Anti-GPRC5D antibody and composition

Abstract

This disclosure provides antigen-binding proteins that specifically bind to GPRC5D, as well as antibodies in enhanced ADCC format, and methods for treating cancers such as multiple myeloma using them.

Description

[Technical Field]

[0001] Multiple myeloma (MM) is the second most common hematological malignancy, characterized by uncontrolled proliferation of plasma cells in the bone marrow. Patients present with symptoms such as weakness, fatigue, anemia, weight loss, immunosuppression, hypercalcemia, and bone pain. Although treatments for MM have improved in recent years, it remains a difficult-to-treat heterogeneous disease and is considered incurable. [Background technology]

[0002] GPRC5D is an orphan G protein-coupled receptor (GPCR) that is strongly expressed on malignant plasma cells in patients with MM. Nearly 80% of MM tumor cells express GPRC5D, but normal tissue expression is mainly limited to plasma cells and hair follicles. Due to its limited expression in healthy tissue, GPRC5D has been identified as a potential immunotherapeutic target for the treatment of MM. [Overview of the project] [Means for solving the problem]

[0003] The present invention provides antigen-binding proteins, particularly antibodies or antigen-binding moieties comprising the HCDR1-3 and LCDR1-3 amino acid sequences of SEQ ID NOs. 1-6, respectively. In some embodiments, the antigen-binding protein comprises two variable domains, the first variable domain comprising HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 2, and HCDR3 of SEQ ID NO. 3, and the second variable domain comprising LCDR1 of SEQ ID NO. 4, LCDR2 of SEQ ID NO. 5, and LCDR3 of SEQ ID NO. 6. In some embodiments, the antigen-binding protein, particularly the antibody, binds to human GPRC5D, cynomolgus monkey GPRC5D, or both. The present invention also provides antigen-binding proteins, particularly antibodies or binding proteins comprising the antigen-binding moiety thereof.

[0004] In some embodiments, antigen-binding proteins, particularly antibodies described herein, include heavy chain variable domain (VH) amino acid sequences and light chain variable domain (VL) amino acid sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (e.g., at least 90%) identical to the amino acid sequences of SEQ ID NOs. 7 and 8, respectively. In some of these embodiments, HCDR1-HCDR3 and LCDR1-LCDR3 have the amino acid sequences of SEQ ID NOs. 1-6, respectively, i.e., the sequence mutations are outside of HCDR1-HCDR3 and LCDR1-LCDR3. In certain embodiments, antigen-binding proteins, particularly antibodies, include VH containing SEQ ID NOs. 7 and VL containing SEQ ID NOs. 8. According to one embodiment, antigen-binding proteins, particularly antibodies, may be human IgG isotypes, preferably human IgG1 isotype subclasses.

[0005] In some embodiments, antigen-binding proteins, particularly antibodies, include heavy chain (HC) variable domain amino acid sequences and light chain (LC) variable domain amino acid sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (e.g., at least 90%) identical to the amino acid sequences of SEQ ID NOs. 9 and 11, respectively. In some of these embodiments, HCDR1-HCDR3 and LCDR1-LCDR3 have the amino acid sequences of SEQ ID NOs. 1-6, respectively, i.e., the sequence mutations are outside of HCDR1-HCDR3 and LCDR1-LCDR3. In certain embodiments, antigen-binding proteins, particularly antibodies, include HC containing SEQ ID NOs. 9 and LC containing SEQ ID NOs. 11.

[0006] In some embodiments, the antigen-binding protein, and in particular the antibody described herein, contains at least one Fc domain mutation, preferably a mutation that enhances the binding of the antigen-binding protein, and in particular the antibody described herein, to human FcγRIIIa. The Fc domain mutation may be, for example, at position 239 (e.g., S239D), position 332 (e.g., I332E), or both (e.g., S239D and I332E), where the residues are numbered according to Eu numbering (Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969)). According to certain embodiments, the Fc domain mutation is S239D, I332E, or a double mutation S239D and I322E, and more preferably S239D and I332E.

[0007] In some embodiments, antigen-binding proteins, and in particular antibodies described herein, include at least one Fc domain mutation that enhances the stability of the antibody. In certain embodiments, the antibody includes a pair of Fc domain mutations to cysteine, for example, at positions 292 and 302 (e.g., R292C and V302C), where the residues are numbered according to Eu numbering. According to a preferred embodiment, antigen-binding proteins, and in particular antibodies described herein, include Fc domain mutations R292C and V302C.

[0008] According to certain embodiments, the antigen-binding protein, in particular the antibody described herein, comprises Fc mutations S239D, I332E, R292C, and V302C, and the residues are numbered according to Eu numbering.

[0009] In certain embodiments, the antigen-binding protein, particularly the antibodies described herein, each comprises a HC amino acid sequence and a LC amino acid sequence that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% (e.g., at least 90%) identical to the amino acid sequences of SEQ ID NOs: 10 and 11. In some of these embodiments, HCDR1-HCDR3 and LCDR1-LCDR3 each have the amino acid sequences according to SEQ ID NOs: 1-6, i.e., the sequence variations are outside the CDRs. In certain embodiments, the antigen-binding protein, particularly the antibody, comprises a heavy chain comprising SEQ ID NO: 10 and a light chain comprising SEQ ID NO: 11.

[0010] According to certain embodiments, the antigen-binding protein, particularly the antibodies described herein, comprises an Fc that is afucosylated Fc.

[0011] In some embodiments, the antigen-binding proteins described herein, particularly antibodies or antigen-binding portions thereof, or binding proteins comprising said antibodies or antigen-binding portions, have at least one property selected from the following: a) specifically binds to cells expressing human GPRC5D; b) specifically binds to cells expressing cynomolgus GPRC5D; c) binds to cells expressing human FcγRIIIa; d) binds to cells expressing cynomolgus FcγRIIIa; e) has the amino acid sequence of SEQ ID NO: 12 or has a maximum of 20 amino acid N-terminal deletions, such as N-terminal deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids, and preferably specifically binds to human GPRC5D; f) preferably specifically binds to SEQ ID NO: 15; g) preferably specifically binds to SEQ ID NO: 16; h) preferably specifically binds to SEQ ID NO: 17; i) promotes NK cell engagement; j) promotes NK cell degranulation; k) Induces NK cell activation; l) Induces primary plasma cell depletion; m) Promote the lysis of multiple myeloma cells in the presence of NK cells; n) Inducing a 5-fold or less increase in IFN-γ, IL-6, and / or TNF-α in peripheral blood mononuclear cells in the presence of GPRC5D-expressing cells; o) Can be quantified in plasma in vivo for at least 28 days; p) Having an in vivo terminal phase elimination half-life of at least 9 days; q) Does not cause elevated IFN-γ, IL-8, and TNFα levels in vivo; r) Inhibit tumor growth in vivo; s) Specifically binds to the GPRC5D dimer; t) Exhibits cross-reactivity in mice; and u) It shows cross-reactivity in cynomolgus monkeys.

[0012] Antigen-binding proteins, particularly antibodies, antigen-binding moieties, or binding proteins, may possess any combination or all of the aforementioned properties.

[0013] In some embodiments, the antigen-binding protein or the antibody described herein or the binding protein comprising its antigen-binding moiety is monospecific, bispecific (e.g., bispecific T-cell engager), or multispecific. In some embodiments, the antigen-binding protein is a fusion protein, such as a chimeric antigen receptor (CAR). In some embodiments, the antigen-binding protein is a T-cell receptor (TCR) or a portion of a TCR.

[0014] The present invention also provides pharmaceutical compositions comprising an antigen-binding protein, particularly an antibody, an antigen-binding moiety or binding protein as described herein, and a pharmaceutically acceptable excipient.

[0015] The present invention further provides isolated nucleic acid molecules comprising one or more nucleotide sequences encoding an antigen-binding protein, particularly an antibody or antigen-binding fragment thereof as described herein. Preferably, the nucleotide sequences include a first variable domain, e.g., a heavy chain variable domain or a protein comprising a heavy chain, and the same or different nucleotide sequences encoding a second variable domain, e.g., an antigen-binding moiety, particularly an antibody or a protein comprising the light chain or light chain variable domain of the antigen-binding moiety thereof as described herein, or a nucleotide sequence encoding a binding protein as described herein. Further provided are vectors comprising isolated nucleic acid molecules, the vectors may include expression regulatory sequences. Also provided are one or more host cells comprising isolated nucleic acid molecules, and a method for producing an antigen-binding protein, particularly an antibody or antigen-binding moiety thereof as described herein, comprising preparing host cells, culturing the host cells under conditions suitable for the expression of the antigen-binding protein, and isolating the obtained antigen-binding protein from the culture.

[0016] The present invention further provides pharmaceutical compositions comprising an antigen-binding protein, an isolated nucleic acid molecule, a vector, or a host cell as described herein, and a pharmaceutically acceptable excipient.

[0017] The present invention further provides antigen-binding proteins, isolated nucleic acid molecules, vectors, host cells, or pharmaceutical compositions described herein for use as pharmaceuticals. According to a preferred embodiment, the pharmaceutical is for use in the treatment of cancer, and more preferably, the cancer is multiple myeloma, non-Hodgkin lymphoma, renal cancer, breast cancer, ovarian cancer, or pancreatic cancer. According to a particularly preferred embodiment, the cancer is multiple myeloma (MM). For example, the cancer may be smoldering (asymptomatic) multiple myeloma or active (symptomatic) multiple myeloma. The multiple myeloma may be hyperdiploid (HMM), non-hyperdiploid, or hypodiploid. The myeloma subtype may be, for example, IgG, IgA, IgM, IgE, or IgD myeloma. Further preferred forms of myeloma include light chain myeloma, non-secretory myeloma, solitary plasmacytoma, multiple solitary plasmacytoma, extramedullary myeloma, and monoclonal gammaglobulinemia of unknown significance (MGUS). In a particularly preferred embodiment, the cancer is GPRC5D-positive cancer.

[0018] This disclosure also provides a method for treating cancer in a patient, particularly in a human patient requiring treatment for cancer, comprising administering to the patient a therapeutically effective amount of an antigen-binding protein described herein, particularly an antibody, its antigen-binding moiety, binding protein, or pharmaceutical composition. In certain embodiments, the cancer is multiple myeloma, non-Hodgkin lymphoma, renal cancer, breast cancer, ovarian cancer, or pancreatic cancer. In certain embodiments, the cancer is multiple myeloma.

[0019] This disclosure is also understood to provide antibodies, their antigen-binding moieties, binding proteins, or pharmaceutical compositions described herein for use in treating a person in need thereof in the therapeutic methods described herein. Also provided are the use of antigen-binding proteins, in particular antibodies, their antigen-binding moieties, binding proteins, isolated nucleic acid molecules, vectors, host cells, or pharmaceutical compositions described herein for manufacturing pharmaceuticals for treating a patient, preferably a patient having cancer. In certain embodiments, cancer is multiple myeloma, non-Hodgkin lymphoma, renal cancer, breast cancer, ovarian cancer, or pancreatic cancer. In certain embodiments, cancer is multiple myeloma.

[0020] Other features, purposes, and advantages of the present invention will become apparent in the detailed description below. However, it should be understood that this detailed description, while illustrating embodiments and aspects of the present invention, is provided for illustrative purposes only and not limiting. Various modifications and alterations within the scope of the present invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawing]

[0021] [Figure 1] This histogram shows the affinity (apparent KD or "appKD") of cynomolgus monkey ("cy") and human ("hu") FcγRIIIa-expressing HEK293T cells for clone 11 in defucosylated ("afco") and DE-DSB format, and for the control IgG1 wild-type ("wt") format anti-GPRC5D antibody benchmark 2 ("BM2"). The data represents the average of three experiments. [Figure 2] This line graph shows the median fluorescence intensity (MFI) of antibodies bound to MM.1R (a GPRC5D-expressing MM cell line) at different concentrations, as evaluated by FACS. 11-Afco:: Clone 11 in a defucosylated format. 11-DE-DSB: Clone 11 in a DE-DSB format. BM1-Afco: Benchmark 1 antibody in a defucosylated format. BM2-IgG1-wt: Benchmark 2 antibody in an IgG1 wild-type format. IC: IgG1 isotype control. [Figure 3] This line graph shows the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of clone 11 in defucosylated ("afco") and DE-DSB formats at different concentrations against MM.1R cells, as evaluated by lysis percentage (calcein release readout). Isotype control IgG1 DE-DSB (IC) was used as a negative control. Benchmark antibodies 1 and 2 in defucosylated formats (BM1 afco and BM2 afco, respectively) were also evaluated as reference. The data shown are representative from one of five experiments. [Figure 4] This line graph shows the ADCC activity of clone 11 in defucosylated ("afco") and DE-DSB formats at different concentrations against the MM cell line EJM, as evaluated by lysis percentage (calcein release readout). Isotype control IgG1 DE-DSB (IC) was used as a negative control. Benchmark antibodies 1 and 2 in defucosylated formats (BM1 afco and BM2 afco, respectively) were also evaluated as reference. The data shown are representative from one of five experiments. [Figure 5] This line graph shows the ADCC activity on day 4 of clone 11 in different concentrations of defucosylated ("afco") and DE-DSB format in MM.1R MM cells, as evaluated by lysis percentage (Incucyte imaging readout). Isotype control IgG1 DE-DSB (IC) was used as a negative control. Benchmark antibodies 1 and 2 of the defucosylated format (BM1 afco and BM2 afco, respectively) were also evaluated as reference. The data shown are representative from one of two experiments. [Figure 6] This line graph shows the ADCC activity of clone 11 in defucosylated ("afco") and DE-DSB format against MM.1R MM cells, assessed by MM.1R counts normalized to day 0 over four days. Isotype control IgG1 DE-DSB (IC) was used as a negative control. Benchmark antibodies 1 and 2 of the defucosylated format (BM1 afco and BM2 afco, respectively) were also evaluated as reference. The data shown are from one representative experiment after overnight incubation in the presence of NK cells and 1 ng / mL of antibody. [Figure 7]This is a set of histograms showing the release of cytokines IFNγ (Figure 7A), IL-6 (Figure 7B), and TNFα (Figure 7C) induced by clone 11 in DE-DSB format in a PBMC environment in the presence of MM.1R MM cells. Isotype control IgG1 DE-DSB (IC) was used as a negative control. CD3-BCMA T cell engagers ("TCE") were evaluated as control subjects. The average of 4-5 experiments is shown. [Figure 7-1] Same as above. [Figure 7-2] Same as above. [Figure 8] This is a Kaplan-Meier plot showing the survival rate of NK humanized hIL15tg-NOG mice transplanted with seeded human MM.1R cells after treatment with defucosylated ("Afco") and DE-DSB format clone 11, as well as with the defucosylated format benchmark 1 antibody (BM1-Afco). Isotype control IgG1 wild type (IC IgGwt) was used as a negative control. [Figure 9] These are paired line graphs showing the mean (Figure 9A) and individual (Figure 9B) pharmacokinetic (PK) profiles after a single 30-minute intravenous infusion of 5 mg / kg or 25 mg / kg of clone 11 in DE-DSB format to female cynomolgus monkeys, or after subcutaneous administration of 25 mg / kg. [Figure 9-1] Same as above. [Figure 10] The principle of the ex vivo study on a patient sample of newly diagnosed primary multiple myeloma (NDMM) in Example 11 is schematically shown. [Figure 11] Figure 11A shows plasma cell lysis after ex vivo treatment with 11-DE-DSB in primary bone marrow samples from NDMM patients. Figure 11B shows the induction of CD107 on NK cells after overnight treatment with anti-GPRC5D in fresh BM MM patient samples. [Figure 12]Dose-response curves of ADCP activity of 11DE-DSB in MM.1R cells from five healthy donors are shown. Black circles: 11-DE-DSB, black triangles: anti-CD38-IgG1, black squares: isotype control. X axis: concentration (pM), Y axis: % of lysis. A: healthy donor 1, B: healthy donor 2, C: healthy donor 3, D: healthy donor 4, and E: healthy donor 5. [Figure 12-1] Same as above. [Figure 12-2] Same as above. [Modes for carrying out the invention]

