Antibody or fragment thereof that binds to FCRL1

Abstract

The present invention relates to: a monoclonal antibody or a fragment thereof that binds to an extracellular region of FCRL1; a hybridoma that produces the antibody; a nucleic acid having a base sequence that encodes the antibody or a fragment of the antibody; cells transformed by a vector including the nucleic acid; a method for producing the antibody or a fragment of the antibody, the method using the hybridoma or the transformed cells; an antibody-drug conjugate including the antibody or a fragment of the antibody; a therapeutic drug and a diagnostic drug which include the antibody or a fragment of the antibody; and a method for treating and a method for diagnosing an FCRL1-related disease which use the antibody or a fragment of the antibody or an antibody-drug conjugate including the antibody or a fragment of the antibody.

Description

Antibody or antibody fragment thereof that binds to FCRL1

[0001] The present invention relates to a monoclonal antibody or antibody fragment that binds to the extracellular region of Fc receptor-like protein 1, a hybridoma that produces the antibody, a nucleic acid having a base sequence encoding the antibody or the antibody fragment, a transformed cell obtained by introducing a vector containing the nucleic acid into a host cell, a method for producing the antibody or the antibody fragment using the hybridoma or the transformed cell, an antibody-drug conjugate containing the antibody or the antibody fragment, a therapeutic agent and a diagnostic agent containing the antibody or the antibody fragment, and a method for treating and diagnosing an Fc receptor-like protein 1-associated disease using the antibody or the antibody fragment, or an antibody-drug conjugate containing the antibody or the antibody fragment.

[0002] Fc receptor-like protein 1 (hereinafter sometimes referred to as FCRL1) is a membrane protein belonging to the immunoglobulin superfamily, also known by other names such as CD307a, FCRH1, IFGP1, and IRTA5. The amino acid sequence of human FCRL1 was identified in 2001 (Non-Patent Document 1).

[0003] FCRL1 is a type I transmembrane protein expressed in B cells. It has three extracellular immunoglobulin-like domains, two intracellular immunoreceptor tyrosine activation motifs, and a transmembrane region (Non-Patent Document 1). To date, the endogenous ligand of FCRL1 has not been identified.

[0004] It has been reported that FCRL1 is expressed not only in normal B cells but also in cancer cells such as chronic lymphocytic leukemia, follicular lymphoma, hairy cell leukemia, and mantle cell lymphoma (Non-Patent Documents 2 and 3).More recently, it has been reported that FCRL1 contributes to cancer proliferation (Non-Patent Document 4).

[0005] Known monoclonal antibodies against FCRL1 include E3 and E9 (Non-Patent Document 2), 2G5, 7G8, 5A2 (Patent Document 1), 1F9, 2A10 (Patent Document 2), and 5A3 (Patent Document 3). It is also known that conjugating an immunotoxin to an anti-FCRL1 antibody exerts cytotoxic activity against cancer cell lines (Non-Patent Document 4).

[0006] International Publication No. WO 2005 / 097185 U.S. Patent Application Publication No. 2006 / 0216232 International Publication No. WO 2006 / 037048

[0007] Davis RS., et al. “Identification of a family of Fc receptor homologs with preferential B cell expression” Proceedings of the National Academy of Sciences of the United States of America 98. 17 (2001): 9772-9777.Du X., et al. “FCRL1 on chronic lymphocytic leukemia, hairy cell leukemia, and B-cell non-Hodgkin lymphoma as a target of immunotoxins” Blood 111. 1 (2008): 338-343.Auat M., et al. 1 as a Promising Target for “Immunotherapeutic Interventions of B-Cell-Related Disorders” Biomarker Insights 14 (2018): 1-9.

[0008] The present invention aims to provide a novel monoclonal antibody or antibody fragment that binds to the extracellular domain of FCRL1, a hybridoma that produces the antibody, a nucleic acid having a base sequence encoding the antibody or the antibody fragment, a transformed cell obtained by introducing a vector containing the nucleic acid into a host cell, a method for producing the antibody or the antibody fragment using the hybridoma or the transformed cell, an antibody-drug conjugate comprising the antibody or the antibody fragment, a therapeutic agent and a diagnostic agent comprising the antibody or the antibody fragment, and a method for treating and diagnosing an FCRL1-related disease using the antibody or the antibody fragment, or an antibody-drug conjugate comprising the antibody or the antibody fragment.

[0009] The present invention relates to the following 1 to 26: 1. A monoclonal antibody or an antibody fragment thereof that binds to Fc receptor-like protein 1 (hereinafter abbreviated as FCRL1), wherein the antibody or the antibody fragment is any one of antibodies selected from the following (a) to (g): (a) an antibody in which complementarity determining regions (CDRs) 1 to 3 of the heavy chain variable region (VH) comprise the amino acid sequences set forth in SEQ ID NOS: 20 to 22, respectively, and CDRs 1 to 3 of the light chain variable region (VL) comprise the amino acid sequences set forth in SEQ ID NOS: 24 to 26, respectively; (b) an antibody in which CDRs 1 to 3 of the VH comprise the amino acid sequences set forth in SEQ ID NOS: 28 to 30, respectively, and CDRs 1 to 3 of the VL comprise the amino acid sequences set forth in SEQ ID NOS: 32 to 34, respectively; (c) an antibody in which CDR1-3 of VH comprise the amino acid sequences set forth in SEQ ID NOS: 36-38, respectively, and CDR1-3 of VL comprise the amino acid sequences set forth in SEQ ID NOS: 40-42, respectively; (d) an antibody in which CDR1-3 of VH comprise the amino acid sequences set forth in SEQ ID NOS: 44-46, respectively, and CDR1-3 of VL comprise the amino acid sequences set forth in SEQ ID NOS: 48-50, respectively; (e) an antibody in which CDR1-3 of VH comprise the amino acid sequences set forth in SEQ ID NOS: 52-54, respectively, and CDR1-3 of VL comprise the amino acid sequences set forth in SEQ ID NOS: 56-58, respectively; and (f) an antibody in which CDR1-3 of VH comprise the amino acid sequences set forth in SEQ ID NOS: 60-62, respectively, and CDR1-3 of VL comprise the amino acid sequences set forth in SEQ ID NOS: 64-66, respectively. (g) An antibody in which CDR1 to CDR3 of VH comprise the amino acid sequences set forth in SEQ ID NOs: 36 to 38, respectively, and CDR1 to CDR3 of VL comprise the amino acid sequences set forth in SEQ ID NOs: 40, 71, and 42, respectively. 2. A monoclonal antibody or an antibody fragment thereof that binds to FCRL1, wherein the antibody is any one antibody selected from the following (2b-1) to (2b-4), (2c-1), (2c-2), and (2g-1).(2b-1) An antibody whose VH comprises the amino acid sequence set forth in SEQ ID NO: 72 and whose VL comprises the amino acid sequence set forth in SEQ ID NO: 68. (2b-2) An antibody whose VH comprises the amino acid sequence set forth in SEQ ID NO: 73 and whose VL comprises the amino acid sequence set forth in SEQ ID NO: 74. (2b-3) An antibody whose VH comprises the amino acid sequence set forth in SEQ ID NO: 72 and whose VL comprises the amino acid sequence set forth in SEQ ID NO: 74. (2b-4) An antibody whose VH comprises the amino acid sequence set forth in SEQ ID NO: 73 and whose VL comprises the amino acid sequence set forth in SEQ ID NO: 68. (2c-1) An antibody whose VH comprises the amino acid sequence set forth in SEQ ID NO: 75 and whose VL comprises the amino acid sequence set forth in SEQ ID NO: 76. (2c-2) An antibody whose VH comprises the amino acid sequence set forth in SEQ ID NO: 77 and whose VL comprises the amino acid sequence set forth in SEQ ID NO: 76. (2g-1) An antibody whose VH comprises the amino acid sequence set forth in SEQ ID NO: 77 and whose VL comprises the amino acid sequence set forth in SEQ ID NO: 78. 3. 3. The antibody or antibody fragment thereof according to 1 or 2 above, wherein the heavy chain constant region of the antibody is an IgG heavy chain constant region. 4. The antibody or antibody fragment thereof according to 3 above, wherein the heavy chain constant region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 79 or 80. 5. The antibody or antibody fragment thereof according to any one of 1 to 4 above, wherein the antibody is a recombinant antibody. 6. The antibody or antibody fragment thereof according to 5 above, wherein the recombinant antibody is one selected from the group consisting of a chimeric antibody, a humanized antibody, and a human antibody. 7. The antibody fragment is Fab, Fab', or F(ab'). ２8. The antibody fragment according to any one of 1 to 6 above, which is one selected from the group consisting of a single chain antibody (scFv), a dimerized V region (diabody), a disulfide-stabilized V region (dsFv), and a peptide comprising CDR. 8. A hybridoma producing the antibody according to any one of 1 to 6 above. 9. A nucleic acid having a base sequence encoding the antibody or antibody fragment according to any one of 1 to 7 above. 10. A vector comprising the nucleic acid according to 9 above. 11. A transformed cell obtained by introducing the vector according to 10 above into a host cell. 12. A method for producing the antibody or antibody fragment according to any one of 1 to 7 above, which comprises culturing the hybridoma according to 8 above or the transformed cell according to 11 above in a medium and collecting the antibody or antibody fragment from the culture. 13. An antibody-drug conjugate comprising the antibody or antibody fragment according to any one of 1 to 7 above. 14. The antibody-drug conjugate according to 13 above, which comprises the antibody or antibody fragment linked to a drug via a linker. 15. A composition comprising the antibody or antibody fragment thereof described in any one of above 1 to 7, or the antibody-drug conjugate described in above 13 or 14. 16. A reagent for detecting or measuring FCRL1, comprising the antibody or antibody fragment thereof described in any one of above 1 to 7, or the antibody-drug conjugate described in above 13 or 14. 17. A diagnostic agent for an FCRL1-related disease, comprising the antibody or antibody fragment thereof described in any one of above 1 to 7, or the antibody-drug conjugate described in above 13 or 14. 18. The diagnostic agent according to above 17, wherein the FCRL1-related disease is cancer, an autoimmune disease, or an inflammatory disease. 19. A therapeutic agent for an FCRL1-related disease, comprising the antibody or antibody fragment thereof described in any one of above 1 to 7, or the antibody-drug conjugate described in above 13 or 14. 20. The therapeutic agent according to above 19, wherein the FCRL1-related disease is cancer, an autoimmune disease, or an inflammatory disease. 21. 21. A method for diagnosing an FCRL1-related disease using the antibody or antibody fragment thereof described in any one of 1 to 7, or the antibody-drug conjugate described in 13 or 14. 22. A method for treating an FCRL1-related disease, comprising administering the antibody or antibody fragment thereof described in any one of 1 to 7, or the antibody-drug conjugate described in 13 or 14.23. Use of the antibody or antibody fragment thereof described in any one of 1 to 7 above, or the antibody-drug conjugate described in 13 or 14 above, for the manufacture of a diagnostic agent for an FCRL1-related disease. 24. Use of the antibody or antibody fragment thereof described in any one of 1 to 7 above, or the antibody-drug conjugate described in 13 or 14 above, for the manufacture of a therapeutic agent for an FCRL1-related disease. 25. The antibody or antibody fragment thereof described in any one of 1 to 7 above, or the antibody-drug conjugate described in 13 or 14 above, for use as a diagnostic agent for an FCRL1-related disease. 26. The antibody or antibody fragment thereof described in any one of 1 to 7 above, or the antibody-drug conjugate described in 13 or 14 above, for use as a therapeutic agent for an FCRL1-related disease.

[0010] The monoclonal antibody or antibody fragment of the present invention selectively binds to the extracellular domain of human FCRL1. In particular, the monoclonal antibody or antibody fragment of the present invention exhibits superior effects compared to existing FCRL1 antibodies when used in antibody-drug conjugates (hereinafter also referred to as ADCs). Therefore, the monoclonal antibody or antibody fragment of the present invention can be used as a therapeutic agent or diagnostic agent for human FCRL1-related diseases.

[0011] Figure 1 shows the results of measuring the antitumor effect of an antibody-drug conjugate in which a known anti-human FCRL1 antibody is linked to the payload linker SG3249 in a mouse model subcutaneously implanted with SU-DHL-6 cells. The vertical axis of Figure 1 represents tumor size (mm ３) are shown. The horizontal axis of Figure 1 indicates the number of days after administration of ADC to a mouse model subcutaneously implanted with SU-DHL-6 cells. E9, 1F9, and 7G8 were used as known anti-human FCRL1 antibodies. An anti-2,4-dinitrophenol (DNP) IgG1 antibody was used as a negative antibody. Figure 2A shows the results of measuring the effect on SU-DHL-6 cell viability of an ADC in which a novel anti-human FCRL1 antibody is conjugated to the payload linker SG3249. The vertical axis of Figure 2A indicates cell viability (%), with the number of cells without ADC treatment set to 100%. The horizontal axis of Figure 2A indicates the concentration of ADC added to SU-DHL-6 cells. DK1142, DK1164, DK681, DK1166, and DK1141 were used as novel anti-human FCRL1 antibodies. 7G8 was used as the known anti-human FCRL1 antibody. Figure 2B shows the results of an assay similar to that shown in Figure 2A, except that DK610 was used as the novel anti-human FCRL1 antibody. Figure 3A shows the results of assaying the effect on Ramos cell viability of an ADC in which the novel anti-human FCRL1 antibody is conjugated to the payload linker SG3249. The vertical axis of Figure 3A represents cell viability (%), with the number of cells in the absence of ADC treatment set to 100%. The horizontal axis of Figure 3A represents the concentration of ADC added to Ramos cells. DK1142, DK1164, DK681, DK1166, and DK1141 were used as novel anti-human FCRL1 antibodies. 7G8 was used as the known anti-human FCRL1 antibody. Figure 3B shows the results of an assay similar to that shown in Figure 3A, except that DK610 was used as the novel anti-human FCRL1 antibody. Figure 4 shows the results of measuring the antitumor effect of an ADC in which the payload linker SG3249 is conjugated to a novel anti-human FCRL1 antibody in a mouse model subcutaneously implanted with SU-DHL-6 cells and a mouse model subcutaneously implanted with Ramos cells. The results are shown 10 days after drug administration. The vertical axis of Figure 4 indicates the relative tumor size, with the tumor size in mice administered with 7G8 set as 1. DK1142, DK1164, DK681, DK1166, DK1141, and DK610 were used as novel anti-human FCRL1 antibodies. 7G8 was used as a known anti-human FCRL1 antibody.Figure 5 shows the results of measuring the antitumor effect of an ADC in which a novel anti-human FCRL1 antibody is conjugated to the payload linker SG3249 in a mouse model subcutaneously implanted with Ramos cells. The results are shown 42 days after drug administration. The vertical axis of Figure 5 represents tumor size (mm). ３) are shown. As novel anti-human FCRL1 antibodies, DK1142, DK1164, DK681, DK1166, DK1141, and DK610 were used. As known anti-human FCRL1 antibody, 7G8 was used. Figure 6 shows the results of measuring the internalization of novel anti-human FCRL1 antibodies in Ramos cells. The vertical axis of Figure 6 shows fluorescence intensity. As novel anti-human FCRL1 antibodies, DK1142, DK1164, DK681, DK1166, DK1141, and DK610 were used. As known anti-human FCRL1 antibody, 7G8 was used. Figure 7A shows the results of measuring the effect on the viability of SU-DHL-6 cells of an ADC in which the novel anti-human FCRL1 antibody is conjugated to the payload linker SG3249. The vertical axis of Figure 7A represents cell viability (%), with the number of cells without ADC treatment set to 100%. The horizontal axis of Figure 7A represents the concentration of ADC added to SU-DHL-6 cells. DK681 was used as the novel anti-FCRL1 chimeric antibody, and DK681 F11, DK681 F12, DK681 F13, and DK681 F14 were used as the novel anti-FCRL1 humanized antibodies. 7G8 was used as the known anti-human FCRL1 antibody. Figure 7B shows the results of measurements similar to those in Figure 7A, except that DK1142 was used as the novel anti-FCRL1 chimeric antibody, and DK1142 F21, DK1142 F22, and DK1142 F24 were used as the novel anti-FCRL1 humanized antibodies. Figure 8A shows the results of measuring the effect on Ramos cell viability of an ADC in which a novel anti-human FCRL1 antibody is conjugated to the payload linker SG3249. The vertical axis of Figure 8A represents cell viability (%), with the number of cells without ADC treatment set to 100%. The horizontal axis of Figure 8A represents the concentration of ADC added to Ramos cells. DK681 was used as the novel anti-FCRL1 chimeric antibody, and DK681 F11, DK681 F12, DK681 F13, and DK681 F14 were used as the novel anti-FCRL1 humanized antibodies. 7G8 was used as the known anti-human FCRL1 antibody.Figure 8B shows the results of measurements similar to those in Figure 8A, using DK1142 as the novel anti-FCRL1 chimeric antibody and DK1142 F21, DK1142 F22, and DK1142 F24 as the novel anti-FCRL1 humanized antibodies. Figure 9 shows the results of measuring the antitumor effect of an ADC in which the novel anti-human FCRL1 antibody is conjugated to the payload linker SG3249 in a mouse model subcutaneously implanted with SU-DHL-6 cells and a mouse model subcutaneously implanted with Ramos cells. The results are shown 7 days after drug administration. The vertical axis of Figure 9 shows the relative tumor size, with the tumor size in mice administered with 7G8 set to 1. As novel anti-human FCRL1 antibodies, DK681 F11, DK681 F12, DK681 F13, DK681 F14, DK1142 F21, DK1142 F22, and DK1142 F24 were used. As a known anti-human FCRL1 antibody, 7G8 was used.

[0012] The present invention relates to a monoclonal antibody or an antibody fragment thereof that binds to human FCRL1.

[0013] FCRL1 is also called CD307a, FCRH1, IFGP1, and IRTA5. FCRL1 is a type 1 membrane protein that belongs to the immunoglobulin superfamily and consists of 413 amino acids.

[0014] FCRL1 contains two immunoreceptor tyrosine-based activation motifs (ITAMs) intracellularly. Therefore, it is expected that ligand binding will transmit an activation signal into the cell. However, at present, the endogenous ligand of FCRL1 has not been identified, and the function of FCRL1 remains unclear. Recent experiments using cancer cell lines have reported that FCRL1 is involved in cancer cell proliferation by regulating the expression of apoptosis-related molecules.

[0015] In the present invention, human FCRL1 includes a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or the amino acid sequence of NCBI Accession No. NP_443170; a polypeptide consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 3 or the amino acid sequence of NCBI Accession No. NP_443170 and having the function of human FCRL1; and a polypeptide consisting of an amino acid sequence which has 60% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more similarity to the amino acid sequence of SEQ ID NO: 3 or the amino acid sequence of NCBI Accession No. NP_443170 and having the function of human FCRL1.

[0016] A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 3 or the amino acid sequence set forth in NCBI Accession No. NP_443170 can be produced by site-directed mutagenesis [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989); Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997); Nucleic Acids Research, 10, 6487 (1982); Proc. Natl. Acad. Sci. USA, 79, 6409 (1982); Gene, 34, 315 (1985); Nucleic Acids Research, 13, 4431 (1985); Proc. Natl. Acad. Sci. USA, 82, 488 (1985); (1985)] or the like, can be obtained by introducing site-specific mutations into DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:3.

[0017] The number of amino acids to be deleted, substituted or added is not particularly limited, but is preferably one to several tens, for example, one to twenty, more preferably one to several, for example, one to five amino acids.

[0018] Examples of genes encoding human FCRL1 include the nucleotide sequence set forth in SEQ ID NO: 1 and the nucleotide sequence of NCBI Accession No. NM_052938. The genes encoding human FCRL1 of the present invention also include genes comprising DNA that consists of a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequence of NM_052938 and encodes a polypeptide having the function of human FCRL1; genes comprising DNA that consists of a nucleotide sequence that has at least 60% similarity, preferably 80% similarity, and more preferably 95% similarity, to the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequence of NM_052938 and encodes a polypeptide having the function of human FCRL1; and genes that consist of DNA that hybridizes under stringent conditions to DNA comprising the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequence of NM_052938 and encodes a polypeptide having the function of human FCRL1.

[0019] The DNA that hybridizes under stringent conditions refers to hybridizable DNA obtained by colony hybridization, plaque hybridization, Southern blot hybridization, DNA microarray, or the like, using DNA containing the nucleotide sequence set forth in SEQ ID NO: 1 or the nucleotide sequence of NM_052938 as a probe.

