Nanobody coupled to GPRC5D and its use

Abstract

Provided herein are nanobodies that bind to GPRC5D, antibodies containing nanobodies, immune complexes, and pharmaceutical compositions, isolated nucleic acids encoding nanobodies, host cells expressing nanobodies, and related methods and applications.

Description

Technical Field

[0001] The present disclosure relates to the field of biomedical technologies, and particularly to nanobodies that bind to GPRC5D and their use.

Background Art

[0002] Multiple myeloma (MM) is a malignant proliferative disease of plasma cells. The clonal plasma cells in the bone marrow of MM patients abnormally proliferate and secrete monoclonal immunoglobulins or their fragments, causing myeloma-related organ or tissue disorders (ROTI) such as hypercalcemia, renal insufficiency, anemia, and bone pain. MM accounts for about 10% of hematological malignancies and is the second most common hematological malignancy in most countries of the world. Currently, MM is still considered an incurable disease. For patients who are relatively young (e.g., under 65 years old) and suitable for hematopoietic stem cell transplantation, the combined use of autologous stem cell transplantation and drug therapy can effectively extend the survival of these patients. For patients who are relatively old (e.g., over 65 years old) and / or not suitable for transplantation, a more effective drug therapy regimen is urgently needed to extend the survival period and improve the quality of life. In recent years, immunomodulatory drugs (IMiD), proteasome inhibitors (PI), antibody drugs and their derivatives, small molecule targeted therapeutic drugs, etc. have shown good prospects for the treatment of MM. However, after the disease recurs or drug resistance occurs, the effect of the drug therapy regimen previously received by MM patients will inevitably gradually decrease. Therefore, the treatment of MM faces the problems of limited available drugs and the urgent research and development of new drugs.

[0003] G protein-coupled receptor class C group 5 member D (GPRC5D) is a G protein-coupled orphan receptor that is expressed limitedly in normal tissues and highly expressed in MM cells. GPRC5D has been reported to serve as a potential target for the treatment of MM.

[0004] Compared to conventional antibodies, nanobodies have the advantages of a smaller molecular weight, easier tissue penetration, and easier access to tumor areas. Nanobodies can achieve higher effective therapeutic concentrations over a wider tumor area, thereby improving therapeutic efficacy. At the same time, the absence of light chains in nanobodies helps avoid light chain mismatch in bispecific / multispecific antibodies. More importantly, nanobodies are hydrophilic and crosslinkable, allowing multiple nanobodies to be linked together to form the desired type of bispecific / multispecific antibody. Therefore, the development of nanobodies and their derivative drugs against GPRC5D will have a positive impact on advancing drug therapy for MM. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Daopeng Yuan, Zhongmin Liu, Jonas Kaindl, Shoji Maeda, Jiawei Zhao, Xiaoou Sun, Jun Xu, Peter Gmeiner, Hong-Wei Wang, Brian K. Kobilka. Activation of the 2B adrenergic receptor by the sedative sympatholytic dexmedetomidine. Nature chemical biology (2020) (https: / / doi.org / 10.1038 / s41589-020-0492-2) [Non-Patent Document 2] Bazan J, Calkosinski I, Gamian A. Phage display--a powerful technique for immunotherapy: 1. Introduction and potential of therapeutic applications. Hum Vaccin Immunother. 2012 Dec 1;8(12):1817-28 (doi: 10.4161 / hv.21703. Epub 2012 Aug 21) [Overview of the project] [Means for solving the problem]

[0006] According to a first aspect of this disclosure, a nanobody is provided that specifically binds to a G protein-coupled receptor, class C, group 5, member D (GPRC5D). The nanobody may include a combination of three complementarity-determining regions (CDRs). The combination may include CDR1 having the amino acid sequence described in SEQ ID NO: 13, CDR2 having the amino acid sequence described in SEQ ID NO: 14, and CDR3 having the amino acid sequence described in SEQ ID NO: 15.

[0007] In some embodiments, the nanobody may include a VHH chain having the amino acid sequence described in SEQ ID NO: 3.

[0008] In some embodiments, nanobodies can be used as pharmaceuticals.

[0009] In some embodiments, nanobodies may be used to treat disorders or diseases associated with GPRC5D. Furthermore, nanobodies may be used to treat multiple myeloma (MM).

[0010] A second aspect of this disclosure provides a nanobody that specifically binds to a G protein-coupled receptor, class C, group 5, member D (GPRC5D). The nanobody may include a combination of three complementarity-determining regions (CDRs). The combination may include CDR1 having the amino acid sequence described in SEQ ID NO: 16, CDR2 having the amino acid sequence described in SEQ ID NO: 17, and CDR3 having the amino acid sequence described in SEQ ID NO: 18.

[0011] In some embodiments, the nanobody may include a VHH chain having the amino acid sequence described in SEQ ID NO: 4.

[0012] In some embodiments, nanobodies can be used as pharmaceuticals.

[0013] In some embodiments, nanobodies may be used to treat disorders or diseases associated with GPRC5D. Furthermore, nanobodies may be used to treat multiple myeloma (MM).

[0014] A third aspect of this disclosure provides a nanobody that specifically binds to a G protein-coupled receptor, class C, group 5, member D (GPRC5D). The nanobody may include a combination of three complementarity-determining regions (CDRs). The combination may include CDR1 having the amino acid sequence described in SEQ ID NO: 19, CDR2 having the amino acid sequence described in SEQ ID NO: 20, and CDR3 having the amino acid sequence described in SEQ ID NO: 21.

[0015] In some embodiments, the nanobody may include a VHH chain having the amino acid sequence described in SEQ ID NO: 5.

[0016] In some embodiments, nanobodies can be used as pharmaceuticals.

[0017] In some embodiments, nanobodies may be used to treat disorders or diseases associated with GPRC5D. Furthermore, nanobodies may be used to treat multiple myeloma (MM).

[0018] A fourth aspect of this disclosure provides a nanobody that specifically binds to a G protein-coupled receptor, class C, group 5, member D (GPRC5D). The nanobody may include a combination of three complementarity-determining regions (CDRs). The combination may include CDR1 having the amino acid sequence described in SEQ ID NO: 22, CDR2 having the amino acid sequence described in SEQ ID NO: 23, and CDR3 having the amino acid sequence described in SEQ ID NO: 24.

[0019] In some embodiments, the nanobody may include a VHH chain having the amino acid sequence described in SEQ ID NO: 6.

[0020] In some embodiments, nanobodies can be used as pharmaceuticals.

[0021] In some embodiments, the nanobody can be used in treating disorders or diseases associated with GPRC5D. Further, the nanobody can be used in treating multiple myeloma (MM).

[0022] According to a fifth aspect of the present disclosure, a nanobody that specifically binds to G protein-coupled receptor, class C, group 5, member D (GPRC5D) is provided. The nanobody can include a combination of three complementarity-determining regions (CDRs). The combination can include CDR1 having the amino acid sequence set forth in SEQ ID NO: 25, CDR2 having the amino acid sequence set forth in SEQ ID NO: 26, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 27.

[0023] In some embodiments, the nanobody can include a VHH chain having the amino acid sequence set forth in SEQ ID NO: 7.

[0024] In some embodiments, the nanobody can be used as a medicament.

[0025] In some embodiments, the nanobody can be used in treating disorders or diseases associated with GPRC5D. Further, the nanobody can be used in treating multiple myeloma (MM).

[0026] According to a sixth aspect of the present disclosure, an anti-GPRC5D antibody is provided. The antibody can include a nanobody.

