Single domain high affinity antibodies and methods of use thereof

Abstract

The present application relates to single domain antibodies and constructs comprising single domain antibodies, both of which exhibit affinity and specificity for the CD16a activating receptor on natural killer cells, and antigens on cancer cells, bacteria, parasites, or viruses.

Description

[Technical field]

[0001] The present application relates to single domain antibodies and constructs comprising single domain antibodies, both of which exhibit affinity and specificity for the CD16a activating receptor on natural killer cells, or antigens on cancer cells, bacteria, parasites, or viruses. [Background technology]

[0002] Crystallizable fragment (Fc) gamma receptors (FcγRs) expressed on the surface of leukocytes bind immunoglobulin G (IgG) antibodies and are essential for the efficacy of many antibody-based drugs. FcγRs are classified into activating receptors (FcγRI / CD64, FcγRIIa / CD32a, FcγRIIc / CD32c, FcγRIIIa / CD16a, FcγRIIIb / CD16b) and inhibitory receptors (FcγRIIb / CD32b) (1). These receptors bind IgG but with different affinities (2). CD16a is the main receptor for the Fc region of monoclonal antibodies (mAbs) with low affinity and is the only FcγR expressed on the surface of natural killer (NK) cells. CD16a on NK cells binds to antibody-coated cells (e.g., cancer cells) and triggers antibody-dependent cellular cytotoxicity (ADCC).

[0003] Due to this function, CD16a-expressing NK cells are currently being investigated in clinical trials for cancer therapy (e.g., NCT04673617 and NCT03383978). It is well established that by increasing the binding affinity of CD16a to the antibody Fc region, NK cell cytotoxicity and clinical outcomes can be significantly improved (1,3). However, current antibodies in the field generally lack specificity for CD16a, which can hinder therapeutic efficacy and result in off-target toxicity. For example, antibodies that also bind to the CD16b activating receptor (expressed on neutrophils) have been shown to limit the ADCC activity of NK cells against cancer cells (4). Furthermore, nonspecific binding of antibodies to the inhibitory CD32b receptor (expressed on B cells) has also been shown to inhibit B cell maturation and macrophage activation (4,5). CD32b binds to CD8 + It is also expressed on a subset of T cells, where it may limit T cell survival by activating the caspase 3 and 7 pathways ( 6 ).

[0004] CD16a-expressing NK cells may also be involved in immune responses against pathogens such as bacteria, parasites, or viruses, and therefore improving the ADCC activity of NK cells may also improve the clinical outcome of patients with bacterial or viral infections.

[0005] The present application addresses the above-mentioned challenges and other problems associated with activating and redirecting natural killer cells to efficiently target surface antigens and lyse target cells, such as cancer cells, bacteria, parasites, or viruses. Summary of the Invention

[0006] In a first aspect, a construct is provided, comprising a first single domain antibody having an amino acid sequence of one of SEQ ID NOs: 14 and 15, exhibiting specificity and affinity for the CD16a receptor on the surface of natural killer (NK) cells, without cross-reacting with CD16b (e.g., CD16b-NA1) or CD32b. The construct also comprises a second single domain antibody having an amino acid sequence exhibiting specificity and affinity for an antigen associated with a cancer cell, a bacterium, a parasite, or a virus, wherein the first and second single domain antibodies are fused to each other with or without a linker.

[0007] In another embodiment, the antigen is associated with a cancer cell, where the antigen is selected from the group consisting of HER2, HER1, HER3, HER4, EGFR, VEGFR, CD47, FGFR, carcinoembryonic antigen (CEA), bladder tumor antigen (BTA), CA125, PDGFR, IGFR, CA15-3 / CA27.29, CA19-9, CA27.29, programmed death-ligand 1 (PD-L1), PD-L2, CTL4, CD3, CD19, CD20, CD22, CD25, CD27, CD30, CD33, CD37, CD38, CD40, CD48, CD52, B7-H3, MICA family, RAET1 / ULBP family, HLA-E, TIM-3, LAG-3, V domain of T cell activation IgA, and / or IL-1. suppressor (VISTA), HVEM, ICOS, 4-1BB, OX40, RANKL and GITR, epithelial and mesenchymal markers of circulating tumor cells, prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), soluble mesothelin-related peptide (SMRP), somatostatin receptor (SR), urokinase plasminogen activator (uPA), plasminogen activator inhibitor (PAI-1), TCR (e.g., MHC class I or class II molecules), A2a receptor, glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, RAGE-1, MN-CAIX, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, prostein, PSMA, prostate cancer tumor antigen-1 (PCTA-1), MART-1, MAGE, tyrosinase, TRP-1, TRP-2BAGE, GAGE-1, GAGE-2, RAGE, pl5, ELF2M, neutrophil elastase, ephrin B2, IGF-I receptor, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, TSP-180, pl85erbB2, pl80erbB-3, nm-23HI, TAG-72, CA19-9, CA72-4 , CAM17.1, NuMa, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, β-HCG, BCA225, BTAA, CA15-3CA27.29BCAA, CA195, CA242, CA-50, CAM43, CD68P1, CO-029, G250, Ga733EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0008] In another embodiment, the antigen is associated with a bacterium, wherein the antigen is selected from the group consisting of polysaccharide or peptide antigens associated with P. aeruginosa, S. aureus, Clostridium difficile, Acinetobacter baumannii, and Klebsiella pneumonia.

[0009] In another embodiment, the antigen is associated with a virus, wherein the antigen is selected from the group consisting of Epstein-Barr virus antigen EBVA, human papillomavirus (HPV) antigens E6 and E7, coronavirus surface antigens, influenza virus surface antigens, and HIV surface antigens.

[0010] In another aspect, the antigen is associated with a parasite, wherein the antigen is selected from the group consisting of antigens associated with malaria, leishmaniasis, Chagas disease, toxoplasmosis, schistosomiasis, cysticercosis, and strongyloidiasis.

[0011] In another embodiment, the first single domain antibody comprises the amino acid sequence of SEQ ID NO:14 and the second single domain antibody comprises the amino acid sequence of SEQ ID NO:6.

[0012] In another aspect, the second single domain antibody comprises at least one amino acid sequence of SEQ ID NOs: 3-11, and the second single domain antibody exhibits selectivity and affinity for HER2 and facilitates recognition of HER2-expressing cancer cells.

[0013] In further embodiments, the HER2-expressing cancer cell is an ovarian cancer cell, a breast cancer cell, a gastric cancer, a gastroesophageal cancer, a cervical cancer cell, a bladder cancer cell, a gallbladder cancer cell, a testicular cancer cell, a uterine cancer cell, or any other HER2-expressing cancer cell.

[0014] In another embodiment, the amino acid sequence of the first single domain antibody exhibits high affinity for the CD16a receptor of NK cells.

[0015] In another embodiment, the amino acid sequence of the second single domain antibody exhibits high affinity for an antigen associated with a cancer cell, a bacterium, a parasite, or a virus.

[0016] In another embodiment, the first and second single domain antibodies are fused using a linker.

[0017] In a further embodiment, the linker is a human muscle aldolase (HMA) linker.

[0018] In another embodiment, the construct further comprises at least one additional single domain antibody having an amino acid sequence exhibiting affinity and specificity for another epitope on the same or another antigen, fused, with or without a linker, to at least one of the first or second single domain antibodies.

[0019] In a further embodiment, the at least one further single domain antibody is of the same type as the first single domain antibody.

[0020] In a further embodiment, the at least one further single domain antibody is of the same type as the second single domain antibody.

[0021] In a second aspect, a single domain antibody is provided, the single domain antibody comprising at least one amino acid sequence of SEQ ID NOs: 14 and 15, wherein the single domain antibody selectively binds with high affinity to the CD16a activating receptor on the surface of natural killer (NK) cells without cross-reacting with CD16b-NA1 or CD32b.

[0022] In a third aspect, a single domain antibody is provided, wherein the single domain antibody comprises at least one amino acid sequence of SEQ ID NOs: 3 to 11, and the single domain antibody exhibits selectivity and high affinity for HER2 and facilitates recognition of HER2-expressing cancer cells.

[0023] In a fourth aspect, a method is provided for inhibiting HER2-positive cancer in a subject, in which an effective amount of a construct as described above is administered to the subject, wherein the construct activates NK cells in the subject to recognize target HER2-positive cancer cells in the subject.

[0024] In a fifth aspect, there is provided a method of performing an ELISA assay using a single domain antibody or construct as described above. In this method, a sample containing one or more antigens is immobilized on a solid support, where the one or more antigens are selected from HER2 and CD16a. A single domain antibody is applied to the surface of the sample, where the single domain antibody acts as a primary antibody. A secondary antibody is applied to the surface of the sample, where the secondary antibody is linked to an enzyme and configured to recognize the single domain antibody. A substance is added to the sample, where the substance includes a substrate for the enzyme. The sample is then examined to determine whether there is binding between the single domain antibody and the one or more antigens, where if there is binding by the single domain antibody to the one or more antigens, a subsequent reaction produces a detectable signal in the sample.

[0025] In a sixth aspect, there is provided a method of performing a flow cytometry assay using the single domain antibody described above. In this method, a sample containing cancer cells and the single domain antibody is suspended in a fluid. A secondary antibody linked to a fluorescent probe capable of binding to the single domain antibody is applied to the sample. The fluid containing the sample is injected into a flow cytometer device. The sample is then analyzed in the flow cytometry analyzer to determine whether the cancer cells are HER2+ cancer cells.

[0026] In a seventh aspect, a method for cell imaging using the single domain antibody as described above is provided. In this method, a sample containing suspected cancer cells is fixed on a slide and the single domain antibody is applied to the sample. A secondary antibody linked to a fluorescent probe capable of binding to the single domain antibody is applied to the sample. The sample is then examined by confocal or fluorescent microscopy to detect the presence or absence of HER2 expression on the surface of the suspected cancer cells. In another aspect, the fluorescently labeled secondary antibody is an anti-his tag antibody or an anti-C-myc tag antibody.

[0027] In an eighth aspect, there is provided an in vivo cell tracking and imaging method for tracking allogeneic or autologous NK cells in a subject using the single domain antibody described above. In this method, an imaging agent conjugated to the single domain antibody is administered to the subject. A whole-body imaging method of the subject is performed to generate an image and identify the anatomical location of the NK cells in the image. In another aspect, the whole-body imaging method is selected from the group consisting of magnetic resonance imaging (MRI), positron emission tomography (PET), computed tomography (CT), and single photon emission computed tomography (SPECT).

[0028] In a ninth aspect, there is provided an in vivo cancer phenotyping method for identifying HER2-expressing cancer lesions in a subject. In this method, an imaging agent conjugated to the above-mentioned single domain antibody is administered to the subject. The subject is subjected to a tumor imaging method to generate an image and identify HER2-expressing cancer lesions in the image. In another aspect, the tumor imaging method is selected from the group consisting of magnetic resonance imaging (MRI), positron emission tomography (PET), computed tomography (CT), and single photon emission computed tomography (SPECT). [Brief description of the drawings]

[0029] [Figure 1A] 1 is a schematic diagram showing the structural differences between conventional IgG and camelid IgG: the Fab fragment of conventional IgG is composed of both heavy and light chains, whereas camelid IgG is composed only of heavy chains and has no light chains. [Figure 1B] Schematic diagram of anti-CD16a VHH and anti-antigen VHH linked to each other via a linker. [Figure 1C] A schematic diagram of BiKE, which is composed of anti-CD16a VHH and anti-antigen VHH, activates NK cells to recognize targets (cancer cells, bacteria, viruses). [Figure 1D] Schematic diagram of various configurations of one or more anti-CD16a VHHs linked via a linker to one or more anti-antigen VHHs. Two or more anti-CD16a VHHs can be engineered in tandem to recognize two or more different epitopes on the CD16a antigen. Similarly, two or more anti-antigen VHHs can be engineered in tandem to recognize two or more different epitopes on the target antigen. [Diagram 2] FIG. 1 shows the timeline and methods used to immunize llamas to generate VHHs against the HER2 protein. [Figure 3A] FIG. 1 shows SDS-PAGE analysis of purified HER2 antigen with a theoretical molecular weight of 72,633 Da. [Figure 3B]FIG. 1 shows IgG levels in serum of llamas before and after immunization with HER2 protein, measured by ELISA. [Figure 4A] PBMCs were isolated using the Ficoll-Paque method. RNA was then extracted to generate a cDNA library, and genes were amplified and cloned into pMECS-GG phagemid. The phagemid was used for phage display, and screened candidates were used to infect TG1 bacteria. Colonies were selected, grown, lysed, and the lysate was removed. [Figure 4B] The lysates were then used in ELISA to screen for VHHs with the highest affinity to HER2 and negligible binding to HER1, HER3, and HER4. Bovine serum albumin (BSA) and skim milk were used as controls. [Figure 5A] FIG. 1 shows SDS-PAGE analysis of purified c-myc / his tagged anti-HER2 VHH. [Figure 5B] FIG. 1 shows flow cytometry histograms of HER2+ cancer cells (BT474) labeled with anti-HER2 VHH, trastuzumab, and pertuzumab. [Figure 5C] FIG. 1 shows flow cytometry histograms of HER2+ cancer cells (SKOV-3) labeled with anti-HER2 VHH, trastuzumab, and pertuzumab. [Figure 5D] FIG. 1 shows flow cytometry histograms of HER2-cancer cells (MDA-MB-231) labeled with anti-HER2 VHH, trastuzumab, and pertuzumab. [Figure 5E]Figure 1 shows flow cytometry histograms of HER2- cancer cells (OVASC-1) labeled with anti-HER2 VHH, trastuzumab, and pertuzumab. Equimolar binding sites of purified VHH and FDA-approved anti-HER2 monoclonal antibodies (trastuzumab and pertuzumab) were used to measure HER2 expression on the surface of HER2+ cancer cells. HER2- cancer cells were used as a negative control. Flow cytometry data showed that the selected anti-HER2 VHH could recognize HER2 on the surface of HER2+ cancer cells without binding to HER2- cancer cells. [Figure 6A] FIG. 1 shows evaluation of the toxicity of selected anti-HER2 VHHs, trastuzumab, and pertuzumab against HER2+BT474 cancer cells. [Figure 6B] Figure 1 shows the evaluation of the toxicity of selected anti-HER2 VHHs, trastuzumab and pertuzumab against SKOV-3 cancer cells. Cytotoxicity was measured in a WST-1 cytotoxicity assay and data are presented as mean ± standard deviation. [Figure 7A] FIG. 1 shows the results of anti-HER2 VHH affinity and binding kinetics measurements by using Biolayer Interferometry. [Figure 7B] Figure 1 shows the results of anti-HER2 VHH affinity and binding kinetics measurement by using Biolayer Interferometer. KD (affinity), Kon (association constant), and Koff (dissociation constant) were determined by using Octet Data Analysis HT11.1 software. E5 and A1 clones were selected as high-performance VHH candidates. [Figure 8A] FIG. 13: Confocal microscopy images of SKOV-3 cells treated with anti-HER2 VHH (E5 clone) and taken at different time points. [Figure 8B]Confocal microscopy images of SKOV-3 cells treated with anti-HER2 VHH (A1 clone) and taken at different time points. Cell nuclei are labeled with DAPI (blue) and VHH with FITC (green). Overlay images from the top view (middle slice of the Z-stack) show the time-dependent binding to the cells, while the side view (edge) images show the internalization of the fluorescently labeled VHH. Results of this experiment showed that both A1 (Figure 8A) and E5 (Figure 8B) clones start to be internalized in as little as 1 hour, and the internalization process is complete in 3-5 hours. Published data shows that trastuzumab binds and internalizes in HER2+ cancer cells in as little as 4 hours. [Figure 9A] FIG. 1 shows SDS-PAGE analysis of purified rCD16a (20 μg). [Figure 9B] FIG. 1 shows IgG levels in the serum of llamas before and after immunization with rCD16a protein, measured by ELISA. [Figure 10A] PBMCs were isolated using the Ficoll-Paque method. RNA was then extracted to generate a cDNA library, and genes were amplified and cloned into pMECS-GG phagemid. The phagemid was used for phage display, and screened candidates were used to infect TG1 bacteria. Colonies were selected, grown, lysed, and the lysate was removed. [Figure 10B] Figure 1: Evaluation of the specificity of anti-CD16a VHHs from periplasmic extracts against CD16a and CD16b antigens by using ELISA, skim milk was used as control. [Figure 10C] FIG. 13 shows the evaluation of the binding affinity of anti-CD16a VHH to the CD16a antigen in periplasmic extracts after 1280-fold dilution. [Figure 11]Figure 1. Evaluation of the specificity of C1 and E3 anti-CD16a VHHs for the CD16a antigen. CD16b-NA1, CD16-NA2, CD32b, and skim milk were used as antigen controls, and the commercially available 3G8 (anti-CD16a / b mAb) and eBioCB16 (anti-CD16a / b mAb) mAbs were used as antibody controls. This figure shows that C1 and E3 anti-CD16a VHHs bind specifically to CD16a without cross-reacting with CD1b-NA1 and CD32b. [Figure 12A] FIG. 1 shows the evaluation of the specificity of C1 and E3 anti-CD16a VHHs for the CD16a antigen by flow cytometry using NK92 (CD16+) cells. [Figure 12B] FIG. 1 shows the evaluation of the specificity of C1 and E3 anti-CD16a VHHs for the CD16a antigen by flow cytometry using NK92 (CD16+) cells. [Figure 12C] FIG. 1 shows the evaluation of the specificity of C1 and E3 anti-CD16a VHHs for the CD16a antigen by flow cytometry using neutrophils. [Figure 12D] FIG. 1 shows the evaluation of the specificity of C1 and E3 anti-CD16a VHHs for the CD16a antigen by flow cytometry using neutrophils. [Figure 12E] Figure 12E: Evaluation of the specificity of C1 and E3 anti-CD16a VHHs for the CD16a antigen by flow cytometry using B cells (Figure 12E). The positive rate (PE+), negative rate (PE-) and mean fluorescence intensity (MFI) of labeled cells are shown. This figure shows the specificity of C1 and E3 anti-CD16a VHHs for the CD16a receptor. [Figure 13A] Figure 1 shows the affinity, binding kinetics and specificity of anti-CD16a VHHs determined by using Biolayer Interferometry. KD (affinity), Kon (association constant) and Koff (dissociation constant) were determined by using Octet Data Analysis HT11.1 software. Figure 2 shows the binding affinity of anti-CD16a C1 clone to CD16a antigen. [Figure 13B] Figure 1 shows the affinity, binding kinetics and specificity of anti-CD16a VHHs determined by using Biolayer Interferometry. KD (affinity), Kon (association constant) and Koff (dissociation constant) were determined by using Octet Data Analysis HT11.1 software. Figure 2 shows the binding affinity of anti-CD16a E3 clone to CD16a antigen. [Figure 13C] Figure 1 shows the affinity, binding kinetics and specificity of anti-CD16a VHHs determined by using Biolayer Interferometry. KD (affinity), Kon (association constant) and Koff (dissociation constant) were determined by using Octet Data Analysis HT11.1 software. Figure 2 shows the binding affinity of anti-CD16a C1 clone to CD16b-NA1 antigen. [Figure 13D] Figure 1 shows the affinity, binding kinetics and specificity of anti-CD16a VHHs determined by using Biolayer Interferometry. KD (affinity), Kon (association constant) and Koff (dissociation constant) were determined by using Octet Data Analysis HT11.1 software. Figure 2 shows the binding affinity of anti-CD16a E3 clone to CD16b-NA1 antigen. [Figure 13E] Figure 1 shows that the affinity, binding kinetics, and specificity of anti-CD16a VHHs were determined by using Biolayer Interferometry. KD (affinity), Kon (association constant), and Koff (dissociation constant) were determined by using Octet Data Analysis HT11.1 software. Figure 1 shows the epitope mapping of C1 VHHs and mAbs (Trastuzumab and Pertuzumab) to the CD16a antigen. This figure shows that anti-CD16a VHHs exhibit high affinity to the CD16a antigen and significantly lower affinity to CD16b-NA1. It is also shown that C1 VHHs bind to a different epitope on CD16a than Trastuzumab and Pertuzumab. [Figure 14A]FIG. 13 shows a schematic diagram of anti-HER2 VHH (E5 clone) fused to anti-CD16a VHH (C1 clone) via a human muscle aldolase (HMA) linker to generate BiKE:HER2 / CD16a. [Figure 14B] FIG. 13 shows SDS-PAGE analysis of expressed and purified BiKE:HER2 / CD16a, which has a theoretical molecular weight of 33.48 kDa. [Figure 14C] Figure 1 shows a liquid chromatography mass spectrometry (LC-MS) profile of purified BiKE:HER2 / CD16a (~1 mg / ml). The peptide is shown as a monomer with no dimers or multimers present. [Figure 15A] FIG. 10 shows a comparison of binding between anti-CD16a VHH and BiKE:HER2 / CD16a by ELISA. [Figure 15B] FIG. 10 shows a comparison of binding between anti-HER2 VHH and BiKE:HER2 / CD16a by ELISA. [Figure 15C] FIG. 10: Comparison of binding of anti-CD16a VHHs with BiKE:HER2 / CD16a in NK92 (CD16+) cells by flow cytometry. [Figure 15D] Figures 14A-14D show a comparison of binding of anti-HER2 VHHs to BiKE:HER2 / CD16a in SKOV-3 (HER2+) cells by flow cytometry. These figures show that fusion of anti-CD16a VHHs to anti-HER2 VHHs did not affect binding affinity to the target antigen. [Figure 16A] FIG. 1 shows the binding affinity of E5C1 BiKE (BiKE:CD16a / HER2) to the CD16a antigen. [Figure 16B] FIG. 1 shows the binding affinity of E5C1 BiKE (BiKE:CD16a / HER2) to the CD16b-NA1 antigen. [Figure 16C] FIG. 1 shows the binding affinity of E5C1 BiKE (BiKE:CD16a / HER2) to the CD16b-NA2 antigen. [Figure 17A]Schematic diagram of antibody-induced cytotoxicity. BiKE, trastuzumab, and pertuzumab activate NK cells to recognize target cancer cells. [Figure 17B] FIG. 1 shows SKOV-3 cells treated with haNK92 cells at different E:T (NK:target cell) ratios under non-adherent cell conditions. [Figure 17C] FIG. 1 shows BT474 cells treated with haNK92 cells at different E:T (NK:target cell) ratios under non-adherent cell conditions. [Figure 17D] This is a diagram showing JIMT-1 cells treated with haNK92 cells at different E:T (NK:target cell) ratios under non-adherent cell conditions. ADCC was measured after treating cancer cells with haNK92 cells in combination with pertuzumab (Prz.), trastuzumab (Trz.), or BiKE. This figure shows that BiKE induced significantly higher ADCC compared to Trz. or Prz. [Figure 18A] FIG. 13 shows the measurement of ADCC under adhesive conditions in three HER2+ cancer cell lines using laNK92 cells in combination with BiKE or trastuzumab at different E:T ratios but a fixed antibody concentration (100 nM). [Figure 18B] FIG. 13 shows the measurement of ADCC under adhesive conditions in three HER2+ cancer cell lines using laNK92 cells in combination with BiKE or trastuzumab at different E:T ratios but a fixed antibody concentration (100 nM). [Figure 18C] FIG. 13 shows the measurement of ADCC under adhesive conditions in three HER2+ cancer cell lines using laNK92 cells in combination with BiKE or trastuzumab at different E:T ratios but a fixed antibody concentration (100 nM). [Figure 18D] FIG. 13 shows the measurement of ADCC in three HER2+ cancer cell lines using laNK92 cells in combination with BiKE or trastuzumab at different antibody concentrations but a fixed E:T ratio of 4. [Figure 18E]FIG. 13 shows the measurement of ADCC in three HER2+ cancer cell lines using laNK92 cells in combination with BiKE or trastuzumab at different antibody concentrations but a fixed E:T ratio of 4. [Figure 18F] FIG. 13 shows the measurement of ADCC in three HER2+ cancer cell lines using laNK92 cells in combination with BiKE or trastuzumab at different antibody concentrations but a fixed E:T ratio of 4. [Figure 18G] FIG. 1 shows the results of measuring IFN-γ using ELISA after incubating SKOV-3 cells with laNK92 cells in the presence of BiKE or trastuzumab (Trz.). [Figure 18H] FIG. 1 shows the results of measuring TNF-α using ELISA after incubating SKOV-3 cells with laNK92 cells in the presence of BiKE or trastuzumab (Trz.). [Figure 18I] FIG. 1 shows the results of measuring perforin using ELISA after incubating SKOV-3 cells with laNK92 cells in the presence of BiKE or trastuzumab (Trz.). [Figure 18J] FIG. 1 shows the results of measuring granzyme B using ELISA after incubating SKOV-3 cells with laNK92 cells in the presence of BiKE or trastuzumab (Trz.). [Figure 18K] Figure 1 shows the change in CD107a expression (laNK92 degranulation) measured at different antibody concentrations using flow cytometry. Data are shown as mean ± standard deviation (*t test, p<0.05, ns: not significant). [Figure 19A] FIG. 13 shows the measurement of ADCC under adhesive conditions in three HER2+ cancer cell lines using haNK92 cells in combination with BiKE or trastuzumab at different E:T ratios but a fixed antibody concentration (100 nM). [Figure 19B]FIG. 13 shows the measurement of ADCC under adhesive conditions in three HER2+ cancer cell lines using haNK92 cells in combination with BiKE or trastuzumab at different E:T ratios but a fixed antibody concentration (100 nM). [Figure 19C] FIG. 13 shows the measurement of ADCC under adhesive conditions in three HER2+ cancer cell lines using haNK92 cells in combination with BiKE or trastuzumab at different E:T ratios but a fixed antibody concentration (100 nM). [Figure 19D] FIG. 13 shows the measurement of ADCC in HER2+ cancer cell lines using haNK92 cells in combination with BiKE or trastuzumab at different antibody concentrations but a fixed E:T ratio of 4. [Figure 19E] FIG. 13 shows the measurement of ADCC in HER2+ cancer cell lines using haNK92 cells in combination with BiKE or trastuzumab at different antibody concentrations but a fixed E:T ratio of 4. [Figure 19F] FIG. 13 shows the measurement of ADCC in HER2+ cancer cell lines using haNK92 cells in combination with BiKE or trastuzumab at different antibody concentrations but a fixed E:T ratio of 4. [Figure 19G] FIG. 1 shows the results of measuring IFN-γ using ELISA after incubating SKOV-3 cells with haNK92 cells in the presence of BiKE or trastuzumab (Trz.). [Figure 19H] FIG. 1 shows the results of measuring TNF-α using ELISA after incubating SKOV-3 cells with haNK92 cells in the presence of BiKE or trastuzumab (Trz.). [Figure 19I] FIG. 1 shows the results of measuring perforin using ELISA after incubating SKOV-3 cells with haNK92 cells in the presence of BiKE or trastuzumab (Trz.). [Figure 19J]FIG. 1 shows the results of measuring granzyme B using ELISA after incubating SKOV-3 cells with haNK92 cells in the presence of BiKE or trastuzumab (Trz.). [Figure 19K] Figure 1 shows the change in CD107a expression (haNK92 degranulation) measured at different antibody concentrations using flow cytometry. Data are shown as mean ± standard deviation (*t-test, p<0.05). [Figure 20] This figure shows the measurement of ADCC under adhesive conditions of HER2+SKOV-3 cells incubated with laNK92 cells for 4 hours in the presence of BiKE (10 pM), trastuzumab (Trz.) (10 pM), pertuzumab (Prz.) (10 pM), trastuzumab (10 pM) + pertuzumab (10 pM), BiKE (10 pM) + pertuzumab (10 pM), BiKE (10 pM) + trastuzumab (10 pM), or no antibody (i.e., SKOV3+laNK92 only) (*t-test, p<0.05). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] In accordance with one or more embodiments, the present application relates to single domain antibodies, constructs comprising two or more single domain antibodies; and related cancer cell killing, bacterial killing, parasitic killing, viral killing, imaging and assay methods utilizing said single domain antibodies and constructs comprising said single domain antibodies.