[0020] Specifically, the DNA can be identified by carrying out hybridization at 65°C in the presence of 0.7 to 1.0 mol / L sodium chloride using DNA derived from hybridized colonies or plaques, or a filter or slide glass onto which a PCR product or oligo DNA having the sequence has been immobilized [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989); Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997); DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University, (1995)], followed by washing the filter or slide glass at 65°C using a 0.1 to 2x SSC solution (a 1x SSC solution consists of 150 mmol / L sodium chloride and 15 mmol / L sodium citrate).

[0021] Examples of hybridizable DNA include DNA having at least 60% or more similarity to the base sequence set forth in SEQ ID NO: 1 or the base sequence of NM_052938, preferably DNA having 80% or more similarity, and more preferably DNA having 95% or more similarity.

[0022] Genetic polymorphisms are often found in the nucleotide sequences of genes encoding proteins in eukaryotes. Genes used in the present invention that have small mutations in their nucleotide sequences due to such polymorphisms are also included in the genes encoding human FCRL1 of the present invention. Antibodies of the present invention include antibodies that bind to both human FCRL1 and monkey FCRL1.

[0023] In the present invention, examples of monkey FCRL1 include a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of NCBI accession number XP_015310712; a polypeptide consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of NCBI accession number XP_015310712 and having the function of monkey FCRL1; and a polypeptide consisting of an amino acid sequence that has 60% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more similarity to the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of NCBI accession number XP_015310712 and having the function of monkey FCRL1.

[0024] A polypeptide having an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 4 or the amino acid sequence shown by NCBI Accession No. XP_015310712 can be obtained by introducing site-specific mutations into DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, for example, using site-specific mutagenesis or the like.

[0025] The number of amino acids to be deleted, substituted or added is not particularly limited, but is preferably one to several tens, for example, one to twenty, more preferably one to several, for example, one to five amino acids.

[0026] Examples of genes encoding simian FCRL1 include the nucleotide sequence set forth in SEQ ID NO: 2 and the nucleotide sequence of NCBI Accession No. XM_005541349. The gene encoding simian FCRL1 of the present invention also includes a gene comprising a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence of SEQ ID NO: 2 or the nucleotide sequence of XM_005541349 and which contains DNA encoding a polypeptide having the function of simian FCRL1; a gene comprising a nucleotide sequence having at least 60% similarity, preferably 80% or more similarity, and more preferably 95% or more similarity to the nucleotide sequence of SEQ ID NO: 2 or the nucleotide sequence of XM_005541349 and which encodes a polypeptide having the function of simian FCRL1; and a gene comprising DNA that hybridizes under stringent conditions to DNA comprising the nucleotide sequence of SEQ ID NO: 2 or the nucleotide sequence of XM_005541349 and which encodes a polypeptide having the function of simian FCRL1.

[0027] In the present invention, the similarity between amino acid sequences or nucleotide sequences refers to a numerical value calculated under specific conditions by comparing two amino acid sequences or nucleotide sequences. Specifically, the similarity can be obtained by obtaining an alignment of the two sequences and calculating the proportion of matching or similar residue pairs within the alignment. Algorithms such as the Needleman-Wunsch method, the Smith-Waterman method, the FASTA method, and the BLAST method are used to obtain the alignment. Parameters used in each algorithm include a similarity evaluation index for each residue pair (for amino acid sequences, for example, substitution matrices such as BLOSUM62, BLOSUM50, and PAM30 are used, and for nucleotide sequences, for example, match reward, mismatch penalty, etc. are used), a quantitative evaluation index for gap portions (for example, an affine gap cost function), and the like. An example of the similarity between amino acid sequences or nucleotide sequences in the present invention is the value of identities or positives output in association with an alignment obtained using default parameters by NCBI BLAST, a representative implementation of the BLAST method.

[0028] The binding of the antibody of the present invention to the extracellular domain of human FCRL1 can be confirmed by measuring the binding affinity of the antibody of the present invention to human FCRL1-expressing cells using ELISA, flow cytometry, surface plasmon resonance, etc. Alternatively, this can be confirmed by combining known immunological detection methods [Monoclonal Antibodies—Principles and practice, Third edition, Academic Press (1996), Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory (1988), Monoclonal Antibody Experiment Manual, Kodansha Scientific (1987)], etc.

[0029] Antibody molecules are also called immunoglobulins (hereinafter referred to as Ig), and human antibodies are classified into isotypes IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM depending on differences in molecular structure. IgG1, IgG2, IgG3, and IgG4, which have relatively high similarity in amino acid sequence, are collectively referred to as IgG.

[0030] An antibody molecule is composed of polypeptides called heavy chains (hereinafter referred to as H chains) and light chains (hereinafter referred to as L chains). The H chain is composed of an H chain variable region (also referred to as VH) and an H chain constant region (also referred to as CH) from the N-terminus, while the L chain is composed of an L chain variable region (also referred to as VL) and an L chain constant region (also referred to as CL) from the N-terminus. The CHs are known as α, δ, ε, γ, and μ chains for each Ig isotype. The CHs are further composed of a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain from the N-terminus. A domain refers to a functional structural unit that constitutes each polypeptide of an antibody molecule. The CH2 domain and CH3 domain are collectively referred to as the Fc region or simply Fc. The CLs are known as Cλ chains and Cκ chains.

[0031] The CH1 domain, hinge region, CH2 domain, CH3 domain, and Fc region of the present invention can be identified by the numbering of amino acid residues from the N-terminus according to the EU index (also known as EU numbering) [Kabat et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]. Specifically, CH1 is identified as the amino acid sequence of positions 118 to 215 in the EU index, the hinge as the amino acid sequence of positions 216 to 230 in the EU index, CH2 as the amino acid sequence of positions 231 to 340 in the EU index, and CH3 as the amino acid sequence of positions 341 to 447 in the EU index.

[0032] The monoclonal antibody of the present invention may be an antibody produced by a hybridoma, or a recombinant antibody produced by a transformed cell transformed with an expression vector containing an antibody gene.

[0033] A hybridoma is a cell that produces a monoclonal antibody with a desired antigen specificity, obtained by cell fusion between B cells obtained by immunizing a non-human animal with an antigen and myeloma cells derived from a mouse or the like. Therefore, the variable regions that make up the antibodies produced by hybridomas consist of the amino acid sequence of a non-human animal antibody.

[0034] The antibodies of the present invention particularly include recombinant antibodies such as recombinant mouse antibodies, recombinant rat antibodies, recombinant rabbit antibodies, human chimeric antibodies (hereinafter also simply referred to as chimeric antibodies), humanized antibodies (also referred to as human complementarity-determining region (CDR)-grafted antibodies), and human antibodies produced by genetic engineering.

[0035] A chimeric antibody refers to an antibody consisting of VH and VL of an antibody from an animal other than a human (non-human animal) and CH and CL of a human antibody. Any non-human animal, such as a mouse, rat, hamster, or rabbit, can be used as long as it is possible to produce a hybridoma from it.

[0036] A human chimeric antibody can be produced by obtaining cDNA encoding the VH and VL of a monoclonal antibody from a hybridoma derived from non-human animal cells that produces the monoclonal antibody, and inserting the cDNA into an expression vector for animal cells that contains DNA encoding the CH and CL of a human antibody to construct a human chimeric antibody expression vector, which is then introduced into animal cells for expression.

[0037] A humanized antibody is an antibody in which the amino acid sequences of the CDRs of the VH and VL of a non-human animal antibody have been grafted onto the corresponding CDRs of the VH and VL of a human antibody. The regions other than the CDRs of the VH and VL are called framework regions (hereinafter referred to as FR).

[0038] A humanized antibody can be produced by constructing a cDNA encoding a VH amino acid sequence consisting of the amino acid sequence of the CDR of the VH of a non-human animal antibody and the amino acid sequence of the FR of the VH of any human antibody, and a cDNA encoding a VL amino acid sequence consisting of the amino acid sequence of the CDR of the VL of a non-human animal antibody and the amino acid sequence of the FR of the VL of any human antibody, and inserting each of these into an expression vector for animal cells containing DNA encoding the CH and CL of a human antibody to construct a humanized antibody expression vector, which can then be expressed and produced by introducing it into animal cells.

[0039] Human antibodies originally refer to antibodies that naturally occur in the human body, but also include human antibody phage libraries produced through recent advances in genetic engineering, cell engineering, and developmental engineering technologies, and antibodies obtained from human antibody-producing transgenic animals.

[0040] Human antibodies can be obtained by immunizing mice carrying human immunoglobulin genes (Tomizuka K. et al., Proc Natl Acad Sci USA. 97, 722-7, 2000) with a desired antigen. Alternatively, human antibodies can be obtained without immunization by selecting human antibodies with the desired binding activity using a phage display library in which antibody genes have been amplified from human-derived B cells (Winter G. et al., Annu Rev Immunol. 12:433-55, 1994). Furthermore, human B cells can be immortalized using EB virus to generate cells that produce human antibodies with the desired binding activity, allowing human antibodies to be obtained (Rosen A. et al., Nature 267, 52-54, 1977).

[0041] An antibody present in the human body can be obtained by, for example, immortalizing lymphocytes isolated from human peripheral blood by infecting them with EB virus or the like, and then cloning the immortalized lymphocytes to produce the antibody. The antibody can then be purified from a culture of the lymphocytes.

[0042] A human antibody phage library is a library of phages in which antibody fragments such as Fab and scFv are expressed on the surface by inserting antibody genes prepared from human B cells into the phage gene. Phages expressing antibody fragments with the desired antigen-binding activity can be recovered from the library using their binding activity toward an antigen-immobilized substrate as an indicator. These antibody fragments can also be converted into human antibody molecules consisting of two complete heavy chains and two complete light chains by genetic engineering techniques.

[0043] A human antibody-producing transgenic animal is an animal in which a human antibody gene has been integrated into the chromosome of the host animal. Specifically, a human antibody-producing transgenic animal can be produced by introducing a human antibody gene into a mouse ES cell, transplanting the ES cell into an early embryo of another mouse, and then allowing it to develop. Human antibodies can be produced from human antibody-producing transgenic animals by obtaining a human antibody-producing hybridoma by a hybridoma production method typically used in mammals other than humans, and culturing it to produce and accumulate human antibodies in the culture.

[0044] The amino acid sequences of the VH and VL of the antibody of the present invention may be any of the amino acid sequences of the VH and VL of a human antibody, the amino acid sequences of the VH and VL of a non-human animal antibody, or the amino acid sequences of the VH and VL of a humanized antibody in which the CDRs of a non-human animal antibody are grafted onto the framework of any human antibody.

[0045] The amino acid sequence of CL in the antibody of the present invention may be either the amino acid sequence of a human antibody or the amino acid sequence of a non-human animal antibody. κ or C λ is preferred.

[0046] The CH of the antibody of the present invention may be a CH of any immunoglobulin molecular species, but preferably any of the subclasses belonging to the IgG class, such as γ1 (IgG1; e.g., accession number AAA02914.1), γ2 (IgG2; e.g., accession number AAG00910.2), γ3 (IgG3; e.g., accession number P01860.2), and γ4 (IgG4; e.g., accession number P01861.1), can be used. Furthermore, the CH may be a CH in which one or more amino acids constituting the CH have been deleted, substituted, or added. The number of amino acids to be deleted, substituted, or added is not particularly limited, but is preferably one to several tens, e.g., one to twenty, and more preferably one to several, e.g., one to five amino acids. An example of a CH in which one or more amino acids constituting the CH have been deleted, substituted, or added is an IgG1 CH variant in which serine at position 239 (EU numbering) of the CH of human IgG1 has been substituted with cysteine. More specifically, examples include IgG1 CH variants comprising an amino acid sequence (SEQ ID NO: 80) in which serine at position 239 according to EU numbering in the CH of human IgG1 comprising the amino acid sequence set forth in SEQ ID NO: 79 has been substituted with cysteine.

[0047] The antibodies of the present invention also include Fc fusion proteins in which Fc is linked to an antibody fragment, Fc fusion proteins (also called immunoadhesins) in which Fc is linked to a naturally occurring ligand or receptor, Fc fusion proteins in which multiple Fc regions are fused, etc. Furthermore, Fc regions in which amino acid residues have been modified to stabilize the antibody and control its half-life in blood can also be used in the antibodies of the present invention.

[0048] The antibodies or antibody fragments of the present invention also include antibodies containing any post-translationally modified amino acid residues, such as deletion of a lysine residue at the C-terminus of the heavy chain (lysine clipping) or conversion of a glutamine residue at the N-terminus of a polypeptide to pyroglutamine (pyroGlu) [Beck et al., Analytical Chemistry, 85, 715-736 (2013)].

[0049] In the present invention, the antibody fragment refers to an antibody fragment that binds to the extracellular domain of human FCRL1 and has antigen-binding activity. In the present invention, the antibody fragment includes Fab, Fab', F(ab') ２ , scFv, diabody, dsFv, or a peptide containing CDR. Fab is an antibody fragment obtained by treating an IgG antibody with the protease papain (cleaved at the 224th amino acid residue of the H chain), in which approximately the N-terminal half of the H chain and the entire L chain are linked via a disulfide bond (S-S bond), and has a molecular weight of approximately 50,000 and has antigen-binding activity. The antibody fragment of the present invention is preferably an antibody fragment that binds to the extracellular domain of FCRL1 and induces internalization of FCRL1.

[0050] F(ab') ２ is an antibody fragment having antigen-binding activity and a molecular weight of approximately 100,000, which is slightly larger than that of Fab fragments linked via disulfide bonds in the hinge region, among fragments obtained by treating IgG with the protease pepsin (cleaved at the 234th amino acid residue of the H chain). Fab' is the same as the above F(ab'). ２ It is an antibody fragment with a molecular weight of approximately 50,000, which has antigen-binding activity and is obtained by cleaving the disulfide bonds in the hinge region of the antibody.

[0051] An scFv is an antibody fragment having antigen-binding activity, which is a VH-P-VL or VL-P-VH polypeptide in which one VH and one VL are linked using an appropriate peptide linker (P), such as a linker peptide consisting of any number of linkers (G4S) each consisting of four Gly and one Ser residue.

[0052] Diabodies are antibody fragments formed by dimerization of scFvs with the same or different antigen-binding specificities, and have bivalent antigen-binding activity to the same antigen or specific antigen-binding activity to different antigens.

[0053] dsFv refers to polypeptides in which one amino acid residue in each of VH and VL is substituted with a cysteine ​​residue, and the polypeptides are linked via an S—S bond between the cysteine ​​residues.

[0054] CDR-containing peptides comprise at least one region of the VH or VL CDR. In peptides containing multiple CDRs, the CDRs can be linked directly or via an appropriate peptide linker. The modified antibodies of the present invention can be produced by constructing DNA encoding the VH and VL CDRs, inserting the DNA into a prokaryotic or eukaryotic expression vector, and introducing the expression vector into a prokaryotic or eukaryotic organism for expression. Alternatively, CDR-containing peptides can be produced by chemical synthesis, such as the Fmoc or tBoc method.

[0055] One embodiment of the antibody of the present invention is any one selected from the following (a) to (g): (a) an antibody in which complementarity determining regions (CDRs) 1 to 3 of the heavy chain variable region (VH) comprise the amino acid sequences set forth in SEQ ID NOS: 20 to 22, respectively, and CDRs 1 to 3 of the light chain variable region (VL) comprise the amino acid sequences set forth in SEQ ID NOS: 24 to 26, respectively; (b) an antibody in which CDRs 1 to 3 of the VH comprise the amino acid sequences set forth in SEQ ID NOS: 28 to 30, respectively, and CDRs 1 to 3 of the VL comprise the amino acid sequences set forth in SEQ ID NOS: 32 to 34, respectively; (c) an antibody in which CDR1-3 of VH comprise the amino acid sequences set forth in SEQ ID NOS: 36-38, respectively, and CDR1-3 of VL comprise the amino acid sequences set forth in SEQ ID NOS: 40-42, respectively; (d) an antibody in which CDR1-3 of VH comprise the amino acid sequences set forth in SEQ ID NOS: 44-46, respectively, and CDR1-3 of VL comprise the amino acid sequences set forth in SEQ ID NOS: 48-50, respectively; (e) an antibody in which CDR1-3 of VH comprise the amino acid sequences set forth in SEQ ID NOS: 52-54, respectively, and CDR1-3 of VL comprise the amino acid sequences set forth in SEQ ID NOS: 56-58, respectively; and (f) an antibody in which CDR1-3 of VH comprise the amino acid sequences set forth in SEQ ID NOS: 60-62, respectively, and CDR1-3 of VL comprise the amino acid sequences set forth in SEQ ID NOS: 64-66, respectively. (g) An antibody in which CDR1 to CDR3 of VH comprise the amino acid sequences set forth in SEQ ID NOs: 36 to 38, respectively, and CDR1 to CDR3 of VL comprise the amino acid sequences set forth in SEQ ID NOs: 40, 71, and 42, respectively.

[0056] One embodiment of the antibody of the present invention includes any one selected from the following (1a) to (1f): (1a) an antibody whose VH comprises the amino acid sequence set forth in SEQ ID NO: 19 and whose VL comprises the amino acid sequence set forth in SEQ ID NO: 23, (1b) an antibody whose VH comprises the amino acid sequence set forth in SEQ ID NO: 27 and whose VL comprises the amino acid sequence set forth in SEQ ID NO: 31, (1c) an antibody whose VH comprises the amino acid sequence set forth in SEQ ID NO: 35 and whose VL comprises the amino acid sequence set forth in SEQ ID NO: 39, (1d) an antibody whose VH comprises the amino acid sequence set forth in SEQ ID NO: 43 and whose VL comprises the amino acid sequence set forth in SEQ ID NO: 47, (1e) an antibody whose VH comprises the amino acid sequence set forth in SEQ ID NO: 51 and whose VL comprises the amino acid sequence set forth in SEQ ID NO: 55, and (1f) an antibody whose VH comprises the amino acid sequence set forth in SEQ ID NO: 59 and whose VL comprises the amino acid sequence set forth in SEQ ID NO: 63

[0057] Embodiments of the antibodies of the present invention include the anti-human FCRL1 mouse monoclonal antibodies DK610, DK681, DK1142, DK1141, DK1166, and DK1164, which will be described later in the Examples. Furthermore, embodiment of the antibodies of the present invention include antibodies comprising the variable region of any one of antibodies DK610, DK681, DK1142, DK1141, DK1166, and DK1164. Furthermore, embodiment of the antibodies of the present invention include antibodies having the amino acid sequence of VH CDR1 to 3 and VL CDR1 to 3 of any one of antibodies DK610, DK681, DK1142, DK1141, DK1166, and DK1164.

[0058] An embodiment of the antibody of the present invention includes any one selected from the following (2b-1) to (2b-4), (2c-1), (2c-2), and (2g-1): (2b-1) An antibody whose VH comprises the amino acid sequence set forth in SEQ ID NO: 72 and whose VL comprises the amino acid sequence set forth in SEQ ID NO: 68. (2b-2) An antibody whose VH comprises the amino acid sequence set forth in SEQ ID NO: 73 and whose VL comprises the amino acid sequence set forth in SEQ ID NO: 74. (2b-3) An antibody whose VH comprises the amino acid sequence set forth in SEQ ID NO: 72 and whose VL comprises the amino acid sequence set forth in SEQ ID NO: 74. (2b-4) An antibody whose VH comprises the amino acid sequence set forth in SEQ ID NO: 73 and whose VL comprises the amino acid sequence set forth in SEQ ID NO: 68. (2c-1) An antibody whose VH comprises the amino acid sequence set forth in SEQ ID NO: 75 and whose VL comprises the amino acid sequence set forth in SEQ ID NO: 76. (2c-2) An antibody whose VH comprises the amino acid sequence set forth in SEQ ID NO: 77 and whose VL comprises the amino acid sequence set forth in SEQ ID NO: 76. (2g-1) An antibody in which VH comprises the amino acid sequence set forth in SEQ ID NO: 77 and VL comprises the amino acid sequence set forth in SEQ ID NO: 78.

[0059] One embodiment of the antibodies of the present invention includes humanized antibodies in which the amino acid sequences of CDR1 to CDR3 of the VH and CDR1 to CDR3 of the VL of the DK681 or DK1142 antibody have been grafted into the FR of a human antibody. Examples of such antibodies include DK681 F11, DK681 F12, DK681 F13, DK681 F14, DK1142 F21, and DK1142 F22, which are described below in the Examples. Another example of a humanized antibody in which the amino acid sequences of CDR1 to CDR3 of the VH and CDR1 and CDR3 of the VL of the DK1142 antibody and an amino acid sequence obtained by modifying CDR2 of the VL of the DK1142 antibody have been grafted into the FR of a human antibody is DK1142 F24, which is described below in the Examples.