[0027] In some embodiments, the antibody is bispecific, binds to GPRC5D, and can further bind to a second antigen.

[0028] In some embodiments, the antibody can be used as a medicament.

[0029] In some embodiments, the antibody can be used in treating disorders or diseases associated with GPRC5D. Further, the antibody can be used in treating multiple myeloma (MM).

[0030] According to a seventh aspect of this disclosure, an immune complex is provided. The immune complex may include nanobodies.

[0031] In some embodiments, the immune complex may include antibodies containing nanobodies.

[0032] In some embodiments, the antibody may be conjugated to a cytotoxic agent, a radioisotope, a drug compound, or a peptide.

[0033] In some embodiments, immune complexes can be used as pharmaceuticals.

[0034] In some embodiments, immune complexes may be used to treat disorders or diseases associated with GPRC5D. Furthermore, immune complexes may be used to treat multiple myeloma (MM).

[0035] According to an eighth aspect of this disclosure, isolated nucleic acids are provided. Isolated nucleic acids may encode nanobodies.

[0036] In some embodiments, isolated nucleic acids can be used as pharmaceuticals.

[0037] In some embodiments, isolated nucleic acids may be used to treat disorders or diseases associated with GPRC5D. Furthermore, isolated nucleic acids may be used to treat multiple myeloma (MM).

[0038] According to a ninth aspect of this disclosure, recombinant eukaryotic or prokaryotic host cells are provided. The host cells may produce nanobodies.

[0039] A pharmaceutical composition is provided according to a tenth aspect of this disclosure. The pharmaceutical composition may include nanobodies.

[0040] In some embodiments, the pharmaceutical composition may be used as a pharmaceutical.

[0041] In some embodiments, the pharmaceutical composition may be used to treat disorders or diseases associated with GPRC5D. Furthermore, the pharmaceutical composition may be used to treat multiple myeloma (MM).

[0042] According to an eleventh aspect of this disclosure, a pharmaceutical composition is provided. The pharmaceutical composition may comprise an antibody, an immune complex, or an isolated nucleic acid.

[0043] In some embodiments, the pharmaceutical composition may be used as a pharmaceutical.

[0044] In some embodiments, the pharmaceutical composition may be used to treat disorders or diseases associated with GPRC5D. Furthermore, the pharmaceutical composition may be used to treat multiple myeloma (MM).

[0045] A twelfth aspect of this disclosure provides a method for treating a patient suffering from a disease associated with GPRC5D. The method may include administering to the patient a pharmacologically effective amount of nanobodies, antibodies, immune complexes, isolated nucleic acids, or pharmaceutical compositions.

[0046] In some embodiments, the disease may be multiple myeloma (MM).

[0047] A thirteenth aspect of this disclosure provides a method for detecting the presence of GPRC5D in a sample. The method may include: contacting the sample with nanobodies under conditions that allow for the formation of a composite between the nanobodies and GPRC5D; and determining whether a composite has been formed.

[0048] A fourteenth aspect of this disclosure provides a method for producing anti-GPRC5D nanobodies. The method may include (i) culturing host cells expressing nucleic acids encoding nanobodies, and (ii) purifying anti-GPRC5D nanobodies from the culture medium of the host cells.

[0049] This disclosure will be further described in relation to exemplary embodiments. These exemplary embodiments will be described in detail with reference to the drawings. These embodiments are non-limiting exemplary embodiments, and similar reference figures throughout some of the drawings represent similar structures: [Brief explanation of the drawing]