[0022] The present invention provides novel anti-GPRC5D antigen-binding proteins, antibodies that can be used to treat cancer in patients (e.g., multiple myeloma), and GPRC5D-binding proteins comprising the antibody or the antigen-binding moiety of the antibody. Also provided are pharmaceutical compositions comprising one or more of these antibodies or their antigen-binding moieties or binding proteins, and the use of antigen-binding proteins, particularly antibodies or their antigen-binding moieties, binding proteins, and pharmaceutical compositions for the treatment of cancer (e.g., multiple myeloma). The antigen-binding proteins, particularly antibodies and their antigen-binding moieties, binding proteins, and compositions described herein may be used in methods for treating cancer in patients; may be used in the manufacture of pharmaceuticals for treating cancer in patients; or may be for use for treating cancer in patients. In specific embodiments, the present invention provides antigen-binding proteins, particularly antibodies comprising two variable domains, the first variable domain comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and the second variable domain comprising LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6. The antigen-binding protein specifically binds to GPRC5D having the amino acid sequence described herein, preferably human GPRC5D, and more preferably the amino acid sequence described in Sequence ID No. 12. The antigen-binding protein of the present invention can be used to treat cancer in a patient (e.g., multiple myeloma). The antigen-binding protein of the present invention is preferably a GPRC5D-binding protein such as an antibody or the antigen-binding moiety of said antibody. Pharmaceutical compositions comprising one or more of these antigen-binding proteins, particularly antibodies or their antigen-binding moieties or binding proteins, as well as antigen-binding proteins, particularly antibodies or their antigen-binding moieties, binding proteins, and pharmaceutical compositions for use in the treatment of cancer (e.g., multiple myeloma). Accordingly, the antigen-binding proteins, particularly antibodies and their antigen-binding moieties, binding proteins, and compositions described herein may be used in methods for treating cancer in a patient; may be used in the manufacture of pharmaceuticals for treating cancer in a patient; or may be for use in treating cancer in a patient.

[0023] In some embodiments, the antigen-binding protein of the present invention is an anti-GPRC5D antibody with enhanced ADCC activity, which can be used, for example, as a natural killer (NK) cell engager, i.e., a molecule that can simultaneously bind GPRC5D on target cells (e.g., malignant plasma cells) and cell surface molecules (e.g., Fc receptors) on NK cells. Treatment with such NK cell engagers is expected to be safer than T cell-based therapies (e.g., BiTE (bispecific T cell engager) and CAR-T therapy) because NK cells are less likely to induce cytokine release syndrome (CRS) or graft-versus-host disease (GVHD) or cause neurotoxicity. The safer profile of NK cell engagers may result in more convenient administration, requiring less or no hospitalization and observation during the active treatment period.

[0024] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methods, protocols, and reagents described herein, as these may vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and do not limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as they would ordinarily understood by those skilled in the art.

[0025] Throughout this specification, multiple documents are referenced. Each of the documents referenced herein (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.), whether listed above or below, is incorporated herein by reference in its entirety. Nothing in this specification shall be construed as an acknowledgment that the present invention is not granted prior rights by prior art. In the event of any conflict between the definitions or teachings of such incorporated references and the definitions or teachings enumerated herein, the text of this specification shall prevail.

[0026] To carry out the present invention, unless otherwise indicated, conventional methods of chemistry, biochemistry, and recombinant DNA technology described in the literature of the art will be used (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0027] The following definitions are provided for some terms that are frequently used herein to characterize the present invention. In each example of their use, these terms have their defined and preferred meanings in the remainder of this specification. Throughout this specification and the subsequent claims, unless the context requires otherwise, the word “comprise” and variations such as “comprises” and “contains” mean to include the integer or process or group of integers or processes that are described, but not to exclude any other integer or process or group of integers or processes. When used herein, the use of the term “comprise” also discloses embodiments that do not contain features other than those specifically mentioned (i.e., “consist of”).

[0028] As used herein and in the appended claims, the singular forms "a," "an," and "it" include multiple referents unless otherwise clearly indicated by the context.

[0029] As used in this application, the term “and / or” is a grammatical conjunction that should be interpreted as encompassing the possibility of one or more cases being connected. For example, the phrase “the variation is at position 239 and / or position 332” indicates that the variation is at position 239, or position 332, or both, or at positions 239 and 332.

[0030] Unless otherwise specified, "GPRC5D" refers to human GPRC5D. The human GPRC5D polypeptide sequence is available under UniProt accession number Q9NZD1(GPC5D_Human) (last updated 2000-10-01) or NCBI accession number NP_061124.1, as shown below: [ka]

[0031] As used herein, the term “antigen-binding protein” refers to a polypeptide or a complex of two or more polypeptides containing an antigen-binding site capable of specifically binding to an antigen. As used herein, the term “antigen-binding protein” includes several different formats of antigen-binding proteins described below, e.g., soluble antigen-binding proteins, membrane-bound antigen-binding proteins, monovalent, bivalent, and multivalent antigen-binding proteins, single-specific, bispecific, and multispecific antigen-binding proteins, single-chain antigen-binding proteins, and antigen-binding proteins containing two or more chains, fusion proteins, and chimeric proteins. This term includes, for example, antigen-binding proteins having the overall structure of a T cell receptor (TCR), antibody, or chimeric antigen receptor (CAR), or fusion proteins containing elements of an antibody, CAR, and / or TCR. This term further includes, for example, antigen-binding proteins having the overall structure of a B cell receptor (BCR), single-chain antibody, and single-chain TCR. Antigen-binding proteins according to the present invention include a variable region or domain containing specific complementary determining regions (CDRs) for binding to their respective epitopes. Variable regions include, for example, antibody-based heavy chain variable domains (VH) and light chain variable domains (LH), or TCR-based alpha and beta, or gamma and delta variable domains (V α and V β , or V γ and V δ ) is possible.

[0032] The terms “antibody” (Ab) or “immunoglobulin” (Ig), as used herein, refer to a tetramer comprising two heavy chains (HC) (approximately 50–70 kDa) and two light chains (LC) (approximately 25 kDa) interconnected by one or more disulfide bonds. Each heavy chain consists of a heavy chain variable domain (VH) and a heavy chain constant region (CH). Each light chain consists of a light chain variable domain (VL) and a light chain constant region (CL). The VH and VL domains can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with highly conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs (HCDRs as used herein refer to CDRs from the heavy chain; LCDRs as used herein refer to CDRs from the light chain) and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. The term "antibody" refers to antibodies and their fragments, as well as single-domain antibodies and their fragments, and multispecific antibodies and their fragments, particularly the variable heavy chains of single-domain antibodies, and chimeric, humanized, bispecific, or multispecific antibodies. An antibody fragment or antigen-binding portion includes a part of an intact antibody, particularly the antigen-binding or variable region of the antibody. Non-limiting examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, bispecific, and multispecific antibodies formed from antibody fragments. Antibody fragments can also be single-domain antibodies, such as the heavy chain variable region (VH). Preferably, the “antibody fragment” includes a portion of an intact antibody, and in particular, the antibody fragment includes an antigen-binding site containing at least a variable domain. The antigen-binding protein or antibody fragment performs essentially the same function as the antigen-binding protein or antibody from which they are derived, meaning that the antigen-binding protein or antibody fragment specifically binds to the same target as the antigen-binding protein or antibody from which they are derived.

[0033] The term "T cell receptor" (TCR), in relation to this invention, refers to a heterodimeric cell surface protein of the immunoglobulin superfamily associated with the invariant CD3 complex protein involved in signal transduction mediation. TCRs exist in αβ and γδ forms, which are structurally similar but have considerably different anatomical locations. The extracellular portions of the native heterodimeric αβTCR and γδTCR each contain two polypeptides, each having a near-membrane constant domain and a distal-membrane variable domain. Each of the constant and variable domains contains an intrachain disulfide bond. The variable domain contains a highly polymorphic loop similar to the complementarity-determining region (CDR) of an antibody. The native alpha-beta heterodimeric TCR has an alpha chain and a beta chain. Each alpha chain contains a variable region, a binding region and a constant region, and the beta chain also usually contains a short diversity region between the variable and binding regions, although this diversity region is often considered part of the binding region. The constant regions of the TCRα and β chains, i.e., the C region, are called TRAC and TRBC, respectively (Lefranc, (2001), Curr Protoc Immunol Appendix 1:Appendix 10). Each variable region contains three "complementarity-determining regions" (CDRs) embedded in the framework sequence of the framework region.

[0034] The amino acid numbers in the heavy chain, light chain, or TCR variable domain, as well as the assignment of FR and CDR regions, are as defined by IMGT® (Lefranc et al., Dev Comp Immunol. (2003) 27(1):55-77); or Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)); Chothia & Lesk, J Mol Biol. (1987) 196:901-17; Chothia et al., Nature (1989) 342:878-83; MacCallum et al., J Mol Biol. (1996) 262:732-45; Honegger and Plueckthun, J Mol Biol. (2001) 309(3):657-70; or Abhinandan and It can be assigned according to the definition in Martin, Mol.Immunol. (2008), 45(14):3832-9.

[0035] As used herein, the term “complementary determinant region” (CDR) refers to discontinuous antigen-binding sites found within the variable domains of immunoglobulins, for example, in VH, VL, Vα, and Vβ. The definitions in the cited literature above include overlaps or subsets of amino acid residues when compared to one another. Nevertheless, the application of any definition to refer to CDRs of antibodies or grafted antibodies or their variants or fragments is intended to be within the scope of the definitions and terms used herein. The amino acid residues comprising CDRs, as defined by each of the cited literature above, are illustratively listed in the following table for comparison.

[0036] [Table 1]

[0037] The terms "HCDR1," "HCDR2," and "HCDR3" refer to the first, second, and third CDRs in the heavy chain variable domain of an antigen-binding polypeptide, such as an antibody or a functional fragment thereof. As used herein, the terms "LCDR1," "LCDR2," and "LCDR3" refer to the first, second, and third CDRs in the light chain variable domain of an antigen-binding polypeptide, such as an antibody or a fragment thereof. As used herein, the terms "CDR1," "CDR2," and "CDR3" refer to the first, second, and third CDRs in the variable region of the polypeptide chain of any variable domain of an antigen-binding polypeptide, such as an antibody or its antigen-binding moiety, or of the TCR.

[0038] As used herein, the term “chimeric antigen receptor” (CAR; also known as chimeric immune receptor, chimeric T cell receptor, or artificial T cell receptor) refers to an engineered receptor that imbues immune effector cells, preferably T cells, with arbitrary specificity. Cells are complex membrane receptor molecules that are genetically equipped with CARs that provide both target specificity and T cell activation. The most common form of CAR is a fusion of a monoclonal antibody-derived single-chain variable fragment (scFv) fused to the CD3 transmembrane and endodomain. The CAR targets T cells to desired cellular targets via the antibody-derived binding domain in the extracellular portion, and T cell activation occurs via the intracellular signaling domain when the target is encountered. The transfer of the coding sequences of these receptors to suitable cells, particularly T cells, is generally facilitated by retroviral or lentiviral vectors.

[0039] The term "recombinant antibody" refers to an antibody that does not exist in nature and is expressed from a cell or cell line containing a nucleotide sequence that codes for the antibody, and the nucleotide sequence does not associate with the cell in nature.

[0040] The terms “isolated protein,” “isolated polypeptide,” or “isolated antibody” refer to a protein, polypeptide, or antibody that, by its origin or source, (1) does not associate with its naturally associated components in its natural state; (2) is substantially free of other proteins from the same species; (3) is expressed by cells of a different species; and / or (4) does not occur in nature. Thus, a chemically synthesized polypeptide or a polypeptide synthesized in a cell system different from that of naturally occurring cells is “isolated” from its naturally associated components. Proteins can also be made substantially free of naturally associated components by isolation using protein purification techniques well known in the art.

[0041] The term "affinity" refers to a measure of the attractiveness between an antigen and an antibody. The intrinsic attractiveness of an antibody to an antigen is typically expressed by the binding affinity equilibrium constant (K) of a particular antibody-antigen interaction. D It is expressed as ). Antibodies are bound to K D It is said that when the concentration is ≤1 μM, for example, ≤100 nM or ≤10 nM, it specifically binds to the antigen. D The binding affinity constant can be measured by surface plasmon resonance (SPR) using, for example, the Biacore® T200 system, the IBIS Technologies IBIS MX96 SPR system, or the Carterra LSA SPR platform, or by biolayer interferometry using, for example, the ForteBio Octet® system. D A preferred method for determining the binding of antigen-binding proteins is preferably by SPR using a Biacore® T200 instrument, as described in more detail in Example 2 below. An alternative preferred method for determining the binding of antigen-binding proteins is preferably by a flow cytometry binding assay, as described in more detail in Example 3 below.

[0042] As used herein, the term “epitope” refers to a portion of an antigen (determinant) that specifically binds to an antigen-binding protein such as an antibody or to an associated molecule such as a bispecific binding molecule. Epitope determinants generally consist of a chemically active surface population of molecules such as amino acids or carbohydrates or sugar side chains, and typically possess specific three-dimensional structural properties and specific charge properties. Epitopes can be “linear” or “concrete.” In linear epitopes, all interaction points between a protein (e.g., antigen) and an interacting molecule (e.g., antibody) are linearly located along the primary amino acid sequence of the protein. In concrete epitopes, interaction points are located across amino acid residues on the protein that are separated from each other in the primary amino acid sequence. Once a desired epitope on an antigen is determined, it is possible to generate an antibody against that epitope using techniques well known in the art. For example, an antibody against a linear epitope can be generated, for example, by immunizing an animal with a peptide having the amino acid residues of the linear epitope. Antibodies against structural epitopes can be produced, for example, by immunizing animals with a minidomain containing the relevant amino acid residues of the structural epitope. Antibodies against specific epitopes can also be produced, for example, by immunizing animals with a target molecule of interest (e.g., GPRC5D) or a relevant portion thereof, and then screening for binding to the epitope. Antibodies against specific epitopes can also be produced using phage display.

[0043] As used herein, the term “specifically binds” means that an antigen-binding protein, particularly an antibody or its antigen-binding fragment, binds to an epitope via its antigen-binding domain, and the binding involves some complementarity between the antigen-binding domain and the epitope. Thus, an antigen-binding protein, particularly an antibody, that “specifically binds” to human GPRC5D (SEQ ID NO: 12) may also bind to GPRC5D from other species (e.g., cynomolgus monkey, mouse, and / or rat GPRC5D) and / or GPRC5D proteins produced from other human alleles, however the degree of binding to unrelated non-GPRC5D proteins is less than about 10%, preferably less than 1%, and more preferably less than 0.1%, of the binding of the antibody to the antigen-binding protein, particularly GPRC5D, as measured, for example, by radioimmunoassay (RIA).