[0060] The antibodies of the present invention include antibodies that selectively bind to FCRL1 expressed on the cell surface and induce the internalization of FCRL1, and antibodies that exhibit strong pharmacological effects when conjugated to a drug to form an ADC.

[0061] Whether the antibody of the present invention induces internalization of FCRL1 can be confirmed by, for example, binding a reagent that emits fluorescence in a low pH environment such as intracellular lysosomes to the antibody, adding the antibody to cells, and measuring the fluorescence intensity.

[0062] The antibodies of the present invention also include antibodies into which a chemical structure capable of reacting with a drug or a linker to form a bond has been introduced. Examples include antibodies into which a natural or unnatural amino acid residue having a functional group such as an α,β-unsaturated carbonyl group, an α,β-unsaturated sulfinyl group, an α,β-unsaturated sulfonyl group, a thiol group, a hydroxyl group, an amino group, an amido group, a formyl group, a carboxyl group, an azide group, an alkynyl group, an alkenyl group, a haloalkyl group, or a carbonyl group has been added, inserted, or substituted at the N-terminus, C-terminus, or along the amino acid sequence of the heavy or light chain of the antibody, and antibodies into which a sugar chain having a functional group such as an α,β-unsaturated carbonyl group, an α,β-unsaturated sulfinyl group, an α,β-unsaturated sulfonyl group, a thiol group, a hydroxyl group, an amino group, an amido group, a formyl group, a carboxyl group, an azide group, an alkynyl group, an alkenyl group, a haloalkyl group, or a carbonyl group has been introduced.

[0063] For example, an antibody in which an amino acid residue at a specific position of the antibody has been substituted with cysteine ​​can be used. In an IgG antibody, heavy chain amino acid residues suitable for substitution with cysteine ​​include, for example, serine at position 239 according to EU numbering (Dimasi, N. et al., Molecular Pharmaceutics. 14, 1501-1516, 2017), serine at position 442 (Stimmel, JB. et al., The Journal of Biological Chemistry. 275, 30445-50, 2000), and lysine at position 290 (Graziani, EI. et al., Molecular Cancer Therapeutics. 19, 2068-2078, 2020), threonine at position 114, alanine at position 140, leucine at position 174, leucine at position 179, threonine at position 187, threonine at position 209, valine at position 262, glycine at position 371, tyrosine at position 373, glutamic acid at position 382, ​​serine at position 424, asparagine at position 434, and glutamine at position 438 (WO 2016 / 040856). Furthermore, examples of amino acid residues in the κ light chain suitable for substitution with cysteine ​​include at least one of lysine at position 183 according to EU numbering (Graziani, E. I. et al., Molecular Cancer Therapeutics. 19, 2068-2078, 2020), glutamine at position 124 (Shinmi, D. et al., Bioconjugate Chemistry. 27, 1324-31, 2016), leucine or isoleucine at position 106, arginine at position 108, arginine at position 142, and lysine at position 149 (WO 2016 / 040856).

[0064] Other examples include an antibody into which paraacetylphenylalanine has been introduced (Skidmore, L. et. al., Molecular Cancer Therapeutics 19(9), 1833-1843, 2020), an antibody in which the thiol group of a cysteine ​​residue has been enzymatically converted to a formyl group (U.S. Patent Application Publication No. 2012 / 0183566), and an antibody in which a cysteine ​​has been inserted between serine at position 239 and valine at position 240 according to EU numbering in the heavy chain constant region (U.S. Patent No. 10,744,204).

[0065] ADCs comprising the antibodies of the present invention include molecules in which an antibody and a drug are chemically or genetically linked together, either directly or via a linker. The antibody portion of such an ADC molecule is also included in the antibodies of the present invention.

[0066] The drug (also referred to herein as a payload) contained in the ADC of the present invention may be any physiologically active molecule, and examples thereof include radioisotopes, low-molecular-weight drugs, high-molecular-weight drugs, proteins, antibody drugs, and nucleic acid drugs.

[0067] ADCs can be produced by chemically binding a drug or a linker to the N-terminus, C-terminus, or an appropriate functional group, side chain, or sugar chain in the antibody molecule of the antibody or antibody fragment of the present invention that binds to human FCRL1.

[0068] The antibodies or antibody fragments of the present invention can be conjugated to a linker or drug by known methods (e.g., methods described in S.J. Walsh et al. Chem. Soc. Rev. 2021, 50, 1305-1353; Tumey, L. Nathan (2020). Antibody-Drug Conjugates - Methods and Protocols: New York, Springer; and Laurent Ducry (2013). Antibody-Drug Conjugate: New York, Springer). For example, a functional group such as an α,β-unsaturated carbonyl group, α,β-unsaturated sulfinyl group, α,β-unsaturated sulfonyl group, thiol group, hydroxyl group, amino group, amide group, formyl group, carboxyl group, azide group, alkynyl group, alkenyl group, haloalkyl group, or carbonyl group introduced into an antibody molecule (including a sugar chain bound to the antibody) can be reacted with a functional group contained in a drug or linker under appropriate conditions. The combination of functional groups contained in an antibody and functional groups contained in a drug or linker can be appropriately selected based on publicly known information. For example, a bond can be formed by a nucleophilic reaction between a nucleophilic functional group, such as a thiol group, in an antibody molecule and a Michael acceptor, such as an α,β-unsaturated carboxylic acid, contained in a drug or linker. Alternatively, a bond can be formed by a cyclization reaction between an azide group in an antibody molecule and an alkynyl group in a drug or linker, with or without a catalyst.

[0069] Alternatively, the monoclonal antibody or antibody fragment of the present invention that binds to human FCRL1 can be produced by genetic engineering techniques, in which DNA encoding the monoclonal antibody or antibody fragment thereof is ligated to DNA encoding the protein or antibody drug to be bound, and the resulting mixture is inserted into an expression vector, and the expression vector is introduced into a suitable host cell for expression.

[0070] Examples of radioisotopes include In, I, I, Y, Cu, Tc, Lu, and At. Radioisotopes can be directly bound to antibodies using the chloramine T method or the like. Alternatively, a substance that chelates radioisotopes may be bound to the antibody. Examples of chelating agents include 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA).

[0071] Examples of low-molecular-weight drugs include anticancer drugs such as alkylating agents, nitrosoureas, antimetabolites, antibiotics, plant alkaloids, topoisomerase inhibitors, hormone therapy agents, hormone antagonists, aromatase inhibitors, P-glycoprotein inhibitors, platinum complex derivatives, M-phase inhibitors, and kinase inhibitors [Clinical Oncology, Cancer and Chemotherapy (1996)], steroid drugs such as hydrocortisone and prednisone, non-steroid drugs such as aspirin and indomethacin, immunomodulators such as gold thiomalate and penicillamine, immunosuppressants such as cyclophosphamide and azathioprine, and anti-inflammatory drugs such as antihistamines such as chlorpheniramine maleate and clemacitin [Inflammation and Anti-inflammatory Therapy, Ishiyaku Publishing Co., Ltd. (1992)].

[0072] Examples of anticancer drugs include amifostine (ethyol), cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin), epirubicin, gemcitabine (Gemzar), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, and methotrexate. , 5-fluorouracil, fluorouracil, vinblastine, vincristine, bleomycin, daunomycin, peplomycin, estramustine, paclitaxel (Taxol), docetaxel (Taxotere), aldesleukin, asparaginase, busulfan, carboplatin, oxaliplatin, nedaplatin, cladribine, camptothecin, 10-hydroxy-7-ethyl-camptothecin (SN38), floxuridine, fludarabine, hydroxyurea Rhea, idarubicin, mesna, irinotecan (CPT-11), nogitecan, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, hydroxycarbamide, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, tamoxifen, goserelin, leuprorenin, flutamide, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil, hydrochloride cyclophosphamide, prednisolone, methylprednisolone, vindesine, nimustine, semustine, capecitabine, tomudex, azacitidine, oxaloplatin, pyrrolobenzodiazepine (PBD) derivatives, auristatins (monomethyl auristatin E, monomethyl auristatin F, etc.), amanitin, camptothecin derivatives (deruxtecan, exatecan, SN-38, etc.), gefitinib (Iressa), imatinib (STI571), erlotinib, FMS-like tyrosine kinase 3 (Flt3) inhibitors, vascular endothelial growth factor receptor (VEGFR) inhibitors, fibroblast growth factor receptor (FGFR) inhibitors,Epidermal growth factor receptor (EGFR) inhibitors such as Iressa or Tarceva, radicicol, 17-allylamino-17-demethoxygeldanamycin, rapamycin, amsacrine, all-trans retinoic acid, thalidomide, lenalidomide, anastrozole, fadrozole, letrozole, exemestane, aurothiomalate, D-penicillamine, bucillamine, azathioprine, mizoribine, cyclosporine, hydrocortisone, bexarotene (Targretin), tamoxifen, dexamethasone, progestins, estrogens, anastrozole (Arimidex), leuprin, aspirin , indomethacin, celecoxib, penicillamine, gold thiomalate, chlorpheniramine maleate, chlorpheniramine, clemacitin, tretinoin, bexarotene, arsenic, bortezomib, allopurinol, ibritumomab tiuxetan, targretin, ozogamicin, clarithromycin, leucovorin, ketoconazole, aminoglutethimide, suramin or maytansinoid, dolastatin 10, actinomycin, anthracycline, duocarmycin, duocarmycin dimer (CPI-dimer, etc.), eribulin or a derivative thereof, etc.

[0073] Examples of polymeric drugs include polyethylene glycol (hereinafter referred to as PEG), albumin, dextran, polyoxyethylene, styrene-maleic acid copolymer, polyvinylpyrrolidone, pyran copolymer, hydroxypropyl methacrylamide, etc. By binding these polymeric compounds to antibodies or antibody fragments, effects such as (1) improved stability against various chemical, physical, or biological factors, (2) significant extension of blood half-life, or (3) elimination of immunogenicity or suppression of antibody production can be expected [Bioconjugate Pharmaceuticals, Hirokawa Shoten (1993)].

[0074] For example, a method for conjugating PEG to an antibody includes reacting the antibody with a PEGylation modifying reagent [Bioconjugate Pharmaceuticals, Hirokawa Shoten (1993)]. Examples of PEGylation modifying reagents include a modifying agent for the ε-amino group of lysine (Japanese Patent Laid-Open Publication No. 61-178926), a modifying agent for the carboxyl group of aspartic acid and glutamic acid (Japanese Patent Laid-Open Publication No. 56-23587), or a modifying agent for the guanidino group of arginine (Japanese Patent Laid-Open Publication No. 2-117920).

[0075] The immunostimulant may be a natural product known as an immunoadjuvant, and specific examples of immune-enhancing agents include β(1→3) glucan (e.g., lentinan or schizophyllan) or α-galactosylceramide (KRN7000).

[0076] Examples of proteins include cytokines or growth factors that activate immunocompetent cells such as NK cells, macrophages, or neutrophils, or toxin proteins.

[0077] Examples of cytokines or growth factors include interferon (hereinafter referred to as IFN)-α, IFN-β, IFN-γ, interleukin (hereinafter referred to as IL)-2, IL-12, IL-15, IL-18, IL-21, IL-23, granulocyte colony-stimulating factor (G-CSF), granulocyte / macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), etc. Examples of toxic proteins include ricin and diphtheria toxin, and also include protein toxins in which mutations have been introduced into the protein to regulate toxicity.

[0078] Examples of antibody drugs include antibodies against antigens that induce apoptosis upon antibody binding, antigens involved in the pathogenesis of tumors, antigens that regulate immune function, or antigens involved in angiogenesis at the lesion site.

[0079] Examples of antigens that induce apoptosis upon antibody binding include cluster of differentiation (hereinafter referred to as CD) 19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80 (B7.1), CD81, CD82, CD83, CDw84, CD85, CD86 (B7.2), human leukocyte antigen (HLA)-Class II, and epidermal growth factor receptor (EGFR).

[0080] Examples of antigens involved in the pathogenesis of tumors or antigens of antibodies that regulate immune function include CD4, CD40, CD40 ligand, B7 family molecules (e.g., CD80, CD86, CD274, B7-DC, B7-H2, B7-H3, or B7-H4), ligands of B7 family molecules (e.g., CD28, CTLA-4, ICOS, PD-1, or BTLA), OX-40, OX-40 ligand, CD137, tumor necrosis factor (TNF) receptor family molecules (e.g., DR4, DR5, TNFR1, or TNFR2), TNF-related apoptosis-inducing ligands, and the like. Examples of the receptor include TRAIL family molecules, receptor families of TRAIL family molecules (e.g., TRAIL-R1, TRAIL-R2, TRAIL-R3, or TRAIL-R4), receptor activator of nuclear factor kappa B ligand (RANK), RANK ligand, CD25, folate receptor, cytokines [e.g., IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-10, IL-13, transforming growth factor (TGF) β, or TNFα, etc.] or receptors of these cytokines, and chemokines (e.g., SLC, ELC, I-309, TARC, MDC, or CTACK, etc.) or receptors of these chemokines.

[0081] Antigens for antibodies that inhibit angiogenesis at lesion sites include, for example, vascular endothelial growth factor (VEGF), angiopoietin, fibroblast growth factor (FGF), EGF, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), erythropoietin (EPO), TGFβ, IL-8, ephrin, or SDF-1, or receptors thereof.

[0082] A fusion antibody with a protein or antibody drug can be produced by linking a cDNA encoding an antibody contained in the protein or antibody drug to a cDNA encoding a monoclonal antibody or antibody fragment to construct DNA encoding the fusion antibody, inserting the DNA into an expression vector for prokaryotes or eukaryotes, and introducing the expression vector into a prokaryote or eukaryote to express the fusion antibody.

[0083] Examples of nucleic acid drugs include pharmaceuticals containing nucleic acids such as small interference ribonucleic acid (siRNA) or microRNA, which act on living organisms by regulating gene function. For example, a conjugate with a nucleic acid drug that suppresses RORγt, a master transcription factor of Th17 cells, is conceivable.

[0084] The linker contained in the ADC of the present invention may have any structure as long as it has the function of binding an antibody to a drug. For example, it may have a structure with a special function, such as being cleaved near or inside a target cell or tissue, or a branched structure that allows multiple drugs to be bound. For example, known linkers (e.g., linkers described in SJ Walsh et al. Chem. Soc. Rev. 2021, 50, 1305-1353; Tumey, L. Nathan (2020). Antibody-Drug Conjugates -Methods and Protocols: New York, Springer; and Laurent Ducry (2013). Antibody-Drug Conjugate: New York, Springer, etc.) can be used in the ADC of the present invention. Specifically, for example, peptides, oligosaccharides, -(CH ２ ) -, oxygen atom, sulfur atom, -NH-, -(CH ２ CH ２ Examples of the linker include a linker consisting of any one selected from the group consisting of —O)—, —CO—, —PO—, amino acids, paraaminobenzyl (PAB), cyclic alkyls having 3 to 10 carbon atoms, and structures represented by the following formulas, or a linker containing a structure formed by linking two or more units selected from the above group:

[0085]

[0086] Examples of amino acids constituting the linker include valine (Val), citrulline (Cit), phenylalanine (Phe), lysine (Lys), D-valine (D-Val), leucine (Leu), glycine (Gly), alanine (Ala), and asparagine (Asn).

[0087] Examples of linkers include peptides, oligosaccharides, -(CH ２ ) ｎ -, -(CH ２ CH ２ O) ｎ-, -CO-, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, Ala-PAB, P AB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, Gly-Gly-Phe-Gly-PAB, -Gly-Gly-Phe-Gly-CH ２ -O-CH ２ Examples include a linker containing any one selected from the group consisting of —CO— and structures represented by the following formulas, and a linker containing a structure in which two or more units selected from the above group are linked together.

[0088]

[0089] Here, n represents an integer of 1 to 1000, preferably an integer of 1 to 100, more preferably an integer of 1 to 50, even more preferably an integer of 1 to 20, and most preferably an integer of 1 to 15. Ac represents an acetyl group. Lys(Ac) indicates that the amino group in the side chain of lysine is acetylated.

[0090] As an embodiment of the linker, for example, -(CH ２ ) ｍ -CO-NH-(CH ２ CH ２ O) ｎ -Val-Cit-PAB, -(CH ２ ) ｍ -CO-NH-(CH ２ CH ２ O) ｎ -Val-Ala-PAB, -(CH ２ ) ｍ -CO-NH-(CH ２ CH ２ O) ｎ -Val-Lys(Ac)-PAB, -(CH ２ ) ｍ -CO-NH-(CH ２ CH ２ O) ｎ -Phe-Lys-PAB, -(CH ２ ) ｍ -CO-NH-(CH ２ CH ２ O) ｎ-Phe-Lys(Ac)-PAB, -(CH ２ ) ｍ -CO-NH-(CH ２ CH ２ O) ｎ -Ala-PAB, -(CH ２ ) ｍ -CO-NH-(CH ２ CH ２ O) ｎ -D-Val-Leu-Lys, -(CH ２ ) ｍ -CO-NH-(CH ２ CH ２ O) ｎ -Gly-Gly-Arg, -(CH ２ ) ｍ -CO-NH-(CH ２ CH ２ O) ｎ -Ala-Ala-Asn-PAB, -(CH ２ ) ｍ -NH-CO-cBu-CO-Cit-PAB, -(CH ２ ) ｍ -Gly-Gly-Phe-Gly-CH ２ -O-CH ２ Examples of linkers include linkers containing one of the following: -CO-. Here, m represents an integer of 1 to 10, preferably 1. Each n represents an integer of 1 to 1000, preferably an integer of 1 to 100, more preferably an integer of 1 to 50, even more preferably an integer of 1 to 20, and most preferably an integer of 1 to 15. Furthermore, Ac represents an acetyl group. Lys(Ac) indicates that the amino group in the side chain of lysine is acetylated.

[0091] Before being bound to an antibody, the linker preferably has a functional group capable of binding to the antibody and the drug. Examples of such functional groups include α,β-unsaturated carbonyl groups, α,β-unsaturated sulfinyl groups, α,β-unsaturated sulfonyl groups, thiol groups, amino groups, hydroxyamino groups, hydrazide groups, hydrazyl groups, amide groups, formyl groups, carboxyl groups, azide groups, alkynyl groups, alkenyl groups, and haloalkyl groups. The atoms adjacent to the carbonyl carbon atoms of α,β-unsaturated carbonyl groups, amide groups, and carboxyl groups, and the atoms adjacent to the sulfur atoms of α,β-unsaturated carbonyl groups and α,β-unsaturated sulfinyl groups, include carbon, oxygen, nitrogen, and sulfur atoms. Examples of the α,β-unsaturated carbonyl groups include maleimide groups. Examples of the alkenyl groups include vinylpyridyl groups. Examples of the alkynyl group include a BCN group (Bicyclo "6.1.0" non-4-yne) and a DBCO group (Dibenzocyclooctyne). In the ADC of the present invention, the linker drug portion excluding the antibody is also referred to as a linker payload. Examples of the linker payload of the present invention include PBD dimer payload linkers such as SG3249 (Med. Chem. Lett. 2016, 7, 983-987) represented by the following formula:

[0092]

[0093] When the antibody derivatives of the present invention are used for the detection and measurement of human FCRL1 and the diagnosis of human FCRL1-related diseases, agents that bind to the antibodies include labels used in conventional immunological detection or measurement methods, such as enzymes such as alkaline phosphatase, peroxidase, or luciferase, luminescent substances such as acridinium ester or lophine, or fluorescent substances such as fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (RITC).

[0094] The present invention also includes a composition comprising, as an active ingredient, a monoclonal antibody that binds to human FCRL1 or a fragment of said antibody.

[0095] The present invention also relates to a therapeutic agent for human FCRL1-associated diseases, which comprises, as an active ingredient, a monoclonal antibody that binds to human FCRL1 or a fragment of the antibody. The present invention also relates to a method for treating human FCRL1-associated diseases, which comprises administering a monoclonal antibody that binds to human FCRL1 or a fragment of the antibody.