[0031] definition So that the present invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0032] As used in this specification, including the appended claims, singular words such as "a," "an," and "the" include their corresponding plural references unless the context clearly dictates otherwise.

[0033] "Natural killer" or "NK" cells are a type of cytotoxic lymphocyte that are critical to the innate immune system. NK cells account for approximately 5-20% of all circulating lymphocytes in humans.

[0034] "CD16" refers to type III Fcγ receptor. In humans, it exists in two different forms: FcγRIIIa ("CD16A") and FcγRIIIb ("CD16B"). "CD16A" is an activating receptor expressed on the cell surface of NK cells and macrophages. CD16A can trigger the cytotoxic activity of NK cells and macrophages. The affinity of an antibody for CD16A directly correlates with its ability to trigger the activation of NK cells, so the higher the affinity for CD16A, the lower the dose of antibody required for activation. CD16B is expressed only on neutrophils.

[0035] "CD32", also known as FcγRII or FCGR2, refers to a surface receptor glycoprotein that can be found on the surface of a variety of immune cells. CD32B is one of three major CD32 subtypes and is an inhibitory surface receptor that is part of a large population of B cell coreceptors.

[0036] The term "HER2" or "human epidermal growth factor receptor 2" refers to an oncogene known to be associated with the development and progression of certain aggressive cancers, such as breast or ovarian cancer.

[0037] The term "his tag", "his-tag" or "polyhistidine-tag" refers to an amino acid motif within proteins that generally consists of six or more histidine (His) residues at the N- or C-terminus of the protein.

[0038] The term "imaging agent" or "imaging substance" generally refers to a compound or agent used to enhance the contrast of structures or fluids within the body during medical imaging (e.g., PET, MRI). The term "imaging agent" or "imaging substance" may be used interchangeably with the term "contrast agent."

[0039] The terms "VHH", "nanobody" and "single domain antibody" generally refer to antibody fragments that consist of a monomeric variable single antibody domain capable of selectively binding to a specific antigen.

[0040] The term "single domain antibody construct" generally refers to a construct comprising two or more single domain antibodies, where one or more single domain antibodies bind to an activating receptor on a natural killer cell (i.e., CD16a) and one or more single domain antibodies bind to an antigen on a cancer cell, a bacterial cell, a parasite, or a virus. Single domain antibody constructs may be bispecific, trispecific, tetraspecific, or multispecific.

[0041] The term "BiKE" or "bispecific killer cell engager" generally refers to a construct comprising two or more single domain antibodies that bind to an activating receptor (i.e., CD16a) on natural killer cells and macrophages and an antigen on a cancer cell, bacterial cell, parasite, or virus.

[0042] The term "peptide" as used herein refers to peptides and proteins that are more than two amino acids in length and that may also incorporate non-amino acid molecules.

[0043] The terms "no binding" or "negligible binding" or "no cross-reactivity" are used interchangeably herein and generally refer to binding similar to background (control) binding and / or statistically insignificant binding.

[0044] The phrase "pharmacologically acceptable" or "pharmacologically acceptable" refers to molecular entities and compositions that do not cause harmful, toxic, allergic, inflammatory, or other undesirable reactions when administered to animals or humans. As used herein, "pharmacologically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are pharma- ceutically acceptable and preferably inert as the term is used herein. The use of such media and agents for pharma- ceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. Any conventional media or agent is contemplated for use in the therapeutic compositions, except insofar as it is incompatible with the active ingredient.

[0045] The term "effective amount of imaging agent" as used herein refers to an amount of imaging agent sufficient to obtain a signal suitable for medical imaging of a part of the body. The method of determining the most effective amount of imaging agent may vary depending on the composition used, the purpose of use, and the target cell to be imaged. When the imaging agent described herein is co-administered with another agent, the effective amount may be less than when the agent is used alone. The suitable formulation and administration method of the imaging agent can be easily determined by those skilled in the art.

[0046] Unless otherwise stated, the terms "subject" or "patient" are used interchangeably and refer to mammals, such as human patients and non-human primates, as well as laboratory animals, such as rabbits, rats, and mice, and other animals. Thus, the terms "subject" or "patient" as used herein refer to any mammalian patient or subject to which the compounds of the present disclosure can be administered. In exemplary embodiments of the present disclosure, to identify a subject patient for treatment with the methods of the present disclosure, accepted screening methods are used to determine risk factors associated with the target or suspected disease or condition, or to determine the status of an existing disease or condition in the subject. These screening methods include, for example, conventional workups to identify risk factors that may be associated with the target or suspected disease or condition. These and other routine methods allow clinicians to select patients who require treatment using the methods and compounds of the present disclosure.

[0047] The terms "treat," "treating," or "treatment" of a condition, disorder, or condition include (a) preventing or delaying the appearance of clinical symptoms of a condition, disorder, or condition occurring in a human who may be afflicted with or predisposed to the condition, disorder, or condition, but who has not yet experienced or exhibited clinical symptoms of the condition, disorder, or condition, or (b) inhibiting the condition, disorder, or condition, i.e., preventing, alleviating, or delaying the onset of the disease or its recurrence (in the case of maintenance treatment) or at least one clinical symptom, sign, or test thereof, or (c) palliating the disease, i.e., causing regression of the condition, disorder, or condition, or at least one clinical or subclinical symptom or sign thereof.

[0048] The term "about" or "approximately" can mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on the method of measuring or determining the value, i.e., the limitations of the measurement system, i.e., the degree of precision required for a particular purpose, such as pharmaceutical formulation. For example, "about" can mean within 1 or more than 1 standard deviation, as is customary in the art. Alternatively, "about" can mean within a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, more preferably within 2-fold, of a value. Unless otherwise stated, when a particular value is described in this application and claims, "about" should be assumed to mean within an acceptable error range for the particular value.

[0049] As used herein, the terms "therapeutically effective amount", "therapeutically effective dose" and "effective amount" refer to an amount of a compound or composition sufficient to produce a beneficial or desired result effective to produce a measurable improvement in one or more symptoms associated with a particular disease or condition when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject. A therapeutically effective dose further refers to an amount of a compound sufficient to produce at least a partial improvement of a symptom, e.g., treatment, cure, prevention or amelioration of an associated medical condition, or an increase in the rate of treatment, cure, prevention or amelioration of such a condition. When applied to an individual active ingredient administered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount may also result in an improvement in a subjective measure, when a subjective measure is used to determine the severity of a disease.

[0050] Single Domain Antibodies Single domain antibodies, also known as VHH (variable domain of the heavy chain of a heavy chain-only antibody), are antibody fragments composed of a monomeric variable single antibody domain and lacking the fragment crystallizable (Fc) region. In addition to lacking the Fc region, VHHs have unique properties such as high target specificity, affinity in the nanomolar (sub-nanomolar) range, high stability, and ease of production in both mammalian and E. coli cells. These properties may make them superior to conventional antibodies for imaging and radiotherapy purposes.

[0051] Figure 1A shows a schematic diagram illustrating the structural difference between conventional immunoglobulin (IgG), a type of antibody, and camelid IgG. The Fab fragment of conventional IgG consists of both heavy and light chains, whereas camelid IgG consists only of heavy chains and has no light chains. As shown in Figure 1A, this Fab fragment containing only the heavy chain of camelid IgG represents a single domain antibody (VHH).

[0052] According to one or more embodiments, the single domain antibodies (VHHs) of the present application selectively bind with high affinity (sub-nanomolar range) to a) the CD16a activating receptor on the surface of natural killer (NK) cells, or b) antigens associated with cancer cells, bacteria, parasites, or viruses. Most antibodies bind with low micromolar (10 -6 ) to nanomoles (10 -7 ~10 -9 ) range of K D High affinity antibodies generally have a binding affinity in the low nanomolar range (10 -9 ), whereas very high affinity antibodies are thought to be at picomolar (10 -12 ) range. D Values ​​generally correspond to molar concentrations (sensitivity) as follows: KD value Molar concentration (sensitivity) 10 -4 ~10 -6 Micromolar (μM) 10 -7 ~10 -9 Nanomolar (nM) 10-10 ~10 -12 Picomolar concentration (pM) 10 -13 ~10 -15 Femtomole (fM)

[0053] According to one or more embodiments of the present application, antibodies with a binding affinity of 100 nM or less are considered "high affinity," antibodies with a binding affinity of 100-500 nM are considered "medium affinity," and antibodies with a binding affinity of greater than 500 nM are considered "medium-low affinity."

[0054] For example, in one or more embodiments, the binding affinity of the single domain antibodies of the present application to CD16a or an antigen associated with a cancer cell, bacteria, parasite, or virus is up to 50 nM. In at least one embodiment, the binding affinity of the single domain antibodies of the present application to CD16a or an antigen associated with a cancer cell, bacteria, or virus is in the range of 750 pM to 850 pM, 850 pM to 950 pM, 950 pM to 1 nM, 1 nM to 1.2 nM, 1.2 nM to 1.4 nM, 1.4 nM to 1.6 nM, 1.6 nM to 1.8 nM, 1.8 nM to 2.0 nM, 2.0 nM to 2.2 nM, 2.4 nM to 2.6 nM, 2.6 nM to 2.8 nM, or 2.8 nM to 3.0 nM. In one or more embodiments, the binding affinity of the single domain antibodies of the present application to CD16a or an antigen associated with a cancer cell, a bacteria, a parasite, or a virus is in the range of 3.0 nM to 5.0 nM, 5.0 nM to 10 nM, 10 nM to 15 nM, 15 nM to 20 nM, 20 nM to 25 nM, 25 nM to 30 nM, 30 nM to 35 nM, 35 nM to 40 nM, 40 nM to 45 nM, or 45 nM to 50 nM. Thus, the single domain antibodies of the present application are considered to be "high affinity" antibodies.

[0055] The ability of NK cells to selectively bind to the CD16a receptor and / or other antigens on target cells enhances the ability of NK cells to kill target cells through a mechanism known as antibody-dependent cellular cytotoxicity (ADCC). Through ADCC, NK cells can actively lyse targets (e.g., target cells) whose membrane surface antigens have been bound by specific antibodies.

[0056] The ability of macrophages to selectively bind to the CD16a receptor and / or other antigens on target cells enhances the ability of macrophages to kill target cells through a mechanism known as antibody-dependent cellular phagocytosis (ADCP), through which macrophages can actively lyse targets (e.g., target cells) whose membrane surface antigens have been bound by specific antibodies.

[0057] Furthermore, in one or more embodiments, the single domain antibodies of the present application selectively bind to the CD16a activating receptor on NK cells, but do not cross-react with CD16b-NA1 or CD32b. The specificity of single domain antibodies to CD16a plays an important role in increasing therapeutic efficacy and reducing off-target toxicity. In contrast, other antibodies that bind to the CD16b activating receptor (expressed on neutrophils) have been shown to limit the ADCC activity of NK cells against target cells (e.g., cancer cells, bacteria, parasites, viruses). Furthermore, non-specific binding of antibodies to the inhibitory CD32b receptor (expressed on B cells) has also been shown to inhibit B cell maturation and macrophage activation. CD32b is also expressed on a subset of CD8+ T cells, where it may limit T cell survival by activating the caspase 3 and 7 apoptotic pathways.

[0058] In one or more embodiments, the high affinity and high specificity single domain antibodies of the present application, including single domain antibodies that selectively bind to CD16a ("anti-CD16a VHH"), may be used in flow cytometry, ELISA, and imaging of tumor-infiltrating NK cells.

[0059] In one or more embodiments, the single domain antibodies of the present application exhibit binding affinity and specificity for antigens associated with cancer cells. For example, in at least one exemplary embodiment, the single domain antibodies exhibit affinity for HER2 (i.e., anti-HER2 VHHs), facilitating recognition of HER2-expressing cancer cells. In one or more embodiments, the present application also discloses a method for identifying HER2-expressing cancer lesions in a subject by tumor imaging methods using the single domain antibodies of the present application. HER2-expressing cancers (HER2+ cancers) are aggressive and are associated with metastasis. Thus, the anti-HER2 single domain antibodies and associated methods of the present application provide a reliable approach for identifying such cancers and distinguishing them from HER2-non-expressing lesions. The VHHs and methods of the present application may be used to identify HER2-expressing tumors, such as ovarian tumors, and their metastatic sites, e.g., lung metastatic sites. Since lung metastasis is the second most common site of metastasis in ovarian cancer (e.g., HER2+ ovarian cancer), a reliable quantitative diagnostic approach for biological characterization of such metastatic sites is clinically valuable.