[0044] Whether an antigen-binding protein, particularly an antibody, binds to the same epitope as an antigen-binding protein such as the anti-GPRC5D antibody of this disclosure, or competes for binding, can be determined, without limitation, by using methods known in the art, including competitive assays, epitope binning, and alanine scanning. In some embodiments, an antigen-binding protein such as the anti-GPRC5D antibody of this disclosure can be bound to GPRC5D under saturated conditions, and then the ability of the test antigen-binding protein, such as the antibody, to bind to GPRC5D can be measured. If the test antibody can bind to GPRC5D simultaneously with the reference anti-GPRC5D antibody, the test antibody binds to a different epitope than the reference anti-GPRC5D antibody. However, if the test antibody cannot bind to GPRC5D simultaneously, the test antibody binds to the same epitope, a duplicate epitope, or an epitope adjacent to the epitope bound by the anti-GPRC5D antibody of this disclosure. This experiment can be carried out, for example, using ELISA, RIA, Biacore®, SPR, biolayer interferometry, or flow cytometry. To test whether an anti-GPRC5D antibody cross-competes with another anti-GPRC5D antibody, the above competition method can be used in two directions: to determine whether a known antibody blocks the test antibody, and vice versa. Such cross-competition experiments can be performed, for example, using a Biacore® T200, IBIS MX96, or Carterra LSA SPR instrument or an Octet® system.

[0045] As used herein, the terms “antigen-binding moiety” or “antigen-binding fragment” of an antibody refer to one or more parts or fragments of an antibody that possess the ability to specifically bind to an antigen (e.g., preferably human GPRC5D of SEQ ID NO: 12, or a portion thereof). It has been shown that certain fragments of a full-length antibody can perform the antigen-binding function of the antibody. Accordingly, examples of binding fragments encompassed by the term “antigen-binding moiety” include, but are not limited to, (i) Fab fragments: monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments: bivalent fragments containing two Fab fragments linked by disulfide crosslinking at a hinge region; (iii) Fd fragments consisting of a VH domain and a CH1 domain; (iv) Fv fragments consisting of VL and VH domains of a single arm of the antibody; (v) dAb fragments consisting of a VH domain; and (vi) isolated complementarity-determining regions (CDRs) that can specifically bind to an antigen. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker via recombination, thereby allowing the VL and VH domains to pair and be created as a single protein chain forming a monovalent molecule (e.g., known as single-chain Fv(scFv)). Antigen-binding molecules containing VH and / or VL are also included in this disclosure. In the case of VH, the molecule may also contain one or more CH1, hinge, CH2, or CH3 regions. Such single-chain antibodies are also intended to be included within the term “antigen-binding portion” of the antibody. Other forms of single-chain antibodies, such as diabodies, are also included. A diabody is a bivalent, bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain, but uses a linker that is too short to pair the two domains on the same chain, thereby forcing the domains to pair with complementary domains on another chain, creating two antigen-binding sites.

[0046] Antibody moieties such as Fab and F(ab')2 fragments can be prepared from the whole antibody using conventional techniques, such as papain or pepsin digestion of the whole antibody. Furthermore, the antibody, antibody moieties, and immunoadhesin molecules can be obtained using standard recombinant DNA techniques, such as those described herein.

[0047] The class (isotype) and subclass of an anti-GPRC5D antibody can be determined by any method known in the art. Generally, the class and subclass of an antibody can be determined using antibodies specific to a particular class and subclass of the antibody. Such antibodies are commercially available. The class and subclass can be determined by ELISA or Western blotting, as well as other techniques. Alternatively, the class and subclass can be determined by sequencing all or part of the constant regions of the heavy and / or light chains of the antibody and comparing their amino acid sequences with known amino acid sequences of various classes and subclasses of immunoglobulins.

[0048] As used herein, the term “nucleic acid” is intended to include DNA and RNA such as genomic DNA, cDNA, and mRNA, recombinantly produced molecules, and chemically synthesized molecules. Nucleic acids can be single-stranded or double-stranded. RNA includes in vitro transcription RNA (IVT RNA) or synthetic RNA. According to the present invention, nucleic acids are preferably isolated nucleic acids. According to the present invention, the term “nucleic acid code” and similar expressions mean that when nucleic acids are present in a suitable environment, preferably within a cell, they can be expressed to produce proteins or peptides encoded by the nucleic acid.

[0049] The terms “patient” and similarly “subject” as used herein refer to individuals such as humans, non-human primates (e.g., chimpanzees and other apes and monkey species); domesticated animals such as birds, fish, cattle, sheep, pigs, goats and horses; domesticated mammals such as dogs and cats; and laboratory animals such as rodents such as mice, rats and guinea pigs. The terms do not indicate a specific age or sex and therefore encompass adults, the elderly, children and newborns. According to a preferred embodiment, the patient or subject is a mammal, and more preferably the patient or subject is a human.

[0050] The elements of the present invention are described in more detail below. These elements are listed together with specific embodiments, but it should be understood that they may be combined in any way and in any number to produce additional embodiments. The various examples and preferred embodiments described should not be construed as limiting the invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any substitution and combination of all elements described herein should be considered disclosed by this description unless the context indicates otherwise.

[0051] I. Anti-GPRC5D antigen-binding proteins, especially antibodies and binding proteins The present invention provides antigen-binding proteins, particularly antibodies directed toward GPRC5D and their antigen-binding moieties, as well as GPRC5D-binding proteins comprising the antibody or antigen-binding moiety. In certain embodiments, the antibody may include Fc region modifications to enhance ADCC, improve its stability, and / or enhance its binding affinity to the Fcγ receptor (e.g., FcγRIIIa) expressed on NK cells.

[0052] In certain embodiments, the present invention provides an antigen-binding protein comprising a first variable domain including CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3, and a second variable domain including CDR1 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 6. According to one embodiment, the antigen-binding protein comprises a first variable domain including SEQ ID NO: 7 or a sequence at least 90% identical thereto, and a second variable domain including SEQ ID NO: 8 or a sequence at least 90% identical thereto. According to a preferred embodiment, the antigen-binding protein preferably specifically binds to GPRC5D as described in SEQ ID NO: 12. Accordingly, the present invention provides an antibody and its antigen-binding moiety that specifically bind to GPRC5D, and a GPRC5D-binding protein comprising the antibody or antigen-binding moiety.

[0053] In certain embodiments, antigen-binding proteins, particularly antibodies, may include Fc region modifications to enhance ADCC, improve its stability, and / or increase its binding affinity to Fcγ receptors (e.g., FcγRIIIa) expressed on NK cells.

[0054] In some embodiments, the antigen-binding protein, particularly the antibody or antigen-binding moiety, competes or cross-competes with the antibody comprising VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 8 for binding to human GPRC5D, or binds to the same human GPRC5D epitope as the antibody comprising VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 8.

[0055] In some embodiments, the antibody or its antigen-binding fragment has the heavy chain CDR3 (HCDR-3) amino acid sequence of SEQ ID NO: 3.

[0056] In some embodiments, the antibody or antigen-binding moiety has heavy chains CDR1-3 (HCDR1-3) each containing the amino acid sequences of SEQ ID NOs. 1-3 (i.e., HCDR-1 containing SEQ ID NO. 1, HCDR-2 containing SEQ ID NO. 2, and HCDR-3 containing SEQ ID NO. 3).

[0057] In some embodiments, the antigen-binding protein includes a heavy chain variable domain (VH) amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 7. In some of these embodiments, HCDR1 to HCDR3 each have the amino acid sequence of SEQ ID NOs: 1 to 3, i.e., the sequence mutation is outside of HCDR1 to HCDR3.

[0058] In some embodiments, the antigen-binding protein, particularly the antibody or antigen-binding moiety, has a VH containing the amino acid sequence of SEQ ID NO: 7.

[0059] In some embodiments, the antigen-binding protein, particularly the antibody, has an HC amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 9 or 10. In some of these embodiments, HCDR1 to HCDR3 each have the amino acid sequence according to SEQ ID NOs: 1 to 3, i.e., the sequence mutation lies outside of HCDR1 to HCDR3.

[0060] In some embodiments, the antibody contains the HC amino acid sequence of SEQ ID NO: 9 or 10.

[0061] In some embodiments, the antibody or its antigen-binding fragment has the light chain CDR3 (LCDR-3) amino acid sequence of SEQ ID NO: 6.

[0062] In some embodiments, the antibody or antigen-binding moiety has light chains CDR1-3 (LCDR1-3) each containing the amino acid sequences of SEQ ID NOs. 4-6 (i.e., LCDR-1 containing SEQ ID NO. 4, LCDR-2 containing SEQ ID NO. 5, and LCDR-3 containing SEQ ID NO. 6).

[0063] In some embodiments, the antigen-binding protein, particularly the antibody or antigen-binding moiety, has a light chain variable domain (VL) amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 8. In some of these embodiments, LCDR1 to LCDR3 each have the amino acid sequences of SEQ ID NOs: 4 to 6, i.e., the sequence mutation is outside of LCDR1 to LCDR3.

[0064] In some embodiments, the antigen-binding protein, particularly the antibody or antigen-binding moiety, has a VL containing the amino acid sequence of SEQ ID NO: 8.

[0065] In some embodiments, the antigen-binding protein, particularly the antibody, has an LC amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 11. In some of these embodiments, LCDR1 to LCDR3 each have the amino acid sequence according to SEQ ID NOs: 4 to 6, i.e., the sequence mutation is outside of LCDR1 to LCDR3.

[0066] In this embodiment, the antigen-binding protein has the LC amino acid sequence of SEQ ID NO: 11.

[0067] In certain embodiments, the antigen-binding protein comprises one of the heavy chains and one of the light chains described above.

[0068] In some embodiments, the antibody or antigen-binding moiety of this disclosure comprises the HCDR1-3 and LCDR1-3 amino acid sequences of SEQ ID NOs: 1-6, respectively. In other words, the antibody or antigen-binding moiety is The amino acid sequence of HCDR-1 in SEQ ID NO: 1; The HCDR-2 amino acid sequence of Sequence ID No. 2; The HCDR-3 amino acid sequence of Sequence ID No. 3; The amino acid sequence of LCDR-1 in SEQ ID NO: 4; The amino acid sequence of LCDR-2 of SEQ ID NO: 5: and The amino acid sequence of LCDR-3 in SEQ ID NO: 6 Includes.

[0069] In some embodiments, the antigen-binding proteins of the Disclosure, particularly the antibody or antigen-binding moiety, include VH and VL which are 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical (e.g., 90% identical) to the amino acid sequences of SEQ ID NOs. 7 and 8, respectively. In some of these embodiments, HCDR1-HCDR3 and LCDR1-LCDR3 have the amino acid sequences of SEQ ID NOs. 1-6, respectively, i.e., the sequence mutations are outside of HCDR1-HCDR3 and LCDR1-LCDR3.

[0070] The percentage of identity between two amino acid sequences (or two nucleic acid sequences) can be obtained, for example, by BLAST® using default parameters (available on the US National Library of Medicine's National Center for Biotechnology Information website). In some embodiments, the length of the reference sequence aligned for comparison is at least 60% (e.g., at least 70%, at least 80%, at least 90%, or 100%) of the reference sequence. For example, if a sequence is shown to be at least 90% identical to a given sequence number such as sequence number 7, the sequence is preferably aligned with the sequence number 7 over the entire length of sequence number 7, and the percentage of identity is calculated with respect to the entire length of sequence number 7, in this example being an alignment over 100% of the length of the reference sequence.

[0071] In some embodiments, the antigen-binding protein of the present disclosure, particularly the antibody or antigen-binding moiety, comprises VH and VL, respectively, containing the amino acid sequences of SEQ ID NO: 7 and 8 (i.e., VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 8).

[0072] In some embodiments, the antigen-binding protein of the present invention, particularly the antibody, a) HC containing an amino acid sequence of SEQ ID NO: 9 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9, and LC containing an amino acid sequence of SEQ ID NO: 11 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11; or b) HC containing an amino acid sequence of SEQ ID NO: 10 or a sequence identical to SEQ ID NO: 10 by at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and LC containing an amino acid sequence of SEQ ID NO: 11 or a sequence identical to SEQ ID NO: 11 by at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, respectively. Includes.

[0073] According to a more specific embodiment, the antigen-binding protein of the present invention, in particular the antibody, a) HC containing the amino acid sequence of SEQ ID NO: 9 and LC containing the amino acid sequence of SEQ ID NO: 11; or b) HC containing the amino acid sequence of SEQ ID NO: 10 and LC containing the amino acid sequence of SEQ ID NO: 11 Includes.

[0074] According to a particularly preferred embodiment of the present invention, the antigen-binding protein is an antibody or an antigen-binding fragment thereof.

[0075] The class of an antibody obtained by the method described herein can be changed or switched to another class or subclass. In some embodiments of the present disclosure, nucleic acid molecules encoding VL or VH are isolated using methods well known in the art such that they do not contain nucleic acid sequences encoding CL or CH, respectively. The nucleic acid molecules encoding VL or VH are then operably linked to nucleic acid sequences encoding CL or CH, respectively, derived from immunoglobulin molecules of different classes. This can be achieved using vectors or nucleic acid molecules containing CL or CH sequences, as described above. For example, an antibody that was originally IgM can be class-switched to IgG. Additionally, class-switching can be used to convert one IgG subclass to another, for example, from IgG1 to IgG2. The κ light chain constant region can be changed to, for example, the λ light chain constant region, and vice versa. An exemplary method for producing an antibody of the present disclosure having a desired Ig isotype includes isolating a nucleic acid molecule encoding the heavy chain of the antibody and a nucleic acid molecule encoding the light chain of the antibody, obtaining the variable domain of the heavy chain, ligating the coding sequence of the variable domain of the heavy chain to the coding sequence of the constant region of the heavy chain of the desired isotype, expressing the light chain and the heavy chain encoded by the ligated sequences in a cell, and recovering the antibody having the desired isotype.

[0076] The antibodies of the present disclosure can be IgG, IgM, IgE, IgA, or IgD molecules, but typically are of the human IgG isotype, for example, of the human IgG subclasses IgG1, IgG 2a or IgG 2b , IgG3, or IgG4. In preferred embodiments, the antibody is of the human IgG1 subclass.

[0077] In certain preferred embodiments, the antibodies of the present disclosure are monospecific.

[0078] In some embodiments, antigen-binding proteins, particularly antibodies, may contain at least one mutation in the Fc region. For example, in some embodiments, the antibody is in isotype subclass IgG1 and contains at least one mutation in the Fc region that enhances the lysis of antibody-coated target cells by cells such as natural killer (NK) cells (antibody-dependent cell-mediated cytotoxicity, or ADCC). In certain embodiments, the antigen-binding protein, particularly antibody, may be, for example, an NK cell engager. In certain embodiments, the mutation is located at one or more positions 239 and 332 of the Fc region according to the Eu numbering scheme. The mutation at position 239 is preferably S239D. The mutation at position 332 is preferably I332E. In certain embodiments, the Fc region contained in the antigen-binding protein, particularly antibody, contains both the S239D and I332E mutations.

[0079] In some embodiments, the antibody may contain at least one mutation in the Fc region to enhance stability (e.g., thermal stability). For example, the antibody may contain at least one heavy chain containing a pair of cysteine-mediated manipulated intrachain disulfide bonds that, according to the EU numbering scheme, substitute arginine (R) (R292C) at amino acid position 292 and valine (V) (V302C) at amino acid position 302. The cysteine ​​may be substituted in one or both heavy chains.

