CN112679609A - anti-CD 16a antigen VHH and related dual-characteristic nanobody - Google Patents

Abstract

The invention relates to an anti-human CD16a VHH and bispecific nanobody, which comprises: (a) a first binding domain that specifically binds to a tumor antigen, said first binding domain comprising a VHH that is anti-tumor antigen; (b) a second binding domain that specifically binds to human CD16a, said second binding domain comprising an anti-human CD16a VHH as described herein. The antibody molecule can be expressed in a prokaryotic expression system in a soluble way, is beneficial to subsequent separation and purification, has better thermal stability and shows superior bonding strength with target cells.

Description

anti-CD 16a antigen VHH and related dual-characteristic nanobody

Technical Field

The invention relates to a single domain antibody VHH of anti-CD 16a antigen and a bispecific antibody drug, in particular to a bispecific antibody drug of anti-human tumor antigen CEA and anti-human natural killer cell specific antigen CD 16. The invention also relates to methods and pharmaceutical compositions for treating cancer.

Background

CD16 exists in two subtypes, CD16a and CD16b, which share 96% sequence identity in their immunoglobulin binding regions. CD16a is expressed on NK cells, macrophages and mast cells and is an activating receptor. CD16b is expressed on granulocytes as a GPI-anchored receptor and does not trigger killing of tumor cells. CD16a (Fc γ RIIIa) is a low affinity receptor for the IgG Fc domain, which is involved in antibody-dependent cellular cytotoxicity (ADCC) and is responsible for triggering cytolysis of target cells by Natural Killing (NK). ADCC is one of the major cytotoxic mechanisms employed by Fc γ R-expressing effector cells to eliminate tumor cells. Several tumor antigen-specific antibodies (e.g., herceptin targeting Her2, rituximab targeting CD 20) have been shown to kill tumor cells depending on ADCC. However, due to the lower binding affinity of IgG1 Fc to CD16a on NK cells, methods have been investigated to improve the binding of tumor antigen-specific antibodies to NK cells, including IgG1 Fc mutations that enhance the affinity of IgG1 Fc to CD16a (fcyriiia). Another approach is to use bispecific antibodies targeting tumor cells and immune cells to improve the recruitment of effector cells. Immune effector cells (e.g., T cells, NK cells, macrophages or monocytes) can be recruited to kill tumor cells in a non-MHC-restricted manner by redirecting the effector cells to the tumor cells using different targeting molecules. Bispecific antibodies that bind to NK cells are attractive candidates for cancer immunotherapy because NK cells are effective in killing tumor cells. For NK cell active binding, anti-CD 16a antibodies have been studied and used to construct bispecific antibodies.

In order to recognize cancer cells in a bispecific format, many different tumor antigens have been studied, including CD19, epithelial cell adhesion molecule (Epcam), and many others. One of the well studied tumor antigens is carcinoembryonic antigen (CEA, CEACAM5, CD66 e), a glycosylated human embryonic antigen belonging to the CEA-associated cell adhesion molecule (CEACAM) superfamily. In normal tissues, CEA is expressed at low levels in epithelial cells in a polarized manner, whereas in many malignant cancers it is overexpressed and not polarized. Therefore, CEA has been frequently targeted for cancer therapy. Clinical studies have shown that radiolabeled anti-CEA antibodies or antibody fragments can be used as imaging agents to localize CEA-expressing solid cancers, such as anti-CEA acipimox (CEA-Scan). MEDI-565 is a T cell-directed cytotoxic BiTE antibody against CEA positive tumor cells and is currently in clinical development. Bispecific antibodies using anti-CEA single domain antibodies also showed potent anti-tumor activity in preclinical studies.

Single domain antibodies, also known as nanobodies or VHHs, are derived from the variable domains of heavy chain-only antibodies found in camelidae. The single domain antibody recognizes antigens with a high specificity and affinity similar to IgG antibodies, but may better penetrate tumour tissue due to the smaller size (-15 kD). Furthermore, single domain antibodies are resistant to extremes of pH, heat denaturation, proteolysis, solvents and detergents. They can be expressed and produced in high yield and high solubility.

Previous reports have shown that anti-CD 16a-VHH can be used in a bispecific format to bind to NK cells and kill tumor cells (see Chinese patent publication No. CN106432502A and Dong, Bin, et al, "A novel bispecific antibody, BiSS, with content anti-Cancer activities," Cancer Biology & Therapy (2016):00-00., referred to herein as bis antibody). However, there is still a need to provide more anti-CD 16a VHH for bispecific antibody studies and more bispecific antibodies with stronger affinity for the target antigen.

Disclosure of Invention

The invention provides an anti-human CD16a VHH, wherein a complementarity determining region CDR1 is selected from amino acid sequences shown in ASGDTTSEYWGA (SEQ ID No: 13), VSGYSYSSYCLA (SEQ ID No: 14), VSGYFGRRYCMA (SEQ ID No: 15), ASGNTYRSYSMA (SEQ ID No: 16) and ASGAIVSRSCMG (SEQ ID No: 17); the complementarity determining region CDR2 is selected from the amino acid sequences shown in AILPLSTTPVYAGS (SEQ ID No: 18), AMSVGGGSTYYADS (SEQ ID No: 19), SIDRNGNIDYTES (SEQ ID No: 20), AIASDGSKAYADS (SEQ ID No: 21), and GIFLSDGRTTYADS (SEQ ID No: 22); and the complementarity determining region CDR3 is selected from the amino acid sequences shown in AAARRGTNAFLTHDKYGY (SEQ ID No: 23), ASRRGGGYCYPAYRLDFYH (SEQ ID No: 24), AAPWAGVYCAASLRESDYKF (SEQ ID No: 25), AAKRGRWFLVQSDRVD (SEQ ID No: 26) and AANWRRWLYGATCEATNDYN (SEQ ID No: 27).

Another aspect of the present invention provides a bispecific nanobody comprising: (a) a first binding domain that specifically binds to a tumor antigen, said first binding domain comprising a VHH that is anti-tumor antigen; (b) a second binding domain that specifically binds to human CD16a, said second binding domain comprising any one of the anti-human CD16a VHHs described above.

Another aspect of the present invention also provides a bispecific nanobody comprising: (a) a first binding domain that specifically binds to human CEA, said first binding domain comprising an anti-human CEA VHH; (b) a second binding domain that specifically binds to human CD16a, said second binding domain comprising any one of the anti-human CD16a VHHs described above.

In another aspect of the present invention, a pharmaceutical composition for treating tumor is provided, which comprises the bispecific nanobody and a pharmaceutically acceptable carrier.

In another aspect of the present invention, a pharmaceutical composition for treating tumor is provided, which comprises the bispecific nanobody and the second anticancer agent.

In another aspect of the present invention, there is also provided a nucleotide sequence encoding the bispecific nanobody as described above.

In another aspect of the invention there is also provided a vector comprising the nucleotide sequence as hereinbefore described.

In a further aspect of the invention there is provided a non-human host cell for a vector as hereinbefore described.

Yet another aspect of the present invention provides a method of treating a tumor comprising contacting any one of the bispecific nanobodies of the present invention with a tumor cell. Accordingly, another aspect of the present invention provides a method of treating a tumor in a subject, comprising administering to the subject a therapeutically effective amount of any one of the bispecific nanobodies of the present invention or any one of the pharmaceutical compositions of the present invention.

After immunizing camels with human CD16a protein, phage display screening (phase display screening) was performed and the inventors obtained a variety of anti-CD 16a VHHs that were more potent than the prior art. Further, different bispecific antibodies were constructed by linking the anti-CEA single domain antibody to selected anti-CD 16a VHH. Bispecific antibodies in E.coli: (E. coli) Expressed and produced in high yield and showed high affinity for both CEA and CD16a, both stronger than known BISS antibodies. The bispecific antibody can recruit NK cells to kill CEA positive tumor cells in vitro with high efficiency. In vivo studies have also demonstrated potent antitumor activity. These studies suggest that these anti-CD 16a VHHs are powerful tools for enabling NK cells to participate in cancer therapy.

Drawings

FIG. 1 shows the screening of anti-human CD16a single domain antibodies. ELISA (enzyme-linked immunosorbent assay) analysis was performed using 0.5 μ g of purified and quantified phage. OD₄₅₀As a cut-off value, 1.25 selected 6 clones (black bars).

