

[0112] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0113] In the present invention, the antibodies of the present invention also include conservative variants thereof, which refer to polypeptides in which no more than 10, preferably no more than 8, more preferably no more than 5, and most preferably no more than 3 amino acids are replaced with amino acids having similar or similar properties, compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by making amino acid substitutions according to Table A below.

[0114] Table A

[0115] Anti-CD16A nanobody

[0116] As used herein, the term "CD16A" generally refers to natural or recombinant human CD16A, as well as non-human homologs of human CD16A.

[0117] As used herein, the terms "Nanoantibodies of the present invention", "single domain antibodies of the present invention", and "anti-CD16A Nanoantibodies of the present invention" are used interchangeably and all refer to the Nanoantibodies of the present invention that are highly specific and have high affinity for CD16A.

[0118] Compared with traditional monoclonal antibodies, the nanoantibodies of the present invention have small molecular weight, strong penetration, high sensitivity, strong specificity and good stability; once they enter the body, they can efficiently penetrate cells to quickly capture antigens to achieve therapeutic purposes.

[0119] In the present invention, the anti-CD16A nanobody includes monomers, bivalents (divalent antibodies), tetravalents (tetravalent antibodies), and / or multivalents (multivalent antibodies).

[0120] In a preferred embodiment of the present invention, the amino acid sequence of the VHH chain of the anti-CD16A nanobody is selected from one or more of SEQ ID NOs: 1 to 6. The preferred nanobody names, VHH sequence numbers and corresponding CDR region sequence numbers of the present invention are shown in Table 1:

[0121] Table 1

[0122] Note: Each numerical value in the table represents a sequence number, i.e., "1" represents "SEQ ID NO: 1"; identical sequences are numbered the same, for example, the CDR1 of 9-F05 and the CDR1 sequence of 9-C05.

[0123] Antibody-drug conjugates (ADCs)

[0124] The present invention also provides an antibody-drug conjugate (ADC) based on the Nanobody of the present invention.

[0125] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a therapeutically active drug. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug, or a radionuclide.

[0126] The antibody of the present invention and the effector molecule can be coupled via a coupling agent. Examples of the coupling agent may include any one or more of a non-selective coupling agent, a coupling agent utilizing a carboxyl group, a peptide chain, and a coupling agent utilizing a disulfide bond. The non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde. The coupling agent utilizing a carboxyl group may include any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (where the coupling site is an acylhydrazone).

[0127] Certain residues on antibodies (such as Cys or Lys, etc.) are used to connect to a variety of functional groups, including imaging agents (such as chromophores and fluorescent groups), diagnostic agents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers) and therapeutic agents. Antibodies can be coupled to functional agents to form antibody-functional agent conjugates. Functional agents (such as drugs, detection reagents, stabilizers) are coupled (covalently linked) to antibodies. Functional agents can be directly or indirectly connected to antibodies through linkers.

[0128] Antibodies can be coupled to drugs to form antibody-drug conjugates (ADCs). Typically, ADCs contain a linker positioned between the drug and the antibody. The linker can be a degradable or non-degradable linker. Degradable linkers typically readily degrade in the intracellular environment, for example, at the target site, where the linker degrades, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including linkers containing peptidyl groups that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronide-containing linkers that can be degraded by glucuronidases. Peptide linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at a pH of less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug when the antibody is hydrolyzed by proteases.

[0129] Prior to attachment to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and attachment is achieved via the active reactive group. Thiol-specific active reactive groups are preferred and include, for example, maleimides, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethylketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, where the counter ion is acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. Linkers may include, for example, maleimides attached to the antibody via thiosuccinimide.

[0130] The drug can be any cytotoxic, cytostatic, or immunosuppressive drug. In embodiments, a linker connects the antibody and the drug, and the drug has a functional group capable of forming a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group capable of forming a bond with the linker. In cases where the drug is directly attached to the linker, the drug has a reactive group prior to attachment to the antibody.

[0131] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. In the present invention, drug-linkers can be used to form ADCs in a single step. In other embodiments, bifunctional linker compounds can be used to form ADCs in a two-step or multi-step process. For example, a cysteine ​​residue reacts with a reactive portion of a linker in a first step, and in a subsequent step, the functional group on the linker reacts with the drug to form an ADC.