[0080] In certain embodiments, the antibody has mutations in the Fc domain that 1) alter effector function and 2) enhance stability. In some embodiments, the Fc domain may include any mutation or combination of mutations described in PCT Patent Publication International Publication No. 2022 / 249146, which is incorporated herein by reference in whole. For example, the antibody heavy chain may include 1) a mutation at position 239 (e.g., S239D) and / or position 332 (e.g., I332E), and 2) a pair of cysteine ​​substitutions forming an intrachain disulfide bond, e.g., R292C and V302C (Eu numbering scheme). The mutation in 1) may be on the same or a different heavy chain as the mutation in 2). In some embodiments, the mutations in 1) and 2) are on one heavy chain. In some embodiments, the mutation in 1) is on one heavy chain and the mutation in 2) is on a different heavy chain. In some embodiments, the mutations in 1) and 2) are on both heavy chains. The combination of S239D / I332E / R292C / V302C quadruple mutation is also referred to herein as the "DE-DSB" mutation. In embodiments, the antibody has the heavy chain amino acid sequence of SEQ ID NO: 10.

[0081] In some embodiments, an antibody described herein having any Fc mutation or combination of Fc mutations (e.g., DE-DSB mutation) has enhanced binding to FcγRIIIa (CD 16a; highly expressed on NK cells), enhanced in vivo stability (e.g., increased serum half-life and / or decreased clearance), enhanced NK cell engagement, or any combination thereof (e.g., all three) compared to the same antibody having a wild-type Fc domain. As used herein, the term “in vivo stability” means the ability of an antibody described herein to remain intact (e.g., limited degradation and / or unfolding) and functional (e.g., retention of binding activity) and to maintain a serum concentration high enough to induce measurable activity (e.g., target tumor cell death).

[0082] In some embodiments, antigen-binding proteins, particularly antibodies, can be defucosylated. In certain embodiments, defucosylated antigen-binding proteins, particularly antibodies, have enhanced ADCC compared to the same antibody without defucosylation. The antigen-binding proteins, particularly antibodies, described herein can be prepared by defucosylated Fc domains according to any method known in the art. For example, antigen-binding proteins, particularly antibodies, can be produced according to the method disclosed in Pereira et al., MAbs(2018)10(5):693-711. In some embodiments, antigen-binding proteins, particularly antibodies, having defucosylated Fc domains can be produced using cell engineering, for example, by knockdown of the FUT8 gene encoding the enzyme responsible for core fucosylation of IgG Fc (Yamane-Ohnuki et al., Biotechnol Bioeng.(2004)87:614-22). In some embodiments, antigen-binding proteins, particularly antibodies, having a defucosylated Fc domain can be generated using heterologous enzymes that redirect fucose synthesis and deplete the intracellular fucose pool (GlymaxX® technology, ProBioGen). In some embodiments, antigen-binding proteins, particularly antibodies, having a defucosylated Fc domain can be generated by adding one or more fucosylation inhibitor molecules, such as kifunensin, to a cell culture (Krahn et al., PHASCI (2017) 96:428-39). In certain embodiments, antigen-binding proteins, particularly antibodies, are defucosylated using GlymaxX® technology (ProBioGen).

[0083] In any of the embodiments described herein, antigen-binding proteins, in particular antibodies, may have any Fc mutation described herein that 1) alters effector function and / or 2) enhances stability (e.g., DE-DSB mutation), and may be further defucosylated. According to further embodiments, antigen-binding proteins, in particular antibodies, having an Fc region containing an Fc mutation described herein, such as the DE-DSB mutation, do not contain a defucosylated Fc region.

[0084] In some embodiments, the antigen-binding proteins described herein, particularly antibodies, or binding proteins comprising such antibodies or their antigen-binding moieties, have at least one of the following characteristics (e.g., all of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21): a) Preferably specifically binds to cells expressing human GPRC5D; b) Preferably specifically binds to cells expressing cynomolgus monkey GPRC5D; c) Preferably specifically binds to cells expressing human FcγRIIIa; d) Preferably specifically binds to cells expressing cynomolgus monkey FcγRIIIa; e) Preferably specifically binds to human GPRC5D as described in Sequence ID No. 12, which has an N-terminal deletion of up to 20 amino acids, such as an N-terminal deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids; f) Preferably binds specifically to sequence number 15; g) Preferably specifically bind to SEQ ID NO: 16; h) Preferably specifically binds to SEQ ID NO: 17; i) Promote NK cell engagement; j) Promote NK cell degranulation; k) Induces NK cell activation; l) Induces primary plasma cell depletion; m) Promoting the lysis of cancer cells (e.g., multiple myeloma cancer cells) in the presence of NK cells; n) Reduce or eliminate the induction of cytokine release (IFN-γ, IL-6, and / or TNF-α, etc.) by peripheral blood mononuclear cells (PBMCs) in the presence of GPRC5D-expressing cells (e.g., multiple myeloma cells); for example, reduce the cytokine increase by 1, 2, 3, 4, or 5 times or less; o) Can be quantified in plasma in vivo for at least 28 days (for example, when administered to cynomolgus monkeys as a single IV or SC dose, e.g., 25 mg / kg); p) Having a terminal phase elimination half-life of at least 9 days in vivo (for example, when administered as a single IV dose to cynomolgus monkeys, e.g., 25 mg / kg); q) Does not cause an increase in IFN-γ, IL-8, and TNFα levels in vivo (for example, when administered to cynomolgus monkeys as a single IV or SC dose, e.g., 25 mg / kg); r) Inhibit tumor growth in vivo (for example, for multiple myeloma tumors); s) Preferably and specifically binds to the GPRC5D dimer; t) Exhibits cross-reactivity in mice; and u) It shows cross-reactivity in cynomolgus monkeys.

[0085] If an antibody contains an Fc mutation and / or glycosylation pattern that enhances the binding affinity of the Fc domain to FcγRIIIa on NK cells, the antibody is also referred to herein as an NK cell engager. In some preferred embodiments, the NK cell engager herein is anti-GPRC5D IgG1 having the DE-DSB mutation described herein. In other preferred embodiments, the NK cell engager herein is anti-GPRC5D IgG1 having a defucosylated Fc domain. In a further preferred embodiment, the NK cell engager herein is anti-GPRC5D IgG1 having the DE-DSB mutation and / or a defucosylated Fc domain. Therefore, particularly preferred antigen-binding proteins of the present invention are antibodies comprising 1) an HC containing the amino acid sequence of SEQ ID NO: 9 and an LC containing the amino acid sequence of SEQ ID NO: 11, or 2) an HC containing the amino acid sequence of SEQ ID NO: 10 (DE-DSB mutation) and an LC containing the amino acid sequence of SEQ ID NO: 11. According to a particularly preferred embodiment, the Fc domain of antibody 1) is defucosylated.

[0086] In some embodiments, anti-GPRC5D antigen-binding proteins described herein, particularly antibodies or their antigen-binding moieties, or binding proteins containing such antibodies or antigen-binding moieties, may inhibit tumor growth in vivo and / or induce tumor regression. In some embodiments, anti-GPRC5D antigen-binding proteins, particularly antibodies or their antigen-binding moieties, or binding proteins described herein, may delay or reverse metastasis in cancer patients. In some embodiments, anti-GPRC5D antigen-binding proteins, particularly antibodies or their antigen-binding moieties, or binding proteins described herein, may reduce or eliminate cancer recurrence in cancer patients. In some embodiments, anti-GPRC5D antigen-binding proteins, particularly antibodies or their antigen-binding moieties, or binding proteins described herein, may extend the survival time of cancer patients. In certain embodiments, the cancer patient has multiple myeloma. Any combination of the above properties is also intended.

[0087] The binding proteins of this disclosure may be the anti-GPRC5D antibody or its antigen-binding moiety described herein, or may include the derivatized anti-GPRC5D antibody or antigen-binding moiety linked to another molecule (e.g., another peptide or protein). Generally, the antibody or its antigen-binding moiety is derivatized or linked in such a way that the GPRC5D binding is not adversely affected by the derivatization or linkage. Therefore, the binding proteins of this disclosure are intended to include both the intact and modified forms of the anti-GPRC5D antibody or antigen-binding moiety described herein. For example, the antibody or antibody moiety of this disclosure may be functionally linked (by chemical bonding, gene fusion, non-covalent bonding or other means) to one or more other molecular moieties, such as another antibody (e.g., a bispecific antibody), a label (e.g., a radioactive or fluorescently detectable marker), or a therapeutic agent (e.g., a cytotoxin or a therapeutically useful radioisotope).

[0088] In some embodiments, the binding protein is a fusion protein, where an anti-GPRC5D antibody or its antigen-binding portion is linked to another polypeptide (e.g., an Fc polypeptide that dimerizes to form an Fc domain). In certain embodiments, only the variable domain of the anti-GPRC5D antibody is linked to the polypeptide. In certain embodiments, the VH domain of the anti-GPRC5D antibody is linked to a first polypeptide, and the VL domain of the anti-GPRC5D antibody is linked to a second polypeptide that associates with the first polypeptide in such a way that the VH and VL domains can interact with each other to form an antigen-binding site. In some embodiments, the VH domain is separated from the VL domain by a linker (e.g., a single-chain antibody) so that the VH and VL domains can still interact with each other. The VH linker-VL antibody (e.g., scFv) is then linked to the polypeptide of interest (e.g., an Fc polypeptide). In scFv, VH can be the N-terminus or C-terminus of VL, and the linker between them can be a flexible linker such as a Gly / Ser-rich linker. For example, the linker may contain one or more (e.g., 2, 3, 4, or 5) GGGGS (SEQ ID NO: 13) motifs.

[0089] In some embodiments, the binding protein is a bispecific binding molecule. In certain embodiments, the bispecific binding molecule further has binding specificity to another distinct anti-GPRC5D antibody or a different protein such as a cancer antigen, another cell surface molecule whose activity mediates a disease condition such as cancer, or an antibody that targets a cell surface molecule on an immune cell (e.g., an NK cell or a T cell). In some embodiments, the bispecific binding molecule binds to GPRC5D and CD3. In some embodiments, the binding protein is a bispecific T cell engager.

[0090] In some embodiments, the binding protein is a chimeric antigen receptor (CAR). Such CARs can be used in CAR-T therapy, in which T cells are engineered to express a CAR that targets GPRC5D.

[0091] In some embodiments, the binding protein is a fusion antibody or an immunoadhesin.

[0092] The antigen-binding protein of the present invention may be, for example, a single-chain antigen-binding protein, a double-chain antigen-binding protein, or an antigen-binding protein containing three or more polypeptide chains.

[0093] The present invention specifically provides anti-GPRC5D IgG1 comprising HC containing the amino acid sequence of SEQ ID NO: 10 (DE-DSB mutation) and LC containing the amino acid sequence of SEQ ID NO: 11. The present invention further specifically provides anti-GPRC5D IgG1 comprising LC containing the amino acid sequence of SEQ ID NO: 9 and SEQ ID NO: 11, wherein the Fc domain is defucosylated.

[0094] The antigen-binding proteins of the present invention preferably exhibit cross-reactivity in mice and / or monkeys, particularly cynomolgus monkeys. According to a particularly preferred embodiment, the antigen-binding protein exhibits this cross-reactivity if it is in antibody format or antibody fragment format such as anti-GPRC5D IgG1 as described herein. This cross-reactivity can be particularly advantageous in assays and preclinical studies, such as in vitro and in vivo studies in or with samples of each animal.

[0095] The antigen-binding proteins described herein are particularly suitable for use as pharmaceuticals. According to preferred embodiments, the pharmaceuticals are for use in the treatment of cancer, preferably multiple myeloma, non-Hodgkin lymphoma, renal cancer, breast cancer, ovarian cancer, or pancreatic cancer. According to particularly preferred embodiments, the cancer is multiple myeloma. For example, the cancer may be smoldering (asymptomatic) multiple myeloma or active (symptomatic) multiple myeloma. The multiple myeloma may be hyperdiploid (HMM), non-hyperdiploid, or hypodiploid. The myeloma subtype may be, for example, IgG, IgA, IgM, IgE, or IgD myeloma. Further preferred forms of myeloma include light chain myeloma, nonsecretory myeloma, solitary plasmacytoma, multiple solitary plasmacytoma, extramedullary myeloma, and monoclonal gammaglobulinemia of unknown significance (MGUS). In a particularly preferred embodiment, the cancer is a GPRC5D-positive cancer.

[0096] II. Preparation of Antibodies and Binding Proteins The antigen-binding proteins of the present invention, particularly antibodies and their antigen-binding moieties, or binding proteins containing the antibody or antigen-binding moiety, can be recombinantly produced using isolated nucleic acid molecules such as expression constructs. Each variable polypeptide chain, such as heavy and light chains, may be encoded by nucleotide sequences on the same or different isolated nucleic acid molecules. Biomolecules (e.g., nucleic acids or polypeptides) referred to herein as “isolated” or “purified” are (1) isolated from their source biomolecules (e.g., nucleic acids or polypeptides of genomic DNA or cellular RNA); and / or (2) not naturally occurring. The coding sequence of each polypeptide chain may be cloned into a single vector or into separate vectors. Thus, the present invention also provides one or more nucleic acid molecules encoding the antigen-binding proteins of the present invention. According to one embodiment, the nucleic acid molecule encoding the antigen-binding protein of the present invention is DNA or RNA, preferably mRNA. The nucleic acid molecule of the present invention encodes one or more polypeptide chains of the antigen-binding protein. The nucleic acid encoding two or more peptide chains may be polycistronic nucleic acids. Each RNA is preferably obtained by in vitro transcription.

[0097] Nucleic acids, such as RNA encoding antigen-binding proteins according to the present invention, can be introduced into T cells or other lytic cells, particularly lymphocytes. Therefore, the present invention also includes the introduction, i.e., transfection, of one or more nucleic acid molecules encoding antigen-binding proteins into cells such as T cells, either in vitro or in vivo. According to the present invention, it is preferable to administer the nucleic acid molecules encoding antigen-binding proteins in a naked form or in a carrier. Each carrier intended for use in the present invention, such as a lipid carrier, is any substance or vehicle capable of associating with nucleic acid molecules, such as RNA, by forming a complex with the nucleic acid or by forming vesicles in which the nucleic acid is encapsulated or contained. This advantageously improves the stability of the nucleic acid compared to naked nucleic acids. In particular, the stability of nucleic acids in blood can be increased. For example, nanoparticle RNA formulations having a defined particle size can be used, such as lipoplexes derived from RNA and liposomes, e.g., lipoplexes containing DOTMA and DOPE or DOTMA and cholesterol.

[0098] According to the present invention, nucleic acid molecules may exist alone or in combination with other nucleic acids, which may be homologous or non-homologous. In a preferred embodiment, a nucleic acid is functionally linked to one or more expression regulatory sequences, which may be homologous or non-homologous with respect to the nucleic acid. The term "homologous" means that the nucleic acid is functionally linked in nature, and the term "non-homologous" means that the nucleic acid is not functionally linked in nature. The nucleic acid and the expression regulatory sequence are "functionally" linked to each other if they are covalently linked in such a way that the expression or transcription of the nucleic acid is under the control or influence of the expression regulatory sequence. Antigen-binding proteins of the present invention, in particular antibodies or their antigen-binding moieties or binding proteins, can be produced, for example, in mammalian host cells using a suitable expression construct. Suitable host cells according to the present invention can be identified by those skilled in the art. Mammalian cell lines available as hosts for expression include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, in particular, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, 293Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and several other cell lines. Other cell lines that may be used include insect cell lines such as Sf9 or Sf21 cells, and yeast cell lines. Cell lines may be selected based on their expression levels. Antigen-binding proteins may be isolated and purified from host cell cultures using well-known methods such as centrifugation, ultracentrifugation, protein A, protein G, protein A / G, or protein L purification, and / or ion-exchange chromatography.