[0060] According to one or more embodiments, the single domain antibodies of the present application may exhibit binding affinity and specificity for one or more other antigens associated with cancer cells. {1} For example, in one or more embodiments, the single domain antibodies may be directed to HER1 (anti-HER1 antibody), HER3 (anti-HER3 antibody), HER4 (anti-HER4 antibody), EGFR (anti-EGFR antibody), VEGFR (anti-VEGFR antibody), CD47 (anti-CD47 antibody), FGFR (anti-FGFR antibody), carcinoembryonic antigen (CEA) (anti-CEA antibody), bladder tumor antigen (BTA) (anti-BTA antibody), CA125 (anti-CA125 antibody), PDGFR (anti-PDGFR antibody), IGFR (anti-IGFR antibody), CA15-3 / CA27.29 (anti-CA15-3 / CA27.29 antibody), CA19-9 (anti-CA19-9 antibody), CA27.29 (anti-CA27.29 antibody), programmed cell death ligand 1 (PD-L1) (anti-PD-L1 antibody), PD-L2 (anti-PD-L2 antibody) CTL4 (anti-CTL4 antibody), CD3 (anti-CD3 antibody), C D19 (anti-CD19 antibody), CD20 (anti-CD20 antibody), CD22 (anti-CD22 antibody), CD25 (anti-CD25 antibody), CD27 (anti-CD27 antibody), CD30 (anti-CD30 antibody), CD33 (anti-CD33 antibody), CD37 (anti-CD37 antibody), CD38 (anti-CD38 antibody), CD40 (anti-CD40 antibody), CD48 (anti-CD48 antibody), CD52 (anti- CD52 antibody), B7-H3 (anti-B7-H3 antibody), TIM-3 (anti-TIM-3 antibody), LAG-3 (anti-LAG-3 antibody), V domain Ig suppressor of T cell activation (VIS TA) (anti-VISTA antibody), HVEM (anti-HVEM antibody), ICOS (anti-ICOS antibody), 4-1BB (anti-4-1BB antibody), OX40 (anti-OX40 antibody), RANKL (anti-RANKL antibody) and GITR (anti-GITR antibody), epithelial and mesenchymal markers of circulating tumor cells, prostatic acid phosphatase (PAP) (anti-PAP antibody), prostate specific antigen (PSA) (anti-PSA antibody), soluble mesothelin-related peptide (SMRP) (anti-SMRP antibody), somatostatin receptor (SR) (anti-SR antibody), urokinase plasminogen activator (uPA) (anti-uPA antibody), plasminogen activator inhibitor (PAI-1) (anti-PAI-1 antibody), TCR (e.g., MHC class I or class II molecules) (anti-MHC The antibody can exhibit affinity and specificity for cancer-associated antigens, including, but not limited to, A2a receptor (anti-A2aR antibody), MICA family (anti-MICA / B antibody), RAET1 / ULBP family (anti-RAET1 / ULBP antibody), and HLA-E (anti-HLA-E antibody).

[0061] According to one or more embodiments, the single domain antibodies of the present application are directed to glioma-associated antigens, carcinoembryonic antigen (CEA), beta human chorionic gonadotropin, thyroglobulin, RAGE-1, MN-CAIX, RU1, RU2 (AS), intestinal carboxylesterase, mut The present invention can exhibit binding affinity and specificity for other antigens associated with cancer cells, including, but not limited to, hsp70-2, M-CSF, prostase, prostate specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, prostein, PSMA, prostate cancer tumor antigen-1 (PCTA-1), MART-1, MAGE, TRP-1, TRP-2BAGE, GAGE-1, GAGE-2, RAGE, pl5, ELF2M, neutrophil elastase, ephrin B2, IGF-I receptor, mesothelin, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, and other protein-based antigens, including TSP-180, pl85erbB2, pl80erbB-3, c-met, nm-23HI, TAG-72, CA 19-9, CA72-4, CAM17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, β-HCG, BCA225, BTAA, CA 15-3CA27.29BCAA, CA195, CA242, CA-50, CAM43, CD68P1, CO-029, G250, Ga733, EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, TPS, and SIRP-α receptor.

[0062] According to one or more embodiments, the single domain antibodies of the present application may exhibit binding affinity and specificity for one or more antigens associated with bacteria, for example, single domain antibodies may exhibit affinity and specificity for bacteria-associated antigens, including but not limited to polysaccharide or peptide antigens associated with P. aeruginosa, S. aureus, Clostridium difficile, Acinetobacter baumannii, STEC infections, Bacillus anthracis, Yersinia pestis, Francisella tularensis, Coxiella burnetii, and Klebsiella pneumonia.

[0063] According to one or more embodiments, the single domain antibodies of the present application may exhibit binding affinity and specificity for one or more antigens associated with a virus, for example, the single domain antibodies may exhibit affinity and specificity for virus-associated antigens, including, but not limited to, Epstein-Barr virus antigen EBVA, human papillomavirus (HPV) antigens E6 and E7, coronavirus surface antigens, influenza virus surface antigens, smallpox virus, viral hemorrhagic fever, and HIV surface antigens.

[0064] According to one or more embodiments, the single domain antibodies of the present application may exhibit binding affinity and specificity for one or more antigens associated with parasites, for example, single domain antibodies may exhibit affinity and specificity for parasite-associated antigens, including but not limited to antigens associated with malaria, leishmaniasis, Chagas disease, toxoplasmosis, schistosomiasis, cysticercosis, and strongyloidiasis.

[0065] In one or more embodiments, these high affinity and high specificity single domain antibodies of the present application, including single domain antibodies (e.g., anti-HER2 VHHs) that exhibit specificity and high affinity for antigens associated with cancer cells, bacteria or viruses, can be used in flow cytometry, ELISA, immunohistochemistry, cell imaging, and ex-vivo cancer phenotyping methods.

[0066] In one or more embodiments, the single domain antibody of the present application may be a human antibody, a humanized antibody, or a chimeric antibody. For example, in one or more embodiments, the single domain antibody may be humanized by the method described in Cecile Vincke et al., THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL.284, NO.5, pp.3273-3284, January 30, 2009, the contents of which are incorporated herein by reference in their entirety. More specifically, in one or more embodiments, the amino acids of the single domain antibody may be humanized in framework regions 1, 3, and 4 (i.e., outside of framework 2 region, positions 42, 49, 50, and 52), generally with neutral or minimal impact on the properties of the single domain antibody. In contrast, certain framework 2 region humanization substitutions, namely Phe-42->Val and Gly / Ala-52->Trp, should generally be avoided during humanization since they may adversely affect antigen affinity due to rearrangements of the H3 loop. However, it should be noted that Glu-49->Gly and Arg-50->Leu substitutions in the framework 2 region may actually increase the stability of the autonomous domain, but may result in reduced solubility.

[0067] Furthermore, in one or more embodiments, amino acids of the VHHs and constructs of the present application can be substituted with homologous amino acids (e.g., polar and non-polar amino acids, hydrophobic and hydrophilic amino acids, positively and negatively charged amino acids, and aromatic amino acids) such that the VHHs and constructs have substantially equivalent biological activity. Furthermore, to maintain substantially equivalent biological activity, preferably amino acids within the functional domains of the VHHs and constructs of the present disclosure are conserved.

[0068] Single domain antibody exhibiting affinity for CD16a (anti-CD16a VHH) According to at least one embodiment of the present application, a single domain antibody (VHH) is provided that exhibits high affinity and specificity for the CD16a receptor and negligible binding to the CD16b-NA1 and CD32b receptors (referred to herein as "anti-CD16a VHH"). In general, VHHs have no light chain, no Fc region, high target specificity, affinity in the nanomolar range (or less), high stability, small size (approximately 15 kDa), and low immunogenicity / toxicity, and can be easily produced in both mammalian and E. coli cells (7-9). Based on these unique characteristics, according to one or more embodiments, the exemplary VHH of the present application was produced by immunizing llamas with recombinant CD16a protein (rCD16a). Then, by using phage display, VHH clones with high affinity and specificity for CD16a and no cross-reactivity with CD16b (e.g., CD16b-NA1) or CD32b were isolated. VHHs can be produced using a variety of materials, as illustrated in the Examples section below (see Examples section 1).

[0069] In one or more embodiments, the single domain antibody may comprise at least one of the amino acid sequences of C1 (SEQ ID NO: 14) and E3 (SEQ ID NO: 15), which exhibit affinity and specificity for CD16a. These amino acid sequences are shown in Table 1 below. [Table 1]

[0070] In one or more embodiments, the anti-CD16a VHH of the present application comprises an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to framework regions 1, 2, 3, and / or 4 of SEQ ID NOs: 14-15, while exhibiting desired binding and functional properties.

[0071] Single domain antibodies that exhibit affinity for cancer cells, bacteria, parasites, or viruses According to at least one embodiment of the present application, single domain antibodies (VHHs) are provided that exhibit high affinity and specificity for antigens associated with cancer cells, bacteria, parasites, or viruses. In one or more embodiments, VHHs can be produced by immunizing camelids (e.g., camels, llamas) with selected antigens associated with a particular type of cancer cell, bacteria, parasite, or virus. Phage display can then be used to isolate VHH clones with high affinity and specificity for the selected antigen. Phage display can be performed, for example, as described in Els Pardon, et al., "A general protocol for the generation of Nanobodies for structural biology", Nat Protoc. 2014 Mar;9(3):674-93.doi:10.1038 / nprot.2014.039, and Kristian Daniel Ralph Roth, et al., "Developing Recombinant Antibodies by Phage Display Against Infectious Diseases and Toxins for Diagnostics and Therapy", Front Cell Infect Microbiol. 2021 Jul 7;11:697876.doi:10.3389 / fcimb.2021.697876, both of which are incorporated herein by reference in their entirety. VHHs can be produced using a variety of materials, as illustrated in the Examples section below (see Examples section 1).

[0072] Single domain antibody with affinity for HER2 (anti-HER2 VHH) According to one or more embodiments, the single domain antibodies (VHHs) of the present application can selectively bind to HER2-expressing cancer cells (referred to herein as "anti-HER2 VHHs"). To address current unmet needs, the single domain antibodies of the present application were produced that exhibit high affinity and specificity for HER2, while exhibiting negligible binding to HER1, HER3, and HER4. According to one or more embodiments, the anti-HER2 VHHs of the present application were produced by immunizing llamas with recombinant HER2 protein. Then, VHH clones with high affinity and specificity for HER2 were isolated by using phage display.

[0073] For example, in one or more embodiments, the anti-HER2 VHH of the present application can be utilized in a method for identifying HER2-expressing cancer lesions in a subject via magnetic resonance imaging (MRI), PET / CT imaging, single photon emission computed tomography (SPECT), or any other radioactive or non-radioactive tracer. Specifically, according to at least one embodiment, a method for identifying HER2-expressing cancer lesions in a subject via MRI and / or PET / CT includes administering to the subject an imaging agent comprising an anti-HER2 VHH, performing MRI and / or PET / CT on the subject to generate an image (e.g., an MRI and / or PET / CT image), and identifying HER2-expressing cancer lesions in the MRI and / or PET / CT image. In a method utilizing MRI imaging, imaging software such as, for example, VivoQuant™ software (ASPECT Imaging) can be used to identify HER2-expressing cancer cells in the MRI image. In methods utilizing PET / SPECT / CT imaging, imaging software such as, for example, Bruker's PMOD software Albira Imaging System may be used to identify HER2-expressing cancer cells in PET / SPECT / CT images.

[0074] In one or more embodiments, the anti-HER2 VHH facilitates the recognition of various types of HER2-expressing cancer cells, such as HER2-expressing ovarian cancer cells (e.g., HER2-expressing ovarian cancer cells from metastatic lesions), breast cancer cells, cervical cancer cells, bladder cancer cells, gallbladder cancer cells, testicular cancer cells, lung cancer, gastric cancer, esophageal cancer, and / or uterine cancer cells.

[0075] In one or more embodiments, the anti-HER2 VHH can be produced using a variety of materials, as illustrated in the Examples section below (see Examples section 2). In one or more embodiments, the anti-HER2 VHH can comprise at least one of the amino acid sequences in Table 2 below. [Table 2] TIFF2025508298000004.tif222159

[0076] In one or more embodiments, the anti-HER2 VHH of the present application comprises an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to framework regions 1, 2, 3, and / or 4 of SEQ ID NOs: 3-11, while exhibiting desired binding and functional properties.

[0077] Constructs Containing Single Domain Antibodies According to one or more embodiments of the present application, there is provided a construct comprising two or more single domain antibodies of the present application, in one or more embodiments, the construct comprises an anti-CD16a single domain antibody ("anti-CD16a VHH") as described above and at least one other single domain antibody ("anti-antigen VHH") that exhibits binding affinity and specificity for an antigen associated with a cancer cell, a bacterium, a parasite, or a virus.

[0078] More specifically, in one or more embodiments, the construct comprises a first single domain antibody having an amino acid sequence that exhibits specificity and high affinity for the CD16a receptor on the surface of natural killer (NK) cells without cross-reacting with CD16b (e.g., CD16b-NA1) or CD32b. In one or more embodiments, the amino acid sequence that exhibits specificity and high affinity for the CD16a receptor without cross-reacting with CD16b (e.g., CD16b-NA1) or CD32b is SEQ ID NO: 14. In at least one embodiment, the amino acid sequence that exhibits specificity and high affinity for the CD16a receptor without cross-reacting with CD16b (e.g., CD16b-NA1) or CD32b is SEQ ID NO: 15. The construct also comprises at least one second single domain antibody having an amino acid sequence that exhibits affinity and specificity for an antigen associated with cancer cells, bacteria, or viruses (anti-antigen VHH). The first and second single domain antibodies are fused to each other with or without a linker. In embodiments where the single domain antibodies are fused together via a linker, the linker can be, for example, a human muscle aldolase (HMA) linker. Exemplary HMA linkers are shown in Table 3 below. In at least one embodiment, the linker can be a (GGGSG)n-based linker (n=1, 2, 3, 4, ...) or any other flexible, semi-flexible, or rigid linker. In one or more embodiments where a first single domain antibody and an additional single domain antibody are fused without a linker, the single domain antibodies can be recombinantly fused sequentially in tandem.

[0079] Schematic diagrams of an exemplary construct featuring a first single domain antibody (anti-CD16a VHH) and a second single domain antibody (anti-antigen VHH) joined by a linker are shown in Figures 1B-1C. As shown in Figure 1C, the anti-CD16a VHH binds to the CD16a receptor on the surface of NK cells, and the anti-antigen VHH binds to a surface antigen on the surface of a target (e.g., cancer cells, bacterial cells, viruses). Thus, this construct not only activates NK cells through binding of the anti-CD16a VHH, but also facilitates recognition of target cancer cells by NK cells by binding of the anti-antigen VHH to the target's surface antigen.

[0080] According to one or more embodiments, the construct may comprise multiple anti-CD16a VHHs fused to each other and to another single domain antibody having an amino acid sequence that exhibits affinity and specificity for an antigen associated with a cancer cell, a bacterium, or a virus. A construct comprising multiple CD16a VHHs may activate one or more NK cells through binding and facilitate recognition of targets (e.g., cancer cells, bacterial cells, parasites, viruses) by the NK cells. Figure ID is a schematic diagram of various configurations of constructs with one or more anti-CD16a VHHs linked to one or more anti-antigen VHHs via a linker.

[0081] In some embodiments, two identical VHHs are fused to each other, one of which is further fused to a different VHH. In some embodiments, two different VHHs are fused to each other, which can optionally be further fused to one or more other VHHs. For example, in a construct comprising two fused VHHs, one VHH can have affinity and specificity for a first immune cell and the other VHH can have affinity and specificity for a second immune cell. Similarly, in one or more embodiments, the first VHH of the construct can have affinity and specificity for a first tumor cell and the second VHH can have affinity and specificity for a second tumor cell. Furthermore, in one or more embodiments, the first VHH of the construct can have affinity and specificity for an immune cell and the second VHH of the construct can have affinity and specificity for, but not limited to, a microorganism, an infected cell, a tumor cell, an inflammatory cell, an apoptotic cell, or a foreign cell. In one or more embodiments, as illustrated in FIG. ID, one or more anti-CD16a VHHs can bind to one or more VHHs ("anti-antigen VHHs") that have affinity and specificity for a microorganism, an infected cell, a tumor cell, an inflammatory cell, an apoptotic cell, or a foreign cell, where the one or more anti-antigens are in the form of a VHH, scFv, mAb, or any other antibody. Also illustrated in FIG. ID, in one or more embodiments, two or more anti-CD16a VHHs can be engineered in tandem to recognize two or more different epitopes on the CD16a antigen. Similarly, two or more anti-antigen VHHs can be engineered in tandem to recognize two or more different epitopes on the target antigen. The VHHs can be specific for the same or different antigens associated with a microorganism, an infected cell, a tumor cell, an inflammatory cell, an apoptotic cell, or a foreign cell.

[0082] In one or more embodiments, the VHH constructs can be produced by the methods described in Ulrich Brinkmann & Roland E. Kontermann (2017), "The making of bispecific antibodies", mAbs, 9:2, pp.182-212, DOI:10.1080 / 19420862.2016.1268307, the contents of which are incorporated herein by reference in their entirety. For example, in one or more embodiments, domains of two or more single domain antibodies can be fused to create a construct molecule, which can be a bivalent, trivalent, or multivalent molecule with one or more specificities (i.e., monospecific, bispecific, trispecific, tetraspecific, multispecific). The terms "bivalent", "trivalent", and "multivalent" refer to the presence of two binding sites, three binding sites, and multiple binding sites, respectively, in an antigen-binding antibody molecule.

[0083] For example, in one or more embodiments, two VHH domains can be fused with a long hinge sequence derived from the upper hinge of llama IgG2a to form a bispecific construct. A flexible linker, such as a shark immunoglobulin neoantigen receptor (VNAR) linker, can be used to combine the two variable domains. In an exemplary embodiment, the linker can comprise the natural shark IgNAR hinge (PGVQPSP (SEQ ID NO: 16)) followed by a GGGGSG (SEQ ID NO: 17) flexible sequence. Other possible linkers include a flexible (G3S)4 linker, a flexible GGGGSGGGS (SEQ ID NO: 18) linker, an HMA linker (see Table 3 below), a (GGGSG)n-based linker (n=1, 2, 3, ...) (GGGSG (SEQ ID NO: 19), or any other flexible, semi-flexible, or rigid linker.

[0084] In at least one embodiment, the construct comprises two VHHs (anti-CD16a VHH and anti-HER2 VHH) fused together with or without a linker to generate a bispecific killer cell engager ("BiKE", specifically "BiKE:HER2 / CD16a") with specificity and affinity for CD16a receptor and HER2, and no cross-reactivity with CD16b (e.g., CD16b-NA1) or CD32b. Thus, BiKE:HER2 / CD16a binds to the CD16a receptor on the surface of NK cells and HER2-expressing cancer cells, thereby activating the NK cells and facilitating the recognition of target HER2+ cancer cells by the NK cells.

[0085] Examples in this application utilizing "BiKE" generally refer to a BiKE:HER2 / CD16a construct, however, according to other embodiments of this application, it is understood that the "BiKE" construct may generally include an anti-CD16a VHH and another single domain antibody (anti-antigen VHH) having an amino acid sequence that exhibits affinity and specificity for a different antigen associated with cancer cells, bacteria, or viruses (Figure 1C).

[0086] In one or more embodiments, the single domain antibodies or constructs comprising single domain antibodies may be administered alone or in combination with other types of therapeutic agents, such as cancer therapeutics, antibiotics, antiparasitic agents, antiviral agents, etc. The single domain antibodies or single domain antibody constructs of the present application may be administered simultaneously or in tandem with other types of therapeutic agents.

[0087] In one or more embodiments, the single domain antibody or construct comprising the single domain antibody may be administered in combination with large molecule drugs (biologics) and / or small molecule drugs and / or cell-based therapeutics for the treatment of cancer. Examples of such drugs include, but are not limited to, anti-HER2 antibodies (such as trastuzumab, pertuzumab, enhertu), anti-ER antibodies, anti-PR antibodies, anti-PDL-1 antibodies, anti-PD-1 antibodies (such as Keytruda), anti-CTL4 antibodies, anti-CD47 antibodies, anti-CD19 antibodies, cisplatin, paclitaxel, irinotecan, 5-FU, tisagenlecleucel, axicabtagene silorucel, brexcabtagene autorucel, and lysocabtagene maralucel, among others.

[0088] In one or more embodiments, the single domain antibody or construct comprising the single domain antibody may be administered in combination with large molecule drugs (biologics) and / or small molecule drugs and / or cell-based therapeutics for the treatment of bacterial infections. Examples of such drugs include, but are not limited to, bezlotoxumab, raxibacumab, obiltoxaximab, subratoxumab, and commonly used antibiotics (penicillins, macrolides, cephalosporins, etc.).

[0089] In one or more embodiments, the single domain antibody or construct comprising the single domain antibody may be administered in combination with large molecule drugs (biologics) and / or small molecule drugs and / or cell-based therapeutics for the treatment of parasitic infections, examples of such drugs include, but are not limited to, metronidazole, tinidazole, and ivermectin.

[0090] In one or more embodiments, the single domain antibody or construct comprising the single domain antibody may be administered in combination with large molecule drugs (biologics) and / or small molecule drugs and / or cell-based therapeutics for the treatment of viral infections. Examples of such drugs include, but are not limited to, remdesivir, baricitinib, palivizumab, zanamivir, peramivir, oseltamivir, baloxavir marboxil, etc.

[0091] The single domain antibody or construct is substantially purified (e.g., substantially free of substances that limit its effectiveness or cause undesirable side effects). Various delivery systems are known and can be surface-decorated with the antibody of the present application, such as liposomes, microparticles, microcapsules, engineered cells, viruses, or other vectors capable of expressing the antibody (see, e.g., Wu, et al., J Biol Chem 262:4429 (1987)).

[0092] The single domain antibody or construct may be administered to a mammal by any acceptable method. Methods of introduction include, but are not limited to, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, epidural, inhalation, and oral routes, including intralesional administration if immunosuppressive treatment is desired. Parenteral injections include intramuscular, intradermal, intravenous, intraarterial, or intraperitoneal administration. The single domain antibody or construct or composition may be administered by any convenient route, such as, for example, infusion or bolus injection, absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may also be administered together with other biologically active agents. Administration may be systemic or local. Additionally, it may be desirable to introduce the therapeutic single domain antibody or construct or composition of the present application into the central nervous system by any suitable route, such as intraventricular and intrathecal injection: intraventricular injection may be facilitated, for example, by an intraventricular catheter attached to a reservoir, such as an Ommaya reservoir. Moreover, the single domain antibody or construct is suitably administered by pulse infusion, particularly with declining doses of the single domain antibody or construct. Preferably, dosing is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.