[0132] Typically, the functional group on the linker is selected to facilitate specific reaction with an appropriate reactive group on the drug moiety. As a non-limiting example, an azide-based moiety can be used to specifically react with a reactive alkynyl group on the drug moiety. The drug is covalently attached to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Technology, 2nd Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that, when a complementary pair of reactive functional groups is selected for selective reaction between the drug moiety and the linker, each member of the complementary pair can be used for both the linker and the drug.

[0133] The present invention also provides a method for preparing an ADC, which may further comprise: combining an antibody and a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).

[0134] In certain embodiments, the methods of the present invention comprise conjugating an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the present invention further comprise conjugating the antibody-linker conjugate to a drug moiety under conditions sufficient to covalently attach the drug moiety to the antibody via the linker.

[0135] Pharmaceutical composition

[0136] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein or its ADC or corresponding CAR-T cell, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary with the properties of the formulated substance and the condition to be treated. The prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0137] The antibody of the present invention can also be expressed in cells by nucleotide sequences for cell therapy, for example, the antibody is used in chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

[0138] The pharmaceutical composition of the present invention can be used to directly bind to CD16A protein molecules, and thus can be used to prevent and treat CD16A-related diseases. In addition, other therapeutic agents can also be used simultaneously.

[0139] The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned monoclonal antibody of the present invention (or its conjugate) and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 μg / kg body weight to about 5 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used in conjunction with other therapeutic agents.

[0140] When using a pharmaceutical composition, a safe and effective amount of the pharmaceutical composition is administered to a mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight. Preferably, the dose is about 10 μg / kg body weight to about 20 mg / kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health condition, which are all within the skill of a skilled physician.

[0141] The main advantages of the present invention include:

[0142] (1) The nanobody of the present invention has high affinity binding to CD16A.

[0143] (2) The nanobody of the present invention can specifically bind to CD16A but not to CD16B, and has high specificity.

[0144] (3) The anti-CD16A nanobody of the present invention has a small molecular weight, strong penetrating power and high sensitivity.

[0145] (4) The anti-CD16A nanobody of the present invention has a simple structure and is easy to prepare, and has broad application prospects in the fields of tumor treatment and immune detection.

[0146] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise indicated, percentages and fractions are by weight.

[0147] Example 1: Screening of CD16A antibodies

[0148] 1.1 Preparation of CD16A recombinant protein

[0149] The extracellular domain sequence of human CD16A (P08637-1) (Gly17-Gln208) was retrieved from the UniProt database. A His tag was added to the C-terminus. After optimization according to human codon preference, the gene was synthesized and subcloned into the pcDNA3.4 vector. After verification by Sanger sequencing, the plasmid was extracted and used for future use. The constructed eukaryotic protein expression vector was transiently transfected into 293F cells, and the protein expression supernatant was collected and purified using a nickel column. Protein purity was assessed by SDS-PAGE.

[0150] The results are shown in Figure 1. The purity of the human CD16A recombinant protein was >90%, indicating that it could be used for subsequent alpaca immunization and antibody screening.

[0151] 1.2 Alpaca Immunity

[0152] Alpaca were immunized with the recombinant protein prepared above, with an immunization interval of 21 days. Ten days after the last immunization, some peripheral blood was collected, and the serum was separated and the immune effect was detected by ELISA.

[0153] 1.3 Detection of immune titer

[0154] Serum was isolated from immunized alpacas, and the serum was diluted according to the dilution gradient in Table 2. The serum was then tested by ELISA on 96-well plates pre-coated with antigens. The experimental method is as follows:

[0155] Collect 5 mL of peripheral blood and place the centrifuge tube containing the blood sample in a 37°C incubator for 1 hour; then transfer the blood sample to 4°C overnight; place the centrifuge tube containing the blood sample in a centrifuge and centrifuge at 5000 rpm for 20 minutes; separate the upper serum and transfer the serum to a new sterile centrifuge tube to collect the immune serum. Use sterile CBS (carbonate buffer) to dilute the target recombinant protein to a final concentration of 1 μg / mL. Take a new 96-well ELISA plate, add 100 μL / well, and coat at 4°C overnight. Remove the antigen coating solution and wash 5 times with PBST (containing 0.05% Tween 20). Add 200 μL / well of 3% MPBS and block at 37°C for 2 hours; after removing the blocking buffer, wash the plate 5 times with PBST; add 100 μL of serially diluted serum (100 μL / well) and incubate at room temperature for 1 hour. The control well is PBS; remove the liquid in the well and wash 5 times with PBST; add 100 μL of HRP anti-Lama IgG (H+L) antibody (1:50000 dilution) and incubate at room temperature for 1 hour; after removing the liquid in the well, wash the plate 5 times with PBST; add 100 μL / well TMB colorimetric solution; incubate at room temperature in the dark for 10-15 minutes; add 50 μL / well stop solution; read the OD in the well using a microplate reader. 450 value.

