Single domain antibody aiming at GPRC5D

Abstract

A single domain antibody having binding specificity for the human GPRC5D protein is provided. Such antibodies, and multispecific counterparts and chimeric antigen receptors thereof, are capable of targeting cancer cells expressing GPRC5D, and thus are useful in the treatment of cancer, particularly hematologic cancer.

Description

Background Technology

[0001] G protein-coupled receptor (GPCR) class C, group 5 member D (GPRC5D) is an orphan, atypical class C GPCR. GPRC5D and other group 5 GPCRs possess an unusually short N-terminal domain found in class C receptors, thus they are expected to be conformationally similar to class A receptors. The functional consequences of GPRC5D activation have not been described, and its ligands remain unknown. The gene has three exons and is located on human chromosome 12p13.3. The GPRC5D receptor is highly conserved across species, with over 90% identity between humans and cynomolgus monkeys.

[0002] GPRC5D mRNA is primarily expressed in all malignant plasma cells of patients with multiple myeloma (MM). GPRC5D expression varies among different patients and is strongly correlated with plasma cell burden and genetic abnormalities such as Rb-1 deficiency. This exclusive expression of GPRC5D across plasma cell lineages makes it an ideal target for anti-myeloma antibodies. In particular, high expression is significantly associated with poor prognosis in both the disease and treatment. Multiple myeloma (MM) is the second most common hematologic malignancy, accounting for 2% of all cancer deaths.

[0003] Given its specific high expression on malignant cells, it has been proposed that specific antibodies against GPRC5D could be used to treat such malignant tumors. Summary of the Invention

[0004] This article identifies single-domain antibodies against GPRC5D. The antibodies disclosed in this article exhibit high specificity and excellent affinity. Some antibodies also show cross-reactivity with human and cynomolgus monkey GPRC5D proteins. Furthermore, due to their single-chain nature and small size, these single-domain antibodies are better suited for use in multifunctional antibodies, including T-cell adaptors, NK-cell adaptors, multifunctional proteins, bispecific or trispecific antibodies, and chimeric antigen receptor (CAR)-related therapies.

[0005] Therefore, according to one embodiment of this disclosure, a single-domain antibody or a polypeptide comprising said single-domain antibody is provided, wherein said single-domain antibody has binding specificity to human G protein-coupled receptor class C group 5 member D (GPRC5D) protein, and comprises antibodies A011, A018, A019, A020, A024, A026, A037, A038, A039, A042, A046, A049, A053, and A05 5. CDR1, CDR2, and CDR3 of any one of A056, A057, A059, B006, B020, B028, B034, B036, B037, B043, B051, B054, B057, B059, B061, B069, B074, B079, B094, B104, B108, B116, B123, B128, B142, B149, and B161. The sequences of these antibodies are provided in Tables 5 and 7, and exemplary CDR sequences (e.g., according to Kabat numbering) are shown in Tables 6 and 8.

[0006] In some embodiments, CDR1, CDR2, and CDR3 respectively comprise the following amino acid sequences: SEQ ID NO: 18, 19, and 20; SEQ ID NO: 21, 22, and 23; SEQ ID NO: 24, 25, and 26; SEQ ID NO: 24, 69, and 26; SEQ ID NO: 24, 70, and 26; SEQ ID NO: 27, 28, and 29; SEQ ID NO: 30, 31, and 32; SEQ ID NO: 33 and 34 and the amino acid sequence RFV; SEQ ID NO: 36, 37, and 38; SEQ ID NO: 39, 40, and 41; SEQ ID NO: 39, 40, and 71; SEQ ID NO: 39, 40, and 72; SEQ ID NO: 39, 40, and 73; SEQ ID NO: 42, 43, and 44; SEQ ID NO: 45, 46, and 47; SEQ ID NO: 48, 49 and 50; SEQ ID NO: 51, 52 and 53; SEQ ID NO: 54, 55 and 56; SEQ ID NO: 57, 58 and 59; SEQ ID NO: 57, 58 and 74; SEQ ID NO: 57, 58 and 75; SEQ ID NO: 60, 61 and 62; SEQ ID NO: 63, 64 and 65; or SEQ ID NO: 66, 67 and 68.