FIG. 2: biochemical properties of the anti-CEA-CD 16a VHH. a) anti-CEA-CD 16a VHH SBC74-79 was constructed by fusing anti-CEA and anti-CD 16a single domain antibodies to a (GGGGS) 3 linker. A His tag was added to the C-terminus for protein detection and purification. b) Purified proteins (SBC 74 (SEQ ID No: 9), SBC75 (SEQ ID No: 10), SBC76 (SEQ ID No: 11), SBC77 (SEQ ID No: 8), SBC78, SBC79 (SEQ ID No: 12)) were Coomassie blue stained after Ni-NTA affinity chromatography. c) Gel filtration of protein tags (upper panel) and SBC77 (lower panel). d) As described in the specific embodiment, DLS experiments were performed at 25 deg.C-75 deg.C using 0.5 mg/ml of purified protein.

FIG. 3: the anti-CEA-CD 16a bispecific antibody can bind CEA and CD16a antigen. a) Flow cytometry analysis of anti-CEA-CD 16a bispecific antibodies was performed using LS174T and SKOV3 cells. The light grey area (blank) indicated that LS174T cells were not stained; dark grey areas (blank) indicate no staining of SKOV3 cells; the dotted line is cells with anti-His-PE staining; the solid line shows anti-CEA-CD 16a VHH, followed by anti-His-PE staining. b) ELISA analysis of different bispecific antibodies, BiSS (grey dashed line) binding to CD16a antigen; anti-CEA-CD 16a bispecific antibody SBC75-79 (solid black line); there is no CD16a (solid grey line). Data are the average of three replicates and error bars represent standard deviations.

FIG. 4: the anti-CEA-CD 16a bispecific antibody induces NK cell mediated cytotoxicity. Different cell lines were treated with anti-CEA-CD 16a bispecific antibody or anti-CD 16a VHH containing freshly isolated NK cells. The ratio of effector cells (NK cells) (25000 cells/well) to target cells LS174T and SKOV3(2500 cells/well) was 10: 1. All data are the mean of triplicate experiments and error bars represent standard deviation (. times.p < 0.001, compared to anti-CD 16a VHH, t-test).

FIG. 5: the anti-CEA-CD 16a bispecific antibody mediates NK cell dependent tumor cell killing. a) Different cell lines were treated with an anti-CEA-CD 16a bispecific antibody with or without freshly isolated NK cells. The ratio of effector cells (NK cells) (25000 cells/well) and target cells LS174T, HT29 and SKOV3(2500 cells/well) was 10: 1. all data are the mean of triplicate experiments and error bars represent standard deviation (. prime.) P < 0.001, t-test compared to tumor + NK). b) SBC77 induced NK cell-mediated cytotoxicity in a dose-dependent manner. The concentration of BiSS, SBC77 and anti-CD 16a VHH (D5) is 0.001 ng/mL to 10 μ g/mL. All data are the mean of triplicate experiments and error bars represent standard deviations.

FIG. 6: SBC77 inhibited tumor growth in vivo. a) Will be provided withNOD/SCIDMice (n = 5/group) were implanted subcutaneously with freshly isolated human PBMCs (5 × 10 per animal)⁶LS174T cells (1X 10 per animal)⁶One). Mice were then dosed intraperitoneally every two days with vehicle PBS (grey line), SBC77 (20 μ g/mouse) (solid triangular line) or SBC77 (5 μ g/mouse) (dashed triangular line). Tumor volumes were then measured. Data represent mean edema in 5 miceTumor volume. Error bars represent standard deviation (. about.. about P <0.001, t-test, vehicle and SBC77 (20 μ g, 5 μ g)). b) Will be provided withNOS/SCIDMice (n = 5/group) were subcutaneously implanted with freshly isolated human NK negative PBMC cells (5 × 10 per animal)⁶) LS174T cells (1X 10 per animal)⁶). The mice were then treated with SBC77 (20 μ g/mouse) or PBS as described in materials and methods.

Detailed Description

Tumor antigens

As used herein, the term "Tumor Antigen (TA)" refers to Tumor-associated antigens as well as Tumor-specific antigens, i.e., any immunogenic epitope (e.g., protein) expressed by Tumor cells. The protein may be expressed by non-tumor cells, but is immunogenic only when expressed by tumor cells. Alternatively, the protein may be expressed by tumor cells, but not normal cells. Preferably, the anti-TA antibodies of the invention bind to the extracellular domain of TA. In a preferred embodiment, the tumor antigen is a human tumor antigen. Exemplary tumor antigens include, but are not limited to, melanoma-associated chondroitin sulfate proteoglycan (MCSP, UniProt Q6UVK1, NCBI accession No. NP _001888), fibroblast activation protein (FAP, UniProt Q12884, Q86Z29, Q99998; NCBI accession No. NP _004451), epidermal growth factor receptor (EGFR, also known as ErbB1 and Her1, UniProt P00533; NCBI accession No. NP _958439, NP _958440), carcinoembryonic antigen (CEA, also known as carcinoembryonic antigen-associated cell adhesion molecule 5 or CD66e; UniProt P06731, NCBI accession No. NP _004354), Her2 (UniProt P04626), and CD33 (also known as gp76 or sialic acid-binding Ig-like lectin 3(Siglec-3, UniProt P20138, NCBI accession No. NP _001076087, NP _ 001171079).

Nanobodies

Hamers et al, 1993, have discovered by chance that camelids have heavy chain antibodies (hcAb) that naturally lack the entire light and heavy chain constant region CH 1. The variable region of heavy chain antibodies (VHH) is the smallest molecular weight antibody fragment found to date with antigen binding function, with a molecular weight of 15kD, which is only 1/10 for conventional antibodies, with a molecular height of about 4.8 nm and a diameter of about 2.2 nm, and is therefore also referred to as nanobody or single domain antibody (sdAb). The antigen binding region of nanobody consists of only 3 hypervariable regions of VHH (H1-H3), forming in space an antigen binding domain that is different from the typical structure of conventional antibodies. Wherein the average length of H3 is longer than that of conventional antibodies, and the H3 can be in a protruding finger structure in space, thereby being capable of binding an epitope which can not be accessed by some conventional antibodies.

The nano-antibody anti-human CD16a VHH of the invention, wherein the complementarity determining region CDR1 is selected from the amino acid sequences shown in ASGDTTSEYWGA (SEQ ID No: 13), VSGYSYSSYCLA (SEQ ID No: 14), VSGYFGRRYCMA (SEQ ID No: 15), ASGNTYRSYSMA (SEQ ID No: 16) and ASGAIVSRSCMG (SEQ ID No: 17); the complementarity determining region CDR2 is selected from the amino acid sequences shown in AILPLSTTPVYAGS (SEQ ID No: 18), AMSVGGGSTYYADS (SEQ ID No: 19), SIDRNGNIDYTES (SEQ ID No: 20), AIASDGSKAYADS (SEQ ID No: 21), and GIFLSDGRTTYADS (SEQ ID No: 22); and the complementarity determining region CDR3 is selected from the amino acid sequences shown in AAARRGTNAFLTHDKYGY (SEQ ID No: 23), ASRRGGGYCYPAYRLDFYH (SEQ ID No: 24), AAPWAGVYCAASLRESDYKF (SEQ ID No: 25), AAKRGRWFLVQSDRVD (SEQ ID No: 26) and AANWRRWLYGATCEATNDYN (SEQ ID No: 27).