[0156] The test results are shown in Table 2 below.

[0157] Table 2 Detection of serum immune titer

[0158] ELISA results showed that the immune serum bound to the CD16A recombinant protein, and the OD values ​​varied with the gradient dilution of the immune serum. When the titer reached 1:64K or above, 100 ml of peripheral blood was collected to prepare a cDNA library for the construction of the antibody display library.

[0159] 1.4 PBMC isolation and VHH antibody fragment cloning

[0160] 1. Collect 100 mL of peripheral blood and separate PBMCs using lymphocyte separation medium.

[0161] 2. Extract RNA using PrimeScript TM Perform reverse transcription using the II 1st Strand cDNA Synthesis Kit to prepare cDNA. Prepare the reaction mixture Mix 1 shown in Table 3 in 200 μL PCR:

[0162] Table 3 Reaction mixture Mix1 configuration system

[0163] After incubation at 65°C for 5 minutes, cool rapidly on ice. Prepare the reaction solution shown in Table 4 in the above PCR tube:

[0164] Table 4 Reaction solution configuration system

[0165] After mixing by pipetting, the mixture was divided into 80 μL / tubes, placed in a PCR instrument at 42°C for 1 hour, and heat-inactivated at 70°C for 15 minutes. Finally, the cDNA sample was placed on ice or stored at -20°C for a long time.

[0166] 3. Amplification of VHH fragments:

[0167] (1) Prepare the first round PCR reaction system as shown in Table 5 (50 μL / tube):

[0168] Table 5 First round PCR reaction system

[0169] After configuring the PCR reaction system, set up the PCR instrument according to the program in Table 6:

[0170] Table 6 PCR program settings

[0171] (2) Agarose gel electrophoresis analysis of PCR products: PCR products were analyzed by electrophoresis using 1% agarose gel to separate fragments with a molecular weight of approximately 750 bp. PCR products were recovered using a gel recovery kit and their concentrations were determined using NanoDrop.

[0172] (3) Prepare the second-round PCR reaction system as shown in Table 7 (50 μL / tube):

[0173] Table 7 Second round PCR reaction system

[0174] After configuring the PCR reaction system, set up the PCR instrument according to the program shown in Table 8:

[0175] Table 8 PCR program settings

[0176] (4) Agarose gel electrophoresis analysis of the second-round PCR products: PCR products were analyzed by electrophoresis using 1% agarose gel to isolate VHH fragments with a molecular weight of approximately 400 bp. The VHH PCR products were recovered using a gel recovery kit and the concentration was determined using NanoDrop.

[0177] Agarose gel electrophoresis of PCR products is shown in Figure 2. In the first round of PCR, PCR bands of approximately 1000 bp and 750 bp were obtained, respectively. The 750 bp fragment was recovered and used as template for the second round of PCR. A band of approximately 400 bp was obtained in the second round of PCR, representing the VHH fragment. The pDisplay vector and the VHH PCR product obtained above were digested with SfiI, respectively, and recovered from the gel. The digested pDisplay vector and VHH fragment were ligated using T4 ligase, incubated overnight at 16°C, and then transformed into E. coli.

[0178] 1.5 Construction of phage display library

[0179] 1. Construction of single domain antibody phage display vector

[0180] The pDisplay vector and the VHH PCR products obtained above were digested with SfiI, respectively. The digestion systems are shown in Tables 9 and 10:

[0181] Table 9 Enzyme Digestion System

[0182] The enzyme digestion system shown in Table 8 was divided into 100 μL / tube and placed in a PCR instrument at 50°C for overnight digestion;

[0183] Table 10 Enzyme digestion system

[0184] Aliquot 100 μL of the digestion system shown in Table 9 into tubes and digest overnight at 50°C in a PCR instrument. Separate the pDisplay vector fragments using a 1% agarose gel, and excise a 5000 bp fragment. Purify the PCR digestion product using a DNA fragment recovery kit and determine its concentration using a NanoDrop. Ligate the digested pDisplay vector and VHH fragment using T4 ligase at 16°C overnight.