[0099] Host cells used to produce antigen-binding proteins, particularly antibodies, antigen-binding moieties, or binding proteins, are also called “recombinant host cells.” As used herein, the term “recombinant host cell” (or simply “host cell”) means a cell into which a recombinant expression construct has been introduced. By definition, recombinant host cells do not exist in nature. Proteins produced from an expression construct are recombinant proteins.

[0100] The present invention also provides immune effector cells comprising antigen-binding proteins, nucleic acid molecules, or vectors according to the present invention. The immune effector cells used in connection with the present invention are preferably selected from the group consisting of T cells, natural killer (NK) cells, lymphokine-activated killer (LAK) cells, and cytotoxic T lymphocytes (CTLs). Upon activation / stimulation, each of these cytotoxic lymphocytes causes the destruction of target cells. According to one embodiment, the immune effector cells are human immune effector cells.

[0101] III. Pharmaceutical Compositions and Uses Another aspect of the present disclosure is a pharmaceutical composition comprising, as an active ingredient (or sole active ingredient), an antigen-binding protein, particularly an antibody of the present invention or its antigen-binding portion or binding protein, an isolated nucleic acid molecule of the present invention, a vector, or a host cell. The pharmaceutical composition may further comprise one or more pharmaceutically acceptable excipients. "Pharmaceutically acceptable excipients" may include suitable solvents, dispersion media, antimicrobial and antifungal agents, isotonic agents, and the like. Examples of pharmaceutically acceptable excipients are water and physiological saline (e.g., phosphate-buffered saline).

[0102] The pharmaceutical compositions of this specification may be used for the treatment of cancer, for example, GPRC5D-positive cancer. In some embodiments, the cancer may be selected from, for example, multiple myeloma, non-Hodgkin lymphoma, renal cancer, breast cancer, ovarian cancer, and pancreatic cancer. In certain embodiments, the cancer is multiple myeloma. For example, the cancer may be smoldering (asymptomatic) multiple myeloma or active (symptomatic) multiple myeloma. The multiple myeloma may be hyperdiploid (HMM), non-hyperdiploid, or hypodiploid. The myeloma subtype may be, for example, IgG, IgA, IgM, IgE, or IgD myeloma. In some embodiments, the antibodies or their antigen-binding moieties or binding proteins of this disclosure are used for the treatment of light chain myeloma, nonsecretory myeloma, solitary plasmacytoma, multiple solitary plasmacytoma, extramedullary myeloma, and monoclonal gammaglobulinemia of unknown significance (MGUS).

[0103] The "therapeutic effective dose" is the amount of a binding protein (e.g., an anti-GPRC5D antibody or its antigen-binding portion) or a pharmaceutical composition containing it that alleviates, to some extent, one or more symptoms of the disease being treated. A therapeutic effective dose of an anticancer drug may result in, for example, delayed tumor growth, elimination of cancer cells, tumor shrinkage, increased survival, slower or reduced metastasis, or other clinical outcomes desired by the healthcare professional.

[0104] In some embodiments, antibodies or their antigen-binding moieties or binding proteins may be co-administered or formulated with other pharmaceuticals / drugs for the treatment of cancer. Additional therapeutic measures may include, for example, immunostimulants, vaccines, chemotherapeutic agents, antitumor agents, anti-angiogenic agents, corticosteroids, and / or radiotherapy. In some embodiments, additional therapeutic measures may include different anti-cancer antibodies.

[0105] The pharmaceutical compositions described herein may be delivered to patients by parenteral administration, for example, intravenous infusion.

[0106] IV. Diagnostic Uses The antigen-binding proteins of this disclosure, particularly antibodies and antigen-binding moieties or binding proteins, are also useful in diagnostic processes (e.g., in vitro or ex vivo). For example, antigen-binding proteins, antibodies or their antigen-binding fragments, or binding proteins may be used to detect and / or measure the level of GPRC5D in biological samples from patients (e.g., tumor biopsies, tissue samples, or blood samples). Preferred detection and measurement methods include immunological methods such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), chemiluminescence assays, radioimmunoassays, and immunohistochemistry. This disclosure further encompasses kits (e.g., diagnostic kits) containing antigen-binding proteins, antibodies or their antigen-binding moieties, or binding proteins, nucleic acid molecules, or host cells as described herein.

[0107] V. Treatment The present invention further provides antigen-binding proteins, particularly antibodies or their antigen-binding portions, isolated nucleic acid molecules, vectors, host cells, or pharmaceutical compositions, for use as drugs or pharmaceuticals, preferably for use in the treatment of cancer.

[0108] Similarly, the present invention provides the use of antigen-binding proteins, isolated nucleic acid molecules, vectors, host cells, or pharmaceutical compositions according to the present invention for the preparation of pharmaceuticals, preferably for the treatment of cancer.

[0109] The present invention also provides a method for treating a disease, comprising administering a therapeutically effective amount of an antigen-binding protein, isolated nucleic acid molecule, vector, host cell, or pharmaceutical composition according to the present invention to a target. The disease is preferably cancer.

[0110] According to the present invention, the cancer is preferably selected from the group consisting of multiple myeloma, non-Hodgkin lymphoma, renal cancer, breast cancer, ovarian cancer, or pancreatic cancer. Multiple myeloma may be hyperdiploid (HMM), non-hyperdiploid, or hypodiploid. Myeloma subtypes may be, for example, IgG, IgA, IgM, IgE, or IgD myeloma. Further preferred forms of myeloma include light chain myeloma, nonsecretory myeloma, solitary plasmacytoma, multiple solitary plasmacytoma, extramedullary myeloma, and monoclonal gammaglobulinemia of unknown significance (MGUS). According to a particularly preferred embodiment, the cancer is GPRC5D-positive cancer.

[0111] The proteins and compositions described herein may be administered via any conventional route, including parenteral administration by injection or infusion. Administration is preferably parenteral, for example, intravenous, intra-arterial, subcutaneous, intradermal, or intramuscular.

[0112] A composition suitable for parenteral administration typically comprises a sterile aqueous or non-aqueous preparation of the active compound, which is preferably nearly isotonic with respect to the recipient's blood.

[0113] The proteins and compositions described herein are typically administered in therapeutically effective doses.

[0114] In further embodiments, the present invention also relates to the following: 1. A binding protein containing the antigen-binding portion of an antibody that includes the HCDR1-3 and LCDR1-3 amino acid sequences of SEQ ID NOs. 1-6, respectively. 2. The binding protein according to item 1, wherein the antibody comprises a heavy chain variable domain (VH) amino acid sequence and a light chain variable domain (VL) amino acid sequence that are at least 90% identical to the amino acid sequences of SEQ ID NOs. 7 and 8, respectively. 3. The binding protein described in item 2, wherein the antibody comprises VH containing SEQ ID NO: 7 and VL containing SEQ ID NO: 8. 4. The antibody is a human IgG isotype, and the binding protein is one of the proteins listed in any one of items 1-3. 5. The binding protein described in item 4, wherein the antibody is of the human IgG1 isotype subclass. 6. The binding protein according to any one of items 1 to 5, wherein the antibody comprises a heavy chain (HC) amino acid sequence and a light chain (LC) amino acid sequence that are at least 90% identical to the amino acid sequences of SEQ ID NOs. 9 and 11, respectively. 7. A binding protein containing an antibody that includes a heavy chain (HC) containing SEQ ID NO: 9 and a light chain (LC) containing SEQ ID NO: 11. 8. A binding protein described in any one of items 1-6, wherein the antibody contains at least one Fc domain mutation. 9. The binding protein described in item 8, wherein the antibody contains at least one Fc domain mutation that enhances the binding of the antibody to human FcγRIIIa. 10. The binding protein described in item 8, wherein the Fc domain mutation is located at position 239, optionally S239D, or at position 332, optionally I332E, and the residues are numbered according to Eu numbering. 11. The binding protein described in item 8, wherein the antibody contains Fc domain mutations at positions 239 and 332, optionally S239D and I332E, and the residues are numbered according to Eu numbering. 12. The antibody contains at least one Fc domain mutation that enhances the stability of the antibody, as described in any one of items 8-11. 13. The binding protein described in any one of items 1-11, wherein the antibody contains a pair of Fc domain mutations to cysteine, the mutations occur at positions 292 and 302, and the mutations are R292C and V302C, and the residues are numbered according to Eu numbering. 14. A binding protein according to any one of items 8 to 13, wherein the antibody comprises a heavy chain (HC) amino acid sequence and a light chain (LC) amino acid sequence that are at least 90% identical to the amino acid sequences of SEQ ID NOs. 10 and 11, respectively. 15. A binding protein containing an antibody that includes a heavy chain containing SEQ ID NO: 10 and a light chain containing SEQ ID NO: 11. 16. The binding protein described in any one of items 1 to 15, wherein the antibody has been defucosylated. 17. The binding protein described in any one of items 1 to 16, wherein the binding protein is a fusion protein. 18. The binding protein described in any one of items 1 to 17, wherein the binding protein is a bispecific or multispecific binding molecule. 20. An isolated nucleic acid molecule encoding one of the binding proteins listed in items 1-18. 21. A vector comprising an isolated nucleic acid molecule as described in item 20, wherein the vector optionally comprises an expression regulatory sequence. 22. Host cells containing the isolated nucleic acid molecules described in item 20. 19. A pharmaceutical composition comprising a binding protein described in any one of items 1 to 18 and a pharmaceutically acceptable excipient. 23. A method for producing a binding protein, Prepare the host cells as described in item 22. The host cells are cultured under conditions suitable for the expression of the binding protein, and Isolate the obtained binding protein from the culture. A method that includes this. 24. A method for treating cancer in a human patient requiring cancer treatment, comprising administering to the patient a therapeutically effective amount of a binding protein described in any one of items 1 to 18 or a pharmaceutical composition described in item 19. 25. The method according to item 24, wherein the cancer is multiple myeloma, non-Hodgkin lymphoma, renal cancer, breast cancer, ovarian cancer, or pancreatic cancer. 26. The method described in item 24, where the cancer is multiple myeloma. 27. A binding protein according to any one of items 1 to 18 or a pharmaceutical composition according to item 19, for use in the treatment of a human being requiring the method described in any one of items 24 to 26. 28. Use of any one of the binding proteins described in any one of items 1 to 18 to manufacture a pharmaceutical product for treating a human patient who requires it by the method described in any one of items 24 to 26.

[0115] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have meanings generally understood by those skilled in the art. Exemplary methods and materials are described below, but similar or equivalent methods and materials may also be used in the practice or testing of this disclosure. Throughout this specification and the embodiments, the terms “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” are understood to mean including the integer or set of integers described, but not to exclude any other integer or set of integers. Where used herein, the terms “approximately” or “about,” when applied to one or more values, mean a value similar to the reference value described. In certain embodiments, unless otherwise stated or the context makes clear, this term refers to a range of values ​​that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less than either direction (greater than or less than) the given reference value.

[0116] According to this disclosure, backreferences in dependent claims mean abbreviated notation to any direct and obvious disclosure of any combination of claims indicated by that backreference. Any compound disclosed herein may be used in any of the therapeutic methods herein, and the individual being treated is as defined elsewhere herein. Furthermore, the headings herein are provided for ease of organization and are not intended to limit in any way the claimed inventions are limited.

[0117] To better understand the present invention, the following examples are provided. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way. [Examples]

[0118] Example 1: Production of GPRC5D binder and preparation of an antibody engineered for enhanced ADCC activity GPRC5D conjugates were generated by phage display, followed by sequencing and screening in IgG format. Specific binding to stable GPRC5D-expressing cell lines was screened using FACS. Several GPRC5D conjugates, including clone 11, were identified.

[0119] After identifying specific binders, we investigated different “Fc-enhancing” formats to increase affinity to CD16a for enhanced ADCC function. Specifically, IgG was converted to two formats: 1) DE-DSB (S239D / I332E mutation to the Fc region, in addition to the R292C / V302C mutation to manipulate the disulfide bond for molecular stabilization; see, for example, PCT Public International Publication No. 2022 / 249146), and 2) defucosylated format (GlymaxX® technology, Probiogen). Clones 11 of the DE-DSB format are also called 11-DE-DSB, and clone 11 of the defucosylated format are also called 11-Afuco.

[0120] Antibodies directed at unrelated small molecule targets were prepared in different formats to function as isotype controls (ICs). These different format isotype controls are called IC-wt-IgG1, IC-Afco, and IC-DE-DSB.

[0121] For comparative reference, comparative antibodies were prepared using the VH and VL sequences of different anti-GPRC5D antibodies ("Benchmark 1" or "BM1", "Benchmark 2" or "BM2", "Benchmark 3" or "BM3", and "Benchmark 4" or "BM4"). The BM1, BM2, BM3, and BM4 sequences are commonly available sequences for the anti-GPRC5D portion of GPRC5D-binding proteins. Comparative antibodies were prepared in wild-type IgG1 format ("BM1 IgG1wt" or "BM2 IgG1wt", respectively), defucosylated IgG1 format ("BM1-afco" and "BM2-afco", respectively), and DE-DSB format ("BM1-DE-DSB" and "BM2-DE-DSB", respectively). Both BM1 and BM2 were selected as preferred benchmarks because they are complete competitors to the GPRC5D conjugate identified herein (clone 11), while BM3 and BM4 were only partial competitors.

[0122] Example 2: Evaluation of binding to FcγR by surface plasmon resonance (SPR) The interaction between the antibody and the human Fc receptor was analyzed by SPR using a Biacore® T200 instrument under the following test conditions: • Use of biotine CAPture for immobilization of anti-His antibodies on CM5 sensor chips or huFcRn analysis via amine coupling of 7 types of Fcγ receptors: huFcγRI, huFcγRIIa_H131, huFcγRIIa_R131, huFcγRIIb / c, huFcγRIIIa_V158, huFcγRIIIa_F158, and huFcγRIIIb. • Capture of each Fc receptor Determination of affinity constants by single-cycle kinetics (SCK) at concentrations ranging from 0.6 nM to 2000 nM. The interaction with huFcRn was tested at pH 5.9 and pH 7.4.

[0123] Table 2.1 shows the K values ​​of the listed antibodies against huFcγRIIIa (F158 and V158 polymorphisms). DThe values ​​(average of two replicated experiments) are shown. The affinity of FcγRIIIa for both polymorphisms was improved in the DE-DSB and Afco formats compared to wild-type IgG1.

[0124] [Table 2]

[0125] Table 2.2 shows the K values ​​of the listed antibodies against huFcγRIIA (H167 and R167 polymorphisms). D The values ​​(average of two replicated experiments) are shown. Compared to the wild type, no effect on affinity was observed with DE-DSB and Afco format.

[0126] [Table 3]

[0127] Table 2.3 shows the K of the listed antibodies against huFcγRI. D The values ​​(average of two replicated experiments) are shown. Compared to the wild type, no effect on affinity was observed with DE-DSB and Afco format.

[0128] [Table 4]

[0129] Table 2.4 shows the K values ​​of the listed antibodies against huFcRn at pH 5.9 and pH 7.4. D The values ​​(average of two replicated experiments) are shown. Compared to the wild type, no effect on affinity was observed with DE-DSB and Afco format.