In some embodiments, the anti-human CD16a VHH is selected from the following combinations: (a) the amino acid sequence of CDR1 is ASGDTTSEYWGA (SEQ ID No: 13), the amino acid sequence of CDR2 is AILPLSTTPVYAGS (SEQ ID No: 18) and the amino acid sequence of CDR3 is AAARRGTNAFLTHDKYGY (SEQ ID No: 23); (b) the amino acid sequence of CDR1 is VSGYSYSSYCLA ((SEQ ID No: 14)), the amino acid sequence of CDR2 is AMSVGGGSTYYADS (SEQ ID No: 19) and the amino acid sequence of CDR3 is ASRRGGGYCYPAYRLDFYH (SEQ ID No: 24); (c) the amino acid sequence of CDR1 is: VSGYFGRRYCMA (SEQ ID No: 15), the amino acid sequence of CDR2 is: SIDRNGNIDYTES (SEQ ID No: 20) and CDR3 has the amino acid sequence AAPWAGVYCAASLRESDYKF (SEQ ID No: 25); (d) the amino acid sequence of CDR1 is ASGNTYRSYSMA (SEQ ID No: 16), the amino acid sequence of CDR2 is AIASDGSKAYADS (SEQ ID No: 21) and the amino acid sequence of CDR3 is AAKRGRWFLVQSDRVD (SEQ ID No: 26); or (e) the amino acid sequence of CDR1 is: ASGAIVSRSCMG (SEQ ID No: 17), the amino acid sequence of CDR2 is GIFLSDGRTTYADS (SEQ ID No: 22) and the amino acid sequence of CDR3 is AANWRRWLYGATCEATNDYN (SEQ ID No: 27).

In some embodiments, the amino acid sequence of said anti-human CD16a VHH is selected from SEQ ID nos: 1. SEQ ID No: 2. SEQ ID No: 3. SEQ ID No: 4. SEQ ID No: 5.

Bispecific nanobodies

In the present invention, a "bispecific nanobody" or "bispecific antibody" refers to a single polypeptide chain comprising two binding domains, wherein each "binding domain" comprises one nanobody VHH, wherein the VHH of the first binding domain specifically binds to a first molecule of a tumor antigen and the VHH of the second binding domain specifically binds to a second molecule of CD16 a. The two binding domains are optionally linked to each other by a short spacer polypeptide (linker peptide). In some embodiments, the tumor antigen is CEA, Her2, CD19, CD20, epithelial cell adhesion molecule, CEACAM5, or CD66 e. In one embodiment, the anti-tumor antibody is in situ N-terminal to the anti-human CD16a VHH. In another embodiment, said anti-human tumor antigen is located C-terminal to said anti-human CD16a VHH. In the present invention, "at the N-terminus" or "at the C-terminus" is relative and not the absolute N-terminus or C-terminus of a bispecific antibody. As a non-limiting example, a first binding domain "N-terminal to a second binding domain" merely means that the first binding domain is amino-terminal to the second binding domain in a bispecific antibody and does not exclude the possibility that additional sequences (e.g., a tag as described above, or another proteinaceous or non-proteinaceous compound, such as a radioisotope) are located at the final N-terminus of the bispecific antibody.

In one aspect of the invention, a bispecific antibody of the invention comprises: (a) a first binding domain that specifically binds to human CEA, said first binding domain comprising an anti-human CEA VHH; (b) a second binding domain that specifically binds to human CD16a, said second binding domain comprising an anti-human CD16a VHH. The two binding domains are optionally linked to each other by a short spacer polypeptide (linker peptide). In one embodiment, the amino acid sequence of said anti-human CEA VHH is as set forth in SEQ ID No: and 6. In one embodiment, the anti-human CEA VHH is N-terminal to the anti-human CD16a VHH. In another embodiment, said anti-human CEA VHH is C-terminal to said anti-human CD16a VHH. In the present invention, "at the N-terminus" or "at the C-terminus" is relative and not the absolute N-terminus or C-terminus of a bispecific antibody. As a non-limiting example, a first binding domain "N-terminal to a second binding domain" merely means that the first binding domain is amino-terminal to the second binding domain in a bispecific antibody and does not exclude the possibility that additional sequences (e.g., a tag as described above, or another proteinaceous or non-proteinaceous compound, such as a radioisotope) are located at the final N-terminus of the bispecific antibody.

The first and/or second binding domain of the bispecific antibody may be of non-human origin, e.g. may be derived from a murine monoclonal antibody. However, when it is administered to a human patient, the bispecific single chain antibody derived from a murine antibody may be recognized as a foreign substance by the human body. Thus, preferably, the first and/or second binding domain of the bispecific single chain antibody is of human origin, i.e. derived from a human sequence. For example, binding domains that specifically bind to human CEA or human CD16a can be identified by phage display-based techniques. Alternatively, one of the binding domains is of human origin and the other is of non-human origin, thereby producing a chimeric bispecific nanobody.

In one embodiment of the invention, the bispecific antibody is prepared by artificially synthesizing the nucleic acid sequence, expressing the nucleic acid sequence in prokaryotic cells, and purifying the nucleic acid sequence.

It is contemplated that the binding domain of the bispecific antibody of the present invention may carry a "tag" such as a Flag-tag, c-myc-tag, GST-tag or His-tag for e.g. protein expression, purification, detection or enrichment. For example, for Flag-tags, the most widely used today is the hydrophilic octapeptide DYKDDDDK. These tags may be located at the N-terminus or C-terminus of the bispecific antibody.

It is also contemplated that the binding domain of the bispecific antibodies of the invention may carry a signal peptide, which is typically located at the N-terminus of the secreted protein, typically consisting of 15-30 amino acids. When the signal peptide sequence is synthesized and recognized by a Signal Recognition Particle (SRP), protein synthesis is suspended or slowed, the signal recognition particle carries the ribosome to the endoplasmic reticulum, and protein synthesis is restarted. Under the guidance of the signal peptide, the newly synthesized protein enters the lumen of the endoplasmic reticulum, and the signal peptide sequence is cleaved by the action of a signal peptidase. If the termination transport sequence is present at the C-terminus of the nascent peptide chain, it may also be unresectable by a signal peptidase, e.g., the ovalbumin contains an internal signal peptide. Neither its precursor nor mature form is cleaved by signal peptidases. An exemplary signal peptide sequence is MGKKIWLALAGLVLAFSASA.

The term "specifically binds" or "specifically binds to …" refers to the ability of the first and/or second binding domain of the bispecific antibody to resolve the respective first and/or second molecule to such an extent that only the respective first and/or second molecule is capable of being bound or significantly bound in a pool from a variety of different molecules that may be binding ligands. This binding can be determined by conventional methods such as ELISA, FACS analysis, etc. on a Biacore instrument, for example.

Specifically, the first binding domain of the bispecific antibody of the present invention binds to human CEA (carcinoembryonic antigen, carcinoembryonic antigen-associated cell adhesion molecule 5, CEACAM5, CD66 e) and the second binding domain binds to human CD16 a.

By "specifically binds" is meant that the bispecific antibodies of the invention are capable of specifically interacting with at least two, three, four, five, six, seven, eight or more amino acids of each human target molecule. The "specific binding" of an antibody is characterized primarily by two parameters: qualitative (binding epitope or antibody binding site) and quantitative (binding affinity or binding strength). Antibody binding epitopes can be determined by FACS, peptide dot epitope mapping, mass spectrometry, or peptide ELISA. The Biacore method and/or ELISA method can determine the binding strength of an antibody to a particular epitope. Signal to noise ratios are often used as a representative assay calculation of binding specificity. In such a signal-to-noise ratio, the signal represents the strength of binding of the antibody to the target epitope, while the noise represents the strength of binding of the antibody to other non-target epitopes. Preferably, an antibody being evaluated can be considered to bind to a target epitope in a specific manner, i.e., "specifically bind," when the signal-to-noise ratio for the target epitope is about 50.

Positively expressed tumors

"CEA-positive expressing tumor" or "CEA-positive tumor" refers to a tumor cell that expresses CEA on the cell surface. In the present invention, the CEA-positive tumor to be treated may be gastrointestinal adenocarcinoma, breast cancer or lung cancer. The gastrointestinal adenocarcinoma may be selected from colorectal, pancreatic, esophageal or gastric adenocarcinoma. As previously mentioned, the bispecific antibodies of the invention are particularly suitable for treating patients with progressive tumors, metastatic tumors, recurrent tumors, advanced epithelial tumors, high epithelial tumor load, or patients with CEA serum concentrations above 100 ng/mL (e.g. as determined by ELISA). In some of these tumor patients, high levels of soluble CEA antigen were present in the plasma. Alternatively, high levels of soluble CEA are present around these tumor cells. It will be appreciated that in many antibody-based therapeutic approaches, serum CEA inhibits the binding of antibodies to CEA on the tumor cell membrane and blocks the activity of the antibodies, thereby hindering the success of anti-tumor therapy. However, due to the specific structure of the bispecific antibodies of the present invention, the bispecific antibodies of the present invention are not limited thereto. As used herein, "inhibiting" or "treating" includes delaying the development of symptoms associated with a disease and/or lessening the severity of those symptoms at which the disease is about to or expected to develop. The term also includes alleviation of existing symptoms, prevention of additional symptoms, and alleviation or prevention of the underlying causes of these symptoms. Thus, the term refers to vertebrate subjects that have been assigned beneficial results to a disease.