[0096] Human FCRL1-associated diseases may be any diseases involving human FCRL1 or a human FCRL1 ligand, including cancer, autoimmune diseases, and inflammatory diseases. Examples of cancer diseases include diffuse large B-cell lymphoma, follicular lymphoma, B-cell lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, mantle cell lymphoma, marginal zone lymphoma, and small lymphocytic lymphoma. Examples of autoimmune diseases or inflammatory diseases include rheumatoid arthritis, multiple sclerosis, chronic obstructive pulmonary disease, systemic lupus erythematosus, lupus nephritis, asthma, atopic dermatitis and inflammatory bowel disease, Crohn's disease, and Behcet's disease.

[0097] Therapeutic agents containing the antibody or antibody fragment of the present invention may contain only the antibody or antibody fragment as the active ingredient, but are usually preferably provided as pharmaceutical formulations prepared by mixing the antibody or antibody fragment with one or more pharmacologically acceptable carriers and producing the same by any method known in the technical field of pharmaceuticals.

[0098] The administration route is preferably the most effective for treatment, and includes oral administration, or parenteral administration such as oral, respiratory, rectal, subcutaneous, intramuscular, or intravenous administration, preferably intravenous administration. Examples of administration forms include sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments, and tapes.

[0099] The dosage or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 10 μg / kg to 10 mg / kg per day for adults.

[0100] The present invention relates to a reagent for detecting or measuring FCRL1, comprising a monoclonal antibody that binds to human FCRL1 or a fragment of the antibody. The present invention also relates to a method for detecting or measuring FCRL1 using a monoclonal antibody that binds to human FCRL1 or a fragment of the antibody. In the present invention, the method for detecting or measuring human FCRL1 can be any known method, such as an immunological detection or measurement method.

[0101] Immunological detection or measurement methods are methods that use labeled antigens or antibodies to detect or measure the amount of antibodies or antigens, including, for example, radioimmunoassay (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescent immunoassay, Western blotting, and physicochemical techniques.

[0102] The present invention relates to a diagnostic agent for FCRL1-associated diseases, which comprises a monoclonal antibody that binds to human FCRL1 or a fragment of the antibody, or a diagnostic method for FCRL1-associated diseases, which comprises detecting or measuring FCRL1 using a monoclonal antibody that binds to human FCRL1 or a fragment of the antibody. Diseases associated with human FCRL1 can be diagnosed by detecting or measuring cells expressing human FCRL1 using the monoclonal antibody or the antibody fragment of the present invention according to the above-mentioned method.

[0103] In the present invention, biological samples to be used for detecting or measuring human FCRL1 include, for example, tissues, cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture medium, and are not particularly limited as long as they may contain human FCRL1 or cells expressing human FCRL1.

[0104] A diagnostic agent containing the monoclonal antibody or antibody fragment of the present invention may contain a reagent for carrying out an antigen-antibody reaction and a reagent for detecting the reaction, depending on the intended diagnostic method. Reagents for carrying out the antigen-antibody reaction include buffers, salts, etc. Detection reagents include reagents used in conventional immunological detection or measurement methods, such as a labeled secondary antibody that recognizes the monoclonal antibody or the antibody fragment, or a substrate corresponding to the label.

[0105] The present invention also relates to use of an anti-human FCRL1 monoclonal antibody or a fragment thereof for the production of a therapeutic or diagnostic agent for an FCRL1-associated disease.

[0106] The method for producing the antibody, the method for treating a disease, and the method for diagnosing a disease of the present invention will be specifically explained below.

[0107] 1. Antibody Production Method (1) Antigen Preparation Human FCRL1 or human FCRL1-expressing cells serving as antigens can be obtained by introducing an expression vector containing a cDNA encoding full-length or partial human FCRL1 into E. coli, yeast, insect cells, animal cells, or the like. Human FCRL1 can also be obtained by purifying human FCRL1 from various human cell lines, human cells, human tissues, and the like that express large amounts of human FCRL1. These human cell lines, human cells, human tissues, and the like can also be used as antigens directly. Furthermore, synthetic peptides containing a partial sequence of human FCRL1 can be prepared by chemical synthesis methods such as the Fmoc method or the tBoc method and used as antigens. Human FCRL1 or synthetic peptides containing a partial sequence of human FCRL1 may have known tags such as FLAG or His attached to the C-terminus or N-terminus.

[0108] The human FCRL1 used in the present invention can be produced by expressing DNA encoding the human FCRL1 in host cells using methods described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) or Current Protocols In Molecular Biology, John Wiley & Sons (1987-1997), for example, by the following method.

[0109] First, a recombinant vector is prepared by inserting a full-length cDNA containing a portion encoding human FCRL1 downstream of the promoter of an appropriate expression vector. Instead of the full-length cDNA, a DNA fragment of an appropriate length containing a portion encoding a polypeptide, prepared based on the full-length cDNA, may be used. Next, the resulting recombinant vector is introduced into a host cell compatible with the expression vector to obtain a transformant that produces the polypeptide.

[0110] Any expression vector can be used as long as it is capable of autonomous replication in the host cell or integration into the chromosome and contains a suitable promoter at a position where the DNA encoding the polypeptide can be transcribed. Any host cell can be used as long as it can express the target gene, such as a microorganism belonging to the genus Escherichia, such as E. coli, yeast, insect cells, or animal cells.

[0111] When a prokaryote such as Escherichia coli is used as a host cell, the recombinant vector is preferably a vector capable of autonomous replication in the prokaryote and containing a promoter, a ribosome binding sequence, DNA containing a portion encoding human FCRL1, and a transcription termination sequence. Although the recombinant vector does not necessarily require a transcription termination sequence, it is preferable to position the transcription termination sequence immediately downstream of the structural gene. Furthermore, the recombinant vector may also contain a gene that controls the promoter.

[0112] As the recombinant vector, it is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence (also called SD sequence), which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases).

[0113] Furthermore, in the base sequence of the DNA encoding said human FCRL1, bases can be substituted to provide optimal codons for expression in a host, thereby improving the yield of the desired human FCRL1.

[0114] Any expression vector can be used as long as it can function in the host cell to be used. Examples of such vectors include pBTrp2, pBTac1, and pBTac2 (all manufactured by Roche Diagnostics), pKK233-2 (manufactured by Pharmacia), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured by Qiagen), pKYP10 (Japanese Patent Laid-Open Publication No. 58-110600), pKYP200 [Agricultural Biological Chemistry, 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], and pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306]. (1985)], pBluescript II SK(-) (Stratagene), pTrs30 [prepared from E. coli JM109 / pTrS30 (FERM BP-5407)], pTrs32 [prepared from E. coli JM109 / pTrS32 (FERM BP-5408)], pGHA2 [prepared from E. coli IGHA2 (FERM BP-400), JP 60-221091 A], pGKA2 [prepared from E. coli IGKA2 (FERM BP-400), JP 60-221091 A], pGKA3 [prepared from E. coli IGKA3 (FERM BP-400), JP 60-221091 B], pGKA4 [prepared from E. coli IGKA4 (FERM BP-400), JP 60-221091 B], pGKA5 [prepared from E. coli IGKA5 (FERM BP-400), JP 60-221091 C], pGKA6 [prepared from E. coli IGKA6 (FERM BP-400), JP 60-221091 B], pGKA7 [prepared from E. coli IGKA7 (FERM BP-400), JP 60-221091 C], pGKA8 [prepared from E. coli IGKA8 (FERM BP-400), JP 60-221091 B], pGKA9 [prepared from E. coli IGKA9 (FERM BP-400), JP 60-221091 C], pGKA10 [prepared from E. coli IGKA1 (FERM BP-400), JP 60-221091 B], pGKA11 [pre BP-6798), Japanese Patent Laid-Open Publication No. 60-221091], pTerm2 (U.S. Pat. Nos. 4,686,191, 4,939,094, and 160,735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol., 172, 2392 (1990)], pGEX (Pharmacia), pET system (Novagen), and pME18SFL3 are examples of such vectors.

[0115] The promoter may be any promoter that can function in the host cell used. Examples include promoters derived from Escherichia coli or phages, such as the trp promoter (Ptrp), lac promoter, PL promoter, PR promoter, or T7 promoter. Other examples include artificially designed and modified promoters, such as a tandem promoter with two Ptrp promoters in tandem, the tac promoter, the lacT7 promoter, or the let I promoter.

[0116] Examples of host cells include E. coli XL1-Blue, E. coli XL2-Blue, E. coli DH1, E. coli MC1000, E. coli KY3276, E. coli W1485, E. coli JM109, E. coli HB101, E. coli No. 49, E. coli W3110, E. coli NY49, and E. coli DH5α.

[0117] Any method for introducing a recombinant vector into a host cell can be used as long as it is a method for introducing DNA into the host cell to be used, and examples thereof include a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979)].

[0118] When animal cells are used as hosts, any expression vector can be used as long as it can function in animal cells. Examples of such vectors include pcDNAI, pCDM8 (Funakoshi Co., Ltd.), pAGE107 [JP Patent Publication No. 3-22979; Cytotechnology, 3, 133 (1990)], pAS3-3 (JP Patent Publication No. 2-227075), pCDM8 [Nature, 329, 840 (1987)], pcDNAI / Amp (Invitrogen Co., Ltd.), pcDNA3.1 (Invitrogen Co., Ltd.), pREP4 (Invitrogen Co., Ltd.), and pAGE103 [J. Biochemistry, 101, 1307 (1987)]. (1987)], pAGE210, pME18SFL3, pKANTEX93 (WO 97 / 10354), N5KG1val (U.S. Pat. No. 6,001,358), INPEP4 (Biogen-IDEC) and transposon vectors (WO 2010 / 143698).

[0119] Any promoter can be used as long as it is functional in animal cells, including, for example, the promoter of the immediate early (IE) gene of cytomegalovirus (CMV), the SV40 early promoter, a retrovirus promoter, a metallothionein promoter, a heat shock promoter, an SRα promoter, or a promoter or enhancer of Moloney murine leukemia virus. The enhancer of the IE gene of human CMV may also be used together with the promoter.

[0120] Examples of host cells include human leukemia Namalwa cells, monkey COS cells, and Chinese hamster ovary CHO cells [Journal of Experimental Medicine, 108, 945 (1958); Proc. Natl. Acad. Sci. USA, 60, 1275 (1968); Genetics, 55, 513 (1968); Chromosoma, 41, 129 (1973); Methods in Cell Science, 18, 115 (1996); Radiation Research, 148, 260 (1997); Proc. Natl. Acad. Sci. USA, 77, 4216 (1980); Proc. Natl. Acad. Sci., 60, 1275 (1968); Cell, 6, 121 (1975); Molecular Cell Genetics, Appendix I, II (pp. 883-900)]; CHO cells (CHO / DG44 cells) lacking the dihydrofolate reductase gene (hereinafter referred to as dhfr) [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)]; CHO-K1 (ATCC CCL-61), DUkXB11 (ATCC CCL-9096), Pro-5 (ATCC CCL-1781), CHO-S (Life Technologies, Cat#11619), Pro-3; rat myeloma cells YB2 / 3HL.P2.G11.16Ag. 20 (also called YB2 / 0), mouse myeloma cells NSO, mouse myeloma cells SP2 / 0-Ag14, Syrian hamster cells BHK or HBT5637 (Japanese Patent Laid-Open Publication No. 63-000299), and the like.

[0121] Any method for introducing a recombinant vector into a host cell can be used as long as it is a method for introducing DNA into an animal cell, and examples thereof include the electroporation method [Cytotechnology, 3, 133 (1990)], the calcium phosphate method (Japanese Patent Laid-Open Publication No. 2-227075), and the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].

[0122] Human FCRL1 can be produced by culturing the thus obtained transformant derived from a microorganism or animal cell harboring a recombinant vector incorporating DNA encoding human FCRL1 in a medium, producing and accumulating the human FCRL1 in the culture medium, and collecting it from the culture medium. The method for culturing the transformant in a medium can be carried out according to a conventional method used for culturing hosts.

[0123] When expressed in eukaryotic cells, human FCRL1 having sugar or a sugar chain added thereto can be obtained.

[0124] When culturing a microorganism transformed with a recombinant vector using an inducible promoter, an inducer may be added to the medium as needed. For example, isopropyl-β-D-thiogalactopyranoside or the like may be added to the medium when culturing a microorganism transformed with a recombinant vector using a lac promoter, and indoleacrylic acid or the like may be added to the medium when culturing a microorganism transformed with a recombinant vector using a trp promoter.

[0125] Examples of media for culturing transformants obtained using animal cells as hosts include commonly used RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [Science, 122, 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 (1959)], 199 medium [Proc. Soc. Exp. Biol. Med., 73, 1 (1950)], Iscove's Modified Dulbecco's Medium (IMDM) medium, and media supplemented with fetal bovine serum (FBS) or the like. Culture is typically performed for 1 to 7 days under conditions such as pH 6-8, 30-40°C, and 5% CO2. Furthermore, antibiotics such as kanamycin or penicillin may be added to the medium during cultivation, if necessary.

[0126] Methods for expressing the gene encoding human FCRL1 include, for example, direct expression, as well as secretory production and fusion protein expression [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)].

[0127] Methods for producing human FCRL1 include, for example, producing it within host cells, secreting it outside the host cells, or producing it on the outer membrane of the host cells. An appropriate method can be selected by changing the structure of the host cells used or the human FCRL1 to be produced.

[0128] When human FCRL1 is produced inside a host cell or on the outer membrane of the host cell, human FCRL1 can be actively secreted outside the host cell by using the method of Paulson et al. [J. Biol. Chem., 264, 17619 (1989)], the method of Rowe et al. [Proc. Natl. Acad. Sci., USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)], or the methods described in Japanese Patent Application Laid-Open No. 05-336963 or International Publication No. 94 / 23021. Furthermore, the amount of human FCRL1 produced can also be increased by utilizing a gene amplification system using a dihydrofolate reductase gene or the like (Japanese Patent Application Laid-Open No. 2-227075).

[0129] The obtained human FCRL1 can be isolated and purified, for example, as follows: When human FCRL1 is expressed in a soluble state within the cells, the cells are collected by centrifugation after the end of the culture, suspended in an aqueous buffer solution, and then disrupted using an ultrasonic homogenizer, French press, Manton-Gaulin homogenizer, Dynomill, or the like to obtain a cell-free extract. A purified sample can be obtained from the supernatant obtained by centrifuging the cell-free extract using conventional protein isolation and purification methods, i.e., solvent extraction, salting out with ammonium sulfate or the like, desalting, precipitation with an organic solvent, anion exchange chromatography using a resin such as diethylaminoethyl (DEAE)-Sepharose or DIAION HPA-75 (manufactured by Mitsubishi Chemical Corporation), cation exchange chromatography using a resin such as S-Sepharose FF (manufactured by Pharmacia), hydrophobic chromatography using a resin such as butyl Sepharose or phenyl Sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, or electrophoresis such as isoelectric focusing, either alone or in combination.

[0130] When human FCRL1 is expressed as an insoluble body within cells, the cells are recovered and disrupted as described above, and centrifuged to recover the insoluble body of human FCRL1 as a precipitate fraction. The recovered insoluble body of human FCRL1 is solubilized with a protein denaturant. The solubilized solution is diluted or dialyzed to restore the human FCRL1 to its normal three-dimensional structure, after which a purified polypeptide preparation can be obtained by the same isolation and purification method as described above.

[0131] When human FCRL1 or a derivative thereof such as a glycosylated form thereof is secreted extracellularly, the human FCRL1 or a derivative thereof such as a glycosylated form thereof can be recovered from the culture supernatant. The culture is treated by a technique such as centrifugation in the same manner as above to obtain a soluble fraction, and a purified preparation can be obtained from the soluble fraction by the same isolation and purification method as above.

[0132] The polypeptides used in the present invention containing a partial sequence of the amino acid sequence of human FCRL1 can be prepared by methods known to those skilled in the art. Specifically, they can be prepared by deleting a portion of the DNA encoding the amino acid sequence of human FCRL1 and culturing a transformant into which an expression vector containing the deleted portion has been introduced. Furthermore, polypeptides having an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence of human FCRL1 can be obtained in accordance with the above-mentioned methods.

[0133] Human FCRL1 used in the present invention can also be produced by chemical synthesis methods such as the Fmoc method or the tBoc method, or can be chemically synthesized using a peptide synthesizer manufactured by Advanced Chemtech, Perkin-Elmer, Pharmacia, Protein Technology Instruments, Synthecel-Vega, Perceptive, Shimadzu Corporation, or the like.

[0134] (2) Immunization of animals and preparation of antibody-producing cells for fusion Animals such as mice, rats, or hamsters aged 3 to 20 weeks are immunized with the antigen obtained in (1), and antibody-producing cells are collected from the spleen, lymph nodes, and peripheral blood of the animals. Alternatively, mouse FCRL1 knockout mice can also be used as immunized animals.

[0135] Immunization is carried out by administering the antigen to the animal subcutaneously, intravenously, or intraperitoneally together with an appropriate adjuvant, such as Freund's complete adjuvant, or aluminum hydroxide gel and pertussis vaccine. When the antigen is a partial peptide, it is conjugated with a carrier protein such as BSA (bovine serum albumin) or KLH (keyhole limpet hemocyanin) and used as an immunogen.

[0136] After the first administration, the antigen is administered 5 to 10 times at 1-2 week intervals. Blood is collected 3 to 7 days after each administration, and the serum antibody titer is measured using an enzyme immunoassay [Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988)] or similar. Animals whose serum shows a sufficient antibody titer against the antigen used for immunization are used as the source of antibody-producing cells for fusion.

[0137] Three to seven days after the final administration of the antigen, tissues containing antibody-producing cells, such as the spleen, are removed from the immunized animal, and the antibody-producing cells are collected. When spleen cells are used, the spleen is minced and disaggregated, then centrifuged, and red blood cells are removed to obtain antibody-producing cells for fusion.

[0138] (3) Preparation of Myeloma Cells As myeloma cells, established cell lines obtained from mice are used, such as 8-azaguanine-resistant mouse (BALB / c-derived) myeloma cell lines P3-X63Ag8-U1 (P3-U1) [Current Topics in Microbiology and Immunology, 18, 1 (1978)], P3-NS1 / 1-Ag41 (NS-1) [European J. Immunology, 6, 511 (1976)], SP2 / 0-Ag14 (SP-2) [Nature, 276, 269 (1978)], P3-X63-Ag8653 (653) [J. Immunology, 123, 1548 (1979)], or P3-X63-Ag8 (X63) [Nature, 256, 495 (1975)] are used.

[0139] The myeloma cells were passaged in normal medium [RPMI 1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, FBS, and 8-azaguanine], and then passaged in normal medium 3 to 4 days before cell fusion. On the day of fusion, 2 × 10 ７ Ensure that there are at least 100 cells.

[0140] (4) Cell fusion and preparation of monoclonal antibody-producing hybridomas. The antibody-producing cells for fusion obtained in (2) and the myeloma cells obtained in (3) are thoroughly washed with Minimum Essential Medium (MEM) or PBS (1.83 g disodium phosphate, 0.21 g monopotassium phosphate, 7.65 g sodium chloride, 1 liter of distilled water, pH 7.2), mixed so that the cell numbers of antibody-producing cells for fusion:myeloma cells = 5-10:1, centrifuged, and the supernatant removed. The precipitated cells are thoroughly loosened, and a mixture of polyethylene glycol-1000 (PEG-1000), MEM medium, and dimethyl sulfoxide is added to the mixture while stirring at 37°C. 1-2 mL of MEM medium is added several times every 1-2 minutes, followed by the addition of MEM medium until the total volume reaches 50 mL. After centrifugation, the supernatant is removed. After gently loosening the precipitated cells, the antibody-producing cells for fusion are gently suspended in HAT medium (normal medium supplemented with hypoxanthine, thymidine, and aminopterin) and cultured in a 5% CO incubator at 37°C for 7 to 14 days.

[0141] After culturing, a portion of the culture supernatant is removed, and a cell population that reacts with an antigen containing human FCRL1 but does not react with an antigen not containing human FCRL1 is selected by a hybridoma selection method such as the binding assay described below. Next, cloning is performed by limiting dilution, and those that show a stable and strong antibody titer are selected as monoclonal antibody-producing hybridomas.

[0142] (5) Preparation of Purified Monoclonal Antibodies: Pristane-treated mice (0.5 mL of 2,6,10,14-tetramethylpentadecane (pristane) is administered intraperitoneally and then housed for two weeks) at 8 to 10 weeks of age or nude mice are injected intraperitoneally with the monoclonal antibody-producing hybridoma obtained in (4). The hybridoma develops into ascites tumors within 10 to 21 days. Ascites fluid is collected from the mice and centrifuged to remove solids. The fluid is then salted out with 40 to 50% ammonium sulfate, and purified using caprylic acid precipitation, a DEAE-Sepharose column, a protein A column, or a gel filtration column. The IgG or IgM fraction is collected and used as purified monoclonal antibodies.