[0130] [Table 5]

[0131] Table 2.5 shows the K of the listed antibodies against huFcγRIIIb. DThe values ​​(average of two replicated experiments) are shown. Affinity for FcγRIIIb was improved in the DE-DSB and Afco formats compared to wild-type IgG1.

[0132] [Table 6]

[0133] Example 3: Cell binding of ADCC-enhancing antibody to human and cynomolgus monkey FcγRIIIa-overexpressing HEK cells. The antibody's ability to bind to cells expressing human or cynomolgus monkey FcγRIIIa on their surface was tested using FACS. The cell lines used were human FcγRIIIa V158 and recombinant stable HEK293T cells expressing cynomolgus monkey FcγRIIIa, in order to evaluate cynomolgus monkey cross-reactivity.

[0134] Flow cytometry-coupled assays were performed using a MACSQuant16 (Miltenyi Biotech) flow cytometer in a 96-well plate.

[0135] For dose-response curves, cells were incubated with serial dilutions of the antibody (0.0001 μg / mL to 100 μg / mL) at 4°C for 1 hour. After one wash in PBS buffer, the cells were incubated with a secondary detection antibody (goat anti-human IgG(H+L) conjugated to A488 fluorochrome; Jackson ImmunoResearch, catalog no. 109-546-088), diluted 1 / 1000 according to the manufacturer's recommendations, at 4°C for 30 minutes, followed by two washes. For live / dead cell viability assessment, cells were stained with DAPI staining solution (4',6-diamino-2-phenylindole, dihydrochloride; Miltenyi Biotech, catalog no. 130-111-170) according to the manufacturer's protocol.

[0136] Analysis was performed using VenturiOne® Software. Cells were first gated with FSC / SSC. Single cells and subsequent viable cells were gated from the resulting cell population. Binding was analyzed using the median fluorescence intensity (MFI) of the secondary antibody.

[0137] Appearance K D The site-specific binding 4-parameter model was used with GraphPad Prism Software to plot an appropriate nonlinear regression curve for each molecule (Figure 1).

[0138] Table 3.1 shows FACS data regarding antibody binding to cynomolgus monkey and human FcγRIIIa-expressing HEK293T cells. The control anti-GPRC5D antibody benchmark 2 (BM2) was introduced in IgG1 wild-type format as a reference. The average of three experiments is reported.

[0139] [Table 7]

[0140] Clones 11 in both formats (DE-DSB and Afco) showed similar affinity (0.76–1.4 nM) to human and cynomolgus monkey FcγRIIIa. Affinity for both human and cynomolgus monkey FcγRIIIa was improved in both formats compared to the wild-type IgG1 format (26- to 48-fold increase).

[0141] Example 4: Conjugation of ADCC-enhancing antibody to multiple myeloma MM.1R cell line and HEK cells overexpressing human GPRC5D (hGPRC5D). Binding to cell lines FACS analysis was performed to determine the ability of the ADCC-enhancing antibody to bind to cells expressing GPRC5D on their surface. The cell lines used were: - Multiple myeloma cell line MM.1R (purchased from ATCC - American Type Culture Collection), - Recombinant, stable Flp-In(trademark)-293 cells expressing human and cynomolgus monkey GPRC5D to evaluate cross-reactivity in cynomolgus monkeys. - Stable HEK FITR cells expressing human GPRC5D (full length or a truncated form with N-terminal deletion of the first 15 or 20 residues) to describe the GPRC5D binding domain, and FreeStyle™ 293-F cells expressing a human GPRC5A / GPRC5D chimera in which the N-terminal domain and extracellular loop of GPRC5D, which describe the GPRC5D binding domain, are replaced with those of GPRC5A. That was the case.

[0142] Binding to REC-1 cell lines (purchased from ATCC) that are negative for GPRC5D expression was also tested to evaluate the specificity of the listed antibodies.

[0143] Flow cytometry-coupled assays were performed using an iQue Screener flow cytometer with 384-well plates.

[0144] For dose-response curves, cells were incubated at 4°C for 1 hour with serial dilutions of several antibody concentrations. After three washes, cells were incubated with a secondary detection antibody (goat anti-human IgG A488) at 4°C for 30 minutes, followed by three washes. To distinguish between live and dead cells, cells were stained with DAPI according to the manufacturer's protocol.

[0145] The analysis was performed using Forecyt® Software. Cells were first gated with FSC (forward scattering) / SSC (side scattering). Single viable cells were gated from the resulting cell population. Binding was analyzed as the median fluorescence intensity (MFI) of the secondary antibody. Curves and apparent K D ("App K D The calculation was generated using GraphPad Prism Software (Figure 2).

[0146] Table 4.1 shows FACS data regarding the binding of the listed antibodies to cynomolgus monkey and human GPRC5D-expressing Flp-In™-293 cells. Binding specificity to GPRC5D was evaluated using untransfected Flp-In™-293 cells. Wild-type format isotype control IgG1 ("IC-IgG1-wt") was used as a negative control. BM1 and BM2 were introduced as references.

[0147] [Table 8]

[0148] All 11-Afco, BM1-Afco, BM2-IgG1-wt, and BM3-IgG1-wt antibodies bound to human GPRC5D expressed on Flp-In(trademark)-293 cells. 11-Afco, BM1-Afco, and BM2-IgG1-wt antibodies also bound to cynomolgus monkey GPRC5D expressed on Flp-In(trademark)-293 cells, with BM2-IgG1-wt showing the lowest affinity. BM3-IgG1-wt did not bind to cynomolgus monkey GPRC5D expressed on Flp-In(trademark)-293 cells.

[0149] Table 4.2 shows FACS data regarding the binding of the listed antibodies to truncated variants of human GPRC5D expressed in HEK FITR cells. IC-IgG1-wt was used as a negative control. BM1, BM2, and BM3 were introduced as references.

[0150] [Table 9]

[0151] These results highlight the differences in how 11-Afco antibodies bind to GPRC5D compared to BM1-Afco antibodies.

[0152] Table 4.3 shows FACS data regarding the binding of the listed antibodies to cells expressing the GPRC5D / GPRC5A chimeric protein. IC-IgG1-wt was used as a negative control. BM1 and BM2 were introduced as references. Sequence number codes.

[0153] [Table 10]

[0154] These results highlight the differences in how the 11 antibodies (DE-DSB and Afco) bind to GPRC5D compared to the BM1-Afco and BM2-Afco antibodies.

[0155] Table 4.4 shows FACS data regarding the binding of the listed antibodies to GPRC5D-positive multiple myeloma MM.1R cells. BM1, BM2, and BM3 are included for reference.

[0156] [Table 11]

[0157] These results demonstrate that 11-DE-DSB and 11-Afco antibodies bind to GPRC5D expressed on MM.1R cells with significantly higher affinity than BM1-Afco, BM2-IgG1-wt, or BM3-IgG1-wt antibodies.

[0158] Example 5: In vitro short-term ADCC activity in MM cell lines with different GPRC5D densities To determine the number of GPRC5D molecules per cell, GPRC5D surface expression was quantified using a flow cytometry-based GPRC5D receptor assay, followed by selection of MM cell lines suitable for downstream analysis with GPRC5D antibodies. MM.1R and EJM cell lines were selected from a panel of MM cell lines for high and intermediate levels of GPRC5D expression, respectively.

[0159] In short, to determine the GPRC5D density, we used the CELLQUANT Calibrator (BioCytex, catalog number 7208) as recommended by the manufacturer. MM cells were incubated with mouse anti-human GPRC5D antibody (monoclonal mouse IgG2B clone number 571961, R&D System) at a dilution of 1 / 500. After two consecutive washes in reagent R1, the cells were labeled with anti-mouse IgG FITC secondary antibody (reagent 3 in the BioCytex kit, catalog number 7208) diluted to 1 / 10 in reagent R1. In parallel, calibration beads were added to a dedicated calibration well and treated under the same staining conditions as the cells. The 96-well plate was then incubated at 4°C for 20 minutes, protected from light.

[0160] After incubation and washing, followed by centrifugation, the cells were resuspended in reading buffer and then read using MACSQuant® Analyzer 10 (Miltenyi Biotec).

[0161] To determine the GPRC5D density on MM cell lines (based on FITC fluorescence), a linear calibration curve was created using calibration bead information. The GPRC5D density per cell was calculated using the following formula: GPRC5D density=10 (log(FITC GPRC5D)*a+b) -10 (log(FITC アイソタイプ)*a+b)

[0162] Calculations of GPRC5D density per cell in a panel of MM cell lines revealed that the MM.1R cell line had the highest level of GPRC5D density on the cell surface (approximately 30,500 antigens per cell) compared to other MM cell lines used in the panel, while the EJM cell line had a moderate level of GPRC5D density (approximately 8,500 antigens per cell).

[0163] Table 5.1 lists all MM cell lines tested and their GPRC5D densities (the data shown are based on the average of 6-7 experiments).

[0164] [Table 12]

[0165] To measure the short-term functionality of anti-GPRC5D-enhanced ADCC antibodies, in vitro NK cell cytotoxicity assays were performed. Anti-GPRC5D ADCC-enhanced antibodies in different Fc formats (Fc-defucosylated, Fc-DE-DSB) were evaluated for their ability to promote lysis of MM tumor cells in the presence of purified NK cells from healthy donors. Two additional anti-GPRC5D antibodies were tested in parallel as benchmarks (BM1 and BM2).

[0166] In detail, human NK cells were purified from healthy donor buffy coats provided by Etablissement Francais du Sang (EFS, the French blood service, Rungis). Peripheral mononuclear cells (PBMCs) were isolated from the buffy coats by Ficoll density gradient centrifugation. Human NK cells were purified from PBMCs using a bead-based negative selection kit from Miltenyi Biotec (MACSxpress® Cell Isolation kit, catalog no. 130-098-185). MM.1R and EJM MM cell lines were purchased from ATCC. Cells were cultured in complete RPMI medium (RPMI-1640 containing 10% FBS and 2 mM L-glutamine). For cytotoxicity assays, target tumor cells were alternatively loaded with calcein (Invitrogen catalog no. C3100MP) as recommended by the supplier. All antibodies and isotype controls (ICs) were tested in the concentration range of 0.00001–100 μg / mL. All tested antibodies and corresponding ICs were distributed into appropriate wells of a round-bottom 96-well plate. Human NK cells (effector cells, approximately 100,000 cells) (fresh or overnight) derived from a healthy donor and labeled tumor cells (target cells, approximately 10,000 cells) were then added to obtain a 10:1 E:T ratio (effector:target cell ratio). In some wells, TRITON X-100 (Sigma-Aldrich) was added as a positive control for tumor cell lysis, as it induced 100% cell lysis. After 2 hours of co-incubation at 37°C and 5% CO2, the supernatant was transferred to a black 96-well plate, and the amount of released calcein was assessed using a TECAN Infinite® 1000 counter. Specific lysis percentages were calculated using the following formula: Specific lysis (%) = (treated cells - individual cells) / (Triron-treated cells - individual cells) × 100

[0167] The EC50 for each molecule was determined by plotting an appropriate nonlinear regression curve (selection of a "log(agonist) vs. response-variable gradient (4 parameters)" model) using GraphPad Prism Software. The results are expressed in AFU = arbitrary fluorescence units.

[0168] Table 5.2 shows the ADCC activity of the listed antibodies against MM.1R MM cells (Figure 3). Lysis percentage was evaluated using calcein release readout. BM1 and BM2 Afco were introduced as benchmark references.

[0169] [Table 13]

[0170] Clones 11 in the DE-DSB and defucosylated formats exhibit high picomolar ADCC activity against MM1.R cells in the presence of NK cells (EC50 of 40.1 and 36 pM, respectively). This activity is superior to that found in anti-GPRC5D benchmarks BM2-afco and BM1-afco (relEC50 of 154 and 1,663 pM, respectively).

[0171] Table 5.3 shows the ADCC activity of the listed antibodies against EJM MM cells (Figure 4). Lysis percentage was evaluated using calcein release readout. BM1 and BM2 Afco were introduced as benchmark references.

[0172] [Table 14]

[0173] Clones 11 in the DE-DSB and defucosylated formats exhibit high picomolar ADCC activity against EJM cells in the presence of NK cells (EC50 of 259 and 141 pM, respectively). This activity is superior to that found in anti-GPRC5D benchmarks BM2-afco and BM1-afco (relEC50 of 489 and 3,460 pM, respectively).

[0174] Example 6: In vitro long-term ADCC activity in MM.1R MM cell line To evaluate the long-term functionality of anti-GPRC5D-enhanced ADCC antibodies, in vitro NK cell cytotoxicity assays were performed over 4 days using the Incucyte® S3 Live-Cell Analysis Instrument (Sartorius). Anti-GPRC5D ADCC-enhanced antibodies in different Fc formats (Fc-defucosylated, Fc-DE-DSB) were evaluated for their ability to promote lysis of MM tumor cells in the presence of purified NK cells from healthy donors. Two additional anti-GPRC5D antibodies were tested in parallel as benchmarks (BM1 and BM2).

[0175] In short, human NK cells were purified from healthy donor buffy coats provided by Etablissement Francais du Sang (EFS, the French blood service, Rungis). Peripheral mononuclear cells (PBMCs) were isolated from the buffy coats by Ficoll density gradient centrifugation. Human NK cells were purified from the PBMCs using a bead-based negative selection kit from Miltenyi Biotec (MACSxpress Whole Blood NK Cell Isolation kit; catalog number 130-127-695). The purified human NK cells were incubated overnight in complete RPMI medium (RPMI-1640 containing 10% FBS and 2 mM L-glutathione). The MM.1R MM cell line was purchased from ATCC. Stable MM.1R-RFP (red fluorescent protein) cell lines were established by infection with a third-generation lentivirus HIV-based VSV-G pseudotyped lentivirus (mKate2 Lentivirus Reagent; Sartorius; catalog number 4625) encoding RFP. MM.1R-RFP cell selection was performed by adding puromycin dihychloride hydrate (Thermo Scientific, Denmark, catalog number 10781691) at a final concentration of 1 μg / mL in complete RPMI culture medium (RPMI-1640 containing 10% FBS and 2 mM L-glutamine). For cytotoxicity assays, all antibodies were tested in the concentration range of 0.00001 to 100 μg / mL. Diluted molecules, labeled target cells (approximately 6,000 cells), and static human NK cells from a healthy donor (approximately 18,000 cells) were sequentially added to each well of a 384-well black plate to obtain an E:T ratio (effector:target cell ratio) of 3:1. The plate was incubated at 37°C and 5% CO2 in an Incucyte® S3 Live-Cell Analysis Instrument (Sartorius) for 4 days, taking images every 4 hours (phase and red image channels; 20X magnification). Results are expressed as the number of red objects per image, normalized to 0d0h0m (%).The percentage of specific dissolution was calculated using the following formula: Specific lysis (%) = (Untreated cells without NK - Cells treated with NK) / (Untreated cells without NK) × 100

[0176] The relative EC50 for each molecule was determined by plotting an appropriate nonlinear regression curve (selection of a "log(agonist) vs. response-variable gradient (4 parameters)" model) using GraphPad Prism Software.

[0177] Table 6.1 shows the ADCC activity of the listed antibodies against MM.1R MM cells. Lysis percentages were evaluated using Incucyte imaging readout. BM1 and BM2 Afco were introduced as benchmark references.