The term "therapeutically effective amount" or "effective amount" as used herein refers to an amount of a bispecific antibody of the invention which, when administered or administered in combination with an additional therapeutic agent to a cell, tissue or subject, is effective to prevent or alleviate the disease or condition to be treated. A therapeutically effective dose further refers to an amount of the compound sufficient to cause a reduction in symptoms, such as treatment, cure, prevention, or reduction of a related medical condition, or to increase the rate of treatment, cure, prevention, or reduction of the condition. When administering an active ingredient administered alone to an individual, a therapeutically effective amount refers to the individual ingredient. When a combination is administered, a therapeutically effective amount refers to the amount of the combination of active ingredients that produces a therapeutic effect, whether administered in combination, sequentially or simultaneously. A therapeutically effective amount will reduce symptoms, typically by at least 10%; usually at least 20%; preferably at least about 30%; more preferably at least 40% and most preferably at least 50%.

Pharmaceutical compositions and therapies

"pharmaceutical composition" refers to a pharmaceutical formulation for use in humans. The pharmaceutical composition comprises a bispecific antibody of the invention and a suitable formulation of a carrier, stabilizer and/or excipient. The invention includes pharmaceutical formulations of bispecific nanobodies described herein. To prepare a pharmaceutical or sterile composition, the antibody is admixed with a pharmaceutically acceptable carrier or excipient. Formulations of therapeutic and diagnostic agents in the form of, for example, lyophilized powders, slurries, aqueous solutions or suspensions may be prepared by mixing with physiologically acceptable carriers, excipients or stabilizers.

Toxicity and therapeutic efficacy of antibody compositions administered alone or in combination with an immunosuppressive agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining LD50 (the dose lethal to 50% of the population) and ED50 (the dose effective to treat 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio of LD50 to ED 50. The data obtained from these cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. The dosage of the compound is preferably within a range of circulating concentrations that include ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

Suitable routes of administration include parenteral (e.g. intramuscular, intravenous or subcutaneous) and oral administration. The antibodies or compositions of the invention can be administered in a variety of conventional ways, such as by oral ingestion, inhalation, topical administration, or by cutaneous, subcutaneous, intraperitoneal, parenteral, intraarterial, or intravenous injection. In one embodiment, the bispecific nanobody or pharmaceutical composition of the present invention is administered intravenously. In another embodiment, the bispecific nanobody or pharmaceutical composition of the present invention is administered subcutaneously. Alternatively, one may administer the antibody in a local rather than systemic manner (typically as a long acting or sustained release formulation), for example via direct injection of the antibody to the site of action. In addition, one may administer the antibody in a targeted drug delivery system.

Suitable dosages are determined by the clinician, for example, using parameters or factors known or suspected to affect the treatment or expected to affect the treatment in the art. Generally, the initial dose is slightly lower than the optimal dose, and thereafter increased by a small amount until the desired or optimal effect is achieved relative to any adverse side effects. Important diagnostic measures include measuring, for example, inflammatory symptoms or levels of inflammatory cytokines produced.

The antibodies, antibody fragments and cytokines may be provided by continuous infusion or by administration at intervals, for example 1-7 times a day, week or week. The dosage may be provided intravenously, subcutaneously, intraperitoneally, transdermally, topically, orally, nasally, rectally, intramuscularly, intracerebrally, intraspinally, or by inhalation. A preferred dosage regimen is one that includes a maximum dose or frequency of administration that avoids significant undesirable side effects. The total weekly dose is typically at least 0.05 μ g/kg body weight, more typically at least 0.2 μ g/kg, most typically at least 0.5 μ g/kg, typically at least 1 μ g/kg, more typically at least 10 μ g/kg, most typically at least 109 μ g/kg, preferably at least 0.2 mg/kg, more preferably at least 1.0 mg/kg, most preferably at least 2.0 mg/kg, ideally at least 10 mg/kg, more ideally at least 25 mg/kg, and most ideally at least 50 mg/kg. The required dose of the small molecule therapeutic, e.g., a peptidomimetic, a natural product, or an organic chemical, is approximately the same as the dose of the antibody or polypeptide, based on mole/kg calculations.

The pharmaceutical compositions of the present invention may also contain other agents, including but not limited to cytotoxic, cytostatic, antiangiogenic or antimetabolic agents, tumor-targeting agents, immunostimulants or immunomodulators or antibodies conjugated to cytotoxic, cytostatic or other toxic agents. The pharmaceutical compositions may also be administered with other forms of treatment, such as surgery, chemotherapy, and radiation. Typical veterinary, experimental or research subjects include monkeys, dogs, cats, rats, mice, rabbits, guinea pigs, horses and humans.

In particular, the bispecific nanobodies of the present invention may be used with or in combination with a second anticancer agent. In particular embodiments, the second anti-cancer agent and the nanobody of the invention are administered at substantially the same time. Individuals sometimes use both the second anti-cancer agent and the bispecific nanobody of the present invention. In one embodiment, the second anti-cancer agent or other agent typically administered to a cancer patient and the bispecific nanobody of the present invention may be combined into a pharmaceutical composition; in other embodiments, the two are administered separately.

The term "second anticancer agent" refers to any anti-neoplastic drug, including but not limited to: tyrosine kinase inhibitors such as Tarceva (erlotinib); anthracyclines such as daunorubicin (including micro-lipidic daunorubicin), doxorubicin (including micro-lipidic doxorubicin), epirubicin (epirubicin), idarubicin (idarubicin), and valrubicin (valrubicin); streptomyces-related agents, such as bleomycin (bleomycin), actinomycin (actinomycin), mithramycin (mithramycin), mitomycin (mitomycin), and pfomycin (porfiromycin); and anthraquinones (anthraendiones), such as mitoxantrone (mitoxantrone) and pixantrone (pixantrone). The second anticancer agent can also be a therapeutic antibody, such as a monoclonal antibody with anti-tumor activity, including but not limited to: murine, chimeric or partially or fully humanized monoclonal antibodies. Therapeutic antibodies include, but are not limited to: antibodies against tumor or cancer antigens on the surface of or within cells. Therapeutic antibodies also include, but are not limited to: antibodies directed against a target or pathway directly or indirectly associated with CEA. Therapeutic antibodies may further include, but are not limited to: antibodies directed against a target or pathway that interacts directly with a target or pathway associated with a compound of the invention. In one embodiment, therapeutic antibodies include, but are not limited to: alemtuzumab (Alemtuzumab), Bevacizumab (Bevacizumab), Cetuximab (Cetuximab), Ibritumomab (Ibritumomab tiuxetan), Rituximab (Rituximab) Pertuzumab (Pertuzumab), or Trastuzumab (Trastuzumab).

One aspect of the present invention provides a method of treating a tumor, the method comprising administering to a subject having a tumor a therapeutically effective amount of a bispecific nanobody of the present invention. In some embodiments, the bispecific nanobody comprises: (a) a first binding domain that specifically binds to a tumor antigen, said first binding domain comprising a VHH that is anti-tumor antigen; (b) a second binding domain that specifically binds to human CD16a, said second binding domain comprising an anti-human CD16a VHH of the invention. In some embodiments, the bispecific nanobody comprises: (a) a first binding domain that specifically binds to human CEA, said first binding domain comprising an anti-human CEA VHH; (b) a second binding domain that specifically binds to human CD16a, said second binding domain comprising an anti-human CD16a VHH of the invention. In some embodiments, the bispecific nanobody amino acid sequence is selected from SEQ ID nos: 8. SEQ ID No: 9. SEQ ID No: 10. SEQ ID No: 11. SEQ ID No: 12. In some embodiments, the subject is a mammal, preferably a human.

Another aspect of the present invention provides a method of treating a tumor, the method comprising administering to a subject having a tumor a therapeutically effective amount of a pharmaceutical composition of the present invention. In some embodiments, the pharmaceutical composition comprises a bispecific nanobody of the present invention and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a bispecific nanobody of the invention and a second anticancer agent. In some embodiments, the subject is a mammal, preferably a human.