[0143] Alternatively, the monoclonal antibody-producing hybridoma obtained in (4) can be cultured in RPMI 1640 medium or the like supplemented with 10% FBS, the supernatant removed by centrifugation, and the cells suspended in Hybridoma SFM medium for 3 to 7 days. The resulting cell suspension can be centrifuged, and the resulting supernatant purified using a protein A column or protein G column to collect the IgG fraction, thereby obtaining a purified monoclonal antibody. The Hybridoma SFM medium can also be supplemented with 5% Daigo GF21.

[0144] The antibody subclass is determined by enzyme immunoassay using a subclass typing kit. The protein amount is quantified by the Lowry method or calculated from absorbance at 280 nm.

[0145] (6) Selection of Monoclonal Antibodies Monoclonal antibodies are selected by measuring the binding affinity of the antibody to human FCRL1-expressing cells using flow cytometry as described below. Human FCRL1-expressing cells may be any cells that express human FCRL1 on their cell surface, including, for example, human cells, human cell lines, and the human FCRL1-expressing cell lines obtained in (1).

[0146] Human FCRL1-expressing cells are dispensed into a plate such as a 96-well plate, and then a test substance such as serum, hybridoma culture supernatant, or purified monoclonal antibody is dispensed as a first antibody and allowed to react. After the reaction, the cells are thoroughly washed with PBS containing 1 to 10% bovine serum albumin (BSA) (hereinafter referred to as BSA-PBS), and then an anti-immunoglobulin antibody labeled with a fluorescent reagent or the like is dispensed as a second antibody and allowed to react. After thorough washing with BSA-PBS, the amount of fluorescence from the labeled antibody is measured using a flow cytometer to select a monoclonal antibody that specifically reacts with human FCRL1-expressing cells.

[0147] Furthermore, antibodies that bind to human FCRL1 in competition with the antibodies of the present invention can be obtained by adding a test antibody to the above-mentioned flow cytometry assay system and allowing it to react. That is, by screening for antibodies that inhibit the binding of the antibody of the present invention to human FCRL1 when the test antibody is added, it is possible to obtain monoclonal antibodies that compete with the antibodies of the present invention for binding to the amino acid sequence or three-dimensional structure of human FCRL1.

[0148] Furthermore, an antibody that binds to an epitope containing the epitope bound by the monoclonal antibody that binds to human FCRL1 of the present invention can be obtained by identifying the epitope of the antibody obtained by the above-mentioned screening method using a known method, preparing a synthetic peptide containing the identified epitope or a synthetic peptide that mimics the three-dimensional structure of the epitope, and then immunizing the subject.

[0149] Furthermore, an antibody that binds to the same epitope as that bound by the monoclonal antibody that binds to human FCRL1 of the present invention can be obtained by identifying the epitope of the antibody obtained by the above-mentioned screening method, preparing a partial synthetic peptide of the identified epitope or a synthetic peptide that mimics the three-dimensional structure of the epitope, and then immunizing the antibody.

[0150] 2. Production of recombinant antibodies As examples of recombinant antibody production, the methods for producing human chimeric antibodies and humanized antibodies are shown below. Recombinant mouse, rat, and rabbit antibodies can also be produced by similar methods.

[0151] (1) Construction of recombinant antibody expression vector A recombinant antibody expression vector is an expression vector for animal cells incorporating DNA encoding the CH and CL of a human antibody, and can be constructed by cloning the DNA encoding the CH and CL of a human antibody into an expression vector for animal cells.

[0152] The C region of a human antibody can be any human antibody CH and CL. For example, a CH of the γ1 subclass and a CL of the κ class of human antibodies can be used. Although cDNA is used to encode the CH and CL of a human antibody, chromosomal DNA consisting of exons and introns can also be used. By adding, inserting, or substituting codons encoding amino acid residues into the DNA encoding the CH or CL of a human antibody, amino acid residues can be added, inserted, or substituted at the corresponding positions. Any expression vector for animal cells can be used, as long as it can incorporate and express a gene encoding the C region of a human antibody. For example, pAGE107 [Cytotechnol., 3, 133 (1990)], pAGE103 [J. Biochem., 101, 1307 (1987)], pHSG274 [Gene, 27, 223 (1984)], pKCR [Proc. Natl. Acad. Sci. USA, 78, 1527 (1981)], pSG1bd2-4 [Cytotechnol., 4, 173 (1990)], or pSE1UK1Sed1-3 [Cytotechnol., 13, 79 (1993)] may be used. Examples of promoters and enhancers for animal cell expression vectors include the SV40 early promoter [J. Biochem., 101, 1307 (1987)], Moloney murine leukemia virus LTR [Biochem. Biophys. Res. Commun., 149, 960 (1987)], and immunoglobulin H chain promoter [Cell, 41, 479 (1985)] and enhancer [Cell, 33, 717 (1983)].

[0153] For recombinant antibody expression vectors, tandem recombinant antibody expression vectors in which the antibody H and L chains are present on the same vector are used [J. Immunol. Methods, 167, 271 (1994)] because of ease of construction of the recombinant antibody expression vector, ease of introduction into animal cells, and balance of expression levels of the antibody H and L chains in the animal cells. However, recombinant antibody expression vectors in which the antibody H and L chains are present on separate vectors can also be used. Examples of tandem recombinant antibody expression vectors include pKANTEX93 (WO 97 / 10354) and pEE18 [Hybridoma, 17, 559 (1998)].

[0154] (2) Obtaining cDNA encoding the V region of an antibody derived from a non-human animal and analyzing the amino acid sequence. Obtaining cDNA encoding the VH and VL of a non-human antibody and analyzing the amino acid sequence can be performed as follows.

[0155] mRNA is extracted from hybridoma cells producing a non-human antibody, and cDNA is synthesized. The synthesized cDNA is cloned into a vector such as a phage or plasmid to prepare a cDNA library. From the library, recombinant phages or recombinant plasmids carrying cDNA encoding VH or VL are isolated using DNA encoding the C or V region of a mouse antibody as a probe. The entire nucleotide sequence of the VH or VL of the target mouse antibody on the recombinant phage or recombinant plasmid is determined, and the entire amino acid sequence of the VH or VL is deduced from the nucleotide sequence.

[0156] Non-human animals used to produce hybridoma cells that produce non-human antibodies include mice, rats, hamsters, and rabbits, but any animal can be used as long as it is possible to produce hybridoma cells.

[0157] Total RNA from hybridoma cells can be prepared by the guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymol., 154, 3 (1987)] or by using a kit such as RNA easy kit (Qiagen).

[0158] Preparation of mRNA from total RNA can be performed using the oligo(dT)-immobilized cellulose column method [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)] or a kit such as the Oligo-dT30<Super>mRNA Purification (registered trademark) Kit (manufactured by Takara Bio Inc.). Alternatively, mRNA can be prepared from hybridoma cells using a kit such as the Fast Track mRNA Isolation (registered trademark) Kit (manufactured by Invitrogen) or the QuickPrep mRNA Purification (registered trademark) Kit (manufactured by Pharmacia).

[0159] For cDNA synthesis and cDNA library construction, known methods [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, Supplement 1, John Wiley & Sons (1987-1997)] or kits such as SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (Invitrogen) or ZAP-cDNA Synthesis (registered trademark) Kit (Stratagene) can be used.

[0160] When preparing a cDNA library, any vector can be used to incorporate the cDNA synthesized using mRNA extracted from hybridoma cells as a template, as long as it can incorporate the cDNA. For example, ZAP Express [Strategies, 5, 58 (1992)], pBluescript II SK(+) [Nucleic Acids Research, 17, 9494 (1989)], λZAPII (Stratagene), λgt10, λgt11 [DNA Cloning: A Practical Approach, I, 49 (1985)], Lambda BlueMid (Clontech), λExCell, pT7T3-18U (Pharmacia), pCD2 [Mol. Cell. Biol., 3, 280 (1983)], or pUC18 [Gene, 33, 103 (1985)] may be used.

[0161] Any E. coli strain capable of introducing, expressing, and maintaining a cDNA library constructed using a phage or plasmid vector can be used for the introduction of the cDNA library, such as XL1-Blue MRF' [Strategies, 5, 81 (1992)], C600 [Genetics, 39, 440 (1954)], Y1088, Y1090 [Science, 222, 778 (1983)], NM522 [J. Mol. Biol., 166, 1 (1983)], K802 [J. Mol. Biol., 16, 118 (1966)], or JM105 [Gene, 38, 275 (1985)].

[0162] To select cDNA clones encoding the VH or VL of a non-human antibody from a cDNA library, methods such as colony hybridization using isotope- or fluorescently-labeled probes or plaque hybridization [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)] are used.

[0163] Alternatively, cDNA encoding VH or VL can be prepared by preparing primers and performing the polymerase chain reaction method (hereinafter referred to as PCR; Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989); Current Protocols in Molecular Biology, Supplement 1, John Wiley & Sons (1987-1997)) using cDNA synthesized from mRNA or a cDNA library as a template.

[0164] The selected cDNA is digested with an appropriate restriction enzyme or the like, and then cloned into a plasmid such as pBluescript SK(-) (Stratagene), and the nucleotide sequence of the cDNA is determined by a commonly used nucleotide sequence analysis method, for example, by performing a reaction such as the dideoxy method [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)], followed by using an automatic nucleotide sequence analyzer such as an ABI PRISM 3700 (PE Biosystems) or an A.L.F. DNA Sequencer (Pharmacia).

[0165] The entire amino acid sequences of VH and VL are deduced from the determined nucleotide sequence and compared with the entire amino acid sequences of VH and VL of known antibodies [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)] to confirm whether the obtained cDNA encodes the complete amino acid sequences of VH and VL of the antibody, including the secretory signal sequence. The complete amino acid sequences of VH and VL of the antibody, including the secretory signal sequence, can be deduced by comparing them with the entire amino acid sequences of VH and VL of known antibodies [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)], thereby determining the length and N-terminal amino acid sequence of the secretory signal sequence and further determining the subgroup to which they belong. The amino acid sequences of each CDR of VH and VL can also be determined by comparing them with the amino acid sequences of VH and VL of known antibodies [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)].

[0166] Furthermore, the obtained complete amino acid sequences of VH and VL can be used to perform a similarity search using a BLAST method [J. Mol. Biol., 215, 403 (1990)] or the like against any database, such as SWISS-PROT or PIR-Protein, to confirm whether the complete amino acid sequences of VH and VL are novel.

[0167] (3) Construction of a human chimeric antibody expression vector A human chimeric antibody expression vector can be constructed by cloning cDNA encoding the VH or VL of a non-human antibody upstream of the gene encoding the CH or CL of a human antibody in the recombinant antibody expression vector obtained in (1).

[0168] To link the 3'-end of the cDNA encoding the VH or VL of a non-human antibody to the 5'-end of the CH or CL of a human antibody, VH and VL cDNAs are prepared so that the nucleotide sequence of the linking portion encodes the appropriate amino acids and is an appropriate restriction enzyme recognition sequence. The prepared VH and VL cDNAs are cloned upstream of the respective genes encoding the CH or CL of the human antibody into the recombinant antibody expression vector obtained in (1) so that they are expressed in an appropriate manner, thereby constructing a human chimeric antibody expression vector.

[0169] Alternatively, the cDNA encoding the non-human antibody VH or VL can be amplified by PCR using synthetic DNA containing appropriate restriction enzyme recognition sequences at both ends, and then cloned into the recombinant antibody expression vector obtained in (1).

[0170] (4) Construction of cDNA Encoding V Region of Humanized Antibody cDNA encoding VH or VL of a humanized antibody can be constructed as follows.

[0171] The amino acid sequence of the FR of the VH or VL of a human antibody is selected for grafting the amino acid sequence of the CDR of the VH or VL of a non-human antibody. Any amino acid sequence derived from a human antibody can be used for the selected FR. For example, the amino acid sequence of the FR of a human antibody registered in a database such as the Protein Data Bank, or the consensus amino acid sequence of each subgroup of FR of a human antibody [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)], etc., can be used. To minimize a decrease in the binding activity of the antibody, the amino acid sequence of the FR is selected to have as high a similarity as possible (at least 60% or more) to the amino acid sequence of the FR of the VH or VL of the original antibody.

[0172] Next, the amino acid sequences of the CDRs of the original antibody are grafted onto the amino acid sequences of the FRs of the VH or VL of the selected human antibody, respectively, to design the amino acid sequences of the VH or VL of the humanized antibody. The designed amino acid sequences are converted into DNA sequences taking into account the codon usage frequency found in the nucleotide sequence of the antibody gene [Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services (1991)], and DNA sequences encoding the amino acid sequences of the VH or VL of the humanized antibody are designed.

[0173] Based on the designed DNA sequence, several synthetic DNAs each about 100 bases long are synthesized and used in a PCR reaction. In this case, taking into account the reaction efficiency of the PCR reaction and the length of DNA that can be synthesized, preferably six synthetic DNAs each are designed for VH and VL. Furthermore, by introducing appropriate restriction enzyme recognition sequences into the 5' or 3' end of the synthetic DNAs located at both ends, the cDNA encoding the VH or VL of the humanized antibody can be easily cloned into the recombinant antibody expression vector obtained in (1).

[0174] After the PCR reaction, the amplified products are each cloned into a plasmid such as pBluescript SK(-) (Stratagene), and the nucleotide sequence is determined in the same manner as described in (2) to obtain a plasmid having a DNA sequence encoding the amino acid sequence of VH or VL of the desired humanized antibody.

[0175] Alternatively, full-length VH and full-length VL can be synthesized as single long-chain DNAs based on the designed DNA sequences and used instead of the PCR amplification products. Furthermore, by introducing appropriate restriction enzyme recognition sequences into both ends of the synthetic long-chain DNAs, the cDNA encoding the VH or VL of the humanized antibody can be easily cloned into the recombinant antibody expression vector obtained in (1).

[0176] (5) Modification of the Amino Acid Sequence of the V Region of a Humanized Antibody When only the CDRs of the VH and VL of a non-human antibody are grafted onto the FRs of the VH and VL of a human antibody, the antigen-binding activity of the humanized antibody is reduced compared to the original non-human antibody [BIO / TECHNOLOGY, 9, 266 (1991)]. In the amino acid sequences of the FRs of the VH and VL of a human antibody, the amino acid residues directly involved in binding to the antigen, the amino acid residues that interact with the amino acid residues of the CDR, and the amino acid residues that indirectly participate in binding to the antigen while maintaining the three-dimensional structure of the antibody are identified, and these amino acid residues are substituted with amino acid residues from the original non-human antibody to increase the reduced antigen-binding activity.

[0177] To identify the amino acid residues in the FRs involved in antigen-binding activity, the three-dimensional structure of an antibody can be constructed and analyzed using X-ray crystallography [J. Mol. Biol., 112, 535 (1977)] or computer modeling [Protein Engineering, 7, 1501 (1994)]. Alternatively, several variants of each antibody can be prepared and their correlation with the antigen-binding activity can be repeatedly examined through trial and error to obtain a humanized antibody with the required antigen-binding activity.

[0178] The amino acid residues in the FRs of the VH and VL of a human antibody can be modified by carrying out the PCR reaction described in (4) using synthetic DNA for modification. The nucleotide sequence of the amplified product after the PCR reaction is determined by the method described in (2) to confirm that the desired modification has been made.

[0179] (6) Construction of humanized antibody expression vectors A humanized antibody expression vector can be constructed by cloning cDNA encoding VH or VL of the constructed recombinant antibody upstream of the gene encoding CH or CL of the human antibody in the recombinant antibody expression vector obtained in (1).

[0180] For example, by introducing a recognition sequence for an appropriate restriction enzyme into the 5' or 3' end of the synthetic DNA located at both ends of the synthetic DNA used to construct the VH or VL of the humanized antibody obtained in (4) and (5), the synthetic DNA can be cloned upstream of the respective genes encoding the CH or CL of the human antibody in the recombinant antibody expression vector obtained in (1) so that they can be expressed in an appropriate form.

[0181] (7) Transient expression of recombinant antibodies The recombinant antibody expression vectors obtained in (3) and (6) or modified expression vectors thereof can be used to transiently express recombinant antibodies, and the antigen-binding activities of the various human chimeric antibodies and humanized antibodies produced can be efficiently evaluated.

[0182] Any host cells can be used to introduce the expression vector as long as they are capable of expressing a recombinant antibody. For example, COS-7 cells [American Type Culture Collection (ATCC) No.: CRL1651] are used [Methods in Nucleic Acids Res., CRC Press, 283 (1991)].

[0183] To introduce an expression vector into COS-7 cells, the DEAE-dextran method [Methods in Nucleic Acids Res., CRC Press (1991)] or the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] can be used.

[0184] After introduction of the expression vector, the expression level and antigen-binding activity of the recombinant antibody in the culture supernatant are measured using enzyme immunoassay [Monoclonal Antibodies - Principles and practice, Third edition, Academic Press (1996); Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988); Monoclonal Antibody Experiment Manual, Kodansha Scientific (1987)] or the like.

[0185] (8) Obtaining a transformant capable of stably expressing a recombinant antibody and preparing the recombinant antibody. A transformant capable of stably expressing a recombinant antibody can be obtained by introducing the recombinant antibody expression vector obtained in (3) and (6) into suitable host cells. The expression vector can be introduced into host cells using electroporation (Japanese Patent Laid-Open Publication No. 2-257891, Cytotechnology, 3, 133 (1990)).

[0186] Any host cells capable of expressing a recombinant antibody can be used as the host cell into which the recombinant antibody expression vector is introduced, including, for example, CHO-K1 (ATCC CCL-61), DUKXB11 (ATCC CCL-9096), Pro-5 (ATCC CCL-1781), CHO-S (Life Technologies, Cat#11619), and rat myeloma cell YB2 / 3HL.P2.G11.16Ag. 20 (ATCC number: CRL1662, also referred to as YB2 / 0), mouse myeloma cells NS0, mouse myeloma cells SP2 / 0-Ag14 (ATCC number: CRL1581), mouse P3X63-Ag8.653 cells (ATCC number: CRL1580), and CHO cells (CHO / DG44 cells) lacking the dihydrofolate reductase gene (hereinafter referred to as dhfr) [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)] are used.

[0187] Furthermore, host cells in which the activity of proteins such as enzymes involved in the synthesis of the intracellular sugar nucleotide GDP-fucose, proteins such as enzymes involved in sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the reducing end of an N-glycosidically linked complex sugar chain, or proteins involved in the transport of the intracellular sugar nucleotide GDP-fucose to the Golgi apparatus, are reduced or deleted, can also be used, for example, CHO cells deficient in the α1,6-fucosyltransferase gene (WO 2005 / 035586, WO 02 / 31140), and lectin-resistant Lec13 [Somatic Cell and Molecular Genetics, 12, 55 (1986)].

[0188] After introduction of the expression vector, transformants that stably express the recombinant antibody are selected by culturing them in an animal cell culture medium containing a drug such as G418 sulfate (hereinafter referred to as G418) (Japanese Patent Laid-Open Publication No. 2-257891).

[0189] Media for animal cell culture include RPMI 1640 medium (Invitrogen), GIT medium (Nihon Pharmaceutical Co., Ltd.), EX-CELL 301 medium (JRH), IMDM medium (Invitrogen), or Hybridoma SFM medium (Invitrogen), or media containing various additives such as FBS. The resulting transformed strain is cultured in the medium, resulting in the expression and accumulation of the recombinant antibody in the culture supernatant. The expression level and antigen-binding activity of the recombinant antibody in the culture supernatant can be measured by ELISA or other methods. Furthermore, the expression level of the recombinant antibody produced by the transformant can be improved by using a dhfr gene amplification system (Japanese Patent Publication No. 2-257891).

[0190] Recombinant antibodies are purified from the culture supernatant of transformed strains using a protein A column [Monoclonal Antibodies - Principles and practice, Third Edition, Academic Press (1996); Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988)]. Protein purification methods such as gel filtration, ion exchange chromatography, and ultrafiltration can also be combined.

[0191] The molecular weight of the H chain, L chain, or entire antibody molecule of the purified recombinant antibody can be measured using polyacrylamide gel electrophoresis [Nature, 227, 680 (1970)] or Western blotting [Monoclonal Antibodies—Principles and practice, Third Edition, Academic Press (1996), Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory (1988)], or the like.