[0093] Pulmonary administration can also be used, for example, by use of an inhaler or nebulizer and formulation with an aerosolizing agent. Single domain antibodies or constructs can also be administered to the lungs of a patient in the form of a dry powder composition (see, for example, U.S. Patent Application Publication No. 6,514,496).

[0094] In certain embodiments, it may be desirable to administer the therapeutic single domain antibodies or constructs or compositions of the present application locally to the area in need of treatment; this may be accomplished, for example, but not limited to, by local infusion, topical application, injection, using a catheter, using a suppository, or using an implant (made of porous, non-porous, or gelatinous materials, including membranes such as silicone membranes or fibers). Preferably, when administering the single domain antibodies or constructs of the present application, care should be taken to use materials to which the protein does not absorb.

[0095] In another embodiment, the single domain antibody or construct may be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527 (1990); Treat, et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein, et al., eds., pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-27; see generally ibid.).

[0096] In yet another embodiment, the single domain antibody or construct can be delivered in a controlled release system, in one embodiment, a pump may be used (see Langer, Science 249:1527 (1990); Sefton, CRC Crit Ref Biomed Eng 14:201 (1987); Buchwald, et al., Surgery 88:507 (1980); Saudek, et al., N Engl J Med 321:574 (1989)). In another embodiment, polymeric materials may be used (see Medical Applications of Controlled Release, Langer, et al., eds., CRC Press (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen, et al., eds., Wiley (1984); Ranger, et al., J Macromol Sci Rev Macromol Chem 23:61 (1983); see also Levy, et al., Science 228:190 (1985); During, et al., Ann Neurol 25:351 (1989); Howard, et al., J Neurosurg 71:105 (1989)). In yet another embodiment, the controlled release system can be placed in proximity to the therapeutic target.

[0097] The present application also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a single domain antibody or construct and a physiologically acceptable carrier. In certain embodiments, the term "physiologically acceptable" means approved by a federal or state regulatory agency for use in animals, and more specifically in humans, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle used in administering a therapeutic agent. Such physiological carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like. The compositions can also contain small amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The compositions can be formulated as suppositories with conventional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. Such compositions contain an effective amount of the antibody, preferably in purified form, together with a suitable amount of carrier, so as to provide a form suitable for administration to the patient. The formulation should suit the mode of administration.

[0098] In at least one embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition suitable for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also contain a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the injection site. Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example as a dry lyophilized powder or water-free concentrate in an air-tight sealed container, such as an ampoule or sachet, indicating the amount of active agent. When the composition is administered by infusion, it can be dispensed in an infusion bottle containing sterile water or sterile saline of pharmaceutical grade. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0099] In one or more embodiments, the single domain antibodies (VHHs) and constructs of the present application can be conjugated to a prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, therapeutic agent, pharmaceutical agent, or PEG. For example, the single domain antibodies can be conjugated or fused to a therapeutic agent, which can include, but is not limited to, a detectable label, such as a radioactive label, an immunomodulatory agent, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent which can be a drug or a toxin, an ultrasound enhancing agent, a non-radioactive label, or a combination thereof. The single domain antibodies can be detectably labeled by linking to a chemiluminescent compound. The presence of the chemiluminescent tagged antigen binding polypeptide is determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of suitable chemiluminescent labeling compounds include, but are not limited to, luminol, isoluminol, imidazole, acridinium salt, theromatic acridinium ester, and oxalate ester.

[0100] The application also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical composition. Such container(s) may optionally be accompanied by a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceutical or biological products, the notice indicating approval by the governmental agency of the manufacture, use, or sale for administration to humans.

[0101] Further, in one aspect of the present application, a method of treating a disease in an individual is provided, the method comprising administering to the individual an effective amount of a pharmaceutical composition comprising a single domain antibody or construct of the present application as described above. In some embodiments, the disease is cancer. In some embodiments, the cancer is breast cancer, ovarian cancer, ovarian carcinoma, renal cancer, melanoma, head and neck cancer, lung cancer, glioblastoma, prostate cancer, bladder cancer, or lymphoma. In some embodiments, the disease is a respiratory disease, an inflammatory disease, or an autoimmune disease. In some embodiments, the disease is an infectious disease caused by a microorganism, such as a virus, including an RNA virus and a DNA virus, a gram-positive bacterium, a gram-negative bacterium, a protozoan, or a fungus.

[0102] Constructs Comprising Single Domain Antibodies Exhibiting Affinity for CD16a and HER2 According to one or more embodiments, the bispecific single domain antibody construct of the present application can selectively bind to CD16a on NK cells and HER2 on HER2-expressing cancer cells (referred to herein as "BiKE:HER2 / CD16a"). Thus, in one or more embodiments, two VHHs can be fused together with or without a linker to create a BiKE with specificity and affinity for the CD16a receptor and HER2 without cross-reacting with CD16b (e.g., CD16b-NA1) or CD32b. In one or more embodiments, the linker can be an HMA linker, for example as shown in Table 3 below. In one or more embodiments, the linker can be a (GGGSG)n-based linker (n=1, 2, 3, ...) or any other flexible, semi-flexible, or rigid linker.

[0103] In one or more embodiments, BiKE:HER2 / CD16a of the present application can be utilized to engage NK cells and kill HER2+ cancer cells. BiKE, with its specificity and affinity for the CD16a receptor and HER2, can address the aforementioned challenges and other issues related to effective killing of HER2-expressing cancer cells. In one or more embodiments, BiKE:HER2 / CD16a can include at least one of the amino acid sequences in Table 1 and at least one of the amino acid sequences in Table 2. Examples are shown in Table 3 below. [Table 3]

[0104] Assays and Methods of Use for Single Domain Antibodies In one or more embodiments, the single domain antibodies (VHH) of the present application can be used in a variety of physicochemical and biological applications.

[0105] For example, in one or more embodiments, the single domain antibody can be used in an enzyme-linked immunosorbent assay (ELISA). For example, in at least one embodiment of the present application, an ELISA is performed in which a sample containing one or more antigens (e.g., HER2 or CD16a) is immobilized on a solid support. A single domain antibody of the present application can then be applied to the surface of the sample as a primary antibody, thereby binding to the antigen(s). A secondary antibody (e.g., an anti-his tag antibody or an anti-HA tag antibody) linked to an enzyme such as HRP (horseradish peroxidase) can be used to recognize the antigen-bound VHH. In a final step, a substance containing a substrate for the enzyme is added. If there is binding by the single domain antibody to the antigen(s), the subsequent reaction produces a detectable signal (such as a color change) in the antigen.

[0106] In one or more embodiments, the single domain antibody of the present application can be used in flow cytometry.In one or more embodiments, the flow cytometry method can be used with the single domain antibody of the present invention as a primary antibody to determine the presence of one or more antigens on the cell surface.For example, in at least one embodiment, the flow cytometry method can be used with the single domain antibody of the present invention as a primary antibody to specifically detect HER2 or CD16a protein on the surface of cancer cells.

[0107] Specifically, according to one or more embodiments, a flow cytometry method is performed in which a sample containing cancer cells and anti-HER2 VHH (or NK cells and anti-CD16a VHH) is suspended in a fluid and injected into a flow cytometer device. The flow cytometry analyzer provides quantifiable data from the sample, such as whether the cancer cells are HER2+ cancer cells or the NK cells are CD16a+ NK cells. The sample can include a sample of cancer cells suspected to be HER2+ cancer cells, and an anti-HER2 single domain antibody of the present application can be applied to bind to the HER2 antigen on the cell surface. Similarly, the sample can include a sample of NK cells suspected to be CD16a+ NK cells, and an anti-CD16a single domain antibody of the present application can be applied to bind to the CD16a antigen on the cell surface. A secondary antibody (e.g., an anti-his tag antibody) linked to a fluorescent probe can then be used to bind to the single domain antibody. The sample can then be analyzed by a flow cytometer to detect expression of HER2 or CD16a. In other words, the anti-HER2 VHH in the sample is used to measure HER2 expression on the surface of the cancer cells.

[0108] The same flow cytometry methods as above can be used to detect antigens on the surface of other targets, such as bacteria, viruses, or other types of cancer cells, by substituting another target (bacteria, viruses, or other types of cancer cells) for the suspected HER2+ cancer cells in the sample, and substituting a single domain antibody specific for the other target in place of the anti-HER2 VHH. Other exemplary methods of flow cytometry of the VHHs of the present application are described in the Examples section.

[0109] In one or more embodiments, the single domain antibodies (VHHs) of the present application are used in immunohistochemistry applications, such as immunohistochemistry to identify HER2+ cancer cells in tissue. In one or more embodiments, the VHHs of the present application are also used in ex-vivo cancer phenotyping methods using histopathology. For example, in ex-vivo cancer phenotyping by immunohistochemistry, suspected tumor tissues can be cryosectioned and fixed onto tissue slides. The tissue sections can then be stained with anti-HER2 VHHs, followed by application of a fluorescently labeled secondary antibody (e.g., anti-his tag or anti-HA tag). Photomicroscopy can be performed using a microscope (e.g., Leica) to detect the presence or absence of HER2 expression in the tissue sections. The ex-vivo cancer phenotyping methods of the present invention can also be performed for other types of cancers by utilizing single domain antibodies specific for other cancer antigens (e.g., EGFR, VEGFR).

[0110] In one or more embodiments, the VHHs of the present application are used for cell imaging applications. For example, in one or more embodiments, a confocal or fluorescent microscope can be used to determine the presence of an antigen on the surface of a target cell or other target (e.g., a virus) using a single domain antibody of the present application as a primary antibody. For example, in one or more embodiments, a microscopy method can be used in which an anti-HER2 single domain antibody of the present application is used as a primary antibody to bind to the HER2 protein on the surface of a cancer cell. A secondary antibody (e.g., an anti-his tag antibody) linked to a fluorescent probe can then be used to bind to the single domain antibody. The sample can then be examined by confocal or fluorescent microscope to detect the expression of HER2 and examine internalization. In other words, the anti-HER2 VHHs in the sample are used to measure HER2 expression on the surface of the cancer cell. The same microscopy method can be performed to detect antigens on the surface of other targets, such as bacteria, viruses, or other types of cancer cells, by replacing the suspected HER2+ cancer cell sample with another sample (bacteria, viruses, or other types of cancer cells) and utilizing a single domain antibody specific for another target instead of the anti-HER2 VHH.

[0111] In one or more embodiments, the single domain antibodies (e.g., anti-HER2 VHHs) of the present application are used in in vivo cancer phenotyping applications to identify different types of cancer lesions in a subject via magnetic resonance imaging (MRI), PET / SPECT / CT imaging, or any other radioactive or non-radioactive tracer. For example, according to at least one embodiment, a method for identifying HER2-expressing cancer lesions in a subject via MRI and / or PET / SPECT / CT comprises administering to the subject an imaging agent conjugated to an anti-HER2 VHH, performing MRI and / or PET / SPECT / CT of the subject to generate images (e.g., MRI and / or PET / SPECT / CT images), and identifying HER2-expressing cancer lesions in the MRI and / or PET / SPECT / CT scans. The in vivo cancer phenotyping methods of the present invention can also be performed on other types of cancers by utilizing single domain antibodies specific for other cancer antigens (e.g., EGFR, VEGFR).

[0112] In at least one embodiment, the anti-CD16a VHH of the present application is used in an in vivo NK cell tracking method. In one or more embodiments, the labeled anti-CD16a VHH of the present application is used for in vivo NK cell tracking in a subject via magnetic resonance imaging (MRI), PET / SPECT / CT imaging, or any other radioactive or non-radioactive tracer. For example, a method for tracking allogeneic or autologous NK cells in a subject via MRI and / or PET / SPECT / CT includes administering to the subject an imaging agent conjugated to an anti-CD16a VHH, performing MRI and / or PET / CT of the subject to generate images (e.g., MRI and / or PET / SPECT / CT images), and identifying the anatomical location of the NK cells in the MRI and / or PET / CT scan. In one or more embodiments, the in vivo cell tracking method described above can also be utilized to track CD16a+ T cells, macrophages, monocytes, mast cells, and basophils.

[0113] In one or more embodiments, a construct for treating HER2-positive cancer is also provided. The construct exhibits significant anti-cancer activity against HER2-positive cancer and can be used as part of an immunotherapy regimen. In at least one embodiment, the construct can include one or more anti-HER2 single domain antibodies (VHH) fused to one or more anti-CD16a VHHs of the present application to engage NK cells and facilitate killing of HER2+ cancer cells.

[0114] Additional features and aspects of the VHHs (single domain antibodies) and VHH-containing constructs and related methods of the present application are further described in the Examples section below. It should be understood that the embodiments described in the Examples are merely illustrative and do not limit the scope of the invention.

[0115] Example - Section 1 Materials and Methods The following are materials used for the generation, isolation, and characterization of the VHHs used in the following experiments. A list of cells, antibodies, and reagents used in this study is shown in Table 4. [Table 4] TIFF2025508298000007.tif238159TIFF2025508298000008.tif240159TIFF2025508298000009.tif22159

[0116] HER2 protein expression and purification The gene encoding the extracellular domain of HER2 (Uniprot ID P04626) was designed and synthesized by VectorBuilder (IL, USA) and cloned into piggyback plasmid vector (eHER2bac) downstream of EF1α promoter. A secretion signal was designed at the N-terminus of the protein sequence and a 12XHis tag (SEQ ID NO: 2) was designed at the C-terminus to facilitate secretion of the expressed protein into the culture medium and purification by Ni-NTA chromatography (Table 5). Since human HER2 protein is highly glycosylated, FreeStyle™ 293-F cell system was selected to perform protein expression. On the day of transfection, 293-F cells were counted and transfected into a 250 mL reusable spinner flask containing fresh FreeStyle™ 293 expression medium at approximately 3x10 cells per 100 mL. 6 The cells were resuspended at a density of 1000 cells / ml and incubated at 37°C (5% CO2) for 30 min. To transfect 293-F cells, the plasmid DNA (eHER2bac) was added to PEI (10 6The cells were complexed with pDNA:PEI at a 1:4 ratio (1 μg per cell) and incubated at room temperature (RT) for 20 min. The pDNA:PEI complex was then added dropwise to the 293-F cells under constant agitation. The flask containing the transfected 293-F cells was transferred to a CO2 incubator and agitated at 90 rpm for 24 h. The next day, 2x fresh medium (200 mL) was added to the transfected cells and protein expression was allowed to continue for 8–10 days until cell viability dropped below 85% (determined by trypan blue). The cells were then harvested by centrifugation (10,000 g, 10 min, 4 °C) and the supernatant containing the protein of interest was transferred to a new tube and incubated with 300 μL of Ni-NTA resin overnight at 4 °C with shaking. The next day, the resin was loaded onto a chromatography column and washed with 20 mL of wash buffer (500 mM NaCl, 20 mM Na2HPO4, 50 mM Tris, 7.5 mM imidazole, pH 7.4). The purified HER2 protein was eluted using 500 μL of elution buffer (500 mM NaCl, 20 mM Na2HPO4, 50 mM Tris, 250 mM imidazole, pH 7.4). The purity of the eluted HER2 protein was assessed by SDS-PAGE. [Table 5]

[0117] Immunizing llamas with HER2 protein The purified HER2 protein was sent to Capralogics Inc. (Gilbertville, MA) for immunization of llamas (Figure 2). A 1.5-year-old female llama was immunized six times (every 2 weeks) using 500 μg of HER2 protein per injection. In four of the six injections, the HER2 protein was mixed with complete or incomplete Freund's adjuvant to maximize the immune response. In the fourth and sixth injections, purified HER2 was injected without any adjuvant. On day 52, 10 days after the fourth injection, 50 ml of serum was collected and an ELISA was performed to measure IgG levels in the llama serum. Once an IgG response was confirmed, immunization was continued for another 4 weeks. Five days after the last injection, 600 ml of llama blood was collected and diluted with 600 ml of DPBS supplemented with 3% FBS.

[0118] Isolation of peripheral blood mononuclear cells (PBMCs) Total PBMCs were isolated using the Ficoll (Histopaque®-1077) method. Briefly, 10 mL of Ficoll was added to a 50 ml tube. Then, 20 ml of diluted blood was dropped in such a way that the blood-Ficoll interface was not disturbed. Then, centrifugation (400 g, 20 min, room temperature) was performed with the brake off to separate red blood cells and granulocytes from the PBMCs. This process resulted in an enrichment of PBMCs in the layer between serum and Ficoll. The serum was slowly removed and the PBMCs were collected in a new tube and washed twice with DPBS supplemented with 3% FBS. The isolated PBMCs were used to generate libraries.

[0119] Creating a library Total RNA was extracted (RNeasy Mini Kit) from isolated PBMCs and cDNA was synthesized (SuperScript™ IV First-Strand Synthesis System) using oligo dT and random hexamers. VHH cDNA was amplified by nested PCR (Q5® High-Fidelity DNA polymerase) using two sets of primers (Table 6) and amplification protocol (Table 7). PCR products were cloned into pMECS-GG phagemid (kindly provided by Dr. S. Muyldermans, Belgium) using SapI restriction enzyme and T4 DNA ligase using Golden Gate cloning protocol. Recombinant phagemid was then transformed into TG1 bacteria by electroporator using 0.1 cm gap electroporation cuvette. Briefly, 5 μL of recombinant phagemid carrying VHH sequence is mixed with 50 μL of competent TG1 cells and transferred to electroporation cuvette. After 20 min on ice, electroporation was performed using voltage settings of 2.5 kV, resistance of 200 Ω, and capacitance of 25 μF. To maintain the diversity of the library, 10 vials of competent TG1 were transformed. Then, 1 ml of SOC medium was added and transferred to a 50 ml tube. After 60 min of incubation at 37 °C, the bacteria were harvested at 6,000 x g for 15 min. The pellet was transferred to six flasks containing 100 mL LB supplemented with 100 μg / mL carbenicillin. The next day, the bacteria from all six flasks were spun down and resuspended in 20 mL of fresh LB medium. After adding 15% glycerol, the library was stored at -80 °C for phage display. [Table 6] [Table 7]

[0120] Phage display To arrive at a specific VHH library, four rounds of panning were performed. Each round was performed as follows: First, approximately 2OD 600nm (1OD 600nm =2.66x10 9 The VHH library (100 cells / mL) was inoculated into 100 mL of LB broth. OD 600 When the β-actin concentration reached 0.6–0.8, 500 μL of VCSM13 helper phage was added and incubated at 37°C for 1–2 h without shaking. Then, 50 μg / ml kanamycin was added and bacterial culture was continued overnight. For phage display, 100 μL of HER2 (1 μg / mL) protein was used to coat a 96-well plate and incubated at 4°C overnight. The next day, the bacteria were pelleted by centrifugation (10,000 g, 20 min, 4°C). The supernatant was transferred to a pre-cooled sterile 50 mL tube, 10% PEG-NaCl was added, and incubated at 4°C for 1–2 h. Then, the phages were collected by centrifugation (3,000 g, 20 min, 4°C). The collected phages were washed twice using DPBS. Next, the ELISA plate was blocked using 2% skim milk buffer, and the recombinant phages were incubated in blocking buffer with shaking. Then, 100 μL of recombinant phages displaying the VHH of interest were added to the HER2-coated plate and incubated for 1 h at room temperature (RT) with shaking at 700 rpm. After 10 washes with DPBS / 0.1% Tween and 10 washes with DPBS, bound phages were enzymatically harvested using trypsin (0.25%). 10 μL of harvested phages were then incubated with 100 μL of exponentially growing TG1 bacteria at 37°C for 1 h. 100 μL of infected TG1 were then transferred to 100 mL LB medium containing 100 μg / mL carbenicillin and bacterial culture was continued overnight.

[0121] Preparation of periplasmic extracts and evaluation of affinity and specificity by ELISA After four rounds of phage display, 190 colonies were screened to find the clone with the highest affinity for HER2. First, 1 mL of LB broth supplemented with 100 μg / mL carbenicillin was added to a Deep-Well 96-well plate. To minimize the possibility of contamination and evaporation, the plate was covered by a breathable adhesive plate seal. Then, one colony was inoculated into each well and incubated at 37 °C overnight. The next day, 10 μL of the overnight culture was transferred to a new Deep-Well 96-well plate containing 1 mL of LB broth supplemented with 100 μg / mL carbenicillin and incubated at 37 °C for 4 h. Protein expression was then induced by adding 1 mM IPTG, and the bacterial culture was grown overnight at 28 °C. The next day, the plate was spun at 10,000 x g for 10 min at 4 °C, and the supernatant was discarded. To prepare periplasmic extracts, pelleted bacteria were subjected to three freeze-thaw cycles (30 min at -20°C, followed by 10 min at room temperature). Then, 500 μL of DPBS was added to each pellet and incubated for 30 min at room temperature with shaking. Cell debris was removed by centrifugation and 400 μL of supernatant was gently transferred to a new 96-well plate and stored at 4°C.