Immunologic adjuvant

Bispecific nanobodies of the present invention may be used in combination with other recombinant proteins and/or peptides (e.g., tumor antigens or cancer cells) in order to enhance the immune response to these proteins (i.e., in vaccination protocols). For example, bispecific antibodies and antibody fragments thereof can be used to stimulate antigen-specific immune responses by co-administering the bispecific antibody with an antigen of interest (e.g., a vaccine). Accordingly, the present invention provides in a further aspect a method of enhancing an immune response to an antigen in a subject, the method comprising administering to the subject: (i) an antigen; and (ii) a bispecific antibody of the invention, or antigen-binding portion thereof, in order to enhance the immune response of a subject to an antigen. For example, the antigen can be a tumor antigen, a viral antigen, a bacterial antigen, or an antigen from a pathogen. Non-limiting examples of such antigens include, but are not limited to, tumor antigens or antigens from viruses, bacteria, or other pathogens.

Other combination therapies

As noted above, a bispecific antibody of the invention can be co-administered with one or more other therapeutic agents (e.g., a cytotoxic agent, a radiotoxic agent, or an immunosuppressive agent). The antibody may be linked to the agent (as an immunocomplex), or may be administered separately from the therapeutic agent. In the latter case (separate administration), the antibody may be administered before, after, or concurrently with the administration of the therapeutic agent, or may be co-administered with other known therapies.

Antibodies may also be used in vivo diagnostic assays. Antibodies are typically labeled with a radionuclide (e.g., 111In, 99Tc, 4C, 31I, 125I, 3H, 32P, 35S, or 18F) so that the antigen or antibody-expressing cell can be located using immunoimaging or positron emission tomography.

Linker sequences or connecting peptides

The bispecific antibodies of the present invention also include a linker sequence, typically a short peptide of 4-20 amino acids, between the first and second binding domains. These linker sequences allow for the rational positioning between the components to achieve the functional activity of each component.

Preferably, the linker sequence comprises a 2 to 20 amino acid sequence, more preferably 5 to 20 amino acids. The linker sequence is preferably a flexible linker sequence, so that it does not restrict the effector molecule or polypeptide in a single undesired conformation. The linker sequence is preferably composed mainly of amino acids with small side chains, such as glycine, alanine and serine, to provide said flexibility. Preferably, a proportion of more than about 80% or more of the amino acids of the linker sequence are glycine, alanine or serine residues, in particular glycine and serine residues. Examples of suitable linker sequences are GGGGS (G)₄S), Gly Gly Gly Gly Ser; or G₄SG₄SG₄S (SEQ ID No: 7), for example for linking anti-human CEA VHH and anti-human CD16a VHH in the present invention. Other different linker sequences may also be used, including a variety of flexible linker designs that have been successfully used to link different antibody variable regions. The size and sequence composition of the linker sequence can be determined by conventional computer modeling and techniques.

In the present invention, "polypeptide" refers to a polymer of any length consisting essentially of any number of the 20 natural amino acids. Although "protein" or "protein" generally refers to a polymer of greater amino acid length and "peptide" generally refers to a polymer of lesser amino acid length, there is generally no clear boundary between the two terms and there is often an overlap in the ranges.

In the present invention, a "vector" is a nucleic acid molecule capable of autonomous replication in a host cell and accepting foreign DNA. The vector carries its own origin of replication, restriction enzyme recognition sites for insertion of foreign DNA, and often a selectable marker (e.g., a gene encoding antibiotic resistance), often together with recognition sequences (e.g., promoters and enhancers) for expression of the inserted DNA. Common vectors include plasmid vectors and phage vectors.

The invention will be more fully understood by reference to the following examples. However, these examples should not be construed as limiting the scope of the invention. All documents and patent citations mentioned herein are expressly incorporated herein by reference.

Example 1 construction and screening of immune VHH phage display libraries

To generate single domain antibodies against CD16a, CD16a-His protein (Acrobiosystems, cat # CDA-H5220) was used to immunize camelids. Briefly, after four rounds of immunization in a camel, high titers were obtained by ELISA, peripheral blood cells were extracted and separated by gradient centrifugation. RNA was completely isolated from lymphocytes by Trizol reagent (invitrogen). After reverse transcription into the first strand of cDNA, the VHH fragment was amplified and ligated into the pMECS phagemid vector. By converting the ligation product to XL1-BlueE.coliCells to create a VHH phage library.

To amplify the phage library, a 200 μ L CD16a-VHH phage library was cultured in 40 mL of super broth medium (10 g MOPS, Sigma; 30 g tryptone, BD-Bioscience; 20 g yeast extract, BD-Bioscience; with ddH) containing 100 μ g/mL ampicillin and 10 μ g/mL tetracycline at 37 ℃ at 220 rpm/min₂O to 1 liter total volume) until the OD600 reached 0.6-0.8. Then-1.4 x 10 is added¹²cfu (colony forming units) helper phage VCSM13, incubated at 37 ℃ for 15 minutes without agitation, and then incubated at 220 rpm/min for about 1.5-2 hours. After incubation, the phage were collected by centrifugation at 4000 rpm/min for 10 minutes, then resuspended in 40 mL of fresh broth containing 100 μ g/mL ampicillin, 10 μ g/mL tetracycline, and 50 μ g/mL kanamycin, and cultured overnight at 30 ℃. Bacterial cells were then discarded by centrifugation at 4000 rpm/min for 10 minutes at 4 ℃. Phages were precipitated from the supernatant with 5 × PEG/NaCl (20% PEG/2.5M NaCl), then resuspended in 1 mL PBS, and then pelleted again to completely remove bacterial cells. The phage was then resuspended in 100-.

Plate panning (Plate panning) was used to select CD16a specific VHH binders. Briefly, human CD16a-His antigen was coated in 96-well microplates. Will then contain about 10¹²Phage library of cfu phage (referred to as input) with the coatedPlates were incubated at 37 ℃ for 15 minutes. Weakly bound phage or excess non-bound phage were washed 5 times with 0.1% PBST. Specifically bound phage were eluted with glycine-BSA buffer (pH 2.2) and immediately neutralized with 2M Tris buffer (pH 9.0). The resulting phage pool was named output. Use of eluted phage conjugates for infection competenceE. coli XL1-blue (OD 600= 0.6) and amplified for the next round of panning. To enrich for positive binders, the stringency was increased by panning for 4 cycles by including a lower concentration of CD16a (1 ug to 100 ng) in each cycle. Positive clones were randomly selected by 4 rounds of phage panning and amplified in 96-well deep-well plates (deep block) and rescued by adding VCSM13 helper phage. Phage ELISA was performed with phage-containing medium supernatants to further confirm positive clones. Positive phage clones were then precipitated by PEG/NaCl solution and resuspended in PBS. By measuring at A₂₈₀The OD of (d) to determine the phage concentration.

As a result: to obtain single domain anti-hCD 16a antibodies, camelids were immunized 4 times with 300 μ g hCD16a-His protein and phage libraries were constructed from lymphocytes isolated from immunized camelids. After 4 rounds of panning (table 1) with increased stringency by reducing the coated antibody from 1 μ g to 100 ng, 54 positive clones were obtained. Positive phage clones were then precipitated by PEG/NaCl solution and resuspended in PBS. By the reaction of₂₈₀OD was measured to determine phage concentration. These purified and quantified 54 clones were then used for another quantitative ELISA. ELISA experiments showed that 34 of these phages could specifically recognize CD16a-His protein. After analysis of the sequences, 23 different sequences were obtained. Based on the diversity and Elisa results, 6 of the 23 clones were further analyzed (fig. 1).