[0007] In some embodiments, CDR1, CDR2, and CDR3 respectively comprise the following amino acid sequences: SEQ ID NO: 100, 101, and 102; SEQ ID NO: 103, 104, and 105; SEQ ID NO: 106, 107, and 108; SEQ ID NO: 109, 110, and 111; SEQ ID NO: 109, 172, and 111; SEQ ID NO: 109, 173, and 111; SEQ ID NO: 109, 174, and 111; SEQ ID NO: 112, 113, and 114; SEQ ID NO: 115, 116, and 117; SEQ ID NO: 118, 175, and 120; SEQ ID NO: 118, 176, and 120; SEQ ID NO: 118, 177, and 120; SEQ ID NO: 118, 119, and 120; SEQ ID NO: SEQ ID NO: 121, 122, and 123; SEQ ID NO: 124, 125, and 126; SEQ ID NO: 127, 128, and 129; SEQ ID NO: 130, 131, and 132; SEQ ID NO: 133, 134, and 135; SEQ ID NO: 136, 137, and 138; SEQ ID NO: 139, 140, and 141; SEQ ID NO: 142, 143, and 144; SEQ ID NO: 145, 146, and 147; SEQ ID NO: 148, 149, and 150; SEQ ID NO: 151, 152, and 153; SEQ ID NO: 154, 155, and 156; SEQ ID NO: 157, 158, and 159; SEQ ID NO: 160, 161, and 162; SEQ ID NO: 163, 164 and 165; SEQ ID NO: 166, 167 and 168; SEQ ID NO: 166, 178 and 168; SEQ ID NO: 166, 179 and 168; or SEQ ID NO: 169, 170 and 171.

[0008] In some embodiments, the antibody or polypeptide comprises an amino acid sequence selected from SEQ ID NO:1-17, or an amino acid sequence having at least 80% sequence identity with a sequence selected from SEQ ID NO:1-17 and retaining the corresponding sequence CDR. In some embodiments, the antibody or polypeptide comprises an amino acid sequence selected from SEQ ID NO:1-17.

[0009] In one embodiment, CDR1 comprises the amino acid sequence of SEQ ID NO:39, CDR2 comprises the amino acid sequence of SEQ ID NO:40, and CDR3 comprises the amino acid sequence of SEQ ID NO:41, 71, 72, or 73. In one embodiment, the antibody or peptide comprises the amino acid sequence of SEQ ID NO:8, or has at least 85% sequence identity with SEQ ID NO:8 and retains the amino acid sequence of the CDR.

[0010] In one embodiment, CDR1 comprises the amino acid sequence of SEQ ID NO:54, CDR2 comprises the amino acid sequence of SEQ ID NO:55, and CDR3 comprises the amino acid sequence of SEQ ID NO:56. In one embodiment, the antibody or polypeptide comprises the amino acid sequence of SEQ ID NO:13, or has at least 85% sequence identity with SEQ ID NO:13 and retains the amino acid sequence of the CDR.

[0011] In one embodiment, CDR1 comprises the amino acid sequence of SEQ ID NO:57, CDR2 comprises the amino acid sequence of SEQ ID NO:58, and CDR3 comprises the amino acid sequence of SEQ ID NO:59, 74, or 75. In one embodiment, the antibody or peptide comprises the amino acid sequence of SEQ ID NO:14, or has at least 85% sequence identity with SEQ ID NO:14 and retains the amino acid sequence of the CDR.

[0012] In one embodiment, CDR1 comprises the amino acid sequence of SEQ ID NO:63, CDR2 comprises the amino acid sequence of SEQ ID NO:64, and CDR3 comprises the amino acid sequence of SEQ ID NO:65. In one embodiment, the antibody or polypeptide comprises the amino acid sequence of SEQ ID NO:16, or has at least 85% sequence identity with SEQ ID NO:16 and retains the amino acid sequence of the CDR.

[0013] In some embodiments, the antibody or polypeptide comprises an amino acid sequence selected from SEQ ID NO: 76-99, or an amino acid sequence having at least 80% sequence identity with a sequence selected from SEQ ID NO: 76-99 and retaining the corresponding sequence CDR. In some embodiments, the antibody or polypeptide comprises an amino acid sequence selected from SEQ ID NO: 76-99.

[0014] In various implementations, chimeric antigen receptors (CARs), bispecific antibodies, trispecific antibodies, multifunctional proteins, and cells engineered to express any of these proteins are also provided.