[0122] To measure affinity and specificity by ELISA, 100 μL of 1 μg / mL HER2 protein and three other members of the heregulin superfamily, including HER1, HER3, and HER4, were used to coat Nunc MaxiSorp™ high protein binding capacity 96-well ELISA plates and incubated overnight at 4° C. The next day, each well was washed three times with washing buffer (DPBS+0.1% Tween 20) and incubated with blocking buffer (2% nonfat dry milk) for 2 hours at room temperature to block free binding sites. The blocking buffer was then replaced with 100 μL of the above periplasmic extract and incubated for 1 hour at room temperature with shaking at 700 rpm. Each well was washed six times with washing buffer (DPBS+0.1% Tween 20), after which 100 μL of HRP anti-HA tag antibody (1:10000 dilution) was added and incubated for 1 hour at room temperature with shaking at 700 rpm. Each well was washed again six times, then 50 μL of 1-Step™ Turbo TMB-ELISA Substrate Solution was added and incubated at room temperature for 15 minutes (in the dark without shaking). The reaction was stopped by adding 50 μL of stop solution (0.1 mM sulfuric acid) and the plate was read at 450 nm using 630 nm as the reference (450 / 630 nm).

[0123] Expression and purification of anti-HER2 VHHs DNA sequences encoding the selected VHHs were codon-optimized, synthesized, and cloned into the pHEN6c plasmid (kindly provided by Dr. S. Muyldermans, Belgium) by GeneWiz (NJ, USA). The recombinant pHEN6c plasmid carrying the VHH sequences was transformed into WK6 E. coli using the heat shock method. 750 mL TB medium was supplemented with 0.1% glucose, 1 mM MgCl2, and 100 μg / mL carbenicillin. The medium was then inoculated with 10 mL of WK6 bacterial culture and grown at 37°C. The OD 600Protein expression was induced by 0.5 mM IPTG when the β-terminal tail reached 0.5-0.8. Protein expression was allowed to continue overnight at 28 °C with shaking at 180 rpm. The next day, the bacterial culture was centrifuged (10,000 x g, 10 min, 4 °C) to pellet the bacteria. The pellet was resuspended in 10 mL of TES buffer (200 mM Tris-HCl, pH 8.0, 500 mM sucrose, 1 mM EDTA) and incubated on ice for 1 h with shaking. Then, 15 mL of TES buffer was diluted with distilled water to a total volume of 60 mL, added to the cell suspension and incubated on ice for an additional 45 min with shaking. The bacteria were pelleted by centrifugation at 15,000 x g, 60 min, 4 °C. The supernatant was then eluted with Ni 2+ The column was loaded and washed with 30 mL of wash buffer (500 mM NaCl, 20 mM Na2HPO4, 50 mM Tris, 25 mM imidazole, pH 7.4). Finally, the protein was eluted with elution buffer (500 mM NaCl, 20 mM Na2HPO4, 50 mM Tris, 250 mM imidazole, pH 7.4). The purity of the eluted protein (VHH) was assessed by SDS-PAGE.

[0124] Assessment of the ability of VHHs to recognize HER2+ cells by using flow cytometry BT474 (Hybri-Care medium / 10% FBS), SKOV-3 (McCoy's 5A / 10% FBS), MDA-MB-231 (Leibovitz's L-15 medium / 10% FBS), and OVASC-1 (RPMI-1640 / 15% FBS / 2.5 μg / mL insulin) cancer cells were cultured in the corresponding culture media. OVASC-1 are ascites-derived epithelial ovarian cancer cells obtained from a patient with advanced metastatic disease (11,12). The VHHs expressed were HER2 + (BT474, JIMT-1, and SKOV-3) and HER2 - (MDA-MB-231) cancer cell line. 6Cells were counted and washed using DPBS-1% FBS. Trastuzumab, Perjeta, or an amount equivalent to 100 nM binding sites of VHH were then added to the cells and incubated on ice for 60 min. After washing, FITC-conjugated anti-human IgG or anti-His tag antibodies were added and incubated on ice for 60 min. Cells were washed three times and analyzed using a Beckman-Coulter Gallios flow cytometer at the Rutgers Flow Cytometry Core Facility.

[0125] Measurement of VHH binding affinity and kinetics using Biolayer Interferometry Binding affinity of VHH (K D ) and constant association rate (K on ) and dissociation rate (K offTo evaluate the activity of HER2-positive ... For the dissociation step, the wells were soaked in wash buffer (DPBS+0.05% Tween20) for 10 minutes and data was acquired (Table 8). Data was then analyzed using Octet Data Analysis HT 11.1 software. For data analysis, sensorgrams were reference subtracted and fitted to 1:1 and 2:1 binding models. Finally, affinity and kinetics were analyzed using "association and dissociation". All BLI data acquisition and analysis studies were performed at the Biophysics Core Facility in the Department of Chemistry at Princeton University. [Table 8]

[0126] Cytotoxicity assay The toxicity of the anti-HER2 VHHs, Herceptin® and Perjeta®, was evaluated using BT-474 and MDA-MB-231 cancer cells. 6Cells were seeded in 96-well plates. The next day, cells were treated with VHHs or antibodies at 1333.3 nM, 13.3 nM, 13 nM, 0.13 nM, 0.013 nM, and 0 nM. After 72 hours, the medium was removed and 100 μL of fresh medium supplemented with 10% WST-1 reagent was added. After 4 hours, the plates were read at 440 nm and 600 nm as reference wavelengths. All experiments were performed in triplicate and data are presented as mean ± standard deviation.

[0127] Evaluation of the application of anti-HER2 VHHs in cellular imaging The internalization of VHHs after binding to the HER2 receptor in SK-BR-3 cancer cells was examined using confocal microscopy. SK-BR-3 cells were plated on Nunc Lab-Tek™ chamber slides at 2.5x10 4 The cells were seeded at a density of 100 μg / well. The next day, cells were treated with 20 nM VHH or Herceptin® (Trastuzumab) and incubated on ice for 60 min. The antibody solution was then discarded and all wells were washed three times using ice-cold DPBS. The cells were fixed with 3.7% paraformaldehyde for 15 min at 37°C, followed by three steps of washing. To investigate the internalization rate at different time points, RPMI medium supplemented with 5% FBS was added to the wells and incubated for 1, 2, and 4 h at 37°C, followed by washing and fixing. The cells were then permeabilized with 0.1% TritonX100 for 15 min at RT. The cells were incubated with blocking buffer (DPBS with 2% BSA) for 2 h at room temperature. The secondary antibody was then added and incubated for 1 h at room temperature. After three steps of washing, the cells were stained with 300 nM DAPI for 3 min, followed by three washes. Finally, HER2 internalization was observed using immersion oil with a Leica TCS SP8 confocal microscope (Leica Microsystems GmbH) and a 63x objective. Z-stack images were taken at 500 nm intervals, respectively. The images of the different Z-stacks were processed by Leica software.

[0128] Results and Discussion HER2 expression and llama immunity Using standard genetic engineering techniques, HER2 antigen was expressed in HEK293 cells and purified. From the results of SDS-PAGE, the purity of the purified HER2 was estimated to be about 95% (Figure 3A). The theoretical molecular weight of HER2 (without secretion signal) is 72,633 Da, but the protein migrates closer to the 100 kDa marker. This indicates that the expressed HER2 was glycosylated. The purified HER2 was used to immunize llamas. Blood was collected 4 weeks after immunization, and then ELISA was performed, showing a significant increase in IgG levels (OD after 1 / 20,000 dilution). 450 The immunological response against the injected HER2 antigen was demonstrated by the IL-1 / IL-2 antibody (Figure 3B).

[0129] PBMC isolation, library generation, phage display, and colony screening On day 75 after immunization of the llamas, blood was collected, PBMCs were isolated, and cDNA and phagemid libraries were generated and used for phage display. After four rounds of phage display, colonies with the highest binding affinity to the HER2 antigen were selected and examined for specificity and selectivity (Figure 4A). The results of these experiments showed that the isolated VHHs were specific and selective for the HER2 antigen and had negligible interactions with HER1, HER3, HER4, BSA, and skim milk (Figure 4B).

[0130] Selection, expression and purification of VHHs All 40 HER2-binding VHHs were sequenced, analyzed, and grouped based on a phylogenetic tree. One sequence with the highest affinity and specificity for HER2 from each group was selected and constructed to have c-myc and his tags at the C-terminus (Table 9). The addition of these two tags allows the VHHs to be recognized by anti-c-myc and anti-His tag antibodies. The best performing VHHs were then expressed in WK6 E. coli and purified. Expression yields were 3-5 mg / l for all VHHs, with estimated purities of >95% (Figure 5A). [Table 9] TIFF2025508298000015.tif238159TIFF2025508298000016.tif55159

[0131] Evaluation of the application of anti-HER2 VHHs in flow cytometry In the next step, we assessed the ability of VHHs to recognize and bind to HER2 on the surface of HER2+ cancer cells. To this end, we measured HER2 expression on the surface of BT474 and SKOV-3 (HER2+) HER2+ cancer cells using purified VHHs and equimolar binding sites of FDA-approved anti-HER2 antibodies (trastuzumab and pertuzumab). - ) cancer cells were used as a negative control. Flow cytometry data demonstrated that the selected VHHs could recognize HER2 on the surface of HER2+ cancer cells, but not HER2 - It was shown that HER2 on the surface of cancer cells could not be recognized (Figures 5B-5E). The total mean fluorescence intensity (MFI) of cells labeled with trastuzumab and pertuzumab appeared to be higher than VHH. The main reason is that the secondary antibodies used to detect trastuzumab and pertuzumab were polyclonal antibodies against light and heavy chains (one antibody / multiple labels), which resulted in a significant signal boost (Table 10). In contrast, the secondary antibodies used to detect the His tag in VHH were monoclonal (one VHH / one label), which generated a lower signal intensity. Nevertheless, the HER2 binding level by VHH, measured by the percentage of labeled cells, was similar to that of trastuzumab and pertuzumab (Table 10).

[0132] Overall, the flow cytometry data demonstrated that the selected VHHs selectively differentiated HER2+ cells into HER2 - These results indicate that the IL-16 receptor agonist (IL-16) can be differentiated from IL-16 cells and can be used as a reagent suitable for cell phenotyping by flow cytometry. [Table 10]

[0133] Evaluation of cytotoxicity of VHHs To examine whether the selected VHHs induce toxicity to HER2+ cells, cytotoxicity assays were performed using BT474 and SKOV-3 cancer cells. Trastuzumab and pertuzumab were used as controls. The results of this experiment revealed that E5 and A1 clones (Figures 6A-6B), similar to trastuzumab and pertuzumab, did not induce statistically significant toxicity to SKOV-3 HER2+ cancer cells even at a high concentration of 133 nM, making them useful agents for cell imaging. However, BT474 cells appeared to be slightly sensitive to E5 and E9 VHHs, with a decrease in viability of about 5% (*t-test, p<0.05) (Figures 6A-6B). This suggests the application of these two clones to antibody-drug conjugates (ADCs) and to construct bifunctional VHHs for antibody-dependent cellular cytotoxicity (ADCC).

[0134] Measurement of VHH affinity using Biolayer Interferometry (BLI) Based on the MFI data in Table 10 and the cytotoxicity data above, the E5 and A1 clones were selected as the top candidates and characterized by BLI to measure their affinity to the HER2 antigen. The BLI data showed that the A1 and E5 clones had affinities of 1.3 nM and 724 pM for HER2, respectively (Figures 7A-7B). Overall, the BLI data indicates that the affinity of the E5 and A1 clones is very high and they do not dissociate after binding to HER2.

[0135] Evaluation of the application of VHHs in cell imaging The selected VHHs were found to be non-toxic to cells and were evaluated for their use as cell imaging tools. For this purpose, SKOV-3 cells were seeded and the binding and internalization of the VHHs into the cells was tested over time by confocal microscopy. The results of this experiment showed that both A1 and E5 clones started to be internalized around 1 h, and the internalization process was completed within 3–5 h (Figure 8A–B). Published data show that trastuzumab binds and is internalized in HER2+ cancer cells around 4 h (13). These results demonstrate the application of the developed VHHs for imaging of HER2+ positive cells.

[0136] Overall, the above examples demonstrate that the current single domain antibodies, including anti-HER2 VHHs, are effective in various imaging and assay methods. HER2 is a receptor that is frequently overexpressed in various aggressive solid tumors and their metastatic lesions, making it a suitable target for preclinical and clinical trials. The present application and the above examples demonstrate that the developed anti-HER2 VHH constructs of the present application are capable of targeting HER2 - It has been demonstrated that it can bind to HER2+ cancer cells with high affinity and specificity, with negligible interaction with the cells.

[0137] The VHHs were constructed with c-myc and His tags incorporated into their sequences, making them suitable for ex-vivo cancer phenotyping, using flow cytometry, ELISA, cell imaging, and immunohistochemistry. Given that the current VHHs are highly stable and can be produced in E. coli, they represent a low-cost alternative to mAbs that are primarily produced and isolated from mammalian cells and animals.

[0138] Example - Section 2 Materials and Methods The following are materials used for the generation, isolation and characterization of the VHHs in the following examples. A list of cells, antibodies and reagents used in the following examples is provided in Table 11. [Table 11] TIFF2025508298000019.tif239159TIFF2025508298000020.tif241159TIFF2025508298000021.tif137159

[0139] Expression and purification of CD16a protein The gene encoding CD16a ectodomain (UniProt ID P08637) was synthesized by VectorBuilder (IL, USA) and cloned into a mammalian expression vector under the EF-1α promoter. The mammalian expression vector was chosen because CD16a ectodomain is a glycosylated protein. A secretion signal sequence was designed into the N-terminus of the gene and a 12xhis tag (SEQ ID NO:2) was designed into the C-terminus, which facilitates purification of the protein from the culture medium by Ni-NTA column chromatography (Table 12). To express CD16a, FreeStyle™ 293-F cells were cultured according to the manufacturer's recommendations and 4x10 5 Cells were seeded at 100 ml of FreeStyle™ 293 Expression Medium in a 250 mL reusable spinner flask at 200 x 10 cells / ml. Cells were passaged every 3 days. On the day of transfection, approximately 3x10 cells were seeded in 100 ml of FreeStyle™ 293 Expression Medium in a 250 mL reusable spinner flask. 6 The cells were seeded at 1000 cells / ml for 30 min. The CD16a expression vector was then complexed with polyethyleneimine (PEI). 6For each cell, 1 μg of plasmid was mixed with 4 μg of PEI (1:4 w / w ratio) in Opti-MEMI and incubated for 20 min at room temperature (RT). The plasmid:PEI complex was then added dropwise to the seeded cells under constant agitation. The flasks were incubated at 37 °C and 5% CO2 for 24 h. The next day, 2x fresh FreeStyle medium was added to the transfected cells. Protein expression continued for 8–10 days or until viability fell below 85%, whichever occurred first. The downstream process started with harvesting the cells using 10,000 g, 10 min, 4 °C. The supernatant was collected and filtered through a 0.45 μm filter to remove cell debris. Then, 300 μL of Ni-NTA resin was washed with equilibration buffer (500 mM NaCl, 20 mM Na2HPO4, 50 mM Tris, pH 7.4), mixed with the supernatant and incubated overnight at 4 °C. The next day, the supernatant:Ni-NTA mixture was poured into a chromatography column and washed with 20 mL of washing buffer (500 mM NaCl, 20 mM Na2HPO4, 50 mM Tris, 7.5 mM imidazole, pH 7.4). Finally, recombinant CD16a (rCD16a) protein was eluted using 500 μL of elution buffer (500 mM NaCl, 20 mM Na2HPO4, 50 mM Tris, 250 mM imidazole, pH 7.4). The purity and molecular weight of the purified protein were estimated by SDS-PAGE. [Table 12]

[0140] Immunization of llamas with CD16a protein Immunization of llamas was performed by Capralogics Inc. (Gilbertville, MA). Female llamas were immunized with rCD16a (500 μg / injection) for six injections every two weeks. To maximize the immune response, the antigen of interest was mixed with complete (first injection) or incomplete Freund's adjuvant (second, third, and fifth injections). The fourth and sixth injections were performed with rCD16a without any adjuvant. After the fourth injection, 50 mL of whole blood was collected and immunization was confirmed using an ELISA with commercially available CD16a (Acrobiosystem, CDA-H82E9-25ug). After immunization confirmation, llama immunization continued for another 10 weeks. Five days after the sixth injection, 600 mL of whole blood was collected and peripheral blood mononuclear cells (PBMCs) were isolated.

[0141] Isolation of peripheral blood mononuclear cells (PBMCs) First, whole blood was diluted 2-fold with DPBS-3% FBS. Diluting the serum significantly improves PBMC recovery. Next, PBMC were isolated using density gradient centrifugation. 35 mL of diluted serum was then gently layered on top of Ficoll (Histopaque®-1077) so as not to disrupt the interphase. The tube was then centrifuged at 400xg for 45 minutes using a swinging bucket rotor with the brake off. The upper layer was removed very gently. PBMC were then collected from the undisturbed interphase. The collected PBMC were transferred to a new 50 mL tube and immediately washed to remove Ficoll residues that are toxic to the cells.

[0142] Creating a library After harvesting and counting the cells using trypan blue exclusion, total RNA was extracted using RNeasy Mini Kit, and then cDNA was synthesized using oligo dT and random hexamers via SuperScript™ IV First-Strand Synthesis System. The generated cDNA was then used as a template for nested polymerase chain reaction (PCR) reactions using the primers in Table 13 and the PCR protocol in Table 14. The first PCR round was performed using CALL001 and CALL002. The products were run on a 1% agarose gel, and the 700 bp band was excised and purified. The first round PCR product was then applied to a second round PCR using VHH-Back and VHH-Forward. The 400 bp band from the agarose gel was excised, purified, and cloned into pMECS-GG phagemid (kindly provided by Dr. S. Muyldermans, Belgium) using the Golden Gate cloning system. Finally, the recombinant phagemids were used to transform TG1 E. coli (electroporator settings: 2.5 kV, 25 μF, 200 Ω). To maintain the diversity of the library, 10 vials of competent TG1 were transformed. Then, 1 ml of SOC medium was added and transferred to a 50 ml tube. After 60 min of incubation at 37 °C, the bacteria were harvested at 6,000 x g for 15 min. Transformants were selected in six flasks containing 100 mL Luria-Bertani (LB) supplemented with 100 μg / mL carbenicillin. The grown bacteria were spun and resuspended in 20 mL of fresh LB medium. After adding 15% glycerol, the library was stored in a -80 °C freezer. [Table 13] [Table 14]

[0143] Phage display To arrive at a specific CD16a library, four rounds of phage display were performed. First, 2OD600nm (1OD 600nm =2.66x10 9 A llama library of 10000 cells / ml was inoculated into 100 mL of LB supplemented with 100 μg / mL carbenicillin. When the OD reached 0.6, 500 μL of helper phage VCSM13 was added to the culture and incubated at 37 °C for 60 min without shaking. Then, 50 μg / mL of kanamycin was added to the culture and incubated at 37 °C overnight with shaking. The next day, the bacteria were spun at 10,000 x g for 20 min at 4 °C, and 10% PEG-NaCl was added to the supernatant and left on ice for 60–120 min. Finally, the recombinant phages were harvested by centrifugation (3,000 x g, 20 min, 4 °C) and then washed in three steps with DPBS.

[0144] The purified phages were used to bind to CD16a protein in a polyclonal phage ELISA. In this experiment, all incubations were at room temperature with a shaking speed of 700 rpm unless otherwise stated. To start panning, 100 μL of streptavidin (5 μg / mL) was coated onto a 96-well plate and left at 4°C overnight. The next day, the coated plate was washed once using DPBS and blocked using 2% nonfat dry milk. Next, 50 μL of biotinylated CD16a (1 μg / mL) was added and incubated at room temperature with shaking for 45 minutes, followed by a three-step wash using DPBS, 0.1% Tween20 (0.1% DPBS-T). Next, about 100 μL of recombinant phages was added. After that, the plate was washed 10 times with 0.1% DPBS-T to remove nonspecific VHHs, followed by a 10-step wash using DPBS. Finally, 50 μL of 0.25% trypsin was used to recover the binders. After neutralizing the trypsin with 4-(2-aminoethyl)benzene-1-sulfonyl fluoride (AEBSF), 100 μL of phage was used to infect 100 μL of TG1 for 1 h at 37° C. Then, 100 μL of infected TG1 was transferred to 100 mL LB medium containing 100 μg / mL carbenicillin, and the bacterial culture was continued overnight.