[0185] 2. Electrotransformation of phage ligation products into E. coli

[0186] Prepare the electroporation cuvette, ligation product, and competent medium and pre-chill them on ice. Add the pre-chilled library construction ligation product to the competent medium and place on ice for 1 minute. Add 300 μL of the DNA / competent medium mixture to each electroporation cuvette and place the cuvette on ice. Perform electroporation at 2500V for 5 ms. Immediately resuspend the cells in SOC medium equilibrated to room temperature and incubate at 37°C on a shaker for 1 hour. Use 15 mL of the bacterial solution directly for phage rescue. Add an equal volume of 50% glycerol to the remaining 5 mL of electroporation product, mix thoroughly, and store at -80°C. Separately, dilute 20 μL of the bacterial solution with 980 μL of 2YT medium. Take 100 μL of this dilution and dilute it again with 900 μL of 2YT medium. Spread 50 μL evenly on an LB plate containing ampicillin and incubate overnight at 37°C. The next day, remove the plate and count the number of colonies generated from each ligation to calculate the library capacity. At the same time, pick 20 single clones from the plate and transfer them to 2YT medium containing ampicillin. Incubate at 37°C with shaking for approximately 6-8 hours. Then send the culture solution for sequencing (sequencing universal primer M13R). Randomly select 20 single clones for sequencing to analyze the diversity of the constructed phage display library.

[0187] The results are shown in Figure 3. Sequencing results showed that the empty rate and antibody duplication rate of the phage display library were no more than 5%. The library capacity of E. coli was 5.36×10 8 .

[0188] 1.6 Phage library preparation and phage precipitation

[0189] The electroporated product was diluted with 2YT to adjust the OD 600 The final concentration of ampicillin was 100 μg / mL, and the cells were cultured in a constant temperature shaker at 37°C and 225 rpm until the OD 600 Stop when the OD value is 0.5; add M13KO7, shake well, let it stand at 37℃ for 30 minutes, and then incubate at 37℃, 225rpm for 1 hour. M13KO7 volume = 10 × volume × OD 600 ×5×10 8 / M13KO7 titer. Centrifuge the bacterial solution at 6000 rpm for 10 minutes, resuspend in 2YT-AK medium, and culture overnight at 25°C and 200 rpm. Centrifuge the bacterial solution at 10000 rpm for 15 minutes. Discard the precipitate and transfer the supernatant to a new centrifuge tube. Add PEG / NaCl (1 / 5 the volume of the bacterial solution) to the tube, mix thoroughly, and incubate at 4°C for 2 hours. The supernatant of the precipitated phage was centrifuged at 10,000 rpm and 4°C for 30 min; the supernatant was discarded and the precipitate (phage) in each 50 mL centrifuge tube was resuspended with 1 mL of sterile PBS; the resuspended phage was transferred to a 1.5 mL EP tube and placed in a centrifuge at 12,000 g and 4°C for 5 min; the supernatant was transferred to a new 1.5 mL EP tube, 250 μL of PEG / NaCl was added to each tube, mixed and allowed to stand at 4°C for 10 min, centrifuged at 12,000 g for 10 min, the supernatant was discarded, 1 mL of PBS was added to resuspend, centrifuged at 12,000 g for 5 min, the precipitate was discarded, the supernatant was transferred to a new 1.5 mL EP tube, centrifuged at 12,000 g for 5 min, and the supernatant was transferred to a new 1.5 mL EP tube to obtain the original phage library. Take 10 μL of precipitate and add 90 μL of 2YT culture medium, recorded as 10 -1 , then dilute 10 times to 10 -9 , take 10 -7 , 10 -8 , 10 -9 Three 20μL dilutions of the sample were added to 200μL of pre-prepared ER2738 at an OD600 of 0.5. Mix thoroughly, place in a 37°C water bath, and let stand for 10 minutes. Each 100μL sample was then plated onto an LB-AMP plate and incubated at 37°C overnight. The next day, the number of spots was counted to determine the titer. Titration calculation: Select plates with 30-300 spots, average the two plates, and multiply the number of spots by the dilution factor, then multiply by 100 to obtain the titer.

[0190] 1.7 Panning of phage display libraries

[0191] Recombinant protein is used for panning, and then the phage display library is incubated with the recombinant protein. The recombinant phage bound to the target antigen is eluted using TEA and amplified. After 3-4 rounds of panning, single clones are selected for sequencing.