[0050] [Figure 1] Figure 1 shows schematic diagrams of anti-GPRC5D nanobodies (Part A) and recombinant antibodies constructed by fusion of anti-GPRC5D nanobodies with IgG-Fc (Part B), respectively, according to several embodiments of the present disclosure; [Figure 2a] This disclosure shows the purity identification results of candidate recombinant antibody 1-F7-FC-fusion constructed by fusion of candidate nanobody 1-F7 with IgG-Fc according to several embodiments of this disclosure; a high-performance liquid chromatography-size exclusion chromatography (HPLC-SEC) graph (detection wavelength 214 nm) of the candidate recombinant antibody; [Figure 2b] The following shows the purity identification results of candidate recombinant antibody 1-F7-FC-fusion constructed by fusion of candidate nanobody 1-F7 with IgG-Fc according to several embodiments of this disclosure; the HPLC-SEC graph of the candidate recombinant antibody (detection wavelength 280 nm); [Figure 2c] The following are purity identification results for candidate recombinant antibodies 1-F7-FC-fusion constructed by fusion of candidate nanobody 1-F7 with IgG-Fc according to several embodiments of this disclosure; and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) graphs of the candidate recombinant antibodies under non-reducing (NR) and reducing (R) conditions. [Figure 3a] This disclosure shows the purity identification results of candidate recombinant antibody 1-E8-FC-fusion constructed by fusion of candidate nanobody 1-E8 with IgG-Fc according to several embodiments of this disclosure; and the HPLC-SEC graph of the candidate recombinant antibody (detection wavelength 214 nm). [Figure 3b]The purity identification results of candidate recombinant antibody 1-E8-FC-fusion, constructed by fusion of candidate nanobody 1-E8 with IgG-Fc, according to several embodiments of this disclosure are shown; the HPLC-SEC graph of the candidate recombinant antibody (detection wavelength 280 nm) is shown; [Figure 3c] The following are purity identification results for candidate recombinant antibody 1-E8-FC-fusion constructed by fusion of candidate nanobody 1-E8 with IgG-Fc according to several embodiments of this disclosure; SDS-PAGE graphs of the candidate recombinant antibody under non-reducing (NR) and reducing (R) conditions; [Figure 4a] This disclosure shows the purity identification results of candidate recombinant antibody 1-D9-FC-fusion constructed by fusion of candidate nanobody 1-D9 with IgG-Fc according to several embodiments of this disclosure; and the HPLC-SEC graph of the candidate recombinant antibody (detection wavelength 214 nm). [Figure 4b] The following are purity identification results for candidate recombinant antibody 1-D9-FC-fusion constructed by fusion of candidate nanobody 1-D9 with IgG-Fc according to several embodiments of this disclosure; an HPLC-SEC graph of the candidate recombinant antibody (detection wavelength 280 nm); [Figure 4c] The following are purity identification results for candidate recombinant antibodies 1-D9-FC-fusion constructed by fusion of candidate nanobody 1-D9 with IgG-Fc according to several embodiments of this disclosure; SDS-PAGE graphs of candidate recombinant antibodies under non-reducing (NR) and reducing (R) conditions; [Figure 5a] This disclosure shows the purity identification results of candidate recombinant antibodies 1-C12-FC-fusion constructed by fusion of candidate nanobody 1-C12 with IgG-Fc according to several embodiments of this disclosure; and the HPLC-SEC graph of the candidate recombinant antibody (detection wavelength 214 nm). [Figure 5b] The purity identification results of candidate recombinant antibodies 1-C12-FC-fusion, constructed by fusion of candidate nanobody 1-C12 with IgG-Fc, according to several embodiments of this disclosure are shown; the HPLC-SEC graph of the candidate recombinant antibody (detection wavelength 280 nm) is also shown; [Figure 5c] The following are purity identification results for candidate recombinant antibodies 1-C12-FC-fusion constructed by fusion of candidate nanobody 1-C12 with IgG-Fc according to several embodiments of this disclosure; SDS-PAGE graphs of candidate recombinant antibodies under non-reducing (NR) and reducing (R) conditions; [Figure 6a] This disclosure shows the purity identification results of candidate recombinant antibody 1-G7-FC-fusion constructed by fusion of candidate nanobody 1-G7 with IgG-Fc according to several embodiments of this disclosure; and the HPLC-SEC graph of the candidate recombinant antibody (detection wavelength 214 nm). [Figure 6b] The following are purity identification results for candidate recombinant antibodies 1-G7-FC-fusion constructed by fusion of candidate nanobody 1-G7 with IgG-Fc according to several embodiments of this disclosure; and an HPLC-SEC graph of the candidate recombinant antibody (detection wavelength 280 nm). [Figure 6c] The following are purity identification results for candidate recombinant antibodies 1-G7-FC-fusion constructed by fusion of candidate nanobody 1-G7 with IgG-Fc according to several embodiments of this disclosure; SDS-PAGE graphs of candidate recombinant antibodies under non-reducing (NR) and reducing (R) conditions; [Figure 7] Figure 7 shows a graph illustrating the binding ability of phage display candidate nanobodies to purified GPRC5D protein, as detected by enzyme-linked immunosorbent assay (ELISA) according to several embodiments of the present disclosure, where the vertical axis represents the absorbance value at 450 nm; [Figure 8] Figure 8 shows a graph illustrating the binding ability of phage display candidate nanobodies to CHOK1 cells transfected to express human GPRC5D (CHOK1-GPRC5D), as detected by ELISA, according to several embodiments of the present disclosure, where the control group is untransfected CHOK1 cells (CHOK1), and the vertical axis represents the absorbance value at 450 nm; [Figure 9]Figure 9 shows a graph illustrating the binding affinity of recombinant antibodies constructed by fusion of candidate nanobodies and Fc fragments to cells expressing human GPRC5D, as detected by ELISA, according to several embodiments of the present disclosure. The vertical axis represents the absorbance value at 450 nm. Cell lines expressing human GPRC5D include CHOK1-C5D, MM1R, and H929, with the control group being untransfected CHOK1 cells (CHOK1). [Figure 10a] The following histograms illustrate the binding ability of phage display candidate nanobodies to human GPRC5D-overexpressing CHOK1-GPRC5D cells, as detected by fluorescence-activated cell sorting (FACS), according to several embodiments of the present disclosure, where the x-axis represents the relative intensity of the fluorescence signal and the y-axis represents the relative count of cells / particles; [Figure 10b] The following histograms illustrate the binding ability of phage display candidate nanobodies to human GPRC5D-overexpressing CHOK1-GPRC5D cells, as detected by fluorescence-activated cell sorting (FACS), according to several embodiments of the present disclosure, where the x-axis represents the relative intensity of the fluorescence signal and the y-axis represents the relative count of cells / particles; [Figure 10c] The following histograms illustrate the binding ability of phage display candidate nanobodies to human GPRC5D-overexpressing CHOK1-GPRC5D cells, as detected by fluorescence-activated cell sorting (FACS), according to several embodiments of the present disclosure, where the x-axis represents the relative intensity of the fluorescence signal and the y-axis represents the relative count of cells / particles; [Figure 10d] The following histograms illustrate the binding ability of phage display candidate nanobodies to human GPRC5D-overexpressing CHOK1-GPRC5D cells, as detected by fluorescence-activated cell sorting (FACS), according to several embodiments of the present disclosure, where the x-axis represents the relative intensity of the fluorescence signal and the y-axis represents the relative count of cells / particles; [Figure 10e]The following histograms illustrate the binding ability of phage display candidate nanobodies to human GPRC5D-overexpressing CHOK1-GPRC5D cells, as detected by fluorescence-activated cell sorting (FACS), according to several embodiments of the present disclosure, where the x-axis represents the relative intensity of the fluorescence signal and the y-axis represents the relative count of cells / particles; [Figure 11a] The following histograms illustrate the binding ability of phage display candidate nanobodies to human GPRC5D-expressing H929 cells, as detected by FACS, according to several embodiments of the present disclosure, where the horizontal axis represents the relative intensity of the fluorescence signal and the vertical axis represents the relative count of cells / particles; [Figure 11b] The following histograms illustrate the binding ability of phage display candidate nanobodies to human GPRC5D-expressing H929 cells, as detected by FACS, according to several embodiments of the present disclosure, where the horizontal axis represents the relative intensity of the fluorescence signal and the vertical axis represents the relative count of cells / particles; [Figure 11c] The following histograms illustrate the binding ability of phage display candidate nanobodies to human GPRC5D-expressing H929 cells, as detected by FACS, according to several embodiments of the present disclosure, where the horizontal axis represents the relative intensity of the fluorescence signal and the vertical axis represents the relative count of cells / particles; [Figure 11d] The following histograms illustrate the binding ability of phage display candidate nanobodies to human GPRC5D-expressing H929 cells, as detected by FACS, according to several embodiments of the present disclosure, where the horizontal axis represents the relative intensity of the fluorescence signal and the vertical axis represents the relative count of cells / particles; [Figure 11e] The following histograms illustrate the binding ability of phage display candidate nanobodies to human GPRC5D-expressing H929 cells, as detected by FACS, according to some embodiments of the present disclosure, where the x-axis represents the relative intensity of the fluorescence signal and the y-axis represents the relative count of cells / particles; and [Figure 12a]The following histograms illustrate the binding ability of phage display candidate nanobodies to human GPRC5D-expressing MM1R cells, as detected by FACS, according to several embodiments of the present disclosure, where the x-axis represents the relative intensity of the fluorescence signal and the y-axis represents the relative count of cells / particles; [Figure 12b] The following histograms illustrate the binding ability of phage display candidate nanobodies to human GPRC5D-expressing MM1R cells, as detected by FACS, according to several embodiments of the present disclosure, where the x-axis represents the relative intensity of the fluorescence signal and the y-axis represents the relative count of cells / particles; [Figure 12c] The following histograms illustrate the binding ability of phage display candidate nanobodies to human GPRC5D-expressing MM1R cells, as detected by FACS, according to several embodiments of the present disclosure, where the x-axis represents the relative intensity of the fluorescence signal and the y-axis represents the relative count of cells / particles; [Figure 12d] The following histograms illustrate the binding ability of phage display candidate nanobodies to human GPRC5D-expressing MM1R cells, as detected by FACS, according to several embodiments of the present disclosure, where the x-axis represents the relative intensity of the fluorescence signal and the y-axis represents the relative count of cells / particles; [Figure 12e] The following histograms illustrate the binding ability of phage display candidate nanobodies to human GPRC5D-expressing MM1R cells, as detected by FACS, according to several embodiments of the present disclosure, where the x-axis represents the relative intensity of the fluorescence signal and the y-axis represents the relative count of cells / particles. [Modes for carrying out the invention]

[0051] To more clearly illustrate the technical solutions related to embodiments of this disclosure, a brief introduction to the drawings referenced in the description of the embodiments is provided below. Obviously, the drawings described below are only some examples or embodiments of this disclosure. Those skilled in the art can apply this disclosure to other similar scenarios according to these drawings without further creative effort. Unless otherwise clearly stated in the context, or unless otherwise illustrated by the context, the same figures in the drawings refer to the same structure or operation.

[0052] While terms such as “first,” “second,” and “third” may be used in this specification to describe various elements, it should be understood that these elements should not be limited by these terms. These terms are simply used to distinguish one element from another. For example, a first product may be called a second product. Similarly, a second product may be called a first product without departing from the scope of the exemplary embodiments of this disclosure.