[0178] [Table 15]

[0179] Clones 11 in both the DE-DSB and defucosylated formats exhibited high picomolar ADCC activity against MM.1R cells in the presence of NK cells after 4 days of incubation (median EC50 of 6 and 3.5 pM, respectively) (Figure 5). In both the DE-DSB and defucosylated formats, clone 11 showed higher cytotoxic activity against MM.1R cells over 4 days than BM1-afco antibody and BM2-afco antibody (Figure 6). Anti-GPRC5D antibody BM2 in the defucosylated format showed similar activity (median relEC50 of 6.6 pM). Anti-GPRC5D antibody BM1 in the defucosylated format showed lower cytotoxic activity with a median relEC50 of 175 pM.

[0180] Example 7: Cytokine release in MM.1R co-cultures in a PBMC environment This experiment evaluated the in vitro effect of clone 11 in the DE-DSB format on cytokine release by peripheral blood mononuclear cells (PBMCs) in the presence of GPRC5D and BCMA-positive cell lines (MM.1R multiple myeloma cells).

[0181] Briefly, for the cytokine release assay, PBMCs were isolated from healthy human donor buffy coat (provided by Etablissement Francais du Sang (EFS, the French blood service, Rungis)) using Ficoll density gradient centrifugation. The MM.1R multiple myeloma cell line was purchased from ATCC. Cells were cultured in complete RPMI medium (RPMI-1640 containing 10% FBS and 2 mM L-glutamine). Since BCMA is expressed on MM.1R cells, an anti-BCMAXCD3 T cell engager was used as a positive control. Clones 11 and controls in DE-DSB format were tested at 20 nM and 20 pM. Target cells (10,000 cells) and healthy donor-derived human PBMC cells (500,000 cells) were sequentially seeded into 96-well plates to obtain an E:T ratio (effector:target cell ratio) of 50:1. The molecules to be tested and the positive control were added to the co-culture. After co-incubation overnight at 37°C and 5% CO2, the plates were centrifuged at 300 g for 5 minutes. Subsequently, 100 μL of supernatant / well was collected for cytokine release analysis. For analysis, the Human Proinflammatory I (4-Plex) Kit V-PLEX was used as recommended by the provider (Mesoscale MSD, catalog number K15052D-1). IFNγ, IL-6, and TNFα cytokines were quantified in the same well. Calibration curves used to calculate the concentration of each analyte were established by fitting the signals from the calibrator to a 4-parameter logistic model. Analyte concentrations (pg / mL) were determined from the ECL signal by inverse fitting to the calibration curve.

[0182] Table 7.1 shows data on cytokine release induced by clone 11 of the DE-DSB format from human PBMCs in the presence of GPRC5D-positive MM.1R cells (average of 5 experiments). Isotype control IgG1DE-DSB(IC) was used as a negative control. Anti-BCMAXCD3 TCE was introduced as a positive control.

[0183] [Table 16]

[0184] Clones 11 of the DE-DSB and defucosylated formats minimally induced cytokine release in human PBMCs in the presence of GPRC5D-expressing MM.1R cells, resulting in 1–3-fold increases in IFNγ, IL-6, and TNFα compared to baseline levels. This induction was significantly lower than the cytokine release induced by anti-BCMA TCE (1.8–42-fold changes) (Figure 7).

[0185] Example 8: In vivo activity of anti-GPRC5D antibody against MM.1R multiple myeloma cells transplanted into NK humanized hIL15tg-NOG mice. The in vivo efficacy of the anti-GPRC5D antibody was evaluated in hIL15tg-NOG immunodeficient mice reconstituted with human NK cells and transplanted with disseminated human MM.1R cells. A hIL15tg-NOG mouse strain possessing the human IL15 gene was used to enable robust development of human NK cells in the mouse microenvironment. Humanization of the mouse NK cells was performed by intravenous adoptive transfer of pre-amplified human NK cells (10 million) after sublethal irradiation (for details of the NK humanization mouse model, see Rettman et al., Cancer Res (2022) 82(12_Supplement): Abstract 4247 and Rettman et al., Cancer Res (2023) 83(7_Supplement): Abstract 2940).

[0186] Tumor cells were intravenously inoculated into NK humanized mice on day 0. Treatment was administered intraperitoneally (IP) on days 1, 4, and 7 after tumor transplantation. Anti-GPRC5D antibodies BM1-afco, 11-DE-DSB, and 11-afco were administered at 10 mg / kg. The control group was treated with a wild-type IgG1 isotype control ("IC IgG wt") at 10 mg / kg.

[0187] Mice were examined, and adverse clinical reactions were observed. The body weight of each individual mouse was measured daily until the end of the experiment (day 110). To avoid animal suffering, mice were euthanized when they reached a mortal state according to pre-defined criteria. Clinical signs considered significant and related to the disease state include quadriplegia, ascites, palpable internal tumor masses, morbidity, or weight loss of 20% or more.

[0188] The primary efficacy endpoints were median survival time (MST) in days, percentage lifespan increase (ILS%), and long-term survival rate (%).

[0189] The individual death date (if any) for each mouse was reported. MST was determined for each group, the ratio ILS was calculated, and expressed as a percentage: ILS% = 100 × (TC) / C In the formula, T = MST of the treatment group and C = MST of the control group.

[0190] The dosage is considered therapeutically active when the ILS% is better than 25%, and highly active when the ILS% is better than 50% (Johnson et al. (2001), Br.J.Cancer, 84(10):1424-31).

[0191] Long-term survival rate is defined as the number of mice in a group that have a survival period at least twice as long as the control group's median survival time (MST), expressed as a percentage of the total number of mice in the group.

[0192] The group treated with IgG wild-type isotype controls showed a median survival time (MST) of 53 days, and there were no long-term survivors.

[0193] The BM1-Afco antibody induced statistically significant activity in disseminated human MM cell line MM.1R transplanted into NK humanized hIL15tg-NOG mice at a dose of 10 mg / kg, resulting in over 100% ILS compared to controls and a 50% long-term survival rate.

[0194] The 11-Afco antibody induced statistically significant activity in disseminated human MM cell line MM.1R transplanted into NK humanized hIL15tg-NOG mice at a dose of 10 mg / kg, resulting in over 100% ILS compared to controls and a long-term survival rate of 61%.

[0195] The 11-DE-DSB antibody induced statistically significant activity in disseminated human MM cell line MM.1R transplanted into NK humanized hIL15tg-NOG mice at a dose of 10 mg / kg, resulting in over 100% ILS compared to controls and a long-term survival rate of 72%.

[0196] In conclusion, all anti-GPRC5D antibodies tested showed robust activity at 10 mg / kg against the disseminated human MM cell line MM.1R transplanted into NK humanized hIL15tg-NOG mice (Figure 8).

[0197] Table 8.1 shows data on the in vivo activity of the listed anti-GPRC5D antibodies against the disseminated human MM cell line MM.1R transplanted into NK humanized hIL15tg-NOG mice.

[0198] [Table 17]

[0199] Example 9: ADCC-enhanced Fc-operated GPRC5D NKCE exhibits a favorable elimination half-life. This example evaluates the pharmacokinetic (PK) profiles and parameters of clone 11 in DE-DSB (11-DE-DSB) and defucosylated (11-afco) formats as a natural killer cell engager (NKCE) after a single intravenous (2.5 mg / kg) administration to female huFcRn Tg32 transgenic mice.

[0200] Materials and methods The mouse experiments were conducted using transgenic Tg 32 (B6.Cg-Fcgrttm,1Dcr Tg(FCGRT)32Dcr / DcrJ) mice derived from C57BL / 6 mice and purchased from Jackson Laboratory (Bar Harbor, Maine). FcRn- / -hFcRn (strain 32)Tg mice possess a null mutation in the mouse gene and a transgene that expresses the hFcRnα chain transgene under the control of its native human promoter. Three Tg32 homozygous naive adult female mice (average body weight 23.7g) were used at the start of the study.

[0201] For the drug regimen, 11-DE-DSB and 11-Afco (1.5 mg / mL) were in situ prepared in 10 mM His, 150 mM NaCl, pH 6, and DPBS buffers, respectively, diluted in the same buffer, and administered intravenously as a single dose of 2.5 mg / kg at a dose volume of 10 mL / kg via the tail vein. Animals were evaluated over a 28-day study period using a continuous sampling approach at 0.083, 4, 24, 72, 168, 336, 504, and 672 hours. At each time point, blood samples (approximately 20 μL - continuous sampling) were collected from the saphenous vein into a K3-EDTA collection device. Immediately after collection, the blood samples were placed on moist ice and then centrifuged. Next, 4 μL of plasma was diluted in 396 μL of PBS / 0.5% BSA (phosphate-buffered saline / 0.05% Tween 20 / 0.5% bovine serum albumin).

[0202] For the analysis, exploratory LBA (ligand binding assay) method was used to determine the concentrations of 11-DE-DSB and 11-Afco in plasma. Both compounds were captured by biotinylation of donkey anti-huIgGFc bound to the streptavidin wells of the MSD plate. Detection was performed using goat anti-huIgG-sulfo tag prior to chemiluminescent reading. The lower limit of quantification (LLOQ) value was 0.2 μg / mL.

[0203] Statistical analysis The individual plasma concentration values (expressed in μg / mL) of 11-DE-DSB and 11-Afco NKCE were summarized by descriptive statistics (mean, standard deviation (SD), and coefficient of variation (CV%)) and tabulated by sampling time. All results were reported with three significant figures, except for CV% without decimal places.

[0204] The individual PK parameters were summarized by descriptive statistics as described above. The individual values and mean values were expressed with three significant figures (except that t max and t last were appropriately rounded for time values, and only the median and range [minimum - maximum] values were reported).

[0205] Results No clinical signs or symptoms were observed during the test.

[0206] The mean and individual values (N = 3) of plasma concentrations (μg / mL) of 11-DE-DSB NKCE obtained after single IV (2.5 mg / kg) administration to female huFcRn Tg32 mice are reported in Table 9.1 below.

[0207]

Table 18

[0208] The mean and individual values (N = 3) of pharmacokinetic parameters of 11-DE-DSB in plasma after single intravenous (2.5 mg / kg) administration are shown in Table 9.2 below.

[0209] [Table 19]

[0210] After intravenous administration of 2.5 mg / kg, the 11-DE-DSB NKCE concentration was quantifiable in plasma for up to 28 days (last sampling time). Estimating plasma clearance at 10.0 mL / day / kg, the steady-state volume of distribution was 139 mL / kg, resulting in a terminal phase elimination half-life (t) of approximately 12 days. 1 / 2 ) was brought about.

[0211] The mean and individual (N=3) plasma concentrations (μg / mL) of 11-AfcoNKCE obtained after a single IV (2.5 mg / kg) administration to female huFcRn Tg32 mice are reported in Table 9.3 below.

[0212] [Table 20]

[0213] The mean and individual values ​​(N=3) of the pharmacokinetic parameters of 11-AfcoNKCE in plasma after a single intravenous administration (2.5 mg / kg) are shown in Table 9.4 below.

[0214] [Table 21]

[0215] After intravenous administration of 2.5 mg / kg, the concentration of 11-AfcoNKCE was quantifiable in plasma for up to 28 days (last sampling time). Estimating a plasma clearance of 4.30 ± 0.525 mL / day / kg, the steady-state volume of distribution was 63.5 ± 10.2 mL / kg, resulting in a terminal phase elimination half-life (t) of approximately 11 days. 1 / 2 ) was brought about.

[0216] Example 10: Combination of exploratory pharmacokinetic (PK) and safety studies after a single intravenous or subcutaneous administration of 11-DE-DSB NKCE in cynomolgus monkeys. The purpose of this exploratory (non-GLP) study was to determine the pharmacokinetics (PK) and tolerance / toxicity profile of 11-DE-DSB when administered to cynomolgus monkeys as a single dose by 1) intravenous (IV) infusion over 30 minutes and 2) subcutaneous (SC) injection, followed by a 4-week observation period.

[0217] A total of 8 female cynomolgus monkeys (Macaca fascicularis, approximately 31 - 33 months old at the start of dosing) were included in the study. The study design is described in Table 10.1 below.

[0218]

Table 22

[0219] Animals were dosed based on their current body weight.

[0220] Intravenous infusion was performed into the saphenous vein using an intravenous catheter, and subcutaneous injection was performed on the back of the animal (one injection site between the scapulae). An intravenous infusion was carried out using a calibrated syringe pump. After dosing, the injection site was carefully observed, and local compression was applied for sufficient time to prevent bleeding.

[0221] Parameters evaluated included mortality, clinical signs, body weight, food consumption, body temperature, examination of the injection site, hematology, coagulation, and clinical chemistry (including CRP). Blood was also collected for PK evaluation and plasma cytokine levels. At the end of the 4-week observation period, all monkeys were returned to the animal colony.

[0222] PK Evaluation Blood samples (0.4 mL in K2-EDTA sampling tubes) were collected from the femoral vein, saphenous vein, and / or cephalic vein according to the following planned days and sampling time points (the vein used for infusion was not used for blood sampling purposes).

[0223]

Table 23

[0224] Blood samples from the control animals (group 1) were discarded immediately after sampling.

[0225] After collection, each blood sample was centrifuged at approximately 4°C at 1500g for 10 minutes. The resulting plasma samples (at least 150 μL) were transferred to labeled polypropylene tubes and frozen at approximately -80°C until analysis.

[0226] An exploratory immunoassay was used to quantify 11-DE-DSB in cynomolgus monkey plasma samples. This method consisted of a stepwise sandwich format. Capture was performed using biotinylated goat anti-human IgG Monkey Ads immobilized on a streptavidin bead column in Gyrolab Bioaffy discs (CD200) containing microstructures. Detection was performed using goat anti-human IgG Monkey Ads AlexaFluor® 647. On-column fluorescence measurements were performed within the Gyrolab platform. The quantification range was 200 ng / mL (lower limit of quantification, LLOQ) to 500,000 ng / mL (upper limit of quantification, ULOQ) using a minimum required dilution (MRD) of 1 / 100.

[0227] WinNonLin software (Phoenix 64 v8.2 Pharsight, Certara Inc.) was used to evaluate the PK of the determined plasma concentrations of 11-DE-DSB and to determine additional PK parameters (dose-proportionality and bioavailability). The mean was calculated for plasma concentrations at each sampling time and for each PK parameter (Figure 9).

[0228] Table 10.2 shows data on individual and mean (n=2) PK parameters in plasma observed after 30-minute intravenous or subcutaneous administration of 11-DE-DSB to female cynomolgus monkeys.

[0229] [Table 24]

[0230] Regardless of the dosage and route of administration, 11-DE-DSB was quantifiable up to the last sampling time (day 28).

[0231] After IV administration, the mean clearance was approximately 6.6 mL / day / kg, the mean volume of distribution was approximately 74 mL / kg, and the terminal phase elimination half-life was approximately 9 days.

[0232] After intravenous infusion of 5 and 25 mg / kg over 30 minutes, exposure increased proportionally with increasing dose.

[0233] After subcutaneous administration at 25 mg / kg, the bioavailability was approximately 65%.

[0234] Cytokine evaluation Blood samples (0.5 mL in a K2-EDTA sampling tube) were collected from the femoral vein, saphenous vein, and / or cephalic vein (veins used for injection were not used for blood collection) before the procedure on day 1, and at 4, 24, and 48 hours after the start of medication.

[0235] The evaluation of IFNγ, IL-6, IL-8, and TNFα in monkey plasma samples was performed using the Mesoscale Discovery (U-PLEX Proinflam Combo 1 (NHP) SECTOR assay kit, catalog number K15070K-2) exploratory ECLIA (electrochemiluminescence immunoassay).