[0192] Take about 6x10 11150 μL of phage library was diluted to about 1 mL with 1% PBSA and added to the ELISA plate coated with the target protein. The plate was incubated at 4°C for 1 hour. The bound phage was eluted with 600 μL 1xTEA for 10 minutes. The eluted product was transferred to a pre-sealed EP tube and neutralized with 300 μL Tris-HCl. 10 μL of the output product was added to 90 μL 2YT medium, recorded as 100, and then diluted 10-fold to 10 -2 , take 101, 100, 10 -1 , 10 -2 Four gradient 20 μL diluted samples were added to 200 μL of pre-prepared OD 600 Add 0.5% ER2738 (101 is to directly add 20 μL of undiluted product to ER2738), mix thoroughly, place in a 37°C water bath, and let it stand for 10 minutes. Apply 100 μL of solution to one LB-AMP solid plate and incubate at 37°C overnight. The next day, count the spots to determine the titration. Titration calculation: Select plates with 30-300 spots, average the two plates, and multiply the number of spots by the dilution factor, then by the elution volume.

[0193] The target antigen recombinant protein was co-incubated with the single-domain antibody phage display library to enrich phage that specifically bind to the target antigen. Four rounds of enrichment were performed. The input and output data for each round were counted, and the enrichment factor for each round was calculated. The results are shown in Table 11.

[0194] Table 11 Enrichment results of each round

[0195] 1.8 Phage ELISA

[0196] Aliquot 2YT-Amp medium into 96-well deep-well plates, 500 μL per well, pick single clones on the output plate, and culture at 37°C, 225 rpm until OD 600 Until 0.5; among them, E12 and F12 wells were not selected for clones, and only culture medium was placed as blank control; G12 and H12 wells were not selected for clones, and the binding of antigen and positive antibody was detected as positive control; at the same time, antigen was coated on ELISA plate with CBS, concentration of 1μg / mL, 100μL / well, 37℃, and coated for 2h; another 96-well deep-well plate was taken, 2YT-A culture medium was dispensed, 500μL per well, and OD was aspirated in sequence with a dispenser. 600 10 μL of the bacterial solution with an OD of 0.5 was added to a newly aliquoted 96-well plate and cultured overnight at 37°C and 225 rpm. This was the bacterial solution for sequencing. 600 Add M13KO7 to the bacterial solution with a concentration of 0.5, mix well, and place at 37°C for 15 minutes; the volume of M13KO7 = 10 × volume × OD600 ×5×10 8 / M13KO7 titer: Place the infected bacterial solution on a shaker and culture at 37°C, 225 rpm for 45 min; place the bacterial solution in a centrifuge and centrifuge at 4000 rpm for 10 min, discard the supernatant, resuspend in 2YT-AK medium, 800 μL per well, return to the shaker, and culture overnight at 30°C, 210 rpm. At the same time, the ELISA plate was shaken off the antigen, washed three times with PBST solution, and then blocked with 3% MPBS at 250μL / well and incubated at 4°C overnight; and an additional blank plate was blocked as BLANK; the next day, the 96-well deep-well plate was placed in a centrifuge and centrifuged at 4000rpm for 10 minutes; the milk in the ELISA plate was discarded and washed 4 times with 200μL PBST; 50μL PBST was added to each well first, and then 50μL of the phage supernatant after centrifugation was added one by one, and incubated at 4°C for 1h; the supernatant was discarded and washed 5 times with PBST; HRP-Anti M13 secondary antibody was diluted with PBST, 100μL per well, incubated at 4°C for 45min, the secondary antibody was washed away, washed 5 times with PBST, TMB was used for color development at room temperature for 10min, hydrochloric acid was used for termination, and the number of reads was read. Clones with an S / N ratio greater than 6 were selected and sent for testing with the preserved bacterial solution for antibody sequence analysis. Phage display library was screened to obtain different antibody sequences, and eukaryotic expression vectors were constructed for verification.

[0197] The output product phage ELISA test results for the fourth round are shown in Figure 4. Plate 1 shows the OD450 value of the target antigen protein-coated monoclonal detection, and Plate 2 shows the OD450 value of the BSA control antigen protein-coated monoclonal detection. The S / N value in Plate 3 is the value in Plate 1 divided by the value at the same position in Plate 2. Clones with an S / N value > 4 were selected for testing.