[0053] Where used in this disclosure and the accompanying claims, the singular forms “a,” “an,” and “the” refer to multiple objects unless the content explicitly indicates otherwise; the plural forms may also be intended to include the singular forms. In general, the terms “comprise,” “comprises,” and / or “comprising,” “include,” “includes,” and / or “including” simply indicate that they include clearly identified steps and elements, and these steps and elements do not constitute an exclusive list. A method or device may also include other steps or elements.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure pertains.

[0055] As used herein, the term “antibody” or “Ab” refers to a molecule comprising at least one immunoglobulin heavy chain (HC) and at least one immunoglobulin light chain (LC). Each heavy chain may include a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain variable region may have three complementarity-determining regions (CDR) and four framework regions (FR). Each light chain may include a light chain variable region (VL) and a light chain constant region (CL). The light chain variable region may have three complementarity-determining regions and four framework regions. Based on the antigenicity of the VH, antibodies can be classified as IgM, IgG, IgA, IgD, and IgE. In some embodiments, antibodies may include, but are not limited to, monoclonal antibodies, bispecific antibodies, multispecific antibodies, dimeric antibodies, trimer antibodies, and multimeric antibodies.

[0056] The term "CDR" or "complementarity-determining region" refers to the region in which an antibody specifically recognizes an antigen.

[0057] The terms "Fc," "Fc fragment," or "fragment crystallizable region" refer to the terminal region of an antibody that can bind to various cell surface receptors (e.g., Fc receptors) and complement proteins.

[0058] The terms "nanobody," "VHH," or "single-domain antibody" refer to antibodies consisting of a single monomeric variable antibody domain, which is the smallest antigen-binding fragment with full functionality.

[0059] The term "peptide" refers to a compound formed by the dehydration and condensation of at least two amino acid molecules.

[0060] The term "purified" refers to nucleic acids or peptides that exist in a state where other biomolecules are substantially absent.

[0061] The term "isolated" refers to a nucleic acid or peptide being separated from one or more other components present in the source of the nucleic acid or peptide (e.g., other genomic nucleic acid sequences, proteins, etc.).

[0062] The term "host cell" refers to a cell capable of introducing and stably maintaining a foreign gene. In some embodiments, host cells include prokaryotic and eukaryotic cells. Non-limiting examples of host cells for expressing nanobodies may include, but are not limited to, Escherichia coli, yeast cells, insect cells, rodent cells, etc.

[0063] The term "identity" refers to the relationship between the sequences of two or more peptide molecules or two or more nucleic acid molecules, determined by aligning and comparing their sequences. "Percent identity" refers to comparing the percentage of identical residues between amino acids or nucleotides in a molecule, and is calculated based on the size of the smallest molecule being compared.

[0064] The term "pharmacologically effective dose" refers to a dosage that is effective in improving the symptoms of a disease.

[0065] The terms "administering" or "dosing" refer to the process of administering a compound (e.g., nanobodies, immune complexes, pharmaceutical compositions, etc.) for use as a pharmaceutical to a subject or patient. In some embodiments, the route of administration may include, but is not limited to, oral administration, intramuscular injection, intradermal injection, subcutaneous injection, intravenous injection, intrapleural or intraperitoneal injection, inhalation administration, implantation administration, etc.

[0066] As mentioned above, the treatment of MM faces the problem of limited available drugs and an urgent need to develop new drugs. GPRC5D can function as a target for the treatment of MM. Novel drugs that can be used to treat MM (e.g., nanobodies targeting GPRC5D) can be designed against GPRC5D. From the standpoint of screening difficulty, it should be known that the screening difficulty of nanobodies that depend on three CDR-binding antigens is far higher than that of conventional antibodies that depend on six CDR-binding antigens. It is difficult for those skilled in the art to obtain nanobodies with high affinity and high specificity. Through detailed and extensive research, this disclosure points to nanobodies and recombinant antibodies having high affinity and high specificity to the GPRC5D protein and various types of cells expressing the GPRC5D protein, using phage display technology. Combined with the characteristics of nanobodies, such as their low immunogenicity and ease of purification, these findings can play an active role in clinical research and pharmaceutical manufacturing of MM in the development of various types of pharmaceuticals or therapies for MM treatment, including, but not limited to, CAR-T cells, nanobodiment monoclonal antibodies, bispecific / multispecific antibodies, and antibody-drug conjugates.

[0067] Some embodiments of this disclosure provide nanobodies that specifically bind to GPRC5D. The nanobodies may include a combination of three CDRs, the combination of which may include CDR1, CDR2, and CDR3.

[0068] In some embodiments, the amino acid sequence of CDR1 may be as described in SEQ ID NO: 13. The amino acid sequence of CDR2 may be as described in SEQ ID NO: 14. The amino acid sequence of CDR3 may be as described in SEQ ID NO: 15.

[0069] In some embodiments, the nanobody may include a VHH chain having the amino acid sequence described in SEQ ID NO: 3.

[0070] In some embodiments, the VHH chain may include conservative substitutions of two amino acids or less, or one amino acid or less. As used herein, the term “conservative substitution” refers to an amino acid substitution that does not adversely affect or alter any essential properties of a protein / peptide, including the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art (e.g., site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis). Conservative amino acid substitutions may include substitutions in which an amino acid residue is replaced by another amino acid residue having a similar side chain, for example, a substitution of a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains are defined in the art. These families may include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (e.g., threonine, valine, and isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, the corresponding amino acid residue may preferably be substituted with another amino acid residue from the same side chain family.

[0071] In some embodiments, the VHH chain may have an amino acid sequence that is at least 90%, 92%, 94%, 96%, or 98% identical in the framework region to the amino acid sequence described in SEQ ID NO: 3. In some embodiments, the VHH chain may have an amino acid sequence that has one or more amino acid additions, deletions, and / or substitutions in the framework region to the amino acid sequence described in SEQ ID NO: 3.

[0072] In some embodiments, the nanobodies may be isolated (or purified). For example, the nanobodies may be produced by immunizing an animal (e.g., a camel) with the human GPRC5D antigen protein and then biochemically isolating it. In other embodiments, the nanobodies may be recombinants. For example, the nucleic acid encoding the nanobodies may be specifically amplified and isolated, the isolated nucleic acid encoding the nanobodies may be cloned into a suitable expression vector, and the nanobodies may be produced by transfecting a suitable host cell with the expression vector and then biochemically isolating it.

[0073] Some embodiments of this disclosure also provide nanobodies that specifically bind to GPRC5D. The nanobodies may include a combination of three CDRs, the combination of which may include CDR1, CDR2, and CDR3.

[0074] In some embodiments, the amino acid sequence of CDR1 may be described in SEQ ID NO: 16. The amino acid sequence of CDR2 may be described in SEQ ID NO: 17. The amino acid sequence of CDR3 may be described in SEQ ID NO: 18.

[0075] In some embodiments, the nanobody may include a VHH chain having the amino acid sequence described in SEQ ID NO: 4.

[0076] In some embodiments, the VHH chain may contain conservative substitutions of two amino acids or less, or one amino acid or less.

[0077] In some embodiments, the VHH chain may have an amino acid sequence that has at least 90%, 92%, 94%, 96%, or 98% identity in the framework region compared to the amino acid sequence described in SEQ ID NO: 4. In some embodiments, the VHH chain may have an amino acid sequence that has one or more amino acid additions, deletions, and / or substitutions in the framework region compared to the amino acid sequence described in SEQ ID NO: 4.

[0078] In some embodiments, the nanobodies may be isolated. In other embodiments, the nanobodies may be recombinants.