[0236] All variability was expressed in comparison to baseline values ​​(i.e., pre-treatment values). Due to analytical variability, an increase in cytokines was considered potentially related to a compound if the value was more than twice the corresponding pre-test value and higher than the highest value observed in the control group. Grading was applied as follows: multiplier changes of 3–10: very slight increase; multiplier changes of 11–100: slight increase; multiplier changes of 101–1000: moderate increase; and multiplier changes of 1001 or more: significant increase.

[0237] Table 10.3 shows data on individual plasma cytokine levels (pg / mL) and their relative changes to baseline after 30-minute intravenous or subcutaneous administration of 11-DE-DSB to female cynomolgus monkeys.

[0238] [Table 25]

[0239] [Table 26]

[0240] No significant cytokine elevations were observed after a single IV administration of 11-DE-DSB up to 25 mg / kg or a single SC administration of 11-DE-DSB at 25 mg / kg. Four hours after 11-DE-DSB administration, only transient, very slight to slight increases in IL-6 were observed, returning to near baseline levels within approximately 24 hours after 11-DE-DSB administration. No 11-DE-DSB-related changes in IFN-γ, IL-8, and TNFα levels were observed in any animal at any dose during the study.

[0241] Administration of 11-DE-DSB to female cynomolgus monkeys via 30-minute IV infusion at 5 or 25 mg / kg and via the SC route at 25 mg / kg was clinically well tolerated. No 11-DE-DSB-related clinical signs were reported, including those related to nail and tongue observation, body temperature, body weight, and clinical observations of the animals. Good local and systemic tolerability was reported. No hematological or clinical chemistry changes were thought to be related to 11-DE-DSB.

[0242] Therefore, under these test conditions, a single 30-minute IV infusion (11-DE-DSB) corresponds to the values ​​of 621 μg / mL and 3,940 μg / mL. max (and AUC) and single SC injection (180 μg / mL and 2610 days * C corresponding to μg / mL values ​​in 11-DE-DSB) maxThe highest dose of 11-DE-DSB tested, 25 mg / kg (as AUC), was well-tolerated and did not induce adverse effects.

[0243] Example 11: Ex vivo activity of anti-GPRC5D antibody and NK cell activation mediated in primary NDMM patient samples. The cumulative effects of anti-GPRC5D antibody were evaluated ex vivo from bone marrow (BM) and peripheral blood samples containing multiple myeloma cells and autoimmune effector cells from newly diagnosed, untreated multiple myeloma (MM) patients. The number of neoplastic plasma cells (PCs) and NK cells were measured, and SS cells were included in the plasma cell count. low / FS high / CD45 low / CD138 + / CD38 + / CD56 ± and CD45 + / CD3 - / CD56 + / CD16 ± Each sample was determined by flow cytometry as defined. The effector:target (E:T) ratio was calculated as NK concentration / PC concentration. The receptor density of GPRC5D on BM-derived neoplastic plasma cells was determined using mouse anti-human GPRC5D clone 571932 (R&D System) and a mouse Ig calibration kit (Biocytex) as recommended by the provider. The volume of the remaining BM sample was divided to perform a killing assay and an NK cell activation and degranulation assay.

[0244] BM samples containing neoplastic plasma cells (PCs) and autoeffector NK cells were incubated with negative isotype control isotype (anti-TNP-DE-DSB) or anti-GPRC5D ADCC-enhanced 11-DE-DSB at 0.01 and 1 μg / ml. At the end of the overnight incubation, cells were stained with the antibody panel shown in Table 11.1 to determine 1) residual viable plasma cells, 2) NK cell activation, and 3) CD107a NK cell degranulation.

[0245] Table 11.1 shows a panel of antibodies used for anti-GPRC5D antibody ex vivo activity and mediated NK cell activation in primary NDMM patient samples.

[0246] [Table 27]

[0247] Flow cytometry data was analyzed using FlowJo software. Myeloma cell death was identified by CD45 low / CD138 + / CD38 + / CD56 ± Cell loss was evaluated. Lysis percentage was calculated relative to isotype-negative controls and normalized to the number of B cells (stable number during the experiment). NK cell degranulation markers on NK cells were identified as CCD3. - / CD56 + This was evaluated by CD107a expression on NK cells.

[0248] Figure 10 schematically illustrates the principle of this ex vivo study. Table 11.2 shows the GPRC5D receptor density (sABC: specific antigen binding ability) NK:PC effector:target (E:T) ratio on neoplastic plasma cells (PCs), and the results of flow cytometry analysis of bone marrow (BM) cells derived from NDMM patients showing PC lysis after ex vivo overnight treatment with 11-DE-DSB.

[0249] [Table 28]

[0250] Figure 11A shows a graph of the results of plasma cell (PC) lysis. Figure 11B shows a graph of CD107 induction on NK cells after overnight treatment with anti-GPRC5D in bone marrow (BM) patient samples.

[0251] Flow cytometry analysis of BM MM patients showed a wide range of GPRC5D receptor densities and E:T ratios. Analysis of BM MM patients treated with anti-GPRC5D ADCC-enhanced 11-DE-DSB antibody showed CD3- / CD56 + The study demonstrated myeloma cell lysis and an increase in activated NK cells, as assessed by CD107a expression on NK cells. These data demonstrate that the anti-GPRC5D ADCC-enhanced 11-DE-DSB antibody activates NK cells ex vivo in primary samples from multiple myeloma cells and NK cells derived from MM patients in an autologous assay using MM patients.

[0252] Example 12: Competition with benchmark molecules for binding to GPRC5D The interaction between the antibody and human GPRC5D on cells was tested by FACS under the following conditions, in competition with a benchmark molecule: • A benchmark antibody (human IgG1 format) was labeled with biotin. The labeling was checked to ensure it did not impair the binding properties. • Labeled benchmark antibodies were added to GPRC5D cells at EC80 concentrations while increasing the dose of the test antibody (human defucosylated IgG1 format). • The binding of benchmark antibodies was measured to determine the level of competition.

[0253] [Table 29]

[0254] Example 13: ADCP macrophage-mediated phagocytic activity against MM.1R MM cell line The antibody-dependent cell-mediated phagocytic (ADCP) activity of the 11-DE-DSB antibody was evaluated using macrophages derived from monocytes purified from human peripheral blood mononuclear cells (PBMCs) isolated from the buffy coats of five different healthy donors provided by MM.1R cells (ATCC ref.CRL-2975) and EFS (Etablissement Francais du Sang).

[0255] First, human PBMCs were isolated from the buffy coat of healthy donors obtained from Etablissement Francais du Sang (EFS): blood was first collected in a 50 mL Falcon tube. 15 mL of Ficoll-Plaque® PLUS 96% (GE Healthcare #17-1440-02) was very gently added to the bottom of a Sepmate tube, then blood was slowly added to the tube and centrifuged at 1300 g for 10 minutes. The PBMC rings were then collected, washed with 50 mL of PBS buffer, and centrifuged again at 300 g for 5 minutes. The washing process was repeated twice, and then the PBMCs were stained with anti-CD14 magnetic beads according to the manufacturer's instructions (Miltenyi; ref 130-050-201): incubated at 4°C for 15 minutes and then positively selected by AutoMACS pro. Monocytes were collected and then washed with 50 mL of PBS. Monocytes were cultured in a T150 flask at 37°C and 5% CO2 for 7 days in RPMI1640 (Invitrogen #11875-093), 10% FBS (Invitrogen #10500-056), 2% human inactivated serum (Fisher Bioreagents #14-490E), and 50 ng / ml granulocyte-macrophage colony-stimulating factor (GM-CSF) (Miltenyi #130-093-866). After 7 days of culture, monocytes differentiated into macrophages.

[0256] To collect macrophages, cells were detached using acutase (ThermoFisher, ref: A1110501) (15 minutes at 37°C), and then their activity was stopped by adding 20 mL of complete culture medium RPMI1640, 10% FBS, and 1% L-glutamine (Invitrogen #25030).

[0257] The harvested cells were centrifuged at 350g for 10 minutes, then washed with 20mL of PBS, and centrifuged again at 350g for 10 minutes.

[0258] 20×10 6Macrophages were resuspended in 2 ml of Diluent C (provided in Sigma Aldrich staining kit #PKH26GL-1KT) and 10 μl of PKH26 (Sigma Aldrich #PKH26GL-1KT) for incubation at 37°C in the dark for 5 minutes. Then, 10% FBS was added to inactivate the staining process for 1 minute, followed by completion with 50 mL of RPMI1640 10% FBS. (1.5 × 10⁶ cells / 100 μl) 6 The volume was adjusted to reach a macrophage concentration of / ml.

[0259] 20×10 6 MM.1R cells were resuspended in 2 ml of Diluent C (provided in Sigma Aldrich staining kit # PKH26GL-1KT) and 10 μl of PKH67 (Sigma Aldrich # PKH67GL-1KT) for incubation at 37°C in the dark for 5 minutes. The staining process was then inactivated for 1 minute with 10% FBS, followed by completion with 50 mL of RPMI1640 10% FBS. (0.5 × 10⁶ cells / 100 μl) 6 The volume was adjusted to reach a macrophage concentration of / ml.

[0260] Next, 5 x 10 4 MM.1R cells / well were incubated with different concentrations of antibodies in a U-bottom 96-well polypropylene plate at 37°C and 5% CO2 for 15 minutes, and then macrophages were added overnight at a macrophage / tumor cell ratio set to 3:1 (150,000 macrophages vs. 50,000 MM.1R). The ADCP assay was performed in the presence of 1 mg / mL human polyclonal IgG (Sigma#I4506) to mimic physiological conditions.

[0261] Compound 11-DE-DSB, anti-CD38-IgG1, and isotype IgG1 DE-DSB were tested at final concentrations ranging from 100 ng / mL to 0.1 pg / mL (1 / 10 serial dilutions using PBS Dulbecco's). ADCP activity of 11-DE-DSB, anti-CD38-IgG1, and isotype IgG1 DE-DSB was read by flow cytometry. The percentage of MM.1R cells phagocytosed by macrophages was determined. Dose-effect curves were fitted using a four-parameter logistic model by Ratkovsky and Reedy (Biometrics. 1986. Sep; 42(3): 575-82). Adjustments were obtained by nonlinear regression using the Marquardt algorithm. Relative EC50 corresponds to the concentration that yields the half-life effect between the maximum and minimum percentages of cell lysis. If the relative EC50 is out of range, it means that, depending on the case, the relative EC50 is either higher than the strongest concentration tested or lower than the weakest concentration tested. EC50 rel The geometric mean of the values ​​was calculated using a 95% confidence interval (CI).

[0262] 11-DE-DSB exhibits ADCP activity against MM.1R cell lines, and EC50 rel The geometric mean was 247.8 pM [CI 95% 81.7; 751.1] (equivalent to 35.93 ng / mL) (n=5). In the same experiment, anti-CD38-IgG1 showed ADCP activity against the MM.1R cell line, and EC50 rel The geometric mean was 21.7 pM [CI95% 2.9;160.2] (equivalent to 3.15 ng / ml) (n=3). Isotype controls showed no ADCP activity. The results are summarized in Tables 13.1 and 13.2; dose-response curves for each donor compound are shown in Figure 12. In conclusion, 11-DE-DSB induced ADCP in GPRC5D-expressing MM.1R cell lines with an EC50rel value of 248 pM [CI95% 82;751].

[0263] [Table 30]

[0264] Table 31

[0265] Table 32

[0266] Table 33

Claims

1. An antigen-binding protein comprising two variable domains: a first variable domain containing HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and a second variable domain containing LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO:

6.

2. The antigen-binding protein according to claim 1, comprising a heavy chain variable domain (VH) amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain (VL) amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

8.

3. Preferably, the antibody is a human IgG isotype antibody, and more preferably, the antibody is a human IgG １ An antigen-binding protein according to claim 1 or 2, which is an isotype subclass.

4. The antigen-binding protein according to any one of claims 1 to 3, comprising a heavy chain (HC) amino acid sequence and a light chain (LC) amino acid sequence, respectively, which are at least 90% identical to the amino acid sequences of SEQ ID NOs. 9 and 11.

5. The antigen-binding protein according to any one of claims 1 to 4, wherein the antibody contains at least one Fc domain mutation.

6. The antigen-binding protein according to claim 5, wherein the antibody comprises at least one Fc domain mutation that enhances the binding of the antibody to human FcγRIIIa, preferably the Fc domain mutation is optionally located at position 239, S239D and / or optionally at position 332, I332E, and the residues are numbered according to Eu numbering.

7. The antigen-binding protein according to claim 5 or 6, wherein the antibody comprises at least one Fc domain mutation that enhances the stability of the antibody.

8. The antigen-binding protein according to claim 5, wherein the antibody comprises a pair of Fc domain mutations to cysteine, optionally, the mutations are located at positions 292 and 302, optionally, the mutations are R292C and V302C, and the residues are numbered according to EU numbering.

9. The antigen-binding protein according to any one of claims 5 to 8, wherein the antibody comprises a heavy chain (HC) amino acid sequence and a light chain (LC) amino acid sequence that are at least 90% identical to the amino acid sequences of SEQ ID NOs. 10 and 11, respectively.

10. An antigen-binding protein according to any one of claims 1 to 9, which is a defucosylated antibody.

11. An antigen-binding protein according to any one of claims 1 to 10, which is a fusion protein.

12. An antigen-binding protein according to any one of claims 1 to 11, which is bispecific or multispecific.

13. An isolated nucleic acid molecule encoding an antigen-binding protein according to any one of claims 1 to 12.

14. A vector comprising an isolated nucleic acid molecule as described in claim 13, wherein the vector optionally comprises an expression control sequence.

15. A host cell comprising an isolated nucleic acid molecule according to claim 13 or a vector according to claim 14.

16. A pharmaceutical composition comprising an antigen-binding protein according to any one of claims 1 to 12, an isolated nucleic acid molecule according to claim 13, a vector according to claim 14 or a host cell according to claim 15, and a pharmaceutically acceptable excipient.

17. An antigen-binding protein according to any one of claims 1 to 12, an isolated nucleic acid molecule according to claim 13, a vector according to claim 14, a host cell according to claim 15, or a pharmaceutical composition according to claim 16, for use as a pharmaceutical, preferably for use in the treatment of cancer, more preferably the cancer being multiple myeloma, non-Hodgkin lymphoma, renal cancer, breast cancer, ovarian cancer, or pancreatic cancer.

18. A method for producing antigen-binding proteins, To prepare the host cells described in claim 15, The host cells are cultured under conditions suitable for the expression of the antigen-binding protein, and A method comprising isolating the obtained antigen-binding protein from the culture.

19. A method for treating cancer in a patient requiring cancer treatment, comprising administering to the patient a therapeutically effective amount of an antigen-binding protein according to any one of claims 1 to 12 or a pharmaceutical composition according to claim 16.

20. The method according to claim 19, wherein the cancer is multiple myeloma, non-Hodgkin lymphoma, renal cancer, breast cancer, ovarian cancer, or pancreatic cancer.

21. Use of an antigen-binding protein according to any one of claims 1 to 12, an isolated nucleic acid molecule according to claim 13, a vector according to claim 14, or a host cell according to claim 15, for the manufacture of a pharmaceutical product for treating a patient who needs it, wherein preferably the patient has cancer, and more preferably the cancer is multiple myeloma, non-Hodgkin lymphoma, renal cancer, breast cancer, ovarian cancer, or pancreatic cancer.

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