[0192] 3. Evaluation of Activity of Purified Monoclonal Antibody or Antibody Fragment The activity of the purified monoclonal antibody of the present invention or the antibody fragment thereof can be evaluated as follows.

[0193] The binding activity of the antibody or antibody fragment of the present invention to human FCRL1 can be measured using flow cytometry as described above in 1-(6), or by fluorescent antibody techniques [Cancer Immunol. Immunother., 36, 373 (1993)].

[0194] The CDC activity or ADCC activity against human FCRL1-expressing cells can be measured by known methods [Cancer Immunol. Immunother., 36, 373 (1993); Current protocols in Immunology, Chapter 7. Immunologic studies in humans, Editor, John E., Coligan et al., John Wiley & Sons, Inc., (1993)].

[0195] 4. Methods for Controlling Antibody Effector Activity Known methods for controlling the effector activity of the monoclonal antibodies of the present invention include controlling the amount of fucose (also known as core fucose) α1,6-linked to N-acetylglucosamine (GlcNAc) present at the reducing end of the N-linked complex sugar chain bound to asparagine (Asn) at position 297 in the Fc region of the antibody (WO 2005 / 035586, WO 2002 / 31140, WO 00 / 61739), or modifying amino acid residues in the Fc region of the antibody. Either method can be used to control the effector activity of the monoclonal antibodies of the present invention.

[0196] Effector activity refers to antibody-dependent activity induced via the Fc region of an antibody, and known examples include ADCC activity, CDC activity, and antibody-dependent phagocytosis (ADP activity) by phagocytes such as macrophages or dendritic cells.

[0197] As a method for measuring effector activity, for example, inflammatory cells as targets, human peripheral blood mononuclear cells (PBMCs) as effectors, and an inflammatory cell-specific antibody are mixed, incubated for about 4 hours, and then the released lactate dehydrogenase (LDH) can be measured as an indicator of cytotoxicity. Alternatively, an antibody that recognizes a blood cell-specific antigen, such as CD20, can be added to human whole blood, and after incubation, the reduction in the number of target blood cells can be measured as effector activity. Alternatively, for example, human whole blood can be mixed with other target cells, and an antibody specific to the target cells can be added and incubated, and then the reduction in the number of target cells can be measured as effector activity. In either case, effector activity can be measured by the free LDH method, the free 51Cr method, flow cytometry, or the like.

[0198] By controlling the content of core fucose in the N-linked complex sugar chains of the Fc of an antibody, the effector activity of the antibody can be increased or decreased. As a method for decreasing the content of fucose bound to the N-linked complex sugar chains bound to the Fc of an antibody, an antibody to which fucose is not bound can be obtained by expressing the antibody using CHO cells lacking the α1,6-fucosyltransferase gene. Antibodies to which fucose is not bound have high ADCC activity.

[0199] On the other hand, as a method for increasing the content of fucose bound to N-linked complex sugar chains bound to the Fc of an antibody, an antibody bound to fucose can be obtained by expressing the antibody using a host cell into which an α1,6-fucosyltransferase gene has been introduced. Antibodies bound to fucose have lower ADCC activity than antibodies not bound to fucose.

[0200] Furthermore, ADCC or CDC activity can be increased or decreased by modifying amino acid residues in the Fc region of an antibody. For example, the CDC activity of an antibody can be increased by using the amino acid sequence of the Fc region described in U.S. Patent Application Publication No. 2007 / 0148165.

[0201] Furthermore, ADCC activity or CDC activity can be increased or decreased by making the amino acid modifications described in U.S. Patent No. 6,737,056, U.S. Patent No. 7,297,775, or U.S. Patent No. 7,317,091. Furthermore, the antibodies of the present invention also include antibodies whose blood half-life is controlled by controlling reactivity to Fc receptors through amino acid modifications described, for example, in Japanese Patent Application Laid-Open No. 2013-165716 or Japanese Patent Application Laid-Open No. 2012-021004, in addition to the amino acid modifications or glycosylation modifications in the antibody constant region described above.

[0202] Furthermore, by combining the above methods and applying them to a single antibody, it is possible to obtain an antibody with controlled effector activity or blood half-life.

[0203] 5. Method for Producing an Antibody-Drug Conjugate Comprising the Anti-FCRL1 Monoclonal Antibody of the Present Invention or the Antibody Fragment Thereof The antibody-drug conjugate comprising the anti-FCRL1 monoclonal antibody of the present invention or the antibody fragment thereof can be produced by linking the monoclonal antibody and a drug by chemical, enzymatic, or genetic engineering techniques.

[0204] (1) Chemical Methods for Conjugating Monoclonal Antibodies to Drugs 2. Using the method described in (1), a reactive substituent is introduced into an antibody by adding, inserting, or substituting an amino acid residue with an appropriate substituent at any position. Alternatively, a bond at any position in the antibody can be cleaved by reduction, hydrolysis, enzymatic degradation, or the like to create a reactive substituent. Furthermore, sugars with reactive substituents can be introduced into sugar chains contained in antibody molecules using enzymes such as glycosidases and glycosyltransferases. Examples of such reactive substituents include α,β-unsaturated carbonyl groups, α,β-unsaturated sulfinyl groups, α,β-unsaturated sulfonyl groups, thiol groups, amino groups, amide groups, formyl groups, carboxyl groups, azido groups, alkynyl groups, alkenyl groups, haloalkyl groups, and carbonyl groups.

[0205] A chemical structure capable of reacting with the reactive functional group introduced into the antibody is introduced into the drug or linker, and the antibody and drug or linker are conjugated by reacting under appropriate reaction conditions. The linker may be conjugated to the drug before or after reaction with the antibody. The linker and drug can be conjugated using known methods (e.g., methods described in SJ Walsh et al. Chem. Soc. Rev. 2021, 50, 1305-1353; Tumey, L. Nathan (2020). Antibody-Drug Conjugates - Methods and Protocols: New York, Springer; and Laurent Ducry (2013). Antibody-Drug Conjugate: New York, Springer, etc.). (2) Method of Conjugating a Monoclonal Antibody and a Drug Using an Enzymatic Method 2. In the method described in (1), for example, an amino acid sequence recognized by a specific enzyme is added or substituted at the C-terminus of the antibody. Examples of such amino acid sequences include CaaX tags (C represents cysteine, a represents any aliphatic amino acid, and X represents the C-terminal amino acid) that are recognized by farnesyltransferase, geranyltransferase, etc. A functional group that is transferred by an enzyme that recognizes the amino acid sequence introduced into the antibody is introduced into the drug or linker, and the antibody is bound to the drug or linker by enzymatically reacting with the amino acid sequence under appropriate conditions. For example, functional groups corresponding to the CaaX tag include prenyl groups such as geranyl and farnesyl groups. The linker may be bound to the drug before or after reaction with the antibody. The linker and drug can be bound by known methods.

[0206] (3) Method of conjugating a monoclonal antibody and a drug by genetic engineering techniques When the drug is a protein or peptide, DNA encoding the protein or peptide can be designed, added, inserted, or substituted at any position in the antibody gene, and expressed in the same manner as in 2, thereby conjugating the monoclonal antibody and the drug.

[0207] 6. Method for Treating Diseases Using the Anti-Human FCRL1 Monoclonal Antibody of the Present Invention or the Antibody Fragment Thereof The monoclonal antibody of the present invention or the antibody fragment thereof can be used to treat any human FCRL1-associated disease, so long as the disease is one in which FCRL1 is expressed.

[0208] Therapeutic agents containing the monoclonal antibodies or antibody fragments of the present invention may contain only the antibodies or antibody fragments as the active ingredient, but are usually provided as pharmaceutical formulations prepared by mixing them with one or more pharmacologically acceptable carriers and using methods known in the technical field of pharmaceuticals.

[0209] Examples of administration routes include oral administration and parenteral administration such as oral, respiratory, rectal, subcutaneous, intramuscular, or intravenous administration. Examples of administration forms include sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments, and tapes.

[0210] Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, and granules.

[0211] Liquid preparations such as emulsions or syrups are produced using additives such as water, sugars such as sucrose, sorbitol, or fructose, glycols such as polyethylene glycol or propylene glycol, oils such as sesame oil, olive oil, or soybean oil, preservatives such as p-hydroxybenzoic acid esters, or flavors such as strawberry flavor or peppermint.

[0212] Capsules, tablets, powders, granules, etc. are produced using additives such as excipients such as lactose, glucose, sucrose, or mannitol; disintegrating agents such as starch or sodium alginate; lubricants such as magnesium stearate or talc; binders such as polyvinyl alcohol, hydroxypropyl cellulose, or gelatin; surfactants such as fatty acid esters; or plasticizers such as glycerin.

[0213] Formulations suitable for parenteral administration include injections, suppositories, and sprays. Injections are prepared using carriers such as saline solutions, glucose solutions, or mixtures of both. Suppositories are prepared using carriers such as cocoa butter, hydrogenated fats, or carboxylic acids.

[0214] Sprays are prepared using carriers that do not irritate the recipient's oral and respiratory mucosa and disperse the monoclonal antibody or antibody fragment of the present invention as fine particles, facilitating absorption. Examples of carriers include lactose and glycerin. Sprays can also be prepared as aerosols or dry powders. Furthermore, the above-mentioned parenteral preparations can also contain the additives listed above for formulations suitable for oral administration.

[0215] 7. Method for Diagnosing Diseases Using the Anti-Human FCRL1 Monoclonal Antibody of the Present Invention or the Antibody Fragment Thereof Human FCRL1-associated diseases can be diagnosed by detecting or measuring human FCRL1 or cells expressing human FCRL1 using the monoclonal antibody of the present invention, the antibody fragment thereof, or the antibody-drug conjugate.

[0216] Diagnosis of human FCRL1-associated diseases such as cancer, autoimmune, and inflammatory diseases can be performed, for example, by detecting or measuring human FCRL1 present in the patient's body by an immunological technique, or by detecting human FCRL1 expressed in cells in the patient's body using an immunological technique such as flow cytometry.

[0217] Immunological techniques are methods for detecting or measuring the amount of antibody or antigen using labeled antigens or antibodies, such as radioactive-labeled immunosorbent assays, enzyme immunoassays, fluorescent immunoassays, luminescent immunoassays, Western blotting, or physicochemical techniques.

[0218] In the radioactive substance-labeled immunoassay, for example, an antibody of the present invention or an antibody fragment thereof is reacted with an antigen or cells expressing the antigen, and then a radiolabeled anti-immunoglobulin antibody or an antibody fragment thereof is reacted, followed by measurement using a scintillation counter or the like.

[0219] In enzyme immunoassays, for example, an antibody or antibody fragment of the present invention is reacted with an antigen or cells expressing the antigen, followed by an anti-immunoglobulin antibody or binding fragment labeled with an enzyme or the like, followed by addition of a substrate and measurement of the absorbance of the reaction solution using an absorptiometer. For example, a sandwich ELISA method is used. The label used in enzyme immunoassays may be a publicly known enzyme label [Enzyme Immunoassay, Igaku Shoin (1987)].

[0220] For example, alkaline phosphatase labeling, peroxidase labeling, luciferase labeling, or biotin labeling may be used. Sandwich ELISA involves binding an antibody to a solid phase, trapping the antigen to be detected or measured, and then reacting the trapped antigen with a second antibody. In this ELISA, two types of antibodies or antibody fragments that recognize the antigen to be detected or measured, each with a different antigen recognition site, are prepared. The first antibody or antibody fragment is pre-adsorbed onto a plate (e.g., a 96-well plate), and the second antibody or antibody fragment is then labeled with a fluorescent substance such as FITC, an enzyme such as peroxidase, or biotin. Cells or their lysates, tissues or their lysates, cell culture supernatant, serum, pleural effusion, ascites, or ocular fluid isolated from a living body are then reacted with a labeled monoclonal antibody or antibody fragment, and a detection reaction appropriate for the labeling substance is carried out. The antigen concentration in the test sample is calculated from a calibration curve prepared by serially diluting a known concentration of antigen. The antibody used in sandwich ELISA may be either a polyclonal antibody or a monoclonal antibody, or an antibody fragment such as Fab, Fab', or F(ab')2. The combination of two types of antibodies used in sandwich ELISA may be a combination of monoclonal antibodies or antibody fragments that recognize different epitopes, or a combination of a polyclonal antibody and a monoclonal antibody or antibody fragment.

[0221] Fluorescence immunoassay is performed according to the methods described in literature such as Monoclonal Antibodies—Principles and Practice, Third Edition, Academic Press (1996) and Monoclonal Antibody Experiment Manual, Kodansha Scientific (1987). Labels used in fluorescence immunoassay include known fluorescent labels such as Fluorescent Antibody Method, Soft Sciences (1983). For example, FITC or RITC can be used.

[0222] Luminescence immunoassay is performed by the method described in the literature [Bioluminescence and Chemiluminescence, Clinical Tests 42, Hirokawa Shoten (1998)], etc. Labels used in luminescence immunoassay include known luminescent labels, such as acridinium ester or lophine.

[0223] In Western blotting, an antigen or cells expressing the antigen are fractionated using SDS (sodium dodecyl sulfate)-PAGE (polyacrylamide gel) [Antibodies - A Laboratory Manual Cold Spring Harbor Laboratory (1988)], the gel is then blotted onto a polyvinylidene fluoride (PVDF) membrane or a nitrocellulose membrane, and the membrane is reacted with an antibody or antibody fragment that recognizes the antigen, followed by reaction with an anti-mouse IgG antibody or binding fragment labeled with a fluorescent substance such as FITC, an enzyme such as peroxidase, or a biotin label, and the label is visualized for measurement.

[0224] An example is shown below. Cells or tissues expressing a polypeptide having the amino acid sequence of SEQ ID NO: 3 or 4 are lysed, and 0.1 to 30 μg of protein per lane is electrophoresed by SDS-PAGE under reducing conditions. The electrophoresed protein is transferred to a PVDF membrane and blocked by reacting it with PBS containing 1 to 10% BSA (hereinafter referred to as BSA-PBS) at room temperature for 30 minutes. The membrane is then reacted with the monoclonal antibody of the present invention, washed with PBS containing 0.05 to 0.1% Tween-20 (hereinafter referred to as Tween-PBS), and then reacted with peroxidase-labeled goat anti-mouse IgG at room temperature for 2 hours. After washing with Tween-PBS, the band bound by the monoclonal antibody is detected using ECL Western Blotting Detection Reagents (Amersham) or the like to detect the polypeptide having the amino acid sequence of SEQ ID NO: 3 or 4. The antibody used for detection by Western blotting is an antibody that can bind to a polypeptide that does not retain its native three-dimensional structure.

[0225] Physicochemical techniques include, for example, forming aggregates by binding the antigen, human FCRL1, with the monoclonal antibody or antibody fragment of the present invention, and then detecting these aggregates. Other physicochemical techniques include capillary tube methods, one-dimensional immunodiffusion, immunoturbidimetry, and latex immunoturbidimetry [Clinical Test Methods Summary, Kanehara Publishing (1998)]. Latex immunoturbidimetry uses a carrier such as polystyrene latex with a particle size of approximately 0.1 to 1 μm sensitized with an antibody or antigen. When an antigen-antibody reaction occurs with the corresponding antigen or antibody, scattered light in the reaction solution increases and transmitted light decreases. This change is detected as absorbance or integrating sphere turbidity, and the antigen concentration in the test sample is measured.

[0226] Cells expressing human FCRL1 can be detected or measured using known immunological detection methods, and among these, immunoprecipitation, immunocytostaining, immunohistostaining, fluorescent antibody staining, or the like is preferably used.

[0227] Immunoprecipitation involves reacting cells expressing human FCRL1 with the monoclonal antibody of the present invention or its antibody fragment, followed by the addition of a carrier capable of specifically binding to immunoglobulin, such as protein G-Sepharose, to precipitate the antigen-antibody complex. Alternatively, immunoprecipitation can be performed by the following method: The monoclonal antibody of the present invention or its antibody fragment is immobilized on a 96-well ELISA plate, followed by blocking with BSA-PBS. When the antibody is in an unpurified state, such as a hybridoma culture supernatant, anti-mouse immunoglobulin, anti-rat immunoglobulin, protein A, protein G, or the like is pre-immobilized on a 96-well ELISA plate, followed by blocking with BSA-PBS, and then the hybridoma culture supernatant is dispensed and allowed to bind. Next, the BSA-PBS is discarded and the plate is thoroughly washed with PBS, after which a lysate of cells or tissues expressing human FCRL1 is reacted. After thorough washing, the immunoprecipitates are extracted from the plate with a sample buffer for SDS-PAGE and detected by Western blotting as described above.

[0228] In immunocytostaining or immunohistostaining, cells or tissues expressing an antigen are treated, optionally with a detergent or methanol to improve antibody permeability, and then reacted with the monoclonal antibody of the present invention. The cells are then further reacted with an anti-immunoglobulin antibody or a binding fragment thereof labeled with a fluorescent label such as FITC, an enzyme label such as peroxidase, or biotin, and the label is visualized and examined under a microscope. Detection can also be performed by a fluorescent antibody staining method [Monoclonal Antibodies—Principles and Practice, Third Edition, Academic Press (1996); Monoclonal Antibody Experimental Manual, Kodansha Scientific (1987)], in which cells are reacted with a fluorescently labeled antibody and analyzed using a flow cytometer. In particular, the monoclonal antibody or antibody fragment of the present invention that binds to human FCRL1 can be used to detect cells expressing the antibody while maintaining its native three-dimensional structure.

[0229] Furthermore, when an FMAT8100HTS system (manufactured by Applied Biosystems) or the like is used in the fluorescent antibody staining method, the amount of antigen or antibody can be measured without separating the formed antibody-antigen complex from the free antibody or antigen that is not involved in the formation of the antibody-antigen complex.

[0230] 8. Method for Treating Diseases Using the Anti-Human FCRL1 Monoclonal Antibody of the Present Invention or the Antibody Fragment Thereof The monoclonal antibody or the antibody fragment thereof, or antibody-drug conjugate of the present invention can be used to treat any human FCRL1-associated disease, so long as the disease is associated with human FCRL1.

[0231] Therapeutic agents containing the monoclonal antibodies or antibody fragments of the present invention may contain only the antibodies or antibody fragments as the active ingredient, but are usually provided as pharmaceutical formulations prepared by mixing them with one or more pharmacologically acceptable carriers and using methods known in the technical field of pharmaceuticals.

[0232] Examples of administration routes include oral administration and parenteral administration such as oral, respiratory, rectal, subcutaneous, intramuscular, or intravenous administration. Examples of administration forms include sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments, and tapes.

[0233] Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, and granules.

[0234] Liquid preparations such as emulsions or syrups are produced using additives such as water, sugars such as sucrose, sorbitol, or fructose, glycols such as polyethylene glycol or propylene glycol, oils such as sesame oil, olive oil, or soybean oil, preservatives such as p-hydroxybenzoic acid esters, or flavors such as strawberry flavor or peppermint.

[0235] Capsules, tablets, powders, granules, etc. are produced using additives such as excipients such as lactose, glucose, sucrose, or mannitol; disintegrating agents such as starch or sodium alginate; lubricants such as magnesium stearate or talc; binders such as polyvinyl alcohol, hydroxypropyl cellulose, or gelatin; surfactants such as fatty acid esters; or plasticizers such as glycerin.

[0236] Formulations suitable for parenteral administration include injections, suppositories, and sprays. Injections are prepared using carriers such as saline solutions, glucose solutions, or mixtures of both. Suppositories are prepared using carriers such as cocoa butter, hydrogenated fats, or carboxylic acids.

[0237] Sprays are prepared using carriers that do not irritate the recipient's oral and respiratory mucosa and disperse the monoclonal antibody or antibody fragment of the present invention as fine particles, facilitating absorption. Examples of carriers include lactose and glycerin. Sprays can also be prepared as aerosols or dry powders. Furthermore, the above-mentioned parenteral preparations can also contain the additives listed above for formulations suitable for oral administration.

[0238] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0239] [Example 1] Preparation of known anti-FCRL1 chimeric antibodies Chimeric antibodies were prepared based on the amino acid sequence information of the variable regions of known antibodies E3 and E9 (Blood. 2008, 111, 338-43), 1F9 and 2A10 (WO 2005 / 063299), and 7G8, 2G5, and 5A2 (WO 2005 / 097185). The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of each antibody are shown in Table 1.