[0079] Some embodiments of this disclosure also provide nanobodies that specifically bind to GPRC5D. The nanobodies may include a combination of three CDRs, the combination of which may include CDR1, CDR2, and CDR3.

[0080] In some embodiments, the amino acid sequence of CDR1 may be as described in SEQ ID NO: 19. The amino acid sequence of CDR2 may be as described in SEQ ID NO: 20. The amino acid sequence of CDR3 may be as described in SEQ ID NO: 21.

[0081] In some embodiments, the nanobody may include a VHH chain having the amino acid sequence described in SEQ ID NO: 5.

[0082] In some embodiments, the VHH chain may contain conservative substitutions of two amino acids or less, or one amino acid or less.

[0083] In some embodiments, the VHH chain may have an amino acid sequence that is at least 90%, 92%, 94%, 96%, or 98% identical in the framework region to the amino acid sequence described in SEQ ID NO: 5. In some embodiments, the VHH chain may have an amino acid sequence that has one or more amino acid additions, deletions, and / or substitutions in the framework region to the amino acid sequence described in SEQ ID NO: 5.

[0084] In some embodiments, the nanobodies may be isolated. In other embodiments, the nanobodies may be recombinants.

[0085] Some embodiments of this disclosure also provide nanobodies that specifically bind to GPRC5D. The nanobodies may include a combination of three CDRs, the combination of which may include CDR1, CDR2, and CDR3.

[0086] In some embodiments, the amino acid sequence of CDR1 may be as described in SEQ ID NO: 22. The amino acid sequence of CDR2 may be as described in SEQ ID NO: 23. The amino acid sequence of CDR3 may be as described in SEQ ID NO: 24.

[0087] In some embodiments, the nanobody may include a VHH chain having the amino acid sequence described in SEQ ID NO: 6.

[0088] In some embodiments, the VHH chain may contain conservative substitutions of two amino acids or less, or one amino acid or less.

[0089] In some embodiments, the VHH chain may have an amino acid sequence that has at least 90%, 92%, 94%, 96%, or 98% identity in the framework region compared to the amino acid sequence described in SEQ ID NO: 6. In some embodiments, the VHH chain may have an amino acid sequence that has one or more amino acid additions, deletions, and / or substitutions in the framework region compared to the amino acid sequence described in SEQ ID NO: 6.

[0090] In some embodiments, the nanobodies may be isolated. In other embodiments, the nanobodies may be recombinants.

[0091] Some embodiments of this disclosure also provide nanobodies that specifically bind to GPRC5D. The nanobodies may include a combination of three CDRs, the combination of which may include CDR1, CDR2, and CDR3.

[0092] In some embodiments, the amino acid sequence of CDR1 may be as described in SEQ ID NO: 25. The amino acid sequence of CDR2 may be as described in SEQ ID NO: 26. The amino acid sequence of CDR3 may be as described in SEQ ID NO: 27.

[0093] In some embodiments, the nanobody may include a VHH chain having the amino acid sequence described in SEQ ID NO: 7.

[0094] In some embodiments, the VHH chain may contain conservative substitutions of two amino acids or less, or one amino acid or less.

[0095] In some embodiments, the VHH chain may have an amino acid sequence that is at least 90%, 92%, 94%, 96%, or 98% identical in the framework region to the amino acid sequence described in SEQ ID NO: 7. In some embodiments, the VHH chain may have an amino acid sequence that has one or more amino acid additions, deletions, and / or substitutions in the framework region to the amino acid sequence described in SEQ ID NO: 7.

[0096] In some embodiments, the nanobodies may be isolated. In other embodiments, the nanobodies may be recombinants.

[0097] Some embodiments of this disclosure also provide anti-GPRC5D antibodies. The antibodies may include nanobodies.

[0098] In some embodiments, the antibody is bispecific and can bind to GPRC5D and also to a second antigen.

[0099] Some embodiments of this disclosure also provide immune complexes. The immune complexes may include nanobodies. In some embodiments, the immune complexes may include antibodies that include nanobodies.

[0100] Various methods can be used to prepare immune complexes. For example, nanobodies or antibodies containing nanobodies can be linked directly to functional molecules or via spacers of appropriate length. Linking to obtain immune complexes can be chemical crosslinking or genetically engineered fusion expression.

[0101] In some embodiments, antibodies may be conjugated to cytotoxic agents, radioisotopes, drug compounds, or peptides. The term “cytotoxic agent” refers to any agent that is harmful to cell growth and proliferation and acts to reduce, inhibit, or kill cells or malignancies. Non-limiting examples of functional molecules to which antibodies are conjugated include angiogenesis inhibitors (e.g., thalidomide), MAPK signaling pathway inhibitors (e.g., metformin), AKT signaling pathway inhibitors (e.g., tanespimycin), PI3K / m-TOR / AKT signaling pathway inhibitors (e.g., bortezomib), kinase inhibitors (e.g., imatinib, dovitinib), HDAC inhibitors (e.g., thidamide), PARP inhibitors (e.g., PJ-34), RNA polymerase inhibitors (e.g., amanitin), and DNA damaging agents (e.g., calichemycin). This may include, but is not limited to, syn), DNA alkylating agents (e.g., melflufen), DNA intercalating agents (e.g., adriamycin), DNA minor groove binding substances (e.g., anthramycin), ribosome inactivators (e.g., geronin), microtubule stabilizers (e.g., epotilon D), microtubule destabilizers (e.g., vincristine), platinum compounds (e.g., carboplatin), topoisomerase inhibitors (e.g., topotecan), strontium-89, samarium-153, and radium-223.

[0102] In some embodiments, the immune complex may be a fusion protein constructed by the fusion of a nanobody and a functional molecule, for example, the functional molecule may be used to extend the half-life of an antibody or have a binding effect on specific effector cells. In some embodiments, the functional molecule used to extend the half-life of an antibody may include serum albumin or its fragments, polyethylene glycol, etc. In some embodiments, the functional molecule having a binding effect on effector cells may include an immunoglobulin Fc fragment. For example, the fusion protein may include a fused nanobody and a human immunoglobulin Fc fragment, which may extend the half-life of the fusion protein in the human body and enhance the relevant effector functions mediated by Fc (e.g., CDC activity, ADCC activity, and ADCP activity). In some embodiments, the fusion protein may be constructed by the fusion of a nanobody and IgG-Fc provided in this disclosure (see Figure 1).

[0103] In certain embodiments, the fusion protein may contain the amino acid sequence described in SEQ ID NO: 8. Furthermore, the amino acid sequence of the fusion protein may be as described in SEQ ID NO: 8.

[0104] In another specific embodiment, the fusion protein may contain the amino acid sequence described in SEQ ID NO: 9. Furthermore, the amino acid sequence of the fusion protein may be as described in SEQ ID NO: 9.

[0105] In further specific embodiments, the fusion protein may include the amino acid sequence described in SEQ ID NO: 10. Furthermore, the amino acid sequence of the fusion protein may be as described in SEQ ID NO: 10.

[0106] In further specific embodiments, the fusion protein may include the amino acid sequence described in SEQ ID NO: 11. Furthermore, the amino acid sequence of the fusion protein may be as described in SEQ ID NO: 11.

[0107] In further specific embodiments, the fusion protein may include the amino acid sequence described in SEQ ID NO: 12. Furthermore, the amino acid sequence of the fusion protein may be as described in SEQ ID NO: 12.

[0108] In some embodiments, the immune complex may be a chimeric antigen receptor (CAR) expressed on immune effector cells, for example, the immune effector cells may include T lymphocytes, NK cells, NKT cells, etc., or combinations thereof. While utilizing the killing effect of nanobodies or antibodies containing nanobodies provided in this disclosure, the chimeric antigen receptor may also enable immune effector cells to have a highly specific cytotoxic effect against myeloma cells expressing GPRC5D.