[0145] Preparation of periplasmic extracts and evaluation of affinity and specificity by ELISA To select the top binders, 190 colonies were randomly selected and cultured in 1 mL LB supplemented with 100 μg / mL carbenicillin and 2% glucose in a Deep-Well 96-well plate. To maintain oxygenation of the culture, a breathable sticky plate was used to cover the plate. The next day, 1 mL fresh LB supplemented with 100 μg / mL carbenicillin was inoculated with 10 μL of the overnight culture. After 4 hours, expression was induced using 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). Expression was continued overnight at 28°C. The next day, bacteria were harvested using centrifugation (10,000xg, 7 min, 4°C). The supernatant was discarded and the pellet was stored at -20°C. Periplasmic extracts were then prepared using three cycles of freeze-thaw (30 min at -20°C, 10 min at room temperature). Then, 500 μL of DPBS was added to the pellet and placed on a shaker for 30 min. Finally, the supernatant (400 μL) was transferred to a new Deep-Well 96-well plate after centrifugation (10,000 x g, 30 min, 4 °C). The periplasmic extracts were used in ELISA to find the top candidates.

[0146] The periplasmic extracts were used in ELISA to find the optimal binders. In this experiment, unless otherwise stated, all washes were performed with 0.1% DPBS-T, the incubation time was 60 min, incubation was performed at room temperature, and the shaking speed was 700 rpm. After each antibody, the plate was washed six times to remove all weak binders. First, the plate was coated with 100 μL of 5 μg / mL streptavidin overnight at 4°C. The next day, after one wash step, 50 μL of biotinylated CD16a and CD16b were added and incubated for 30 min. After three wash steps, 100 μL of periplasmic extracts were added. To detect bound VHHs, the secondary antibody HRP anti-HA tag antibody (1:10000 dilution) was added. For color development, 50 μL of 1-Step™ Turbo TMB-ELISA Substrate Solution was added and incubated in the dark for 15 min. After stopping the reaction, the plates were read at a wavelength of 450 nm. To narrow down the top two candidates, serial dilutions of periplasmic extracts of the top 20 VHHs were used in ELISA as described above. The top two candidates (Clone C1 and Cl E3) were selected based on the highest OD at the top dilution.

[0147] Expression and purification of selected VHHs The selected VHHs were sequenced and cloned into an expression plasmid vector. The plasmids were then chemically transformed into WK6 E. coli. First, the transformants with the highest expression were selected by Western blot. Then, 7.5 mL of overnight culture was inoculated into 750 mL of Terrific Broth (TB) supplemented with 0.1% glucose, 1 mM MgCl2, and 100 μg / mL carbenicillin. After an OD600nm of 0.6-0.8 was obtained, 0.5 mM IPTG was added and expression was initiated. The culture was then incubated overnight at 28°C. The next day, the bacterial cells were spun down using a centrifuge (10,000xg, 10 min, 4°C). The pellet was then resuspended in 10 mL of TES buffer (200 mM Tris-HCl, pH 8.0, 500 mM sucrose, 1 mM EDTA) and incubated for 1 h at 4°C with shaking. Then, 15 mL of TES / 4 buffer (10 mL of TES buffer was added to 30 mL of distilled water) was added and incubated at 4°C for 45 min with shaking. The bacterial cells were then pelleted by centrifugation (40,000xg, 4°C, 30 min). The supernatant was then loaded onto a Ni-NTA column. Non-specific proteins were washed away with 30 mL of washing buffer (500 mM NaCl, 20 mM Na2HPO4, 50 mM Tris, 25 mM imidazole, pH 7.4). The purified protein was eluted using elution buffer (500 mM NaCl, 20 mM Na2HPO4, 50 mM Tris, 250 mM imidazole, pH 7.4). The quality and quantity of the eluted protein were assessed by SDS-PAGE and BCA kit, respectively.

[0148] Assessment of VHH specificity by ELISA The purified VHHs were subjected to functional analysis using ELISA and flow cytometry. For ELISA, 50 μL of biotinylated CD16a, CD16b-NA1, CD16b-NA2, and CD32b were added to streptavidin-coated plates and incubated for 45 min at room temperature. After washing, 100 μL of 100 nM purified VHHs were added and incubated for 1 h at room temperature. Then, 100 μL of anti-HA tag HRP-conjugated secondary antibody was added. Finally, substrate was added for color development and the plate was read using an OD of 450 nm.

[0149] Evaluation of the ability of VHHs to recognize CD16a+haNK92 cells by using flow cytometry Flow cytometry was performed using haNK92 cells (CD16a+) and neutrophils (CD16b + ) were used. CD16a+haNK92 cells were purchased from ATCC and cultured in MEM-α supplemented with 12.5% ​​fetal bovine serum (FBS), 12.5% ​​horse serum, 0.2 mM myoinositol, 0.02 mM folic acid, 100 U / ml penicillin-streptomycin, 0.1 mM 2-mercaptoethanol, and 100 U / ml IL-2. Human neutrophil cells were purchased from HemaCare and cultured according to the manufacturer's protocol. First, both cell lines were harvested and washed once using DPBS supplemented with 2% FBS (2%DPBS-FBS). For each sample, 0.5x10 6 Cells were stained with antibodies for 60 minutes on ice. In this experiment, 3G8 monoclonal antibody was used as a positive control. This antibody binds both CD16a and CD16b. After three washes, a secondary antibody, FITC-labeled anti-His tag antibody, was added. After washing, samples were run on a Cytoflex using the FL2 channel.

[0150] Measurement of VHH binding affinity and kinetics using Biolayer Interferometry To evaluate the binding affinity (KD) and constant association (Kon) and dissociation (Koff) rates of VHHs, an Octet RED96e instrument (Sartorius) Biolayer Interferometer (BLI) and Octet® Streptavidin (SA) biosensor were used. In this experiment, 3G8 was used as a positive control. After soaking the sensor in DPBS for at least 10 min, biotinylated CD16a, CD16b NA1, and CD16b NA2 were loaded onto the streptavidin (SA) biosensor for 10 min. The sensor was then soaked in wash buffer (DPBS + 0.05% Tween 20) for 2 min to reach baseline. The sensor was then submerged in wells containing 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.25nM, and 0nM of purified 3G8 mAb or anti-CD16a VHH for 5 minutes (association step). Next, in the dissociation step, the wells were submerged in washing buffer (DPBS+0.05% Tween20) for 10 minutes and data was obtained (Table 15). Data was then analyzed using Octet Data Analysis HT 11.1 software. For data analysis, the sensorgrams were subtracted from the reference and fitted to 1:1 and 2:1 binding models. Finally, the affinity and kinetics were analyzed using "association and dissociation". [Table 15]

[0151] Results and Discussion Expression of rCD16a and immunization of llamas rCD16a was engineered, expressed in the 293-F mammalian expression system, and then purified. SDS-PAGE analysis of the purified protein revealed a purity of rCD16a of more than 95% (Figure 9A). The observed molecular weight is approximately 48 kDa, while the theoretical molecular weight (expected value) is 21.83 kDa. The difference between the predicted and observed molecular weight is due to five glycosylation sites on the CD16a ectodomain. Correct glycosylation is essential for protein folding and immunogenicity.

[0152] Purified rCD16a was used to immunize llamas. Blood was collected 4 weeks after immunization, and then ELISA was performed, showing a significant increase in IgG levels (OD450 of 1.6 after 1 / 50,000 dilution), indicating a strong immune response to the injected rCD16a antigen. Pre-immune serum was used as a negative control in this experiment. As can be observed, the serum of the immunized llamas had a higher titer of antibodies against rCD16a compared to pre-immunization (Figure 9B). The data indicate that the concentration of injected rCD16a was sufficient to boost the humoral immune response.

[0153] PBMC isolation, library generation, phage display, and colony screening On day 75 after immunization of the llamas, blood was collected, PBMCs were isolated, and cDNA and phagemid libraries were generated and used for phage display. After four rounds of phage display, 20 colonies were selected and lysed to obtain periplasmic extracts. The lysates with the highest binding affinity to the CD16a antigen were selected and then examined for specificity using ELISA (Figure 10A). The results of these experiments revealed that there were several VHH clones with higher binding affinity to CD16a than to the CD16b-NA1 antigen (Figure 10B). To identify high-performing VHH candidates in terms of binding affinity, 1280-fold dilutions of the periplasmic extracts were prepared and used for binding to the CD16a antigen. The ELISA results showed that the remaining VHHs, except for the A1, A2, E4, and G4 clones, had high binding affinity, while the C1 and E3 clones appeared to be the best-performing constructs (Figure 10C). Therefore, these two VHHs were selected for further studies based on their specificity and high affinity.

[0154] Expression and purification of C1 and E3 clones Phagemids encoding the C1 and E3 VHHs were transformed into WK6 E. coli and expressed in the periplasmic space (Table 16) and subsequently purified. The periplasmic space provides an opportunity for the expressed VHHs to fold properly. The purity of the purified VHHs was analyzed by SDS-PAGE. The results of this experiment showed that the purity of both VHHs was greater than 95% with a yield of 2-3 mg / l of culture medium. [Table 16]

[0155] Evaluation of specificity of purified C1 and E3 VHHs ELISA was first performed to determine the specificity of purified C1 and E3 clones to CD16a. CD16b has two dominant alleles, including human neutrophil antigen 1 (NA1) and NA2 (10), so both were used as controls. CD32b and skim milk were also used as antigen controls. Anti-CD16a / b mAbs 3G8 and eBioCB16 were used as antibody controls. Note that there are no commercially available CD16a-selected antibodies that can be used as controls. ELISA results showed that both C1 and E3 VHHs efficiently bound to the CD16a antigen without any significant interaction with the CD32b antigen (Figure 11). CD32b is a receptor expressed on B cells and plays a role in inhibiting B cell activation. One of the drawbacks of mAb-based therapy is that the Fc region binds to CD32b, thereby reducing the activity of humoral immunity. In addition to CD32b, C1 and E3 VHH did not interact with the CD16b-NA1 antigen, but did interact with the CD16b-NA2 antigen (FIG. 11).

[0156] In a next step, the specificity of C1 and E3 VHH was evaluated using flow cytometry. In this experiment, NK92 cells (CD16a + ), neutrophils (CD16b + ), and B cells (CD32b +) was used as a cell control. 3G8 (anti-CD16a / b) and AT10 (anti-CD32a / b) mAbs were used as antibody controls. The results of this experiment showed that the C1 and E3 VHHs bound effectively to NK92 cells but not to neutrophils (Figures 12A-D). Furthermore, the C1 clone did not bind to B cells (Figure 12E). In contrast, the 3G8 mAb bound to both NK92 and neutrophils, while AT10 bound indiscriminately to B cells. This result has broad clinical application, since the C1 and E3 VHHs bind only to NK cells without being shed by neutrophils (4).

[0157] Measurement of affinity of VHHs by using BLI The affinity and specificity of C1 and E3 VHHs were quantified by BLI (Figures 13A-D). The BLI data results showed that C1 and E3 VHHs have high affinity (sub-nanomolar) for CD16a, but at least 100-fold lower affinity for CD16b-NA1. Epitope mapping data by BLI also showed that C1 VHHs have a different binding site on CD16a than trastuzumab and pertuzumab (Figure 13E). Overall, the BLI, ELISA, and flow cytometry data showed that the selected C1 and E3 VHHs interacted specifically with CD16a, without significant interaction with CD16b-NA1 and CD32b.

[0158] Example: Section 3 Materials and Methods Materials used in the production and characterization of E5C1 BiKE (BiKE:HER2 / CD16a) A list of cells, antibodies, and reagents used in this study is shown in Table 17. [Table 17] TIFF2025508298000028.tif240159TIFF2025508298000029.tif72159

[0159] Expression and purification of E5C1 BiKE The E5C1 BiKE construct was synthesized and then cloned into the pET28a expression vector by GenScript (Piscataway, NJ). The plasmid was chemically transformed into SHuffle® T7 Express Competent E. coli. The highest expressing transformants were first selected by Western blot. 750 mL of Terrific Broth (TB) 100 μg / mL carbenicillin was then inoculated with 50 mL of overnight culture. The OD of 2.5-3 was reached. 600nmAfter obtaining a 1 mM IPTG was added to initiate expression. The culture was then incubated at 37°C for 7 hours. The bacteria were then spun down using a centrifuge (10,000xg, 10 min, 4°C). The pellet was stored overnight at -20°C. The next day, the pellet was resuspended using basal purification buffer (1M NaCl, 100mM KCl, 50mM Tris, 20mM phosphate buffer, 0.01% Tween20, 15mM imidazole, pH 8) at 3mL per gram of pellet and incubated at 4°C for 30 min with shaking. The bacterial suspension was then sonicated (5 sec on, 3 sec off, 70% amplitude) on ice for 30 min. The cell debris was then pelleted by centrifugation (40,000xg, 4°C, 30 min). The supernatant was then loaded onto a Ni-NTA column. Nonspecific proteins were washed away with 30 mL of wash buffer (500 mM NaCl, 100 mM KCl, 100 mM Tris, 20 mM phosphate buffer, 25 mM imidazole, pH 8). To elute E5C1 BiKE, the resin was first equilibrated with 20 mL of basal elution buffer (500 mM NaCl, 100 mM KCl, 100 mM Tris, 20 mM phosphate buffer, 7% sucrose, 0.01% Tween 20, pH 8). Finally, the protein was eluted using 20 U / mL of WELQut protease. The quality and quantity of the eluted protein were assessed by SDS-PAGE and BCA kit, respectively. The molecular weight of the purified BiKE and its monomeric state were assessed using liquid chromatography / mass spectrometry (LC-MS) at the core facility of the Rutgers Center for Advanced Biotechnology and Medicine. [Table 18]

[0160] Assessment of VHH specificity by enzyme-linked immunosorbent assay (ELISA) Purified monovalent VHHs, E5 and C1, as well as E5C1 BiKE, were subjected to functional analysis using ELISA and flow cytometry. For ELISA, 50 μL of biotinylated CD16a was added to streptavidin-coated plates and incubated for 45 min at room temperature. After washing, 100 μL of 10-fold serial dilutions of purified VHH / BiKE from 1000 nM to 0 nM were added and incubated for 1 h at room temperature. Then, 100 μL of anti-cMyc tag HRP-conjugated secondary antibody (1:10,000 dilution) was added. Finally, after washing, substrate was added for color development and the plate was read using an OD of 450 nm.

[0161] Evaluation of the ability of VHH / E5C1 BiKE to recognize CD16a+haNK92 cells using flow cytometry For flow cytometry, CD16a + haNK92 cells were purchased from ATCC and cultured in MEM-α supplemented with 12.5% ​​fetal bovine serum (FBS), 12.5% ​​horse serum, 0.2 mM myoinositol, 0.02 mM folic acid, 100 U / ml penicillin-streptomycin, 0.1 mM 2-mercaptoethanol, and 100 U / ml IL-2. First, CD16a+NK92 were harvested and washed once using Dulbecco's phosphate buffered saline (DPBS) supplemented with 2% FBS (2%DPBS-FBS). Then, all cells were fixed with 3.7% paraformaldehyde for 20 min at room temperature and then washed twice. For each sample, 0.5x10 6 Cells were stained with 10-fold serial dilutions of purified VHH / BiKE from 1000 nM to 0 nM for 60 min at RT. After 3 washes, a secondary antibody, FITC-conjugated anti-His tag antibody, was added. After washing, samples were run on a Cytoflex using the FL1 channel.

[0162] Measurement of binding affinity and kinetics of VHH and E5C1 BiKE using BLI Binding affinity of VHH and BiKE (K DTo assess the constant association rate (Kon) and dissociation rate (Koff), an Octet RED96e (Sartorius) Biolayer Interferometer (BLI) was used at the Biophysics Core Facility, Princeton University. Octet® streptavidin (SA) biosensors were immersed in DPBS supplemented with 0.1% casein for at least 10 minutes, and biotinylated CD16a, CD16b-NA1, CD16b-NA2, and HER2 antigens were loaded onto the streptavidin (SA) biosensors until a shift of 1 nm was reached. The sensors were then immersed in wash buffer (DPBS+0.05% Tween 20+0.1% casein) for 2 minutes to reach baseline. The sensors were then submerged in wells containing 60nM, 30nM, 15nM, 7.5nM, 3.75nM, 1.875nM, and 0nM of purified anti-CD16a VHH or BiKE for 5 minutes (association step). Next, in the dissociation step, the sensors were submerged in wash buffer for 5 minutes and data were acquired (Table 5). Data were then analyzed using Octet Data Analysis HT 11.1 software. For data analysis, sensorgrams were subtracted from the reference and fitted to a 1:1 binding model. Finally, the affinity and kinetics were analyzed using "association and dissociation".

[0163] Assessment of ADCC by cytotoxicity assay The ADCC induction capacity of BiKE was evaluated and compared with mAbs trastuzumab and / or pertuzumab. In this experiment, the target cancer cells (i.e., SKOV-3, BT474, and JIMT-1) were either in adherent or non-adherent state. To perform ADCC under adherent conditions, 10 4Cancer cells were seeded into tissue culture-treated 96-well plates. The next day, 100 nM or 10 pM of BiKE or mAb were added to the cells and incubated at 37°C for 30 minutes. Next, laNK92 or haNK92 cells were added to the target cells at E:T ratios of 4, 2, 1, 0.5, 0.25, and 0 and incubated at 37°C for 4 hours. Next, the wells were washed twice using DPBS to remove the NK92 cells. Finally, 10% alamarBlue™ HS Cell Viability Reagent was added to the plates and incubated at 37°C for 1-2 hours. The plates were then read using a Tecan Infinite M Plex plate reader and cell viability was calculated.

[0164] To perform ADCC in non-adherent conditions (in suspension), 5x10 3 Target cancer cells were mixed with 100 nM of mAb or BiKE and then added to a naive 96-well plate. Different numbers of haNK92 cells were then added to each well to achieve E:T ratios of 16, 8, 4, 2, 1, 0.5, and 0. The plates were incubated at 37°C for 4 hours. Then, alamarBlue™ HS Cell Viability Reagent was added and incubated at 37°C for 2-3 hours. Finally, the plates were read on a Tecan Infinite M Plex plate reader and cell viability was measured using the following formula: [(fluorescence intensity of test group - fluorescence intensity of NK cells only) / (fluorescence intensity of target cells only)] x 100

[0165] Assessment of cytokine and cytotoxic protein release from NK92 cells during ADCC SKOV-3 cells were seeded at a density of 10,000 cells / well in tissue culture treated 96 well plates. The next day, 10 nM BiKE or trastuzumab was added to the plates and incubated at 37°C for 30 min. Effector cells (laNK92 or haNK92) were then added at an E:T ratio of 4. After 2 h (perforin and granzyme B) and 24 h (TNF-α and IFN-γ), the plates were centrifuged at 2000 g for 10 min to pellet the cells. The supernatants were then transferred to untreated 96 well plates. Cytokine release was quantified using Quantikine ELISA kits and Perforin Human ELISA kits according to the manufacturer's protocol. Data are presented as mean ± SD (n=3).

[0166] Quantification of degranulation using surface-expressed CD107a (LAMP-1) First, 10 4 SKOV-3 cells were seeded in a 96-well plate and incubated overnight. The next day, serial dilutions of BiKE and trastuzumab were added in the range of 0 to 100 nM and incubated at 37°C for 30 minutes. Next, laNK92 (GFP + ) or haNK92 was added at an E:T ratio of 4 and the plate was incubated at 37°C for 2 hours. Then, Fc Blocker was added to the plate and incubated at 37°C for another hour. After that, APC-conjugated anti-CD107a antibody was added and incubated in an incubator for 1 hour. Finally, the plate was centrifuged and the supernatant was discarded. The cells were washed twice to remove excess antibody and data were acquired on a Beckman Coulter CytoFLEX cytometer. Data analysis included GFP + We gated on (laNK92) to distinguish effector cells from target cells. We then detected surface-expressed CD107a by GFP + Quantification was performed on cell populations.

[0167] Results and Discussion Construction and characterization of E5C1 BiKE Using recombinant engineering, E5C1 BiKE was constructed by fusing C1 anti-CD16a VHH and E5 anti-HER2 VHH via HMA semi-flexible linker (Figure 14A). For simplicity, this construct is shown as E5C1 BiKE. SDS-PAGE data showed that the purified E5C1BiKE was more than 95% pure, and LC-MS graphs showed that the purified BiKE did not contain dimers or multimers (Figures 14B-C). Next, the binding of E5C1 BiKE to CD16a and HER2 antigens was evaluated by ELISA and flow cytometry. C1 anti-CD16a VHH and E5 anti-HER2 VHH were used as controls. Statistical analysis of the data (ELISA and flow cytometry) showed that the affinity of E5C1 BiKE for CD16a and HER2 antigens remained intact and that the fusion of the two VHHs via the HMA linker did not negatively affect binding to CD16a and HER2 antigens (t-test, p>0.05) (Figures 15A-D).