[0015] Treatment methods and uses are also provided. In one embodiment, a method of treating cancer in a patient in need is provided, comprising administering to the patient the antibody or peptide, the CAR, the polynucleotide encoding them, cells engineered to express them, or the antibody-drug conjugate of this disclosure. In some embodiments, the cancer is a blood cancer, such as a B-cell cancer expressing GPRC5D (e.g., multiple myeloma). Attached Figure Description

[0016] Figure 1A -C shows the binding of the test antibody to human GPRC5D expressed on HEK293 cells.

[0017] Figure 2A -C shows the binding of the test antibody to cynomolgus monkey GPRC5D expressed on HEK293 cells.

[0018] Figure 3A -C indicates the binding of the tested antibody to GPRC5D-positive human B lymphoblasts with multiple myeloma.

[0019] Figure 4 The binding of the test antibody to HEK293 cells expressing GPRC5D was shown.

[0020] Figure 5 The test antibody showed no nonspecific binding to GPRC5A and GRPC5C expressed on HEK293 cells.

[0021] Figure 6 The study showed that the tested CAR interacted strongly with wild-type H929 (H929-WT), but the CAR-mediated signal was lower when co-cultured with GPRC5D knockout H929 (H929-GPRC5D-KO).

[0022] Figure 7 The test CAR showed stronger antitumor activity compared to the positive control. Detailed Implementation definition

[0023] It should be noted that the term "a" entity refers to one or more of the same entity; for example, "an antibody" should be understood as one or more antibodies. Therefore, the terms "a," "one or more," and "at least one" are used interchangeably in this document.

[0024] "Sequence identity" of a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) with another sequence having a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) means that, when aligned, that percentage of bases (or amino acids) are identical when comparing the two sequences. This alignment and percentage homology or sequence identity can be determined using software programs known in the art, such as those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, alignment is performed using default parameters. One alignment program is BLAST, using default parameters. Specifically, the procedures are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filtering = none; strands = two; truncation value = 60; expected value = 10; matrix = BLOSUM62; description = 50 sequences; sorting = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Biologically equivalent polynucleotides are those that have the percentage of homology specified above and encode polypeptides with the same or similar biological activities.

[0025] The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having a nucleotide sequence that is homologous or sequence-identical to the nucleotide sequence of the nucleic acid or its complementary sequence. Homologs of double-stranded nucleic acids are intended to include nucleic acids having a nucleotide sequence that is homologous to the nucleic acid or its complementary sequence. In one aspect, the homolog of the nucleic acid is capable of hybridizing with the nucleic acid or its complementary sequence. Similarly, "equivalent polypeptide" refers to a polypeptide having a degree of homology or sequence identity to the amino acid sequence of a reference polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some aspects, the equivalent polypeptide or polynucleotide has one, two, three, four, or five additions, deletions, substitutions, or combinations thereof compared to the reference polypeptide or polynucleotide. In some aspects, the equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridge) of the reference sequence.

[0026] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody or any antigen-binding fragment or single chain. Therefore, the term "antibody" includes any protein or peptide molecule that contains at least a portion of an immunoglobulin molecule and has the biological activity of binding to an antigen. Examples include, but are not limited to: the complementarity-determining region (CDR) of the heavy or light chain or its ligand-binding portion, the variable region of the heavy or light chain, the constant region of the heavy or light chain, the frame region (FR) or any portion thereof, or at least a portion of a binding protein.

[0027] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to a part of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, mirror isoforms, and bispecific antibodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.

[0028] "Single-chain variable fragment" or "scFv" refers to the immunoglobulin heavy chain (V). H ) and light chains (V L A fusion protein with variable regions. In some respects, these regions are linked by short linker peptides of 10 to approximately 25 amino acids. The linkers may be enriched with glycine for flexibility, and serine or threonine for solubility, and may link V... H The N-terminus and V L The C-terminus is the same, and vice versa. Despite the removal of the constant region and the introduction of a linker, the protein retains the specificity of the original immunoglobulin. ScFv molecules are known in the art, for example, as described in U.S. Patent 5,892,019.

[0029] The term antibody encompasses a wide range of biochemically distinguishable classes of polypeptides. Those skilled in the art will understand that heavy chains are classified as γ, μ, α, δ, or ε, with several subclasses (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of the antibody, namely IgG, IgM, IgA, IgG, or IgE. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well-characterized and known to confer functional specificity. Modified versions of these classes and isotypes can be readily identified by those skilled in the art based on this disclosure, and therefore, they are within the scope of this disclosure. All immunoglobulin classes are clearly defined within the scope of this disclosure; the following discussion will generally refer to IgG class immunoglobulin molecules. Regarding IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000 Daltons. These four chains are typically connected in a "Y"-shaped structure by disulfide bonds, where the light chain begins to enclose the heavy chain at the opening of the "Y" and extends all the way to the variable region.