[0109] Some embodiments of this disclosure also provide isolated nucleic acids capable of encoding nanobodies. Various methods can be used to isolate the nucleic acids; for example, the nucleic acids may be prepared by recombinant DNA technology or isolated from other suitable sources.

[0110] Some embodiments of this disclosure also provide pharmaceutical compositions. Pharmaceutical compositions may include nanobodies.

[0111] Some embodiments of this disclosure also provide pharmaceutical compositions. The pharmaceutical compositions may comprise an anti-GPRC5D antibody, an immune complex, or an isolated nucleic acid.

[0112] In some embodiments, the pharmaceutical composition may also include pharmaceutically acceptable carriers such as buffers, antioxidants, surfactants, flavor modifiers, and preservatives. The dosage form of the pharmaceutical composition can be selected as needed and includes, but is not limited to, tablets, injections, sprays, etc.

[0113] Some embodiments of this disclosure also provide pharmaceutical uses of nanobodies, anti-GPRC5D antibodies, immunocomplexes, isolated nucleic acids, or pharmaceutical compositions.

[0114] Some embodiments of this disclosure also provide the use of nanobodies, anti-GPRC5D antibodies, immune complexes, isolated nucleic acids, or pharmaceutical compositions in the treatment of disorders or diseases associated with GPRC5D. In some embodiments, the disease may be MM.

[0115] Some embodiments of this disclosure also provide methods for treating patients suffering from diseases associated with GPRC5D. The methods may involve administering to the patient a pharmacologically effective amount of nanobodies, anti-GPRC5D antibodies, immune complexes, isolated nucleic acids, or pharmaceutical compositions. In some embodiments, the patient's disease may be MM, such as early-stage MM, mid-stage MM, or advanced-stage MM. Furthermore, the methods may treat patients with cancer recurrence or metastasis.

[0116] The nanobodies, anti-GPRC5D antibodies, immune complexes, isolated nucleic acids, or pharmaceutical compositions provided in this disclosure may be administered alone or in combination with other agents as pharmaceuticals for the treatment of MM patients, and this is understood to be not limited to the embodiments.

[0117] Some embodiments of this disclosure also provide methods for detecting the presence of GPRC5D in a sample. The method may include: contacting the sample with nanobodies under conditions that allow for the formation of a complex between the nanobodies and GPRC5D; and determining whether a complex has been formed. In some embodiments, the process for determining whether a complex has been formed may include, but is not limited to, immunochromatography, immunoblotting (IBT), immunofluorescence, and chemiluminescence immunoassay (CLIA).

[0118] Some embodiments of this disclosure also provide recombinant host cells. The host cells may include eukaryotic or prokaryotic host cells that produce nanobodies.

[0119] In some embodiments, isolated nucleic acids may be inserted into the genome of a host cell, for example, using recombinant DNA techniques and gene transfer methods well known in the art, and genes encoding nanobodies may be operably ligated to transcriptional and translational regulatory sequences, and transcription and translation of genes encoding nanobodies may be achieved through the expression system of the host cell, thereby producing the nanobodies provided by this disclosure.

[0120] In some embodiments, the host cells may preferably be eukaryotic cells such as rodent cells or human-derived cells. Suitable host cells for expressing the nanobodies provided herein may include, but are not limited to, NSO, CHO, CHOK1, perC.6, Tk-ts13, BHK, HEK293, COS-7, T98G, CV-1 / EBNA, L cells, C127, 3T3, HeLa, NS1, Sp2 / 0 myeloma cells, and the like.

[0121] Some embodiments of this disclosure also provide methods for producing anti-GPRC5D nanobodies. The methods may include (i) culturing host cells expressing nucleic acids encoding nanobodies, and (ii) purifying anti-GPRC5D nanobodies from the culture medium of the host cells.

[0122] The experimental methods in the following embodiments are conventional methods unless otherwise specified. The experimental materials used in the following embodiments are purchased from conventional biochemical reagent companies unless otherwise specified. In the following embodiments, all quantitative experiments are repeated three times, and the results are averaged. [Examples]

[0123] Examples Antigen preparation For animal immunization, two antigens were prepared: a HEK293 cell line expressing GPRC5D (GPRC5D-expressing HEK293 cells) and purified GPRC5D membrane protein. Specifically, the human GPRC5D gene (SEQ ID NO: 1) was ligated to the expression vector pcDNA® 3.4, and HEK293 cells (Invitrogen) were transfected using the recombinant expression vector pcDNA® 3.4. Transient overexpression was then achieved in the HEK293 cells to obtain a HEK293 cell line expressing GPRC5D. Subsequently, to obtain the flag-tagged GPRC5D membrane protein (SEQ ID NO: 2), isolation and purification may be performed using a surfactant, or the antigen may be stabilized by adding lipid stabilization.

[0124] Various methods can be used to prepare the antigen; see, for example, Daopeng Yuan, Zhongmin Liu, Jonas Kaindl, Shoji Maeda, Jiawei Zhao, Xiaoou Sun, Jun Xu, Peter Gmeiner, Hong-Wei Wang, Brian K. Kobilka. Activation of the 2B adrenergic receptor by the sedative sympatholytic dexmedetomidine. Nature Chemical Biology (2020) (https: / / doi.org / 10.1038 / s41589-020-0492-2).

[0125] Construction of animal immunology and phage antibody libraries After mixing and emulsifying with an adjuvant, GPRC5D membrane protein and HEK293 cell line overexpressing GPRC5D were used as antigens to immunize healthy alpacas. Specifically, alpacas were immunized six times at 14-day intervals, and their blood was collected seven days after the sixth immunization.

[0126] Using the blood of immunized alpaca, peripheral blood lymphocytes (PBMCs) were isolated, total RNA was extracted, cDNA was reverse transcribed and synthesized, and target VHH fragments were amplified and recovered by PCR.

[0127] The target VHH fragment and the phage vector pComb3XTT (QT078) were simultaneously digested using the restriction endonuclease Sac1 / Sal1 (NEB, R3156M / R3138M). The digested products were purified by agarose gel electrophoresis and ligated with T4 ligase (M0202M). Electroporation-competent cells TG1 (Lucigen, 60502) were transformed with the ligated product, and the TG1 cells were infected with helper phage M13K07 to generate a phage antibody library. The target VHH fragment is understood to be inserted upstream of gene III of recombinant phage M13K07 so that it presents the target VHH fragment on the coat protein G3P on the surface of recombinant phage M13K07.

[0128] Screening of anti-GPRC5D nanobodies A phage antibody library was screened in three rounds using purified GPRC5D membrane protein as an antigen, and positive phage display nanobodies were isolated using magnetic beads (Pierce, 88817). The above procedure is understood to achieve the objective of exposing phage display nanobodies to the antigen, thereby isolating and purifying nanobodies that can specifically bind to the antigen. Specifically, phages were blocked with bovine serum albumin (BSA), and the blocked phages were applied to blocked anti-Flag magnetic beads for negative selection. Unbound phages were mixed with purified Flag-tagged GPRC5D membrane protein to form a mixture, which was incubated with blocked magnetic beads, and the phages bound to the magnetic beads were recovered using a magnetic separator (positive selection). The bound phages were eluted with acidic buffer and amplified for two subsequent rounds of negative and positive selection.