[0240]

[0241] The expression vectors for the chimeric antibodies were constructed by inserting the VH region into the pFUSE-CHIg-hG1 plasmid vector and the VL region into the pFUSE2-CLIg-hk plasmid vector. A vector in which serine at position 239 (EU numbering) in the heavy chain constant region was converted to cysteine ​​was used. Human chimeric antibodies were produced using these vectors and the Expi293 Expression System (Life Technologies). The procedure was as follows, following the attached manual.

[0242] 7.5 x 10 per reaction ８ Expi293F cells (Thermo Fisher Scientific) were added to 255 mL of Expi293 Expression Medium (Thermo Fisher Scientific). 200 μg of pFUSE-CHIg-hG1 plasmid vector, 100 μg of pFUSE2-CLIg-hk plasmid vector, and ExpiFectamin 293 Reagent (Thermo Fisher Scientific) were added to Opti-MEM (Thermo Fisher Scientific). After allowing to stand for 20 minutes, the plasmid solution was added to the cell-containing solution. After further overnight culture, ExpiFectamin 293 Transfection Enhancer was added to the cell-containing solution (total culture volume: 300 mL). The cell-containing solution was further cultured for 2 days, and then the culture supernatant was collected.

[0243] MabSelect SuRe (GE Healthcare) was used for antibody purification. The collected culture supernatant was centrifuged, and the resulting culture supernatant was filtered through a filter. 1 mL of carrier was packed into a column, and the buffer was replaced with DPBS. The culture supernatant was added to the column, and the antibody was adsorbed onto the carrier. The column was then washed twice with 10 mL of DPBS. 2.5 mL of Arg-Antibody Elution Buffer (Nacalai Tesque) was added to the column to elute the antibody. The antibody solution was desalted using a NAP column (GE Healthcare) and used for subsequent analysis.

[0244] The resulting antibody is an IgG1 antibody in which the serine at position 239 (EU numbering) in the heavy chain has been replaced with cysteine ​​(hereinafter also referred to as S239C mutation). The heavy chain constant region containing the S239C mutation comprises the amino acid sequence set forth in SEQ ID NO: 80. Note that E3, 2G5, and 5A2 were not subjected to ADC conversion because aggregates were detected when they were produced.

[0245] [Example 2] Preparation of ADC of known anti-FCRL1 chimeric antibody ADC can be prepared by the method described in Bioconjug Chem 2013, 24(7), 1256-1263, or the like. An ADC was prepared by reacting the FCRL1 chimeric antibody having the S239C mutation prepared in Example 1 with SG3249 (Med. Chem. Lett. 2016, 7, 983-987), a PBD dimer payload linker.

[0246] Analysis of the drug-to-antibody ratio (DAR) can be performed using a high-performance liquid chromatography system and a reverse-phase column (reverse-phase HPLC) after converting the ADC into a light chain fragment and a heavy chain fragment by pretreatment with a reducing agent. The DAR is calculated from the peak area ratio of the unreacted light chain, the drug-bound light chain, the unreacted heavy chain, and the drug-bound heavy chain. The drug-to-antibody ratios of all the ADCs prepared were 1.8 to 1.9. 2A10 was excluded from the evaluation because it was difficult to prepare an ADC with a controlled DAR.

[0247] Furthermore, an ADC (anti-DNP antibody-ADC) was prepared in the same manner as in Example 1 using a vector encoding the anti-2,4-dinitrophenol (DNP) IgG1 antibody (S239C mutation) described in Clin Cancer Res 2005, 11(8), 3126-3135. This ADC was used as a negative control in the following tests.

[0248] Example 3 In vivo efficacy evaluation of ADC of known anti-FCRL1 chimeric antibody 1 x 10 SU-DHL-6 cells suspended in Phosphate Buffered Saline (PBS) containing 50 vol% Matrigel (Corning) were injected subcutaneously into the ventral side of 5-week-old male SCID mice. ７ The tumor volume was 120 mm on the 21st day after transplantation. ３ The above individuals were selected and divided into groups.

[0249] The day of grouping was designated day 0, and on day 0, a diluted known FCRL1 chimeric antibody-ADC at 0.3 mg / kg body weight or an anti-DNP antibody-ADC at 0.4 mg / kg body weight was administered into the tail vein. The vehicle composition was 10 mmol / L sodium L-glutamate, 262 mmol / L D-sorbitol, 0.05 mg / mL polysorbate 80, pH 5.5. The tumor volume and body weight of the mice were measured twice a week. The results are shown in Figure 1. The ADC of the known anti-human FCRL1 chimeric antibody used was the ADC prepared in Example 2.

[0250] As shown in Figure 1, 7G8-ADC showed the strongest antitumor activity among the ADCs of known anti-human FCRL1 antibodies. Similarly, 7G8-ADC showed the strongest antitumor activity in a mouse model subcutaneously implanted with Ramos cells (details omitted).

[0251] Example 4: Obtaining novel anti-human FCRL1 mouse antibodies A / J, BALB / c, or C57BL6 mice (all from Japan SLC) were used as immunization hosts, and the immunogens used were plasmid vectors (15-50 μg) expressing the full-length human FCRL1 (NP_443170.1), the full-length cynomolgus monkey FCRL1 (XP_015310712.1), or mutants thereof, fusion proteins (10-25 μg) of the human FCRL1 extracellular domain with a C-terminal His Tag or rabbit IgG1-Fc, or 293T cells (0.5-2.0 × 10) transiently expressing human FCRL1, cynomolgus monkey FCRL1, or mutants thereof. ７ pcs) were used.

[0252] One or more of these immunogens were administered intramuscularly, intradermally, intraperitoneally, or intravenously 3-8 times at 10-50 day intervals based on various regimens. When an adjuvant was used, the Sigma adjuvant system (Sigma-Aldrich) was used. Mice were selected based on the reactivity of the antisera with various antigen-expressing cells, and 3 days after the final immunization, spleen cells were fused with mouse myeloma cells P3U1 to generate monoclonal antibody-producing hybridomas.

[0253] The obtained novel anti-FCRL1 mouse antibodies were screened based on in vitro anti-cellular activity using a second immunotoxin and cross-reactivity with cynomolgus monkey FCRL1, and antibodies suitable for ADC conversion were selected from the viewpoint of whether their amino acid sequences are suitable for antibody engineering modification for ADC conversion. The amino acid sequences of the VH and VL, as well as the heavy chain CDRs 1-3 (HCDRs 1-3) and light chain CDRs 1-3 (LCDRs 1-3) of the selected six antibodies (DK610, DK681, DK1142, DK1141, DK1166, and DK1164) are shown in Table 2.

[0254]

[0255] [Example 5] FCRL1 binding activity of novel anti-human FCRL1 mouse antibodies The antigen-specific reactivity of the novel anti-human FCRL1 mouse antibodies obtained in Example 4 was evaluated by flow cytometry (FCM). FCRL1 antigen-expressing cells were prepared by transiently expressing recombinant FCRL1 antigen on 293T cells. Codon-optimized cDNA was synthesized based on the amino acid sequence of human FCRL1 (NCBI accession number: NP_443170.1) or cynomolgus monkey FCRL1 (XP_015310712). An internal ribosome entry site (IRES) sequence derived from encephalomyocarditis virus (EMCV) and a TagBFP sequence were arranged in tandem downstream of the cDNA. This fragment was cloned into pcDNA3.1hygro to construct an expression plasmid vector for human FCRL1 or cynomolgus monkey FCRL1. The purified expression vector was transfected into 293T cells, and the cells were harvested two days after transfection. Binding to each antibody was confirmed by FCM.

[0256] FCM was performed under the following conditions: The collected cells were diluted to 2 × 10 in Dulbecco's PBS (FCM buffer) containing 5% FBS, 25% DMEM, and 0.1% sodium azide. ６ 25 μL of this cell suspension and 25 μL of an antibody solution prepared by diluting each antibody with PBS to 1 μg / mL were mixed in each well of a 96-well V plate (5×10 ４ The plate was incubated with 100 μg of each MAb (100 μg / well, 500 ng / mL of each MAb) at 4° C. for 30 minutes.

[0257] The plate was centrifuged, the supernatant was removed, and the cells were washed once by adding 200 μL / well of FCM buffer, suspending the cells, and then centrifuging again. The washed cells were resuspended in 25 μL / well of a diluted solution of the secondary antibody in FCM buffer and incubated at 4°C for 30 minutes.

[0258] As the secondary antibody, if the sample is a mouse antibody, R-Phycoerythrin (PE) F(ab') ２Fragment Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch) was used, and if the sample was a human antibody or a human chimeric antibody, R-Phycoerythrin (PE) F(ab') ２ Fragment Goat Anti-Human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch) was used. Each secondary antibody solution was prepared by diluting the stock solution 200-fold with FCM buffer. After one wash, the cells in each well were suspended in 100 μL of FCM buffer, and binding to each antibody was confirmed by flow cytometry (FCM).

[0259] The binding affinity of the antibody was expressed as the apparent affinity for FCRL1-expressing cells in FCM, expressed as the dissociation constant (K Ｄ ) was calculated.

[0260] Cells transiently expressing human FCRL1 or cynomolgus monkey FCRL1 were reacted with 12 three-fold serial dilutions of each antibody starting from 100 nM (15 μg / ml). Data obtained by FCM was analyzed, and the amount of each antibody bound to the cells was calculated as the amount of fluorescence (MFI) derived from the PE-labeled secondary antibody. The antibody concentration and the measured MFI were plotted against the logarithm of the antibody concentration to obtain a saturation binding curve for each antibody, which was then regressed using a four-parameter logistic model. Prism 5 software (GraphPad Software Inc.) was used for fitting, and the obtained 50% effective concentration (EC ５０ ) to obtain the dissociation constant (K Ｄ The binding activity to human or cynomolgus monkey FCRL1 is shown in Table 3.

[0261]

[0262] [Example 6] Preparation of ADCs of novel anti-human FCRL1 chimeric antibodies Based on the amino acid sequence information of the variable regions of the novel anti-human FCRL1 mouse antibody obtained in Example 4, novel anti-FCRL1 antibodies having the S239C mutation and ADCs in which SG3249 was specifically attached to the mutation site of these antibodies were prepared according to the methods of Examples 1 and 2. The drug-antibody ratios of all ADCs were 1.8 to 1.9.

[0263] Example 7 Anti-Cell Test of ADC of Novel Anti-Human FCRL1 Chimeric Antibody SU-DHL-6 cells were seeded at 40 μL / well in a 384-well plate (Greiner-Bio) to achieve a density of 5,000 cells / well. Ramos cells were seeded at 80 μL / well in a 96-well plate (Thermo Fisher Scientific) to achieve a density of 4,000 cells / well. The ADC was diluted √10 times, and 9 or 10 dilutions were prepared with 10,000 ng / mL as the highest concentration (final concentration: 10-10,000 ng / mL). The ADC diluted to the desired final concentration was added to the 384-well plate at 10 μL / well and to the 96-well plate at 20 μL / well.

[0264] After addition of the ADC, the cells were cultured for approximately 4 days in a carbon dioxide incubator set at 37°C. After completion of the culture, CellTiter-Glo Luminescent Cell Viability Assay (Promega) was added to the 384-well plate at 20 μL / well and to the 96-well plate at 100 μL / well, and after approximately 15 minutes of reaction, the luminescence value was measured to determine the number of viable cells. The ADC prepared in Example 6 was used as the ADC of the novel anti-human FCRL1 chimeric antibody, and the ADC prepared in Example 2 was used as the 7G8-ADC.

[0265] The results of the anti-cellular test on SU-DHL-6 cells are shown in Figures 2A and 2B, and the results of the anti-cellular test on Ramos cells are shown in Figures 3A and 3B. As shown in Figures 2A, 2B, 3A, and 3B, ADCs of all novel anti-FCRL1 antibodies were found to have stronger anti-cellular effects on SU-DHL-6 cells and Ramos cells than known anti-FCRL1 antibodies.

[0266] [Example 8] Antitumor test of ADC of novel anti-human FCRL1 chimeric antibody A Ramos cell subcutaneously transplanted mouse model was prepared by the following method. Ramos cells were suspended in PBS and 5 × 10 ６ The tumor was subcutaneously transplanted into the ventral side of SCID mice at a dose of 0.05 ml / head. On the 7th day after transplantation, the tumor volume reached 85 mm ３ The above individuals were selected and divided into groups.

[0267] The Ramos cell subcutaneously transplanted mouse model thus prepared and the SU-DHL-6 cell subcutaneously transplanted mouse model prepared by the method of Example 3 were used in the following antitumor test.

[0268] On the day of grouping (day 0), the novel anti-FCRL1 chimeric antibody ADC or 7G8-ADC diluted to 0.3 mg / kg body weight was administered into the tail vein. The vehicle composition was 10 mmol / L sodium L-glutamate, 262 mmol / L D-sorbitol, 0.05 mg / mL polysorbate 80, pH 5.5. The mean tumor volume of the 7G8-ADC administration group on day 10 was set to 1, and the relative values ​​of the mean tumor volumes of each novel anti-human FCRL1 chimeric antibody-ADC administration group are shown in Figure 4. The novel anti-human FCRL1 chimeric antibody ADC used was the ADC prepared in Example 6, and the 7G8-ADC used was the ADC prepared in Example 2.

[0269] As shown in Figure 4, all ADCs of the novel anti-human FCRL1 chimeric antibodies had stronger anti-tumor activity than 7G8-ADC. Furthermore, Figure 5 shows the tumor size on day 42 in a mouse model subcutaneously implanted with Ramos cells. As shown in Figure 5, tumor growth was observed with 7G8-ADC, whereas all ADCs of the novel anti-human FCRL1 chimeric antibodies were found to exhibit sustained and potent efficacy. These results demonstrate that ADCs of the novel anti-FCRL1 antibodies have superior anti-tumor activity than ADCs of known anti-FCRL1 antibodies.

[0270] Example 9 Internalization of Novel Anti-Human FCRL1 Chimeric Antibody The novel anti-human FCRL1 chimeric antibody prepared in Example 6 was labeled with IncuCyte Human FabFluor-pH Red Antibody Labeling Reagent (Sartorius) according to the attached instructions. Ramos cells were treated with the labeled antibody diluted to a final concentration of 200 ng / mL. After culturing for approximately 4 or 24 hours in a carbon dioxide incubator set at 37°C, the mean fluorescence intensity (MFI) was measured by FCM. Note that the more the antibody is internalized, the higher the MFI.

[0271] The results of internalization are shown in Figure 6. As shown in Figure 6, all of the novel anti-human FCRL1 chimeric antibodies had a higher internalization ability than the known anti-human FCRL1 chimeric antibodies.

[0272] [Example 10] Production of novel anti-human FCRL1 humanized antibodies (1) Design of VH and VL amino acid sequences of DK681 humanized antibody and DK1142 humanized antibody Various VH and VL amino acid sequences of DK681 humanized antibody and DK1142 humanized antibody were designed by the method described below. In the following description, DK681 humanized antibody and DK1142 humanized antibody having various VH and VL amino acid sequences are collectively referred to as hzDK681 antibody and hzDK1142 antibody. The amino acid sequences of the frameworks (hereinafter referred to as FR) of known human antibodies suitable for grafting the amino acid sequences of the CDRs of the variable regions of the DK681 mouse antibody and the DK1142 mouse antibody obtained in Example 4 were selected from the human FR consensus sequences reported by Kabat et al. [Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services (1991)], human antibody germline sequences, and FR sequences derived from human antibody variable regions obtained by similarity searches such as the BLAST method [J. Mol. Biol., 215, 403 (1990)], as follows. Genbank Accession Number: AKU38660.1 and Genbank Accession Number: AAW69164.1 were selected for DK681, and human subgroup H chain I (hereinafter also referred to as hSGHI) and Genbank Accession Number: ABG38363.1 reported in Kabat et al. [Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services (1991)] were selected for DK1142, and CDRs were grafted into these FRs.

[0273] For DK681, the amino acid sequences of CDR1 to 3 of DK681 VH shown in SEQ ID NOS: 28, 29, and 30, respectively, were grafted into appropriate positions in the amino acid sequence of the FR of AKU38660.1 to design hzDK681 HV0 (SEQ ID NO: 67). Furthermore, the amino acid sequences of CDR1 to 3 of DK681 VL shown in SEQ ID NOS: 32, 33, and 34, respectively, were grafted into appropriate positions in the amino acid sequence of the FR of AAW69164.1 (the FR of the DK681 chimeric antibody was used as is for FR4), to design hzDK681 LV0 (SEQ ID NO: 68).

[0274] Computer modeling of the hzDK681 HV0 and hzDK681 LV0 designed as described above identified amino acid residues in the FR that are thought to affect the binding activity of the antibody. As a result, among the amino acid residues in the FR of the variable regions of hzDK681 HV0 and hzDK681 LV0, the following amino acid residues were selected as those that alter the three-dimensional structure of the antigen-binding site and affect the binding activity of the antibody: Val at position 11, Lys at position 12, Arg at position 38, Met at position 48, Arg at position 67, Val at position 68, Ile at position 70, Ala at position 72, Thr at position 74, and Ala at position 97 in the amino acid sequence represented by SEQ ID NO: 67 for VH; Ile at position 21, Pro at position 49, and Val at position 91 in the amino acid sequence represented by SEQ ID NO: 68 for VL. At least one of these selected amino acid residues was substituted with an amino acid residue present at the same position in the DK681 antibody, and humanized antibody VH (sequence numbers 72 and 73) and VL (sequence number 74) with various modifications were designed.

[0275] For DK1142, the amino acid sequences of CDR1 to 3 of DK1142 VH represented by SEQ ID NOS: 36, 37, and 38, respectively, were grafted into appropriate positions in the amino acid sequence of the FR of hSGHI to design hzDK1142 HV0 (SEQ ID NO: 69). Also, the amino acid sequences of CDR1 to 3 of DK1142 VL represented by SEQ ID NOS: 40, 41, and 42, respectively, were grafted into appropriate positions in the amino acid sequence of the FR of ABG38363.1 (the FR of the DK1142 chimeric antibody was used as is for FR4), to design hzDK1142 LV0 (SEQ ID NO: 70).

[0276] Computer modeling of the hzDK1142 HV0 and hzDK1142 LV0 designed as described above identified amino acid residues in the FR that are thought to affect the binding activity of the antibody. As a result, among the amino acid residues in the FR of the variable regions of hzDK1142 HV0 and hzDK1142 LV0, the 11th Val, the 12th Lys, the 38th Arg, the 48th Met, the 67th Arg, the 68th Val, the 70th Ile, the 74th Thr, and the 95th Thy of the amino acid sequence described in SEQ ID NO: 69 were selected as amino acid residues that change the three-dimensional structure of the antigen-binding site and affect the binding activity of the antibody. In VH, the 2nd Ile, the 15th Pro, and the 50th Gln of the amino acid sequence described in SEQ ID NO: 70 were selected. At least one of these selected amino acid residues was substituted with an amino acid residue present at the same position in the DK1142 antibody, and humanized antibody VH (sequence numbers 75 and 77) and VL (sequence number 76) with various modifications were designed.

[0277] In addition to the standard CDR-grafting design described above, VL CDRs were also modified for some hzDK1142 antibody VLs. Specifically, the second Val in the amino acid sequence of CDR2 of VL set forth in SEQ ID NO:41 was replaced with Ile to introduce the CDR2 of VL set forth in SEQ ID NO:71. A humanized antibody VL (SEQ ID NO:78) was also designed, containing the CDR2 of VL set forth in SEQ ID NO:71.

[0278] (2) Design of humanized antibody variable region genes The DK681 humanized antibodies designed in this manner were named DK681 F11, DK681 F12, DK681 F13, and DK681 F14, respectively, and the DK1142 humanized antibodies were named DK1142 F21, DK1142 F22, and DK1142 F24, respectively. The variable regions and CDRs of these humanized antibodies are shown in Table 4. The nucleotide sequences encoding the amino acid sequences of the variable regions of these humanized antibodies were designed using codons frequently used in animal cells.

[0279]

[0280] (3) Preparation of humanized antibodies The gene fragment corresponding to the nucleotide sequence designed in (2) was introduced into an expression vector using seamless cloning to prepare the necessary plasmids. The VL expression vector used was the pCI-OtCMV_hK vector containing a signal sequence and a human κ chain constant region sequence, and the VH expression vector used was the pCI-OtCAG_hG1(S239C) vector containing a signal sequence and a human γ chain constant region sequence. The constant region sequence of the pCI-OtCAG_hG1(S239C) vector is a heavy chain constant region obtained by introducing the S239C mutation into human IgG1. These vectors were prepared by total synthesis using the Promega pCI vector as a common backbone, with the necessary restriction enzyme sites introduced to express human antibody genes. The completed plasmid was prepared in large quantities using the QIAGEN Plasmid Plus Maxi kit (QIAGEN). The humanized antibody of interest was then transiently expressed using the Expi293 Expression System Kit (Thermo Fisher Scientific). The plasmid was introduced according to the instructions in the package insert.