TABLE 1 enrichment data of hCD16a-VHH library

	1 st wheel	Wheel	2	Wheel 3	4 th wheel
							1 µg CD16a-His	500 ng CD16a-His	200 ng CD16a-His	100 ng CD16a-His
Input phage (cfu)	1.4*10^12	3.15*10^11	1.46*10^12	1.01*10^12
					Output phage (cfu)	5.7*10^8	7.26*10^8	7.15*10^9	1.18*10^10
Input/output	4.07*10^-4	2.30*10^-3	4.89*10^-3	1.17*10^-2

Example 2 expression and purification of anti-CEA-CD 16a VHH protein

The selected phage clones were sequenced and cloned, then fused with anti-CEA-VHH (GenBank: ABS 29544.1) and subcloned into pET26b vector (Novagen). A periplasmic protein purification process is performed. Briefly, the anti-CEA-CD 16a VHH plasmid was transformed intoE. coliStrain BL21 (DE 3) was used for inducible expression of 0.1mM isopropyl-. beta. -D-1-thiogalactopyranoside (IPTG). The CEA-CD16a VHH antibody was purified by Ni-NTA affinity chromatography and analyzed by SDS-PAGE.

Gel filtration was performed through Superdex 7510/300 GL (GE health, cat. No. 17-5174-01) at a flow rate of 0.5 ml/min. Protein markers (Sigma Aldrich, cat # MWGF 200) were loaded as standard controls for gel filtration analysis.

Dynamic Light Scattering (DLS): DLS measurements were performed with a DynaPro plate reader (white) operating at a source wavelength of 830 nm and a fixed scattering angle of 150 °. Approximately 50 μ L of the sample was measured at a running temperature of 25 ℃ to 75 ℃.

As a result:

we have previously constructed the bispecific antibody BiSS by linking an anti-CD 16a VHH to an anti-CEA VHH 15. The BiSS bispecific antibody showed potent anti-tumor activity both in vitro and in vivo, indicating that this bispecific antibody format can be used to assess anti-CD 16a VHH activity in a bispecific format. Thus, six different anti-CD 16a VHHs (fig. 1) were used to construct bispecific antibodies (SBC 74-SBC 79) by replacing the anti-CD 16a VHH in bisss with selected anti-CD 16a VHH with a flexible linker (GGGGS) 3 (between the anti-CD 16a VHH and the anti-CEA VHH), respectively (fig. 2 a).

The antibody was purified by Ni-NTA affinity chromatography and exchanged in phosphate buffered saline (PBS, pH 7.4) at 4 ℃ for at least 12 h. The SBC75-79 operates on SDS-PAGE as a single band of the expected molecular weight of about-36 KD of the monomer. SBC74 was almost half the size expected, indicating degradation (figure 2 b). Thus, SBC74 was not further characterized.

To further characterize the purified SBC75-79, gel filtration was performed. The bispecific antibody SBC77 operates as a single peak of about-36 KD molecular size, indicating that most SBC77 is in monomeric form (fig. 2 c). Similar results were observed for the bispecific antibodies SBC75 (), SBC76, SBC78, SBC79 and BiSS.

To further characterize SBC75-79, the melting temperature of the bispecific antibody was measured by DLS. The radius of SBC77 did not change below 50 ℃, but increased significantly above 50 ℃ (fig. 2 d). Similar results were observed for the other bispecific antibodies SBC75, SBC76, SBC78, SBC79 and BiSS.

Example 4 biological Activity of the CEA-CD16a VHH antibody

Cell culture and animals

The CEA positive cancer cell lines LS174T and HT29 (human colon cancer cell lines) and the CEA negative cancer cell line SKOV3 (human ovarian cancer cell line) were purchased from the type culture collection of the national academy of sciences in shanghai. HT29 and SKOV3 were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Life Technologies, China) containing 10% HI fetal bovine serum (Gibco, Life Technologies, USA) and 1% penicillin/streptomycin (HyClone); LS174T was also cultured in RPMI-1640 medium (Gibco, Life Technologies, China), which also contained 10% HI fetal bovine serum and 1% penicillin/streptomycin.

Fresh human Peripheral Blood Mononuclear Cells (PBMC) were prepared from healthy donors by gradient centrifugation with Ficoll-plane Plus (GE health). Fresh NK cells were isolated using EasySep human NK cell enrichment kit (PB) (Stem cell co. ltd, Vancouver, Canada) according to the manufacturer's instructions.

Non-obese diabetic severe combined immunodeficiency disease (NOD/SCID) 4-5 week old 18-22g female mice were purchased from witnessee laboratories ltd (beijing) and housed at the animal testing center of the university of zhongshan (room temperature 20-26 ℃, relative humidity 40% -70%, 12 hour circadian rhythm).

The anti-CEA-CD 16a bispecific antibody is capable of recognizing CEA and CD16 antigen

Flow cytometry analysis: for flow cytometry analysis, LS174T and SKOV3 cells were digested with 0.25% trypsin and collected. 5X 10 per sample collection⁵Cells were then washed twice with 1 mL ice-cold PBS + 0.2% BSA. Pellet was resuspended in 200 μ L ice-cold PBS + 0.2% BSA. In each tube, anti-CEA-CD 16a VHHs were added as primary antibodies. anti-His PE (BioLegend, cat # 652504) was used as a secondary antibody. Flow cytometry analysis was then performed on FC500 (Beckman Coulter).

And (3) testing: ELISA methods were performed to determine the interaction between bispecific antibody and hCD16 a. Briefly, bispecific antibodies SBC75-79 and bisss were coated overnight at 5 μ g per well in 96-well plates containing 100 μ L PBS (pH 7.4) buffer, followed by incubation with blocking buffer (PBS + 0.2% BSA) at 37 ℃ for 2 hours. After 3 washes with PBS containing 0.05% Tween-20 (pH 7.2), CD16a-Avi (Sino Biological, 10389-H27H 1-B) was added and incubated. After 3 washes, detection was performed using anti-streptavidin-HRP (R & D, 1: 40 v/v). The absorbance was measured at 450 nm. Data were analyzed using Graphpad Prism 5.

Results

To determine whether anti-CEA-CD 16a (SBC 75-SBC 79) bispecific antibodies can bind to the tumor antigen CEA, flow cytometry analysis was performed using the CEA-positive cell line LS174T and the CEA-negative cell line SKOV 3. The positive control BiSS and anti-CEA-CD 16a (SBC 75-SBC 79) showed positive staining on the CEA positive cell line LS174T, but no staining on the CEA negative cell line SKOV3 (fig. 3 a).

An ELISA assay was then performed to check whether the bispecific antibody could bind to CD16a protein (fig. 3 b). All bispecific antibodies SBC75, SBC76, SBC77, SBC78 and SBC79 were able to bind CD16a with similar strength (fig. 3 b). The absence of coating CD16a (as a negative control) showed no binding for each bispecific antibody, indicating that the bispecific antibody has specific binding. The anti-CD 16a VHH portion of BiSS (as positive control) had a Kd value for hCD16a of 10X 10^-9mol/L, was lower than that of SBC75-79, indicating that SBC75-79 had a higher binding strength than anti-CD 16 VHH in BiSS.

anti-CEA-CD 16a bispecific antibodies mediate potent cytotoxic activity in CEA-expressing cells

In vitro cytotoxicity assay: briefly, tumor cells were seeded into 96-well plates at about 2500 cells per well and at 37 ℃, 5% CO₂Incubate for 6 hours. 25000 NK cells and varying concentrations of antibody were then mixed with growth medium and added to each well. After 72 hours, Cell Counting Kit-8 reagent (Dojindo, CK 04) was used. After 1-4 hours of incubation, OD450nm was measured by TECAN plate reader. The survival (%) of the target cells was calculated using the following formula: [ (live target cell (sample) -Medium)/(live target cell (control) -Medium)]。

As a result:

to assess whether the anti-CEA-CD 16a bispecific antibody can mediate killing of tumor cells, cytotoxicity assays were performed. No cytotoxicity was observed in CEA negative cells SKOV3 with NK cells (fig. 4). When each anti-CD 16a VHHs was used with NK cells, no cytotoxicity was observed in CEA-positive cells LS174T (fig. 4). For the CEA positive cells LS174T, all bispecific antibodies showed potent tumor cell killing in the presence of NK cells, whereas no cell killing was observed in the absence of NK cells (fig. 5).