[0168] Measurement of binding affinity of E5C1 BiKE by using BLI The affinity and specificity of E5C1 BiKE were measured by BLI. The results of this experiment showed that E5C1 BiKE not only retained high affinity for CD16a, but also low affinity for CD16b-NA1 antigen (Figure 16A-C). These results confirmed that the affinity and specificity of the BiKE construct for CD16a and CD16b-NA1 remained unchanged.

[0169] HER2 in E5C1 BiKE and haNK92 suspensions + Assessment of ability to kill cancer cells E5C1 BiKE expresses HER2 in suspension, which represents circulating cancer cells. +To examine whether E5C1 BiKE can enhance the recognition and killing of cancer cells by haNK92 cells, we performed an ADCC assay under non-adherent conditions (Figure 17A). In this experiment, both cancer cells and haNK92 cells were seeded on a non-adherent plate, and then either E5C1 BiKE, pertuzumab, or trastuzumab was added. The results of this study showed that E5C1 BiKE inhibited HER2 activation by haNK92 cells more effectively than trastuzumab and pertuzumab. + It was shown that BiKE, trastuzumab, and pertuzumab not only activate NK cells but also promote the recognition of target cancer cells by NK cells (Figures 17B to 17D). Figure 17A shows a schematic diagram of antibody-induced cytotoxicity, in which BiKE, trastuzumab, and pertuzumab not only activate NK cells but also promote the recognition of target cancer cells by NK cells.

[0170] Measuring ADCC and effector protein release using E5C1 BiKE, laNK92, and haNK92 To determine whether E5C1 BiKE performs better in terms of ADCC than the currently available best-in-class anti-HER2 mAb (i.e., trastuzumab), a cytotoxicity assay was performed. As effector cells, both laNK92(F176) and haNK92(V176) cells were used. CD16a(V176) has been shown to have a relatively high affinity for the Fc region of the mAb. HER2 +Cancer cell lines SKOV-3, BT474, and JIMT-1 were seeded under adherent conditions and used as target cells. First, cancer cells were treated with laNK92 cells alone, laNK92 and trastuzumab (100 nM equivalent), and laNK92 and E5C1 BiKE (100 nM equivalent), and cell viability was measured. The results of this experiment showed that trastuzumab significantly increased the cytotoxicity of laNK92 cells at most E:T ratios, but the ADCC of E5C1 BiKE was superior to trastuzumab in all three cell lines tested (Figures 18A-C). Both trastuzumab and E5C1 BiKE maintained an E:T ratio of 4 that could kill more than 90% of cancer cells, and ADCC was measured using different concentrations of antibodies. The results of this experiment showed that E5C1 BiKE was approximately 100 times more potent than trastuzumab (Figure 18D-Figure 18F). To evaluate whether cancer cell death was due to stimulation of laNK92 cells by BiKE, the concentrations of cytotoxic proteins and cytokines, such as perforin, granzyme B, IFN-γ, and TNF-α, were measured during the ADCC experiment. Here, SKOV-3 cells were used as target cells because our data and previous literature have shown that SKOV-3 cells have limited expression of NKG2D ligands (i.e., MICA / B) on their surface. As a result, the possibility of stimulating laNK92 cells by NKG2D ligands on SKOV-3 cells (in the absence of BiKE) is significantly reduced. The results of this experiment showed that BiKE plays an important role in stimulating laNK92 cells to release cytotoxic proteins and cytokines at a rate substantially faster than trastuzumab (Figure 18G-Figure 18K). The higher rate of laNK92 stimulation by E5C1 BiKE reveals that the observed ADCC rate is higher in cancer cells treated with laNK92 and E5C1 BiKE than in cancer cells treated with laNK92 and trastuzumab. As expected, the data also showed that incubation of laNK92 cells with SKOV-3 (without BiKE) had limited effect on the release of cytotoxic proteins and cytokines (Figure 18G-K).

[0171] To determine whether E5C1 BiKE would perform better than trastuzumab in terms of ADCC in patients with high affinity NK(V176) cells, a cytotoxicity assay using haNK92(V176) was performed. + Cancer cells were seeded in an adherent state as described above and treated with haNK92 cells alone, haNK92 and trastuzumab (100 nM equivalent), or haNK92 and E5C1BiKE (100 nM equivalent), after which cell viability was measured. Cancer cells were also treated with different concentrations of antibodies under the same conditions and at a fixed E:T ratio. The results of these experiments showed that ADCC by E5C1 BiKE was superior to trastuzumab not only at different E:T ratios (Figures 19A-19C) but also at different concentrations (Figures 19D-19F). Measurements of effector protein release also showed that E5C1 BiKE activated haNK92 cells more significantly than trastuzumab (Figures 19G-19K).

[0172] To determine whether E5C1 BiKE would perform better than the combination of trastuzumab and pertuzumab in terms of ADCC in patients with low-affinity NK cells (F176), a cytotoxicity assay using laNK92 was performed. + SKOV-3 cancer cells were seeded in an adherent state as described above and treated with laNK92 cells alone, laNK92 + trastuzumab (10 pM equivalent), laNK92 + pertuzumab (10 pM equivalent), laNK92 + E5C1 BiKE (10 pM equivalent), laNK92 + trastuzumab + pertuzumab (10 pM equivalent each, 20 pM total), laNK92 + E5C1 BiKE + trastuzumab (10 pM equivalent each, 20 pM total), or laNK92 + E5C1 BiKE + pertuzumab (10 pM equivalent each, 20 pM total), and then cell viability was measured. The results of this experiment showed that E5C1 BiKE alone resulted in significantly higher levels of ADCC compared to trastuzumab, pertuzumab, or the combination of trastuzumab and pertuzumab (Figure 20).

[0173] Exemplary single domain antibodies, methods and uses according to one or more embodiments are presented in the following sections.

[0174] Item 1. A first single domain antibody having an amino acid sequence of one of SEQ ID NOs: 14 and 15, exhibiting specificity and affinity for the CD16a receptor on the surface of natural killer (NK) cells, without cross-reacting with CD16b (e.g., CD16b-NA1) or CD32b; a second single domain antibody having an amino acid sequence exhibiting specificity and affinity for an antigen associated with a cancer cell, a bacterium, a parasite, or a virus, The construct, wherein the first and second single domain antibodies are fused to each other with or without a linker.

[0175] Item 2. The antigen is associated with a cancer cell, wherein the antigen is selected from the group consisting of HER2, HER1, HER3, HER4, EGFR, VEGFR, CD47, FGFR, carcinoembryonic antigen (CEA), bladder tumor antigen (BTA), CA125, PDGFR, IGFR, CA15-3 / CA27.29, CA19-9, CA27.29, programmed death-ligand 1 (PD-L1), PD-L2, CTL4, CD3, CD19, CD20, CD22, CD25, CD27, CD30, CD33, CD37, CD38, CD40, CD48, CD52, B7-H3, MICA family, RAET1 / ULBP family, HLA-E, TIM-3, LAG-3, V-domain Ig suppressor of T-cell activation (VISTA). , HVEM, ICOS, 4-1BB, OX40, RANKL and GITR, epithelial and mesenchymal markers of circulating tumor cells, prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), soluble mesothelin-related peptide (SMRP), somatostatin receptor (SR), urokinase plasminogen activator (uPA), plasminogen activator inhibitor (PAI-1), TCR (e.g., MHC class I or class II molecules), A2a receptor, glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, prostein, PSMA, prostate cancer tumor antigen-1 (PCTA-1), MART-1, MAGE, tyrosinase, TRP-1, TRP-2BAGE, GAGE-1, GAGE-2, RAGE, pl5, ELF2M, neutrophil elastase, ephrin B2, IGF-I receptor, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, TSP-180, pl85erbB2, pl80erbB-3, nm-23HI, TAG-72, CA19-9, CA72-4 , CAM17.1, NuMa, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, β-HCG, BCA225, BTAA, CA15-3CA27.29BCAA, CA195, CA242, CA-50, CAM43, CD68P1, CO-029, G250, Ga733EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0176] Item 3. The construct of item 1, wherein the antigen is associated with a bacterium, wherein the antigen is selected from the group consisting of polysaccharide or peptide antigens associated with P. aeruginosa, S. aureus, Clostridium difficile, Acinetobacter baumannii, and Klebsiella pneumonia.

[0177] Item 4. The construct of item 1, wherein the antigen is associated with a virus, and wherein the antigen is selected from the group consisting of Epstein-Barr virus antigen EBVA, human papillomavirus (HPV) antigens E6 and E7, coronavirus surface antigens, influenza virus surface antigens, and HIV surface antigens.

[0178] Item 5. The construct of item 1, wherein the antigen is associated with a parasite, and wherein the antigen is selected from the group consisting of antigens associated with malaria, leishmaniasis, Chagas disease, toxoplasmosis, schistosomiasis, cysticercosis, and strongyloidiasis.

[0179] Item 6. The construct of item 1 or item 2, wherein the first single domain antibody comprises the amino acid sequence of SEQ ID NO: 14 and the second single domain antibody comprises the amino acid sequence of SEQ ID NO: 6.

[0180] Item 7. The construct according to item 1 or 2, wherein the second single domain antibody comprises at least one amino acid sequence selected from SEQ ID NOs: 3 to 11, and the second single domain antibody exhibits selectivity and affinity for HER2 and facilitates recognition of HER2-expressing cancer cells.

[0181] Item 8. The construct according to item 7, wherein the HER2 expressing cancer cell is an ovarian cancer cell, a breast cancer cell, a gastric cancer cell, a gastroesophageal cancer cell, a cervical cancer cell, a bladder cancer cell, a gallbladder cancer cell, a testicular cancer cell, a uterine cancer cell, or any other HER2 expressing cancer cell.

[0182] Item 9. The construct according to any one of items 1 to 8, wherein the amino acid sequence of the first single domain antibody exhibits high affinity for the CD16a receptor of the NK cell.

[0183] Item 10. The construct of item 1, wherein the amino acid sequence of the second single domain antibody exhibits high affinity for an antigen associated with a cancer cell, a bacterium, a parasite, or a virus.

[0184] Item 11. The construct according to any one of items 1 to 10, wherein the first and second single domain antibodies are fused using a linker.

[0185] Item 12. The construct of item 11, wherein the linker is a human muscle aldolase (HMA) linker.

[0186] Item 13. The construct according to any one of items 1 to 12, wherein the construct further comprises at least one additional single domain antibody having an amino acid sequence exhibiting affinity and specificity for another epitope on the same or another antigen, fused to at least one of the first or second single domain antibodies with or without a linker.

[0187] Item 14. The construct of item 13, wherein the at least one additional single domain antibody is an antibody of the same type as the first single domain antibody.

[0188] Item 15. The construct of item 13, wherein the at least one additional single domain antibody is an antibody of the same type as the second single domain antibody.

[0189] Item 16. A single domain antibody comprising at least one amino acid sequence of SEQ ID NO: 14 and 15, wherein the single domain antibody selectively binds with high affinity to the CD16a activating receptor on the surface of natural killer (NK) cells without cross-reacting with CD16b-NA1 or CD32b.

[0190] Item 17. A single domain antibody comprising at least one amino acid sequence selected from SEQ ID NOs: 3 to 11, which exhibits selectivity and high affinity for HER2 and promotes recognition of HER2-expressing cancer cells.

[0191] Item 18. A method for inhibiting HER2-positive cancer in a subject, comprising: The method includes administering to the subject an effective amount of the construct according to any one of items 6 to 8, so that the construct activates NK cells in the subject and recognizes target HER2-positive cancer cells in the subject.

[0192] Item 19. A method for performing an ELISA assay using the single domain antibody according to item 16 or 17 or the construct according to any one of items 1 to 15, comprising: immobilizing a sample containing one or more antigens on a solid support, wherein the one or more antigens are selected from HER2 and CD16a; applying the single domain antibody to a surface of the sample, the single domain antibody acting as a primary antibody; applying a secondary antibody to the surface of the sample, wherein the secondary antibody is linked to an enzyme and configured to recognize the single domain antibody; adding to the sample a substance comprising a substrate for the enzyme; and testing the sample to determine whether there is binding between the single domain antibody and the one or more antigens, where if there is binding by the single domain antibody to the one or more antigens, a subsequent reaction produces a detectable signal in the sample.

[0193] Item 20. A method for performing a flow cytometry assay using the single domain antibody according to item 17, comprising: suspending a sample comprising cancer cells and said single domain antibody in a fluid; applying to the sample a second antibody linked to a fluorescent probe capable of binding to the single domain antibody; injecting the fluid containing the sample into a flow cytometer device; analyzing the sample with a flow cytometry analyzer; determining whether the cancer cells are HER2+ cancer cells.

[0194] Item 21. A cell imaging method using the single domain antibody according to item 17, comprising: Fixing a sample containing suspected cancer cells on a slide; applying said single domain antibody to said sample; applying to the sample a second antibody linked to a fluorescent probe capable of binding to the single domain antibody; examining the sample by confocal or fluorescent microscopy to detect the presence or absence of HER2 expression on the surface of the suspected cancer cells.

[0195] Item 22. The method of item 21, wherein the fluorescently labeled secondary antibody is an anti-his tag antibody or an anti-C-myc tag antibody.

[0196] Item 23. An in vivo cell tracking and imaging method for tracking allogeneic or autologous NK cells in a subject using the single domain antibody according to item 16, comprising: administering to the subject an imaging agent conjugated to the single domain antibody; performing a whole body imaging method on the subject to generate an image; and identifying an anatomical location of the NK cells within the image.

[0197] Item 24. The method of item 23, wherein the whole-body imaging method is selected from the group consisting of magnetic resonance imaging (MRI), positron emission tomography (PET), computed tomography (CT), and single photon emission computed tomography (SPECT).

[0198] Item 25. An in vivo cancer phenotyping method for identifying HER2-expressing cancer lesions in a subject, comprising: administering to the subject an imaging agent conjugated to the single domain antibody of item 17; performing a tumor imaging method on the subject to generate an image; and identifying a HER2-expressing cancer lesion within the image.

[0199] Item 26. The method according to item 25, wherein the tumor imaging method is selected from the group consisting of magnetic resonance imaging (MRI), positron emission tomography (PET), computed tomography (CT), and single photon emission computed tomography (SPECT).

[0200] Item 27. The single domain antibody of item 17, wherein the single domain antibody comprises the amino acid sequence of SEQ ID NO:6.

[0201] Item 28. The single domain antibody of item 17, wherein the single domain antibody comprises the amino acid sequence of SEQ ID NO:11.

[0202] Item 29. The single domain antibody of item 17, wherein the single domain antibody comprises the amino acid sequence of SEQ ID NO:7.

[0203] Item 30. The single domain antibody of item 17, wherein the HER2-expressing cancer cells are HER2-expressing ovarian cancer cells.

[0204] Item 31. The single domain antibody of item 30, wherein the HER2-expressing ovarian cancer cells are from a metastatic lesion.

[0205] Item 32. The single domain antibody of item 17, wherein the HER2 expressing cancer cell is a breast cancer cell, a cervical cancer cell, a bladder cancer cell, a gallbladder cancer cell, a testicular cancer cell, a uterine cancer cell, or any other HER2 expressing cancer cell.

[0206] Item 33. A first single domain antibody having an amino acid sequence of any one of SEQ ID NOs: 14 and 15; and a second single domain antibody having an amino acid sequence of any one of SEQ ID NOs: 3 to 11, wherein the first and second single domain antibodies are fused to each other with or without a linker, and the construct exhibits specificity and high affinity for the CD16a receptor on the surface of natural killer (NK) cells and HER2, without cross-reacting with CD16b (e.g., CD16b-NA1) or CD32b.

[0207] Item 34. The bispecific single domain antibody construct of item 33, wherein the first single domain antibody comprises the amino acid sequence of SEQ ID NO: 14 and the second single domain antibody comprises the amino acid sequence of SEQ ID NO: 6.

[0208] Item 35. The bispecific single domain antibody construct of item 34, wherein the first and second single domain antibodies are fused using a linker.

[0209] Item 36. The bispecific single domain antibody construct of item 35, wherein the linker is a human muscle aldolase (HMA) linker.

[0210] Item 37. A bispecific single domain antibody construct comprising the amino acid sequence of SEQ ID NO: 20, wherein the construct exhibits specificity and high affinity for the CD16a receptor on the surface of natural killer (NK) cells and HER2, without cross-reacting with CD16b (e.g., CD16b-NA1) or CD32b.

[0211] Item 38. A method for performing an ELISA assay using the single domain antibody according to any one of items 16, 17, and 27 to 32, comprising: immobilizing a sample containing one or more antigens on a solid support, wherein the one or more antigens are selected from HER2 and CD16a; applying the single domain antibody to a surface of the sample, the single domain antibody acting as a primary antibody; applying a secondary antibody to the surface of the sample, wherein the secondary antibody is linked to an enzyme and configured to recognize the single domain antibody; adding to the sample a substance comprising a substrate for the enzyme; and testing the sample to determine whether there is binding between the single domain antibody and the one or more antigens, where if there is binding by the single domain antibody to the one or more antigens, a subsequent reaction produces a detectable signal in the sample.

[0212] Item 39. The method of item 38, wherein the secondary antibody is an anti-his tag antibody, an anti-C-myc antibody, or an anti-HA tag antibody.

[0213] Item 40. The method of item 38, wherein the enzyme is horseradish peroxidase (HRP).

[0214] Item 41. The method of item 38, wherein the detectable signal is a color change.

[0215] Item 42. A method for performing a flow cytometry assay using the single domain antibody according to any one of items 17, 27 to 32, comprising: suspending a sample comprising cancer cells and said single domain antibody in a fluid; applying to the sample a second antibody linked to a fluorescent probe capable of binding to the single domain antibody; injecting the fluid containing the sample into a flow cytometer device; analyzing the sample with a flow cytometry analyzer; determining whether the cancer cells are HER2+ cancer cells.

[0216] Item 43. A method for performing a flow cytometry assay using the single domain antibody according to item 16, comprising: suspending a sample comprising NK cells and said single domain antibody in a fluid; applying to the sample a second antibody linked to a fluorescent probe capable of binding to the single domain antibody; injecting the fluid containing the sample into a flow cytometer device; analyzing the sample with a flow cytometry analyzer; determining whether the NK cells are CD16a+ NK cells.

[0217] Item 44. The method of item 42 or 43, wherein the secondary antibody is an anti-his tag antibody, an anti-C-myc tag antibody, or an anti-HA tag antibody.

[0218] Item 45. A cell imaging method using the single domain antibody according to any one of items 17, 27 to 32, comprising: Fixing a sample containing suspected cancer cells on a slide; applying said single domain antibody to said sample; applying to the sample a second antibody linked to a fluorescent probe capable of binding to the single domain antibody; examining the sample by confocal or fluorescent microscopy to detect the presence or absence of HER2 expression on the surface of the suspected cancer cells.

[0219] Item 46. The method of item 45, wherein the secondary antibody is an anti-his tag antibody or an anti-C-myc tag antibody.

[0220] Item 47. An ex-vivo cancer phenotype analysis by immunohistochemistry, comprising using a single domain antibody according to any one of items 17 and 27 to 32, cryosectioning suspected tumor tissue and fixing said suspected tumor tissue on a slide; staining the suspect tissue with the single domain antibody; applying a fluorescently labeled secondary antibody; performing photomicrographic examination using a microscope to detect the presence or absence of HER2 expression in the suspected tumor tissue.

[0221] Item 48. The method of item 47, wherein the fluorescently labeled secondary antibody is an anti-his tag antibody or an anti-C-myc tag antibody.

[0222] Item 49. An in vivo cell tracking and imaging method for tracking allogeneic or autologous NK cells in a subject using the single domain antibody of item 16, comprising: administering to the subject an imaging agent conjugated to the single domain antibody; performing a whole body imaging method on the subject to generate an image; and identifying an anatomical location of the NK cells within the image.

[0223] Item 50. The method of item 49, wherein the whole-body imaging method is magnetic resonance imaging (MRI).

[0224] Item 51. The method of item 49, wherein the whole-body imaging method is positron emission tomography (PET).

[0225] Item 52. The method of item 49, wherein the whole-body imaging method is computed tomography (CT).

[0226] Item 53. The method of item 49, wherein the whole-body imaging method is single photon emission computed tomography (SPECT).

[0227] Item 54. An in vivo cancer phenotyping method for identifying HER2-expressing cancer lesions in a subject, comprising: administering to the subject an imaging agent conjugated to the single domain antibody according to any one of items 17, 27 to 32; performing a tumor imaging method on the subject to generate an image; and identifying a HER2-expressing cancer lesion within the image.

[0228] Item 55. The method of item 54, wherein the tumor imaging method is magnetic resonance imaging (MRI).

[0229] Item 56. The method of item 54, wherein the tumor imaging method is positron emission tomography (PET).

[0230] Item 57. The method of item 54, wherein the tumor imaging method is computed tomography (CT).

[0231] Item 58. The method of item 54, wherein the tumor imaging method is single photon emission computed tomography (SPECT).