[0281] The light chain expression vector and the heavy chain expression vector were mixed at a ratio of 2:1 and transfected. After the plasmid transfection, the cells were incubated at 37°C, 8% CO ２The cells were cultured for 2 to 4 days at 125 rpm. The cell culture suspension was then centrifuged and the culture supernatant was collected through a 0.2 μm filter. Purified antibodies were obtained from the culture supernatant by affinity purification using MabSelect SuRe (Cytiva).

[0282] Specifically, the resin packed in the column was equilibrated with PBS, and then the culture supernatant was added to the column. The column was washed with PBS, and the antibody was eluted using elution buffer (20 mM citric acid, 50 mM NaCl, pH 3.4). The resulting antibody solution was neutralized by adding 1 / 10 volume of neutralization buffer (1 M phosphoric acid-NaOH, pH 7.0), and the solvent of the antibody solution was replaced with PBS using NAP25 (Cytiva). The antibody solution after buffer replacement was concentrated by ultrafiltration using Amicon Ultra-4 Centrifugal Filter Units (Millipore), and the absorbance A280 was measured using Nanodrop (Thermo Fisher Scientific), and the concentration of the antibody solution was measured and prepared. The extinction coefficient is C.N. The amino acid sequence of each humanized antibody was calculated according to the method of Pace et al. (1995, Prot. Sci. 4:2411-2423). The quality of the purified antibodies was confirmed by analytical gel filtration chromatography (using a column TSKgel SuperSW3000 manufactured by Tosoh Corporation) and SDS-PAGE.

[0283] Example 11 FCRL1-binding activity of novel anti-human FCRL1 humanized antibodies To compare the binding activity to human FCRL1 of the DK681 chimeric antibody (chDK681) and DK1142 chimeric antibody (chDK1142), in which the constant regions of the DK681 and DK1142 mouse antibodies obtained in Example 4 were ligated to a human IgG1 (S239C) constant region, with the anti-FCRL1 humanized antibody obtained in Example 10, the binding activity to hFCRL1 / FcRH1-His (R&D Systems) was measured by surface plasmon resonance (SPR) using a Biacore8K+ (Cytiva).

[0284] The binding activity of the anti-FCRL1 antibody was measured as follows: An anti-human IgG antibody was immobilized on a CM5 sensor chip (Cytiva) using a Human Antibody Capture Kit (Cytiva) according to the attached protocol. An anti-FCRL1 antibody adjusted to 5 μg / mL was added to the flow cell on which the anti-human IgG antibody was immobilized at a flow rate of 10 μL / min for 30 seconds.

[0285] Next, hFCRL1 / FcRH1-His was prepared at 5 concentrations, diluted 3-fold starting from 10,000 ng / mL, and the binding reaction was monitored for 180 seconds and the dissociation reaction for 400 seconds at a flow rate of 30 μL / min. The obtained sensorgram was fitted using Bia Evaluation Software (Cytiva) with a steady state affinity model or a 1:1 binding model to calculate the kinetic constant of each antibody. The calculated binding rate constant (ka), dissociation rate constant (kd) and dissociation constant [KD] of each antibody are shown in Table 5. chDK681 is referred to as DK681 F01, and chDK1142 is referred to as DK 1142 F02.

[0286]

[0287] The above results demonstrated that the anti-human FCRL1 humanized antibody prepared in Example 10 has binding activity equivalent to that of the chDK681 antibody and the chDK1142 antibody.

[0288] Example 12 Preparation of ADCs of Novel Anti-Human FCRL1 Humanized Antibodies Novel anti-FCRL1 antibodies having the S239C mutation and ADCs in which SG3249 was specifically added to the mutation site of these antibodies were prepared according to the methods of Examples 1 and 2. The variable region sequences of the novel anti-human FCRL1 humanized antibodies used were those shown in Table 4. The drug-antibody ratios of all ADCs were 1.7 to 1.8.

[0289] [Example 13] Anticellular test of ADC of novel anti-human FCRL1 humanized antibody According to the method of Example 7, the anticellular effect of the ADC of the novel anti-FCRL1 humanized antibody prepared in Example 12 on SU-DHL-6 cells and Ramos cells was confirmed.

[0290] The results of the anti-cellular test on SU-DHL-6 cells are shown in Figures 7A and 7B, and the results of the anti-cellular test on Ramos cells are shown in Figures 8A and 8B. As shown in Figures 7A, 7B, 8A, and 8B, the ADCs of all novel anti-FCRL1 antibodies exhibited stronger anti-cellular effects on SU-DHL-6 cells and Ramos cells than the ADCs of known anti-FCRL1 antibodies.

[0291] Example 14 Antitumor Test of ADC of Novel Anti-Human FCRL1 Humanized Antibody A mouse model subcutaneously implanted with SU-DHL-6 cells and a mouse model subcutaneously implanted with Ramos cells were prepared according to the methods of Examples 3 and 8 and used in the following antitumor test.

[0292] On the day of grouping (day 0), the novel anti-human FCRL1 humanized antibody ADC or 7G8-ADC diluted to 0.3 mg / kg body weight was administered intravenously to the tail vein. The vehicle composition was 10 mmol / L sodium L-glutamate, 262 mmol / L D-sorbitol, 0.05 mg / mL polysorbate 80, pH 5.5. The mean tumor volume of the 7G8-ADC-administered group on day 7 was set to 1, and the relative values ​​of the mean tumor volumes of each novel anti-human FCRL1 humanized antibody ADC-administered group are shown in Figure 9. The novel anti-human FCRL1 humanized antibody ADC used was the ADC prepared in Example 12, and the 7G8-ADC used was the ADC prepared in Example 2.

[0293] As shown in Figure 9, the ADCs of all novel anti-FCRL1 humanized antibodies had stronger anti-tumor activity than 7G8-ADC. These results demonstrated that the ADCs of novel anti-FCRL1 humanized antibodies have superior anti-tumor effects than ADCs of known anti-FCRL1 antibodies.

[0294] The present invention provides a novel monoclonal antibody or a fragment thereof that binds to the extracellular domain of FCRL1.

[0295] Although the present invention has been described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention. This application is based on a Japanese patent application (Patent Application No. 2022-019051) filed on February 9, 2022, the entirety of which is incorporated by reference. All references cited herein are incorporated in their entirety.

[0296] SEQ ID NO: 1: Gene sequence of human FCRL1 SEQ ID NO: 2: Gene sequence of monkey FCRL1 SEQ ID NO: 3: Amino acid sequence of human FCRL1 SEQ ID NO: 4: Amino acid sequence of monkey FCRL1 SEQ ID NO: 5: Amino acid sequence of VH of E3 SEQ ID NO: 6: Amino acid sequence of VL of E3 SEQ ID NO: 7: Amino acid sequence of VH of E9 SEQ ID NO: 8: Amino acid sequence of VL of E9 SEQ ID NO: 9: Amino acid sequence of VH of 1F9 SEQ ID NO: 10: Amino acid sequence of VL of 1F9 SEQ ID NO: 11: Amino acid sequence of VH of 2A10 SEQ ID NO: 12: Amino acid sequence of VL of 2A10 SEQ ID NO: 13: Amino acid sequence of VH of 7G8 SEQ ID NO: 14: Amino acid sequence of VL of 7G8 SEQ ID NO: 15: Amino acid sequence of VH of 2G5 SEQ ID NO: 16: Amino acid sequence of VL of 2G5 SEQ ID NO: 17: Amino acid sequence of VH of 5A2 SEQ ID NO: 18: Amino acid sequence of VL of 5A2 SEQ ID NO: 19: Amino acid sequence of VH of DK610 SEQ ID NO: 20: Amino acid sequence of HCDR1 of DK610 SEQ ID NO: 21: Amino acid sequence of HCDR2 of DK610 SEQ ID NO: 22: Amino acid sequence of HCDR3 of DK610 SEQ ID NO: 23: Amino acid sequence of VL of DK610 SEQ ID NO: 24: Amino acid sequence of LCDR1 of DK610 SEQ ID NO: 25: Amino acid sequence of LCDR2 of DK610 SEQ ID NO: 26: Amino acid sequence of LCDR3 of DK610 SEQ ID NO: 27: Amino acid sequence of VH of DK681 SEQ ID NO: 28: Amino acid sequence of HCDR1 of DK681 SEQ ID NO: 29: Amino acid sequence of HCDR2 of DK681 SEQ ID NO: 30: Amino acid sequence of HCDR3 of DK681 SEQ ID NO: 31: Amino acid sequence of VL of DK681 SEQ ID NO: 32: Amino acid sequence of LCDR1 of DK681 SEQ ID NO: 33: Amino acid sequence of LCDR2 of DK681 SEQ ID NO: 34: Amino acid sequence of LCDR3 of DK681 SEQ ID NO: 35: Amino acid sequence of VH of DK1142 SEQ ID NO: 36: Amino acid sequence of HCDR1 of DK1142 SEQ ID NO: 37: Amino acid sequence of HCDR2 of DK1142 SEQ ID NO: 38: Amino acid sequence of HCDR3 of DK1142 SEQ ID NO: 39: Amino acid sequence of VL of DK1142 SEQ ID NO: 40: Amino acid sequence of LCDR1 of DK1142 SEQ ID NO: 41: Amino acid sequence of LCDR2 of DK1142 SEQ ID NO: 42: Amino acid sequence of LCDR3 of DK1142SEQ ID NO: 43: Amino acid sequence of VH of DK1141 SEQ ID NO: 44: Amino acid sequence of HCDR1 of DK1141 SEQ ID NO: 45: Amino acid sequence of HCDR2 of DK1141 SEQ ID NO: 46: Amino acid sequence of HCDR3 of DK1141 SEQ ID NO: 47: Amino acid sequence of VL of DK1141 SEQ ID NO: 48: Amino acid sequence of LCDR1 of DK1141 SEQ ID NO: 49: Amino acid sequence of LCDR2 of DK1141 SEQ ID NO: 50: Amino acid sequence of LCDR3 of DK1141 SEQ ID NO: 51: Amino acid sequence of VH of DK1166 SEQ ID NO: 52: Amino acid sequence of HCDR1 of DK1166 SEQ ID NO: 53: Amino acid sequence of HCDR2 of DK1166 SEQ ID NO: 54: Amino acid sequence of HCDR3 of DK1166 SEQ ID NO: 55: Amino acid sequence of VL of DK1166 SEQ ID NO: 56: Amino acid sequence of LCDR1 of DK1166 SEQ ID NO: 57: Amino acid sequence of LCDR2 of DK1166 SEQ ID NO: 58: Amino acid sequence of LCDR3 of DK1166 SEQ ID NO: 59: Amino acid sequence of VH of DK1164 SEQ ID NO: 60: Amino acid sequence of HCDR1 of DK1164 SEQ ID NO: 61: Amino acid sequence of HCDR2 of DK1164 SEQ ID NO: 62: Amino acid sequence of HCDR3 of DK1164 SEQ ID NO: 63: Amino acid sequence of VL of DK1164 SEQ ID NO: 64: Amino acid sequence of LCDR1 of DK1164 SEQ ID NO: 65: Amino acid sequence of LCDR2 of DK1164 SEQ ID NO: 66: Amino acid sequence of LCDR3 of DK1164 SEQ ID NO: 67: Amino acid sequence of hzDK681 HV0 SEQ ID NO: 68: hzDK681 Amino acid sequence of LV0, amino acid sequence of VL of DK681 F11 and DK681 F14 SEQ ID NO: 69: Amino acid sequence of hzDK1142 HV0 SEQ ID NO: 70: Amino acid sequence of hzDK1142 LV0 SEQ ID NO: 71: Amino acid sequence of LCDR2 of DK1142 F24 SEQ ID NO: 72: Amino acid sequence of VH of DK681 F11 and DK681 F13 SEQ ID NO: 73: Amino acid sequence of VH of DK681 F12 and DK681 F14 SEQ ID NO: 74: Amino acid sequence of VL of DK681 F12 and DK681 F13 SEQ ID NO: 75: Amino acid sequence of VH of DK1142 F21 SEQ ID NO: 76: Amino acid sequence of VL of DK1142 F21 and DK1142 F22SEQ ID NO: 77: Amino acid sequence of VH of DK1142 F22 and DK1142 F24 SEQ ID NO: 78: Amino acid sequence of VL of DK1142 F24 SEQ ID NO: 79: Amino acid sequence of CH of human IgG1 SEQ ID NO: 80: Amino acid sequence of CH of IgG1 (S239C)

Claims

1. A monoclonal antibody or an antibody fragment thereof that binds to Fc receptor-like protein 1 (hereinafter abbreviated as FCRL1), wherein the antibody or the antibody fragment is any one of antibodies selected from the following (a) to (g): (a) an antibody in which complementarity determining regions (CDRs) 1 to 3 of its heavy chain variable region (VH) comprise the amino acid sequences set forth in SEQ ID NOS: 20 to 22, respectively, and CDRs 1 to 3 of its light chain variable region (VL) comprise the amino acid sequences set forth in SEQ ID NOS: 24 to 26, respectively; (b) an antibody in which CDR1 to CDR3 of VH comprise the amino acid sequences set forth in SEQ ID NOS: 28 to 30, respectively, and CDR1 to CDR3 of VL comprise the amino acid sequences set forth in SEQ ID NOS: 32 to 34, respectively; (c) an antibody in which CDR1 to CDR3 of VH comprise the amino acid sequences set forth in SEQ ID NOs: 36 to 38, respectively, and CDR1 to CDR3 of VL comprise the amino acid sequences set forth in SEQ ID NOs: 40 to 42, respectively; (d) an antibody in which CDR1 to CDR3 of VH comprise the amino acid sequences set forth in SEQ ID NOs: 44 to 46, respectively, and CDR1 to CDR3 of VL comprise the amino acid sequences set forth in SEQ ID NOs: 48 to 50, respectively; (e) an antibody in which CDR1-3 of VH comprise the amino acid sequences set forth in SEQ ID NOs: 52-54, respectively, and CDR1-3 of VL comprise the amino acid sequences set forth in SEQ ID NOs: 56-58, respectively; and (f) An antibody in which CDR1 to CDR3 of VH comprise the amino acid sequences set forth in SEQ ID NOs: 60 to 62, respectively, and CDR1 to CDR3 of VL comprise the amino acid sequences set forth in SEQ ID NOs: 64 to 66, respectively. (g) An antibody in which CDR1 to CDR3 of VH comprise the amino acid sequences set forth in SEQ ID NOs: 36 to 38, respectively, and CDR1 to CDR3 of VL comprise the amino acid sequences set forth in SEQ ID NOs: 40, 71, and 42, respectively.

2. A monoclonal antibody or an antibody fragment thereof that binds to FCRL1, wherein the antibody is any one antibody selected from the following (2b-1) to (2b-4), (2c-1), (2c-2) and (2g-1): (2b-1) An antibody in which VH comprises the amino acid sequence set forth in SEQ ID NO: 72 and VL comprises the amino acid sequence set forth in SEQ ID NO:

68. (2b-2) An antibody in which VH comprises the amino acid sequence set forth in SEQ ID NO: 73 and VL comprises the amino acid sequence set forth in SEQ ID NO:

74. (2b-3) An antibody in which VH comprises the amino acid sequence set forth in SEQ ID NO: 72 and VL comprises the amino acid sequence set forth in SEQ ID NO:

74. (2b-4) An antibody in which VH comprises the amino acid sequence set forth in SEQ ID NO: 73 and VL comprises the amino acid sequence set forth in SEQ ID NO:

68. (2c-1) An antibody in which VH comprises the amino acid sequence set forth in SEQ ID NO: 75 and VL comprises the amino acid sequence set forth in SEQ ID NO:

76. (2c-2) An antibody in which VH comprises the amino acid sequence set forth in SEQ ID NO: 77 and VL comprises the amino acid sequence set forth in SEQ ID NO:

76. (2g-1) An antibody in which VH comprises the amino acid sequence set forth in SEQ ID NO: 77 and VL comprises the amino acid sequence set forth in SEQ ID NO:

78.

3. The antibody or antibody fragment thereof according to claim 1 or 2, wherein the heavy chain constant region of the antibody is a heavy chain constant region of IgG.

4. The antibody or antibody fragment thereof according to claim 3, wherein the heavy chain constant region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 79 or 80.

5. The antibody or antibody fragment thereof according to claim 1 or 2, wherein the antibody is a recombinant antibody.

6. The antibody or antibody fragment thereof according to claim 5, wherein the recombinant antibody is one selected from the group consisting of a chimeric antibody, a humanized antibody, and a human antibody.

7. The antibody fragment is Fab, Fab', F(ab') ２ 3. The antibody fragment according to claim 1 or 2, which is selected from the group consisting of a single chain antibody (scFv), a dimerized V region (diabody), a disulfide-stabilized V region (dsFv) and a peptide containing CDR.

8. A hybridoma producing the antibody according to claim 1 or 2.

9. A nucleic acid having a base sequence encoding the antibody or antibody fragment thereof according to claim 1 or 2.

10. A vector comprising the nucleic acid of claim 9.

11. A transformed cell obtained by introducing the vector according to claim 10 into a host cell.

12. A method for producing the antibody or antibody fragment of claim 1 or 2, comprising culturing in a medium a transformed cell obtained by introducing into a host cell a hybridoma that produces the antibody of claim 1 or 2, or a vector containing a nucleic acid having a base sequence that encodes the antibody or antibody fragment of claim 1 or 2, and collecting the antibody or antibody fragment from the culture.

13. An antibody-drug conjugate comprising the antibody or antibody fragment thereof according to claim 1 or 2.

14. The antibody-drug conjugate of claim 13, comprising the antibody or antibody fragment thereof linked to a drug via a linker.

15. A composition comprising the antibody or antibody fragment thereof according to claim 1 or 2, or an antibody-drug conjugate comprising the antibody or antibody fragment thereof according to claim 1 or 2.

16. A reagent for detecting or measuring FCRL1, comprising the antibody or antibody fragment thereof according to claim 1 or 2, or an antibody-drug conjugate comprising the antibody or antibody fragment thereof according to claim 1 or 2.

17. A diagnostic agent for an FCRL1-associated disease, comprising the antibody or antibody fragment thereof according to claim 1 or 2, or an antibody-drug conjugate comprising the antibody or antibody fragment thereof according to claim 1 or 2.

18. The diagnostic agent according to claim 17, wherein the FCRL1-associated disease is cancer, an autoimmune disease, or an inflammatory disease.

19. A therapeutic agent for an FCRL1-associated disease, comprising the antibody or antibody fragment thereof according to claim 1 or 2, or an antibody-drug conjugate comprising the antibody or antibody fragment thereof according to claim 1 or 2.

20. The therapeutic agent according to claim 19, wherein the FCRL1-associated disease is cancer, an autoimmune disease, or an inflammatory disease.

21. A method for diagnosing an FCRL1-associated disease, using the antibody or antibody fragment thereof according to claim 1 or 2, or an antibody-drug conjugate comprising the antibody or antibody fragment thereof according to claim 1 or 2.

22. A method for treating an FCRL1-associated disease, comprising administering the antibody or antibody fragment thereof according to claim 1 or 2, or an antibody-drug conjugate comprising the antibody or antibody fragment thereof according to claim 1 or 2.

23. Use of the antibody or antibody fragment thereof according to claim 1 or 2, or an antibody-drug conjugate comprising the antibody or antibody fragment thereof according to claim 1 or 2, for the manufacture of a diagnostic agent for an FCRL1-associated disease.

24. Use of the antibody or antibody fragment thereof according to claim 1 or 2, or an antibody-drug conjugate comprising the antibody or antibody fragment thereof according to claim 1 or 2, for the manufacture of a therapeutic agent for an FCRL1-associated disease.

25. 3. An antibody or antibody fragment thereof according to claim 1 or 2, or an antibody-drug conjugate comprising the antibody or antibody fragment thereof according to claim 1 or 2, for use as a diagnostic agent for an FCRL1-associated disease.

26. 3. An antibody or antibody fragment thereof according to claim 1 or 2, or an antibody-drug conjugate comprising the antibody or antibody fragment thereof according to claim 1 or 2, for use as a therapeutic agent for an FCRL1-associated disease.