To further assess the cytotoxicity of the anti-CEA-CD 16a bispecific antibody, a dose-dependent cell killing effect was investigated (fig. 5). No cytotoxicity was observed in CEA negative cells SKOV3 regardless of the concentration of bispecific antibody SBC75-SBC79 (fig. 5a and 5 b). For CEA positive cells LS174T and HT29, all bispecific antibodies showed potent tumor cell killing in the presence of NK cells (fig. 5 a). Among the 5 different bispecific antibodies, SBC77 showed the strongest killing ability. Thus, dose-dependent cell killing was further evaluated (fig. 5 b). SBC77 showed higher cytotoxicity to both high CEA expressing LS174T cells and low CEA expressing HT29 cells compared to the positive control BiSS antibody (fig. 5 b). For anti-CD 16a VHH with NK cells, no cell killing was observed in both LS17T and HT29 cells (fig. 5 b).

anti-CEA-CD 16a bispecific antibody SBC77 inhibits tumor growth in vivo

In vivo tumor growth inhibition assay: in the co-transplantation model, LS174T human colon cancer cells were harvested, washed twice, and then resuspended in PBS. The cells were then mixed with freshly isolated human Peripheral Blood Mononuclear Cells (PBMC) and injected subcutaneously intoNOD/ SCIDRight abdomen of mice, wherein the total volume of each mouse is 200 μ L1X 10⁶LS174T cells and 5X 10 cells⁶And (4) PBMC cells. After transplantation, antibodies (20 μ g/mouse or 5 μ g/mouse) or vehicle control (PBS) were administered intraperitoneally (i.p.) each group n = 5. The animals were then treated every two days for the next 10 days.

In another study, the EasySep human NK cell enrichment kit (Stem cell Co. Ltd, Vancouv) was useder, Canada) deplete NK cells of freshly prepared PBMCs. The cells (NK negative cells) were then incubated with LS174T cells (total volume of 200 μ L in each mouse, 5X 10 cells)⁶NK negative PBMC cells and 1X 10⁶LS174T cells) and injected subcutaneously into the bodyNOD/SCIDThe right abdomen. When the tumor volume reaches 50-100 mm³Every 2 days SBC77 (20 μ g/mouse) or PBS (n =5 per group) was administered intraperitoneally (i.p.). Animal body weight and tumor volume were measured every two days. Using the formula (width)²X length)/2 tumor volume was calculated.

As a result:

to further investigate whether the anti-CEA-CD 16a bispecific antibody could inhibit tumor cell growth in vivo, SBC77 was used to evaluate anti-tumor activity in Nod/Scid mice. Then, makeNod / ScidMice were transplanted with LS174T cells and freshly isolated human PBMCs. Rapid growth of the tumor was observed. SBC77 showed minimal tumor growth inhibition at the lower dose (5 micrograms per animal), but effective tumor growth inhibition at the high dose (20 micrograms per animal) (figure 6 a). No significant weight loss and toxicity were observed in these mice treated with either low or high dose of SBC 77. These data indicate that SBC77 can effectively inhibit tumor growth in a xenograft mouse model.

To further analyze whether SBC77 requires NK cells for in vivo efficacy, NK cells were depleted from freshly isolated human PBMCs. LS174T cells and NK negative PBMC cells were then transplanted onto NOD/SCID mice. Mice were treated every two days with SBC77 or vehicle PBS. No significant inhibition of tumor growth was observed with treatment of SBC77 (figure 6 b). These data demonstrate that NK cells are required to confer tumor suppression of SBC77 in vivo.

SEQUENCE LISTING

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Ala Asp Ile Ser Ser Gly Asp Gly Arg Thr Thr Asn Tyr Ala Asp Phe

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Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ile Lys Asn Thr Val

65 70 75 80

Phe Leu Arg Met Thr Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Asn Thr Phe Val Ser Phe Val Gly Ile Ala Arg Ser Trp Gly Gln

100 105 110

Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Gly

130 135 140

Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser

145 150 155 160

Gly Tyr Ser Tyr Ser Ser Tyr Cys Leu Ala Trp Phe Arg Gln Ala Pro

165 170 175

Gly Lys Glu Arg Glu Arg Val Ala Ala Met Ser Val Gly Gly Gly Ser

180 185 190

Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Ile Ile Ser Gln Asp

195 200 205

Asn Ala Gln Asn Thr Val Tyr Leu Gln Met Asn Asn Leu Lys Pro Glu

210 215 220

Asp Thr Ala Met Tyr Tyr Cys Ala Ser Arg Arg Gly Gly Gly Tyr Cys

225 230 235 240

Tyr Pro Ala Tyr Arg Leu Asp Phe Tyr His Trp Gly Gln Gly Thr Gln

245 250 255

Val Thr Val Ser Ser

260

<210> 10

<211> 261

<212> PRT

<213> Artificial Sequence

<220>

<223> 5 '-anti-CEA VHH-anti-CD 16a VHH-3'

<400> 10

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Phe Val Gln Ala Gly Glu

1 5 10 15

Ser Leu Thr Leu Ser Cys Thr Ser Ser Thr Leu Thr Phe Thr Pro Tyr

20 25 30

Arg Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Asp Leu Val

35 40 45

Ala Asp Ile Ser Ser Gly Asp Gly Arg Thr Thr Asn Tyr Ala Asp Phe

50 55 60

Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ile Lys Asn Thr Val

65 70 75 80

Phe Leu Arg Met Thr Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Asn Thr Phe Val Ser Phe Val Gly Ile Ala Arg Ser Trp Gly Gln

100 105 110

Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Gly

130 135 140

Gly Ser Val Gln Thr Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser

145 150 155 160

Gly Tyr Phe Gly Arg Arg Tyr Cys Met Ala Trp Phe Arg Gln Val Pro

165 170 175

Gly Lys Glu Arg Glu Gly Val Ala Ser Ile Asp Arg Asn Gly Asn Ile

180 185 190

Asp Tyr Thr Glu Ser Val Arg Gly Arg Phe Ala Ile Ser Lys Asp Asn

195 200 205

Ala Gly Asn Thr Leu Ser Leu Gln Met Asn Ser Leu Lys Pro Asp Asp

210 215 220

Thr Ala Met Tyr Tyr Cys Ala Ala Pro Trp Ala Gly Val Tyr Cys Ala

225 230 235 240

Ala Ser Leu Arg Glu Ser Asp Tyr Lys Phe Trp Gly Gln Gly Thr Gln

245 250 255

Val Thr Val Ser Ser

260

<210> 11

<211> 258

<212> PRT

<213> Artificial Sequence

<220>

<223> 5 '-anti-CEA VHH-anti-CD 16a VHH-3'

<400> 11

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Phe Val Gln Ala Gly Glu

1 5 10 15

Ser Leu Thr Leu Ser Cys Thr Ser Ser Thr Leu Thr Phe Thr Pro Tyr

20 25 30

Arg Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Asp Leu Val

35 40 45

Ala Asp Ile Ser Ser Gly Asp Gly Arg Thr Thr Asn Tyr Ala Asp Phe

50 55 60

Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ile Lys Asn Thr Val

65 70 75 80

Phe Leu Arg Met Thr Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Asn Thr Phe Val Ser Phe Val Gly Ile Ala Arg Ser Trp Gly Gln

100 105 110

Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Gly

130 135 140

Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

145 150 155 160

Gly Asn Thr Tyr Arg Ser Tyr Ser Met Ala Trp Phe Arg Gln Ala Pro

165 170 175

Gly Lys Glu Arg Glu Trp Val Ala Ala Ile Ala Ser Asp Gly Ser Lys

180 185 190

Ala Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Asn

195 200 205

Val Lys Asn Thr Leu Thr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp

210 215 220

Thr Ala Met Tyr Tyr Cys Ala Ala Lys Arg Gly Arg Trp Phe Leu Val

225 230 235 240

Gln Ser Asp Arg Val Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

245 250 255

Ser Ser

<210> 12

<211> 263

<212> PRT

<213> Artificial Sequence

<220>

<223> 5 '-anti-CEA VHH-anti-CD 16a VHH-3'

<400> 12

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Phe Val Gln Ala Gly Glu

1 5 10 15

Ser Leu Thr Leu Ser Cys Thr Ser Ser Thr Leu Thr Phe Thr Pro Tyr

20 25 30

Arg Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Asp Leu Val

35 40 45

Ala Asp Ile Ser Ser Gly Asp Gly Arg Thr Thr Asn Tyr Ala Asp Phe

50 55 60

Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ile Lys Asn Thr Val

65 70 75 80

Phe Leu Arg Met Thr Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Asn Thr Phe Val Ser Phe Val Gly Ile Ala Arg Ser Trp Gly Gln