[0129] Positive phage display nanobodies isolated after three rounds of screening were used as candidate nanobodies. The binding ability of the candidate nanobodies to the GPRC5D membrane protein and GPRC5D-expressing cells was evaluated by ELISA, and their sequences were determined by Sanger sequencing.

[0130] Various approaches can be used for constructing phage antibody libraries and screening antibodies; see, for example, Bazan J, Calkosinski I, Gamian A. Phage display--a powerful technique for immunotherapy: 1. Introduction and potential of therapeutic applications. Hum Vaccin Immunother. 2012 Dec 1;8(12):1817-28 (doi: 10.4161 / hv.21703. Epub 2012 Aug 21).

[0131] Recombinant antibodies constructed by fusing nanobodies and IgG-Fc Based on sequencing results, candidate nanobodies were fused with IgG-Fc to construct the expression vector pcDNA® 3.4. HEK293 cells were transfected with the recombinant expression vector pcDNA® 3.4 and transiently overexpressed in the HEK293 cells. The cell culture supernatant was collected, isolated and purified using protein A resin to obtain candidate recombinant antibodies (also referred to as fusion proteins).

[0132] The candidate recombinant antibodies may be subjected to purity analysis using high-performance liquid chromatography-size exclusion chromatography (HPLC-SEC). Specifically, HPLC-SEC analysis was performed using a TSKgel G3000SWxl SEC column (TOSOH). The conditions were as follows: phosphate buffer (pH 7.4) was used as the mobile phase, and the flow rate was 1 ml / min. The injection volume was 20 μl at a concentration of 1 mg / ml. Each sample was run for 15 minutes and detected at 214 nm and 280 nm.

[0133] The candidate recombinant antibodies may be analyzed for purity using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Specifically, the sample was mixed with loading buffer to a concentration of 1 mg / ml, and DTT was used for reduction. The sample was then boiled at 100°C for 5 minutes, and 10 μg of the sample was loaded into the wells of an SDS-PAGE gel (Bio Rad, 4568086) and separated by electrophoresis at 130 V. The gel was stained with Coomassie brilliant blue for 30 minutes, then destained with water for 1 hour, and scanned with an imaging system (Bio Rad, ChemiDoc®).

[0134] Figures 2(a) to 2(c) show the purity identification results of candidate recombinant antibody 1-F7-FC-fusion constructed by fusion of candidate nanobody 1-F7 with IgG-Fc according to several embodiments of the present disclosure. Figures 3(a) to 3(c) show the purity identification results of candidate recombinant antibody 1-E8-FC-fusion constructed by fusion of candidate nanobody 1-E8 with IgG-Fc according to several embodiments of the present disclosure. Figures 4(a) to 4(c) show the purity identification results of candidate recombinant antibody 1-D9-FC-fusion constructed by fusion of candidate nanobody 1-D9 with IgG-Fc according to several embodiments of the present disclosure. Figures 5(a) to 5(c) show the purity identification results of candidate recombinant antibody 1-C12-FC-fusion constructed by fusion of candidate nanobody 1-C12 with IgG-Fc according to several embodiments of the present disclosure. Figures 6(a)–6(c) show the purity identification results of candidate recombinant antibodies 1-G7-FC-fusion constructed by fusion of candidate nanobody 1-G7 with IgG-Fc according to several embodiments of the present disclosure. From the corresponding HPLC-SEC graphs, it can be analyzed that there is virtually no interference from impurity peaks and the purity is greater than 99%. From the corresponding SDS-PAGE graphs, it can be shown that the detected band sizes are correct and there are no other impurity bands. This indicates that each candidate recombinant antibody after purification has high purity and exists in monomeric form.

[0135] Analysis of the antigen-binding ability of nanobodies and their recombinant antibodies The affinity of candidate phage-displaying nanobodies and candidate recombinant antibodies with Fc fusions to antigens was analyzed using enzyme-linked immunosorbent assay (ELISA). Specifically, candidate phage-displaying nanobodies and candidate recombinant antibodies with Fc fusions were placed on plates and incubated. Detection was performed using a horseradish peroxidase (HRP)-conjugated secondary antibody. Purified GPRC5D membrane protein or cells expressing human GPRC5D (e.g., CHOK1 cells exogenously expressing GPRC5D, MM1R cells endogenously expressing GPRC5D, and H929 cells endogenously expressing GPRC5D) were used as antigens, and the binding ability of the candidate nanobodies and candidate recombinant antibodies was similarly tested. As a result, it is possible to screen for target antibodies with excellent antigen-binding ability and applicability, such as candidate nanobody 1-F7 (SEQ ID NO: 3), candidate nanobody 1-E8 (SEQ ID NO: 4), candidate nanobody 1-D9 (SEQ ID NO: 5), candidate nanobody 1-C12 (SEQ ID NO: 6), candidate nanobody 1-G7 (SEQ ID NO: 7), candidate recombinant antibody 1-F7-FC-fusion (SEQ ID NO: 8), candidate recombinant antibody 1-E8-FC-fusion (SEQ ID NO: 9), candidate recombinant antibody 1-D9-FC-fusion (SEQ ID NO: 10), candidate recombinant antibody 1-C12-FC-fusion (SEQ ID NO: 11), and candidate recombinant antibody 1-G7-FC-fusion (SEQ ID NO: 12).

[0136] Figure 7 shows a graph illustrating the binding ability of phage display candidate nanobodies to purified GPRC5D protein as detected by ELISA, according to several embodiments of the present disclosure. Figure 8 shows a graph illustrating the binding ability of phage display candidate nanobodies to CHOK1 cells transfected to express human GPRC5D (CHOK1-GPRC5D), as detected by ELISA, according to several embodiments of the present disclosure. From Figures 7 and 8, it can be seen that, compared to the control group, candidate nanobodies 1-F7, 1-E8, 1-D9, 1-C12, and 1-G7 exhibit relatively good affinity and specificity to various antigens (e.g., GPRC5D protein, CHOK1 cells expressing human GPRC5D).

[0137] Figure 9 shows a graph illustrating the binding ability of recombinant antibodies constructed by fusion of candidate nanobodies and Fc fragments to cells expressing human GPRC5D, as detected by ELISA, according to several embodiments of the present disclosure. From Figure 9, it can be seen that, compared to the control group, candidate recombinant antibodies 1-F7, 1-E8, 1-D9, 1-C12, and 1-G7 exhibit relatively good affinity and specificity to various antigens (e.g., CHOK1 cells overexpressing human GPRC5D (CHOK1-GPRC5D), MM1R cells expressing human GPRC5D, and H929 cells expressing human GPRC5D).

[0138] The affinity of candidate phage-display nanobodies and candidate recombinant antibodies with Fc fusions to antigens was analyzed using fluorescence-activated cell sorting (FACS). Specifically, cells expressing human GPRC5D (e.g., CHOK1 cells exogenously expressing human GPRC5D (CHOK1-GPRC5D), MM1R cells endogenously expressing human GPRC5D, and H929 cells endogenously expressing human GPRC5D) were used as antigens. After incubation with nanobodies in phage-display and recombinant antibody forms, cells were obtained and blocked with bovine serum albumin (BSA). Detection was performed using an APC (allophycocyanin)-conjugated anti-Flag fluorescent antibody (Biolegend, 637307).

[0139] Figures 10(a)–10(e) show histograms illustrating the binding ability of phage display candidate nanobodies detected by fluorescence-activated cell sorting (FACS) to human GPRC5D-overexpressing CHOK1-GPRC5D cells in several embodiments of the present disclosure. Five candidate nanobodies showed binding to CHOK1-GPRC5D cells (solid lines) compared to untransfected CHOK1 cells (spaced dotted lines).