[0198] The candidate antibody sequences obtained by screening are as follows:

[0199] The CDR region sequences of the candidate antibodies are shown in Table 12.

[0200] Table 12

[0201] 1.9 Amplification, transient transfection, and detection of overlap PCR products

[0202] (1) First round of PCR: amplification of CMV, VHH and FC

[0203] Prepare 50 μL of PCR reaction system as shown in Table 13: CMV fragment amplification primers: CMV-F and PCom-R1; VHH fragment amplification primers: PCom-F1 and PCom-R2; FC fragment amplification primers: PCom-F2 and PGK-R.

[0204] Table 13 PCR reaction system

[0205] The PCR reaction program is shown in Table 14:

[0206] Table 14 PCR reaction program settings

[0207] Take 50 μL of PCR product, add 1 / 10 volume of 10× loading buffer, and perform electrophoresis analysis using 1% agarose. The band size of CMV and FC is about 750 bp, and the band size of VHH is about 560 bp.

[0208] The target band was removed from the gel, and the PCR product was purified and the concentration was determined using NanoDrop (if the concentration was too high, it could be diluted for subsequent reactions).

[0209] (2) Second round of PCR: Overlap Extension PCR was used to connect CMV, VHH, and FC. The PCR reaction system is shown in Table 15.

[0210] Table 15 Second round PCR system

[0211] The PCR reaction program is shown in Table 16:

[0212] Table 16 PCR reaction program

[0213] Add 2 μl each of primers CMV-F and PGK-R. The PCR reaction procedure is shown in Table 17:

[0214] Table 17 PCR reaction program

[0215] Overlap PCR products were purified using TakaRa's DNA Fragment Recovery Kit and the concentration was determined using NanoDrop. A minimum of 10 μg of PCR product was required for subsequent cell transfection verification. The PCR products were transiently transfected into 293F cells, and the supernatant was collected for FACS analysis.

[0216] The results of FACS detection are shown in Figure 5. Clones 7-F11, 9-F05, 9-G05, 9-H03, etc. can bind to Jurkat cells expressing CD16A, but not to Jurkat cells that do not express CD16A.

[0217] 1.10 FACS detection of specific binding of recombinant antibodies to target proteins

[0218] Based on FACS detection, positive clones were selected for antibody preparation, and the prepared antibodies were verified by FACS binding. The experimental method is as follows:

[0219] CHO, CHO-CD16A, and CHO-CD16B (NA1 and NA2 subtypes) cell lines were revived from liquid nitrogen and adjusted to the logarithmic growth phase. Each cell type was divided into several aliquots, with the number of cells in each aliquot being 3×10 5 cells. Incubate the target cells with the expressed antibodies separately, mix thoroughly, and incubate at room temperature for 1 hour. Centrifuge at 800xg for 5 minutes at room temperature, remove the supernatant containing the antibodies, and wash the cells three times with PBS. Add 100uL of PE-labeled anti-human IgG (1:500 dilution), mix thoroughly, and incubate at room temperature in the dark for 30 minutes; centrifuge at 800xg for 5 minutes at room temperature, remove the supernatant containing the secondary antibody, and wash the cells three times with PBS; resuspend the cells in 500uL of PBS for flow cytometry analysis.

[0220] The results are shown in Figure 6. Antibodies such as 9-C05, 9-G05, 9-F05, 7-F11, and 9-H03 can bind to CHO cells expressing CD16A, but not to CHO cells expressing CD16B NA1 or CD16B NA2, reflecting the specific binding of the antibodies of the present invention to the target protein.

[0221] 1.11 Affinity testing

[0222] The affinity of antibodies 9-F05, 9-C05, and 9-G05 was tested using the following assay: Biotin-CD16A-176v-His was immobilized on a SA sensor at a concentration of 5 μg / ml for 60 seconds. The buffer was PBST (PBS + 0.02% tween 20). PCDNA3.4-SDAB210108-9F05-His, PCDNA3.4-SDAB210108-9C05-His, and PCDNA3.4-SDAB210108-9G05-His were diluted to 50, 25, 12.5, 6.25, 3.13, and 0 nM, respectively. Affinity testing was performed with a 60-second equilibrium, 180-second association, and 180-second dissociation cycle. The assay temperature was 25°C.

[0223] The results are shown in FIG7 and Table 18. 9-F05, 9-C05 and 9-G05 all have very high affinities.