100 105 110

Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Gly

130 135 140

Gly Ser Val Gln Ala Gly Gly Ser Leu Thr Leu Ser Cys Ala Ala Ser

145 150 155 160

Gly Ala Ile Val Ser Arg Ser Cys Met Gly Trp Phe Arg Gln Ala Pro

165 170 175

Asp Lys Glu Arg Glu Ala Val Ala Gly Ile Phe Leu Ser Asp Gly Arg

180 185 190

Thr Thr Tyr Ala Asp Ser Val Gln Gly Arg Phe Thr Ile Ser Arg Asp

195 200 205

Thr Ala Lys Asn Thr Leu Thr Leu Gln Met Thr Ser Leu Lys Pro Glu

210 215 220

Asp Thr Ala Met Tyr Tyr Cys Ala Ala Asn Trp Arg Arg Trp Leu Tyr

225 230 235 240

Gly Ala Thr Cys Glu Ala Thr Asn Asp Tyr Asn Leu Trp Gly Gln Gly

245 250 255

Thr Gln Val Thr Val Ser Ser

260

<210> 13

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of anti-CD 16a VHH

<400> 13

Ala Ser Gly Asp Thr Thr Ser Glu Tyr Trp Gly Ala

1 5 10

<210> 14

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of anti-CD 16a VHH

<400> 14

Val Ser Gly Tyr Ser Tyr Ser Ser Tyr Cys Leu Ala

1 5 10

<210> 15

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of anti-CD 16a VHH

<400> 15

Val Ser Gly Tyr Phe Gly Arg Arg Tyr Cys Met Ala

1 5 10

<210> 16

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of anti-CD 16a VHH

<400> 16

Ala Ser Gly Asn Thr Tyr Arg Ser Tyr Ser Met Ala

1 5 10

<210> 17

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of anti-CD 16a VHH

<400> 17

Ala Ser Gly Ala Ile Val Ser Arg Ser Cys Met Gly

1 5 10

<210> 18

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of anti-CD 16a VHH

<400> 18

Ala Ile Leu Pro Leu Ser Thr Thr Pro Val Tyr Ala Gly Ser

1 5 10

<210> 19

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of anti-CD 16a VHH

<400> 19

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

1 5 10

<210> 20

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of anti-CD 16a VHH

<400> 20

Ser Ile Asp Arg Asn Gly Asn Ile Asp Tyr Thr Glu Ser

1 5 10

<210> 21

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of anti-CD 16a VHH

<400> 21

Ala Ile Ala Ser Asp Gly Ser Lys Ala Tyr Ala Asp Ser

1 5 10

<210> 22

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of anti-CD 16a VHH

<400> 22

Gly Ile Phe Leu Ser Asp Gly Arg Thr Thr Tyr Ala Asp Ser

1 5 10

<210> 23

<211> 18

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of anti-CD 16a VHH

<400> 23

Ala Ala Ala Arg Arg Gly Thr Asn Ala Phe Leu Thr His Asp Lys Tyr

1 5 10 15

Gly Tyr

<210> 24

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of anti-CD 16a VHH

<400> 24

Ala Ser Arg Arg Gly Gly Gly Tyr Cys Tyr Pro Ala Tyr Arg Leu Asp

1 5 10 15

Phe Tyr His

<210> 25

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of anti-CD 16a VHH

<400> 25

Ala Ala Pro Trp Ala Gly Val Tyr Cys Ala Ala Ser Leu Arg Glu Ser

1 5 10 15

Asp Tyr Lys Phe

20

<210> 26

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of anti-CD 16a VHH

<400> 26

Ala Ala Lys Arg Gly Arg Trp Phe Leu Val Gln Ser Asp Arg Val Asp

1 5 10 15

<210> 27

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of anti-CD 16a VHH

<400> 27

Ala Ala Asn Trp Arg Arg Trp Leu Tyr Gly Ala Thr Cys Glu Ala Thr

1 5 10 15

Asn Asp Tyr Asn

20

Claims (20)

1. An anti-human CD16a VHH, wherein

The complementarity determining region CDR1 is selected from SEQ ID Nos: 13. SEQ ID No: 14. SEQ ID No: 15. SEQ ID No: 16. SEQ ID No: 17;

the complementarity determining region CDR2 is selected from SEQ ID Nos: 18. SEQ ID No: 19. SEQ ID No: 20. SEQ ID No: 21. SEQ ID No: 22; and

the complementarity determining region CDR3 is selected from SEQ ID Nos: 23. SEQ ID No: 24. SEQ ID No: 25. SEQ ID No: 26. SEQ ID No: 27, or a pharmaceutically acceptable salt thereof.

2. The anti-human CD16a VHH of claim 1 selected from the following combinations:

(a) CDR1 is SEQ ID No: 13, CDR2 is SEQ ID No: 18, and CDR3 is SEQ ID No: 23;

(b) CDR1 is SEQ ID No: 14, CDR2 is SEQ ID No: 19, and CDR3 is SEQ ID No: 24;

(c) CDR1 is SEQ ID No: 15, CDR2 is SEQ ID No: 20, and CDR3 is SEQ ID No: 25;

(d) CDR1 is SEQ ID No: 16, CDR2 is SEQ ID No: 21, and CDR3 is SEQ ID No: 26; (ii) a Or

(e) CDR1 is SEQ ID No: 17, CDR2 is SEQ ID No: 22, and CDR3 is SEQ ID No: 27, or a pharmaceutically acceptable salt thereof; .

3. The anti-human CD16a VHH of claim 2, having an amino acid sequence selected from the group consisting of SEQ ID No: 1. SEQ ID No: 2. SEQ ID No: 3. SEQ ID No: 4. SEQ ID No: 5.

4. A bispecific nanobody comprising:

(a) a first binding domain that specifically binds to a tumor antigen, said first binding domain comprising a VHH that is anti-tumor antigen;

(b) a second binding domain that specifically binds to human CD16a, said second binding domain comprising an anti-human CD16a VHH of any one of claims 1-3.

5. The bispecific nanobody of claim 4, wherein the tumor antigen is MCSP, FAP, EGFR, CEA, Her2, CD33 or Siglec-3.

6. A bispecific nanobody comprising:

(a) a first binding domain that specifically binds to human CEA, said first binding domain comprising an anti-human CEA VHH;

(b) a second binding domain that specifically binds to human CD16a, said second binding domain comprising an anti-human CD16a VHH of any one of claims 1-3.

7. The bispecific nanobody according to claim 6, wherein the amino acid sequence of the anti-human CEA VHH is as set forth in SEQ ID No: and 6.

8. The bispecific nanobody of claim 6 or 7, wherein the anti-human CEA VHH domain is N-terminal to the anti-human CD16a VHH domain.

9. The bispecific nanobody of claim 6 or 7, wherein the anti-human CEA VHH domain is located C-terminal to the anti-human CD16a VHH domain.

10. The bispecific nanobody according to any one of claims 4 to 6, wherein the first binding domain is linked to the second binding domain by a linking peptide.

11. The bispecific nanobody according to claim 10, wherein the amino acid sequence of the linker peptide is shown in SEQ ID No. 7.

12. The bispecific nanobody according to claim 11, having an amino acid sequence selected from the group consisting of SEQ ID No: 8. SEQ ID No: 9. SEQ ID No: 10. SEQ ID No: 11. SEQ ID No: 12.

13. A pharmaceutical composition for treating a tumor comprising the bispecific nanobody of any one of claims 4 to 12 and a pharmaceutically acceptable carrier.

14. A pharmaceutical composition for treating a tumor comprising the bispecific nanobody of any one of claims 4 to 12 and a second anticancer agent.

15. The pharmaceutical composition of claim 13 or 14, wherein the tumor is a progressive tumor, an advanced tumor, or a metastatic tumor.

16. The pharmaceutical composition of claim 13 or 14, comprising the bispecific nanobody of claims 6-12, wherein the tumor is a CEA-positive expressing tumor.

17. The pharmaceutical composition of claim 16, wherein the CEA positive expressing tumor is colorectal cancer, pancreatic cancer, esophageal cancer, gastric adenocarcinoma, breast cancer, or lung cancer.

18. A nucleotide sequence encoding the bispecific nanobody of any one of claims 4 to 12.

19. A vector comprising the nucleotide sequence of claim 18.

20. A non-human host cell comprising the vector of claim 19.

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