[0232] Item 59. A construct for treating HER2-positive cancer, comprising: A bispecific single domain antibody construct according to any one of items 33 to 37; and natural killer (NK) cells expressing a CD16a receptor which engages said single domain antibody; The construct exhibits significant anti-cancer activity against HER2-positive cancer.

[0233] Item 60. A method for inhibiting HER2-positive cancer in a subject, comprising: 38. The method comprising administering to the subject an effective amount of the bispecific single domain antibody construct according to any one of items 33 to 37, and natural killer (NK) cells expressing the CD16a receptor which engages with said single domain antibody.

[0234] Item 61. A method for inhibiting HER2-positive cancer in a subject, the method comprising: 38. The method of claim 37, comprising administering to the subject an effective amount of the bispecific single domain antibody construct according to any one of items 33 to 37, wherein the bispecific single domain antibody construct activates NK cells in the subject to recognize target HER2 positive cancer cells in the subject.

[0235] Item 62. A first single domain antibody having an amino acid sequence of one of SEQ ID NOs: 14 and 15, exhibiting specificity and affinity for the CD16a receptor on the surface of macrophages without cross-reacting with CD16b (e.g., CD16b-NA1) or CD32b; a second single domain antibody having an amino acid sequence exhibiting specificity and affinity for an antigen associated with a cancer cell, a bacterium, a parasite, or a virus, The construct, wherein the first and second single domain antibodies are fused to each other with or without a linker.

[0236] Item 63. The antigen is associated with a cancer cell, wherein the antigen is selected from the group consisting of HER2, HER1, HER3, HER4, EGFR, VEGFR, CD47, FGFR, carcinoembryonic antigen (CEA), bladder tumor antigen (BTA), CA125, PDGFR, IGFR, CA15-3 / CA27.29, CA19-9, CA27.29, programmed death-ligand 1 (PD-L1), PD-L2, CTL4, CD3, CD19, CD20, CD22, CD25, CD27, CD30, CD33, CD37, CD38, CD40, CD48, CD52, B7-H3, MICA family, RAET1 / ULBP family, HLA-E, TIM-3, LAG-3, V-domain Ig suppressor of T-cell activation (VISTA). , HVEM, ICOS, 4-1BB, OX40, RANKL and GITR, epithelial and mesenchymal markers of circulating tumor cells, prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), soluble mesothelin-related peptide (SMRP), somatostatin receptor (SR), urokinase plasminogen activator (uPA), plasminogen activator inhibitor (PAI-1), TCR (e.g., MHC class I or class II molecules), A2a receptor, glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, prostein, PSMA, prostate cancer tumor antigen-1 (PCTA-1), MART-1, MAGE, tyrosinase, TRP-1, TRP-2BAGE, GAGE-1, GAGE-2, RAGE, pl5, ELF2M, neutrophil elastase, ephrin B2, IGF-I receptor, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, TSP-180, pl85erbB2, pl80erbB-3, nm-23HI, TAG-72, CA19-9, CA72-4 , CAM17.1, NuMa, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, β-HCG, BCA225, BTAA, CA15-3CA27.63. The construct of item 62, selected from the group consisting of 29BCAA, CA195, CA242, CA-50, CAM43, CD68P1, CO-029, G250, Ga733EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0237] Item 64. The construct of item 62, wherein the antigen is associated with a bacterium, wherein the antigen is selected from the group consisting of polysaccharide or peptide antigens associated with P. aeruginosa, S. aureus, Clostridium difficile, Acinetobacter baumannii, and Klebsiella pneumonia.

[0238] Item 65. The construct of item 62, wherein the antigen is associated with a virus, wherein the antigen is selected from the group consisting of Epstein-Barr virus antigen EBVA, human papillomavirus (HPV) antigens E6 and E7, coronavirus surface antigens, influenza virus surface antigens, and HIV surface antigens.

[0239] Item 66. The construct of item 62, wherein the antigen is associated with a parasite, and wherein the antigen is selected from the group consisting of antigens associated with malaria, leishmaniasis, Chagas disease, toxoplasmosis, schistosomiasis, cysticercosis, and strongyloidiasis.

[0240] Item 67. The construct of item 62 or 63, wherein the first single domain antibody comprises the amino acid sequence of SEQ ID NO: 14 and the second single domain antibody comprises the amino acid sequence of SEQ ID NO: 6.

[0241] Item 68. The construct according to item 62 or 63, wherein the second single domain antibody comprises at least one amino acid sequence of SEQ ID NOs: 3 to 11, and the second single domain antibody exhibits selectivity and affinity for HER2 and facilitates recognition of HER2-expressing cancer cells.

[0242] Item 69. The construct of item 68, wherein the HER2 expressing cancer cell is an ovarian cancer cell, a breast cancer cell, a gastric cancer cell, a gastroesophageal cancer cell, a cervical cancer cell, a bladder cancer cell, a gallbladder cancer cell, a testicular cancer cell, a uterine cancer cell, or any other HER2 expressing cancer cell.

[0243] Item 70. The construct according to any one of items 62 to 69, wherein the amino acid sequence of the first single domain antibody exhibits high affinity for the CD16a receptor of the NK cell.

[0244] Item 71. The construct of item 62, wherein the amino acid sequence of the second single domain antibody exhibits high affinity for an antigen associated with a cancer cell, a bacterium, a parasite, or a virus.

[0245] Item 72. The construct according to any one of items 62 to 71, wherein the first and second single domain antibodies are fused using a linker.

[0246] Item 73. The construct of item 72, wherein the linker is a human muscle aldolase (HMA) linker.

[0247] Item 74. The construct according to any one of items 62 to 73, wherein the construct further comprises at least one additional single domain antibody having an amino acid sequence exhibiting affinity and specificity for another epitope on the same or another antigen, fused with or without a linker to at least one of the first or second single domain antibodies.

[0248] Item 75. The construct of item 74, wherein the at least one additional single domain antibody is an antibody of the same type as the first single domain antibody.

[0249] Item 76. The construct of item 74, wherein the at least one additional single domain antibody is an antibody of the same type as the second single domain antibody.

[0250] Item 77. A single domain antibody comprising at least one amino acid sequence of SEQ ID NOs: 14 and 15, wherein the single domain antibody selectively binds with high affinity to the CD16a activating receptor on the surface of macrophages without cross-reacting with CD16b-NA1 or CD32b.

[0251] Item 78. A method for inhibiting HER2-positive cancer in a subject, the method comprising: The method includes administering to the subject an effective amount of the construct according to any one of items 67 to 69, wherein the construct activates NK cells in the subject to recognize target HER2-positive cancer cells in the subject.

[0252] Item 79. A first single domain antibody having an amino acid sequence of any one of SEQ ID NOs: 14 and 15; and a second single domain antibody having an amino acid sequence of any one of SEQ ID NOs: 3 to 11, wherein the first and second single domain antibodies are fused to each other with or without a linker, and the construct exhibits specificity and high affinity for the CD16a receptor on the surface of macrophages and HER2, without cross-reacting with CD16b (e.g., CD16b-NA1) or CD32b.

[0253] Item 80. The bispecific single domain antibody construct of item 79, wherein the first single domain antibody comprises the amino acid sequence of SEQ ID NO: 14 and the second single domain antibody comprises the amino acid sequence of SEQ ID NO: 6.

[0254] Item 81. The bispecific single domain antibody construct of item 80, wherein the first and second single domain antibodies are fused using a linker.

[0255] Item 82. The bispecific single domain antibody construct of item 81, wherein the linker is a human muscle aldolase (HMA) linker.

[0256] Item 83. A bispecific single domain antibody construct comprising the amino acid sequence of SEQ ID NO: 20, wherein the construct exhibits specificity and high affinity for the CD16a receptor on the surface of macrophages and HER2 without cross-reacting with CD16b (e.g., CD16b-NA1) or CD32b.

[0257] Item 84. A construct for treating HER2-positive cancer, comprising: A bispecific single domain antibody construct according to any one of items 79 to 83; a macrophage expressing a CD16a receptor which engages said single domain antibody; The construct exhibits significant anti-cancer activity against HER2-positive cancer.

[0258] Item 85. A method for inhibiting HER2-positive cancer in a subject, the method comprising: 84. The method comprising administering to the subject an effective amount of a bispecific single domain antibody construct according to any one of items 79 to 83, and a macrophage expressing the CD16a receptor which engages said single domain antibody.

[0259] Item 86. A method for inhibiting HER2-positive cancer in a subject, the method comprising: 84. The method of claim 8, further comprising administering to the subject an effective amount of the bispecific single domain antibody construct according to any one of items 79 to 83, wherein the bispecific single domain antibody construct activates macrophages in the subject to recognize target HER2 positive cancer cells in the subject.

[0260] All publications, patents, and patent documents are incorporated herein by reference as if individually incorporated by reference. This incorporation-by-reference statement is intended by the applicant to relate, in accordance with 37 CFR § 1.57(b)(1), to all individual publications, patent applications, or patents, each of which is specifically identified, in accordance with 37 CFR § 1.57(b)(2), even if such citation is not immediately adjacent to the dedicated incorporation-by-reference statement. The inclusion of a dedicated incorporation-by-reference statement in the specification does not in any way weaken the general incorporation-by-reference statement. The citation of a reference herein is not an admission that the reference is relevant prior art, nor is it any admission as to the contents or date of these publications or documents. No limitations inconsistent with this disclosure are to be understood therefrom.

[0261] The invention has been described with reference to various specific and preferred embodiments and techniques, but it should be understood that many variations and modifications can be made while remaining within the spirit and scope of the invention.

[0262] Although particular embodiments have been described above with reference to disclosed embodiments and examples, such embodiments are merely illustrative and do not limit the scope of the present invention. References 1.Barb AW.Fc gamma receptor compositional heterogeneity:Considerations for immunotherapy development.J Biol Chem.2021;296:100057. 2.Chenoweth AM,Wines BD,Anania JC,Mark Hogarth P.Harnessing the immune system via FcgammaR function in immune therapy:a pathway to next-gen mAbs.Immunol Cell Biol.2020;98(4):287-304. 3.Koene HR,Kleijer M,Algra J,Roos D,von dem Borne AE,de Haas M.Fc gammaRIIIa-158V / F polymorphism influences the binding of IgG by natural killer cell Fc gammaRIIIa,independently of the Fc gammaRIIIa-48L / R / H phenotype.Blood.1997;90(3):1109-14. 4.Treffers LW,van Houdt M,Bruggeman CW,Heineke MH,Zhao XW,van der Heijden J,et al.FcgammaRIIIb Restricts Antibody-Dependent Destruction of Cancer Cells by Human Neutrophils.Front Immunol.2018;9:3124. 5.Ocana-Guzman R,Vazquez-Bolanos L,Sada-Ovalle I.Receptors That Inhibit Macrophage Activation:Mechanisms and Signals of Regulation and Tolerance.J Immunol Res.2018;2018:8695157. 6.Morris AB,Farley CR,Pinelli DF,Adams LE,Cragg MS,Boss JM,et al.Signaling through the inhibitory Fc receptor FcγRIIB induces CD8+ T cell apoptosis to limit T cell immunity.Immunity.2020;52(1):136-50.e6. 7.Arezumand R,Alibakhshi A,Ranjbari J,Ramazani A,Muyldermans S.Nanobodies as novel agents for targeting angiogenesis in solid cancers.Frontiers in immunology.2017;8:1746. 8.Ackaert C,Smiejkowska N,Xavier C,Sterckx YG,Denies S,Stijlemans B,et al.Immunogenicity risk profile of nanobodies.Frontiers in immunology.2021;12:578. 9.Allegra A,Innao V,Gerace D,Vaddinelli D,Allegra AG,Musolino C.Nanobodies and cancer:current status and new perspectives.Cancer investigation.2018;36(4):221-37. 10.Roberts JT,Barb AW.A single amino acid distorts the Fc gamma receptor IIIb / CD16b structure upon binding immunoglobulin G1 and reduces affinity relative to CD16a.J Biol Chem.2018;293(51):19899-908. 11.Nomani A,Li G,Yousefi S,Wu S,Malekshah OM,Nikkhoi SK,et al.Gadolinium-labeled affibody-XTEN recombinant vector for detection of HER2+ lesions of ovarian cancer lung metastasis using quantitative MRI.Journal of Controlled Release.2021;337:132-43. 12.Malekshah OM,Sarkar S,Nomani A,Patel N,Javidian P,Goedken M,et al.Bioengineered adipose-derived stem cells for targeted enzyme-prodrug therapy of ovarian cancer intraperitoneal metastasis.Journal of Controlled Release.2019;311:273-87. 13.Li JY,Perry SR,Muniz-Medina V,Wang X,Wetzel LK,Rebelatto MC,et al.A biparatopic HER2-targeting antibody-drug conjugate induces tumor regression in primary models refractory to or ineligible for HER2-targeted therapy.Cancer cell.2016;29(1):117-29.

Claims

1. a first single domain antibody having an amino acid sequence of one of SEQ ID NOs: 14 and 15, and exhibiting specificity and affinity for the CD16a receptor on the surface of natural killer (NK) cells, without cross-reacting with CD16b (e.g., CD16b-NA1) or CD32b; a second single domain antibody having an amino acid sequence exhibiting specificity and affinity for an antigen associated with a cancer cell, a bacterium, a parasite, or a virus, The construct, wherein the first and second single domain antibodies are fused to each other with or without a linker.

2. The antigen is associated with a cancer cell, wherein the antigen is selected from the group consisting of HER2, HER1, HER3, HER4, EGFR, VEGFR, CD47, FGFR, carcinoembryonic antigen (CEA), bladder tumor antigen (BTA), CA125, PDGFR, IGFR, CA15-3 / CA27.29, CA19-9, CA27.29, programmed death-ligand 1 (PD-L1), PD-L2, CTL4, CD3, CD19, CD20, CD22, CD25, CD27, CD30, CD33, CD37, CD38, CD40, CD48, CD52, B7-H3, MICA family, RAET1 / ULBP family, HLA-E, TIM-3, LAG-3, V domain Ig subfamily of T cell activation, lesser (VISTA), HVEM, ICOS, 4-1BB, OX40, RANKL and GITR, epithelial and mesenchymal markers of circulating tumor cells, prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), soluble mesothelin-related peptide (SMRP), somatostatin receptor (SR), urokinase plasminogen activator (uPA), plasminogen activator inhibitor (PAI-1), TCR (e.g., MHC class I or class II molecules), A2a receptor, glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, RAGE-1, MN-CAIX, RU1, RU2 (AS), intestinal carboxylesterase, muthsp70-2, M-CSF, prostase, prostate specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, prostein, PSMA, prostate cancer tumor antigen-1 (PCTA-1), MART-1, MAGE, tyrosinase, TRP-1, TRP-2BAGE, GAGE-1, GAGE-2, RAGE, pl5, ELF2M, neutrophil elastase, ephrin B2, IGF-I receptor, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, TSP-180, pl85erbB2, pl80erbB-3, nm-23HI, TAG-72, CA19-9, CA72-4, CAM17.1, NuMa, 2. The construct of claim 1, wherein the construct is selected from the group consisting of β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, β-HCG, BCA225, BTAA, CA15-3CA27.29BCAA, CA195, CA242, CA-50, CAM43, CD68P1, CO-029, G250, Ga733EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

3. 2. The construct of claim 1, wherein the antigen is associated with a bacterium, wherein the antigen is selected from the group consisting of polysaccharide or peptide antigens associated with P. aeruginosa, S. aureus, Clostridium difficile, Acinetobacter baumannii, and Klebsiella pneumonia.

4. 2. The construct of claim 1, wherein the antigen is associated with a virus, wherein the antigen is selected from the group consisting of Epstein-Barr virus antigen EBVA, human papillomavirus (HPV) antigens E6 and E7, coronavirus surface antigens, influenza virus surface antigens, and HIV surface antigens.

5. The construct of claim 1, wherein the antigen is associated with a parasite, wherein the antigen is selected from the group consisting of antigens associated with malaria, leishmaniasis, Chagas disease, toxoplasmosis, schistosomiasis, cysticercosis, and strongyloidiasis.

6. The construct of claim 1 , wherein the first single domain antibody comprises the amino acid sequence of SEQ ID NO: 14 and the second single domain antibody comprises the amino acid sequence of SEQ ID NO:

6.

7. The construct of claim 1, wherein the second single domain antibody comprises at least one amino acid sequence of SEQ ID NOs: 3-11, and the second single domain antibody exhibits selectivity and affinity for HER2 and facilitates recognition of HER2-expressing cancer cells.

8. The construct of claim 7, wherein the HER2-expressing cancer cell is an ovarian cancer cell, a breast cancer cell, a gastric cancer cell, a gastroesophageal cancer cell, a cervical cancer cell, a bladder cancer cell, a gallbladder cancer cell, a testicular cancer cell, a uterine cancer cell, or any other HER2-expressing cancer cell.

9. The construct of claim 1 , wherein the amino acid sequence of the first single domain antibody exhibits high affinity for the CD16a receptor of the NK cell.

10. The construct of claim 1 , wherein the amino acid sequence of the second single domain antibody exhibits high affinity for an antigen associated with a cancer cell, a bacterium, a parasite, or a virus.

11. The construct of claim 1 , wherein the first and second single domain antibodies are fused using a linker.

12. The construct of claim 11 , wherein the linker is a human muscle aldolase (HMA) linker.

13. 2. The construct of claim 1, further comprising at least one additional single domain antibody having an amino acid sequence exhibiting affinity and specificity for another epitope on the same or another antigen, fused to at least one of the first or second single domain antibodies, with or without a linker.

14. The construct of claim 13 , wherein the at least one further single domain antibody is of the same type as the first single domain antibody.

15. The construct of claim 13 , wherein the at least one further single domain antibody is of the same type as the second single domain antibody.

16. A single domain antibody comprising at least one of the amino acid sequences of SEQ ID NOs: 14 and 15, wherein the single domain antibody selectively binds with high affinity to the CD16a activating receptor on the surface of natural killer (NK) cells without cross-reacting with CD16b-NA1 or CD32b.

17. A single domain antibody comprising at least one of the amino acid sequences of SEQ ID NOs: 3 to 11, which exhibits selectivity and high affinity for HER2 and facilitates recognition of HER2-expressing cancer cells.

18. 1. A method for inhibiting HER2 positive cancer in a subject, the method comprising: The method comprises administering to the subject an effective amount of a construct according to any one of claims 6 to 8, wherein the construct activates NK cells in the subject to recognize target HER2-positive cancer cells in the subject.

19. A method for carrying out an ELISA assay using a single domain antibody according to claim 16 or 17 or a construct according to any one of claims 1 to 15, comprising the steps of: immobilizing a sample containing one or more antigens on a solid support, wherein said one or more antigens are selected from HER2 and CD16a; applying the single domain antibody to a surface of the sample, the single domain antibody acting as a primary antibody; applying a secondary antibody to the surface of the sample, wherein the secondary antibody is linked to an enzyme and configured to recognize the single domain antibody; adding to the sample a substance comprising a substrate for the enzyme; and testing the sample to determine whether there is binding between the single domain antibody and the one or more antigens, where if there is binding by the single domain antibody to the one or more antigens, a subsequent reaction produces a detectable signal in the sample.

20. 20. A method for performing a flow cytometry assay using a single domain antibody according to claim 17, comprising: suspending a sample comprising cancer cells and said single domain antibody in a fluid; applying to the sample a second antibody linked to a fluorescent probe capable of binding to the single domain antibody; injecting the fluid containing the sample into a flow cytometer device; analyzing the sample with a flow cytometry analyzer; determining whether the cancer cells are HER2+ cancer cells.

21. A cell imaging method using the single domain antibody according to claim 17, Fixing a sample containing suspected cancer cells on a slide; applying said single domain antibody to said sample; applying to the sample a second antibody linked to a fluorescent probe capable of binding to the single domain antibody; examining the sample by confocal or fluorescent microscopy to detect the presence or absence of HER2 expression on the surface of the suspected cancer cells.

22. The method of claim 21, wherein the fluorescently labeled secondary antibody is an anti-his tag antibody or an anti-C-myc tag antibody.

23. 17. An in vivo cell tracking and imaging method for tracking allogeneic or autologous NK cells in a subject using a single domain antibody according to claim 16, comprising: administering to the subject an imaging agent conjugated to the single domain antibody; performing a whole body imaging method on the subject to generate an image; and identifying the anatomical location of the NK cells within the image.

24. 24. The method of claim 23, wherein the whole-body imaging method is selected from the group consisting of magnetic resonance imaging (MRI), positron emission tomography (PET), computed tomography (CT), and single photon emission computed tomography (SPECT).

25. 1. An in vivo cancer phenotyping method for identifying a HER2-expressing cancer lesion in a subject, comprising: administering to the subject an imaging agent conjugated to the single domain antibody of claim 17; performing a tumor imaging method on the subject to generate an image; and identifying a HER2-expressing cancer lesion within the image.

26. 26. The method of claim 25, wherein the tumor imaging method is selected from the group consisting of magnetic resonance imaging (MRI), positron emission tomography (PET), computed tomography (CT), and single photon emission computed tomography (SPECT).

Images