[0224] Table 18 Antibody affinity test results

[0225] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An anti-CD16A nanobody, characterized in that, The anti-CD16A nanobody has one or more complementarity-determining regions CDRs selected from the group consisting of: (1) CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8, and CDR3 shown in SEQ ID NO:9; (2) CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11, and CDR3 shown in SEQ ID NO:12; (3) CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:13, and CDR3 shown in SEQ ID NO:14; (4) CDR1 shown in SEQ ID NO:15, CDR2 shown in SEQ ID NO:16, and CDR3 shown in SEQ ID NO:17; (5) CDR1 shown in SEQ ID NO:18, CDR2 shown in SEQ ID NO:19, and CDR3 shown in SEQ ID NO:20; (6) CDR1 shown in SEQ ID NO:21, CDR2 shown in SEQ ID NO:22, and CDR3 shown in SEQ ID NO:

23.

2. The anti-CD16A nanobody according to claim 1, wherein, The CDR1, CDR2, and CDR3 are separated by framework regions FR1, FR2, FR3, and FR4 of the VHH chain.

3. The anti-CD16A nanobody according to claim 1, wherein The amino acid sequence of the VHH chain of the anti-CD16A nanobody is as shown in any of SEQ ID NOs: 1-6.

4. The anti-CD16A nanobody according to claim 1, characterized in that, The anti-CD16A nanobody described above includes humanized antibodies, animal-derived antibodies, and chimeric antibodies.

5. A multivalent anti-CD16A antibody or an antibody directed against multiple epitopes, characterized in that, The multivalent antibody or antibody against multiple epitopes includes at least one antibody element against the CD16A epitope, and the antibody element is the anti-CD16A nanobody described in claim 1.

6. A recombinant protein, characterized in that, The recombinant protein has: (i) The anti-CD16A nanobody described in claim 1, or the multivalent antibody against CD16A or antibody against multiple epitopes described in claim 5; and (ii) Optionally, a tag sequence for assisting expression and / or purification.

7. An antibody-drug conjugate, characterized in that, The antibody-drug conjugate contains: (a) The nanobody described in claim 1, the multivalent antibody described in claim 5, or the antibody against multiple epitopes, the recombinant protein described in claim 6; and (b) A conjugate part conjugated to the antibody part, and the conjugate part is selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

8. A chimeric antigen receptor (CAR), characterized in that, The CAR contains an extracellular domain, and the extracellular domain contains the anti-CD16A nanobody described in claim 1, or the anti-CD16A multivalent antibody described in claim 5.

9. A polynucleotide, characterized in that, The polynucleotide encodes a protein selected from the group consisting of: the nanobody described in claim 1, or the multivalent antibody or antibody against multiple epitopes described in claim 5, the recombinant protein described in claim 6, or the CAR described in claim 8.

10. An expression vector, characterized in that, The expression vector contains the polynucleotide described in claim 9.

11. A host cell, characterized in that, The host cell contains the expression vector recited in claim 10, or the polynucleotide recited in claim 9 is integrated into its genome, or expresses the nanobody recited in claim 1, the multivalent antibody recited in claim 5, or the antibody against multiple epitopes, the recombinant protein recited in claim 6, or the CAR recited in claim 8.

12. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: (i) the nanobody recited in claim 1, the multivalent antibody recited in claim 5, or the antibody against multiple epitopes, the recombinant protein recited in claim 6, the antibody-drug conjugate recited in claim 7, or the host cell recited in claim 11; and (ii) a pharmaceutically acceptable carrier.

13. Use of the nanobody recited in claim 1, the multivalent antibody recited in claim 5, or the antibody against multiple epitopes, the recombinant protein recited in claim 6, the antibody-drug conjugate recited in claim 7, or an immune cell expressing the CAR recited in claim 8 for the preparation of a medicament for preventing and / or treating a CD16A-related cancer or disease.

14. The use according to claim 13, characterized in that, The CD16A-related disease or disorder is selected from the group consisting of: hematological malignancies, solid tumors, or a combination thereof.

15. A method for treating a disease, characterized in that, The method comprises administering to a subject in need of treatment: the nanobody recited in claim 1, the multivalent antibody recited in claim 5, or the antibody against multiple epitopes, the recombinant protein recited in claim 6, the antibody-drug conjugate recited in claim 7, or an immune cell expressing the CAR recited in claim 8.

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