[0140] Figures 11(a)–11(e) show histograms illustrating the binding ability of phage display candidate nanobodies to human GPRC5D-expressing MM1R cells, as detected by FACS, according to several embodiments of the present disclosure. Figures 12(a)–12(e) show histograms illustrating the binding ability of phage display candidate nanobodies to human GPRC5D-expressing H929 cells, as detected by FACS, according to several embodiments of the present disclosure. Five candidate nanobodies showed a histogram shift compared to the blank control (dashed line), indicating their binding ability to naturally expressed GPRC5D protein on MM1R and H929 tumor cell lines (solid lines).

[0141] Having explained the basic concepts in this way, it may be rather clear to those skilled in the art, after reading this detailed disclosure, that the aforementioned detailed disclosure is intended to be presented merely as an example and is not limiting. Although not expressly stated herein, those skilled in the art can make various modifications, improvements, and alterations to this disclosure. Such modifications, improvements, and alterations are intended to be suggested by this disclosure and are within the spirit and scope of the exemplary embodiments of this disclosure.

[0142] Furthermore, specific terminology is used to describe embodiments of this disclosure. For example, the terms “one embodiment,” “an embodiment,” and / or “some embodiments” mean that certain features, structures, or characteristics described in relation to that embodiment are included in at least one embodiment of this disclosure. Therefore, it should be emphasized and understood that two or more references to “an embodiment,” “one embodiment,” or “alternative embodiment” in various parts of this specification do not necessarily all refer to the same embodiment. In addition, some features, structures, or characteristics in one or more embodiments of this disclosure can be appropriately combined.

[0143] In some embodiments, numerical values ​​representing quantities or characteristics used to describe and claim specific embodiments of this disclosure should be understood to be modified in some cases by the terms “about,” “approximate,” or “substantially.” For example, “about,” “approximate,” or “substantially” may indicate a variation of ±20% of the value they describe, unless otherwise specified. Thus, in some embodiments, numerical parameters described in the specification and the appended claims are approximations that may vary depending on the desired characteristics sought to be obtained by a particular embodiment. In some embodiments, numerical parameters should be interpreted by taking into account the number of significant figures reported and applying common rounding techniques. Although the numerical ranges and parameters defining a broad range of some embodiments of this disclosure are approximations, the numerical values ​​described in specific embodiments are reported as accurately as possible.

[0144] Each patent, patent application, published patent application, and other materials such as articles, books, specifications, publications, documents, objects, and / or similar materials referenced herein are incorporated herein by reference in their entirety for all purposes, except for all related examination records, any of which conflict with or are inconsistent with this document, or which may have a limited effect on the broadest scope of the claims currently or in the future relating to this document. For example, if there is any inconsistency or inconsistency between the description, definition, and / or use of terms relating to any of the incorporated materials and those relating to this document, the description, definition, and / or use of terms in this document shall prevail.

[0145] In conclusion, the embodiments of the disclosure disclosed herein should be understood as illustrative of the principles of the embodiments of the disclosure. Other modifications that may be adopted may fall within the scope of the disclosure. Accordingly, alternative configurations of the embodiments of the disclosure may be used in accordance with the teachings herein, not as an example but as an example. Accordingly, the embodiments of the disclosure are not strictly limited to those illustrated and described.

Claims

1. A nanobody that specifically binds to a G protein-coupled receptor, class C, group 5, member D (GPRC5D), wherein the nanobody comprises a combination of three complementarity-determining regions (CDRs), and the combination is (i) CDR1 having the amino acid sequence described in SEQ ID NO: 13, CDR2 having the amino acid sequence described in SEQ ID NO: 14, and CDR3 having the amino acid sequence described in SEQ ID NO: 15 (ii) CDR1 having the amino acid sequence described in SEQ ID NO: 16, CDR2 having the amino acid sequence described in SEQ ID NO: 17, and CDR3 having the amino acid sequence described in SEQ ID NO: 18 (iii) CDR1 having the amino acid sequence described in SEQ ID NO: 19, CDR2 having the amino acid sequence described in SEQ ID NO: 20, and CDR3 having the amino acid sequence described in SEQ ID NO: 21 (iv) CDR1 having the amino acid sequence described in SEQ ID NO: 22, CDR2 having the amino acid sequence described in SEQ ID NO: 23, and CDR3 having the amino acid sequence described in SEQ ID NO: 24, (v) CDR1 having the amino acid sequence described in SEQ ID NO: 25, CDR2 having the amino acid sequence described in SEQ ID NO: 26, and CDR3 having the amino acid sequence described in SEQ ID NO: 27 A nanobody selected from the group consisting of the following.

2. The nanobody according to claim 1, comprising a VHH chain having the amino acid sequence described in SEQ ID NOs: 3, 4, 5, 6, or 7.

3. An anti-GPRC5D antibody comprising the nanobody described in claim 1 or 2.

4. The antibody according to claim 3, wherein the antibody is bispecific, binds to GPRC5D, and further binds to a second antigen.

5. An immune complex comprising the nanobody described in claim 1 or 2.

6. The immunocomplex according to claim 5, comprising an antibody containing nanobodies.

7. The immune complex according to claim 6, wherein the antibody is conjugated to a cytotoxic agent, a radioisotope, a drug compound, or a peptide.

8. An isolated nucleic acid encoding a nanobody according to claim 1 or 2.

9. Recombinant eukaryotic or prokaryotic host cells that produce nanobodies according to claim 1 or 2.

10. A pharmaceutical composition comprising the nanobody described in claim 1 or 2.

11. A pharmaceutical composition comprising the antibody according to claim 3 or 4, the immune complex according to claim 5, 6, or 7, or the isolated nucleic acid according to claim 8.

12. A nanobody according to claim 1 or 2 for use as a pharmaceutical.

13. An antibody according to claim 3 or 4, an immune complex according to claim 5, 6, or 7, an isolated nucleic acid according to claim 8, or a pharmaceutical composition according to claim 10 or 11, for use as a pharmaceutical.

14. A nanobody according to claim 1 or 2, an antibody according to claim 3 or 4, an immune complex according to claim 5, 6 or 7, an isolated nucleic acid according to claim 8, or a pharmaceutical composition according to claim 10 or 11, for use in the treatment of disorders or diseases related to GPRC5D.

15. A nanobody according to claim 1 or 2, an antibody according to claim 3 or 4, an immune complex according to claim 5, 6 or 7, an isolated nucleic acid according to claim 8, or a pharmaceutical composition according to claim 10 or 11 for use in the treatment of multiple myeloma.

16. A method for treating patients suffering from a disease associated with GPRC5D, The process of administering to a patient a pharmacologically effective amount of the nanobody described in claim 1 or 2, the antibody described in claim 3 or 4, the immune complex described in claim 5, 6, or 7, the isolated nucleic acid described in claim 8, or the pharmaceutical composition described in claim 10 or 11. Methods that include...

17. The method according to claim 16, wherein the disease is multiple myeloma.

18. A method for detecting the presence of GPRC5D in a sample, A step of bringing a sample into contact with the nanobody described in claim 1 or 2 under conditions that enable the formation of a composite between the nanobody and GPRC5D; and A process to determine whether a complex has been formed. Methods that include...

19. A method for producing anti-GPRC5D nanobodies, (i) A step of culturing host cells that express nucleic acids encoding nanobodies according to claim 1 or 2, and (ii) A step to purify anti-GPRC5D nanobodies from the culture medium of host cells. Methods that include...

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