[0030] The antibodies, antigen-binding peptides, variants, or derivatives disclosed herein include, but are not limited to: polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, primate-like antibodies or chimeric antibodies, single-chain antibodies, epitope-binding fragments (e.g., Fab, Fab', and F(ab')2, Fd, Fv, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), fragments containing VK or VH domains, fragments generated by Fab expression libraries, and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies against the LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0031] Single-domain antibodies (sdAbs), also known as nanobodies, are antibody fragments composed of a single monomeric variable antibody domain. Nanobodies produced by camels and some other animals are also called VHH fragments. Like intact antibodies, nanobodies can selectively bind to specific antigens. Single-domain antibodies have a molecular weight of only 12-15 kDa, much smaller than ordinary antibodies (150-160 kDa).

[0032] "Specific binding" or "specific to" generally refers to an antibody binding to an epitope through its antigen-binding domain, and this binding requires a certain complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to it more easily than to a random, unrelated epitope. The term "specific" is used in this document to define the relative affinity of an antibody for a given epitope. For example, antibody "A" can be considered to have higher specificity for a given epitope than antibody "B," or antibody "A" may have higher binding specificity to epitope "C" than to its binding specificity to related epitope "D."

[0033] As used herein, the terms "treatment" or "management" refer to therapeutic treatment and preventative or preventative measures aimed at preventing or slowing (alleviating) undesirable physiological changes or conditions, such as the progression of cancer. Beneficial or desired clinical outcomes include, but are not limited to: symptom relief, reduction in disease severity, stable disease state (i.e., no worsening), delayed or slowed disease progression, improved or alleviated disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" can also refer to extended survival compared to expected survival without treatment. Individuals requiring treatment include those who already have the condition or disorder, those susceptible to the condition or disorder, or those seeking to prevent the condition or disorder.

[0034] The terms "subject / object," "individual," "animal," "patient," or "mammal" refer to any subject requiring diagnosis, prognosis, or treatment, particularly mammalian subjects. Mammal subjects include humans, domestic animals, farm animals, and zoo, sporting, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and dairy cows.

[0035] As used herein, phrases such as "patient requiring treatment" or "subject requiring treatment" include subjects, such as mammalian subjects, who would benefit from the administration of the antibodies or compositions of this disclosure, for example, in the context of detection, diagnostic procedures, and / or treatment.

[0036] Where two or more definitions of a term exist that are used and / or accepted in the art, the definitions of terms used herein are intended to include all such meanings unless explicitly stated otherwise. A specific example is the use of the term "complementarity-determining region" ("CDR") to describe discontinuous antigen-binding sites found within the variable regions of heavy and light chain polypeptides. This particular region has been described by Kabat et al., US Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and Chothia et al., J. MoI. Biol. 196:901-917 (1987), the entire contents of which are incorporated herein by reference. The CDR definitions by Kabat and Chothia include overlapping or subsets of amino acid residues when compared with each other. However, the application of either definition to refer to the CDR of an antibody or a variant thereof is intended to fall within the scope of the terminology defined and used herein. The following table lists the appropriate amino acid residues of the CDRs covered according to each of the above-cited references for comparison. The exact residue numbering containing a specific CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues constitute a specific CDR given the amino acid sequence of the variable region of an antibody.

[0037] Kabat et al. also defined a numbering system for variable-domain sequences applicable to any antibody. Those skilled in the art can explicitly assign this "Kabat numbering" system to any variable-domain sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described in Kabat et al., USDept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983). Anti-GPRC5D single-domain antibody

[0038] By screening a humanized single-domain antibody library, the inventors were able to identify a large number of single-domain antibodies against human GPRC5D protein, including A003, A004, A007, A009, A011, A018, A019, A020, A022, A024, A026, A036, A037, A038, A039, A042, A046, A049, A053, A054, A055, A056, A057, A058, and A059. These antibodies underwent cell-based binding assays, including tests with HEC293 cells engineered to express human GPRC5D protein, cynomolgus monkey GPRC5D protein, and GPRC5D-positive human B lymphoblasts with multiple myeloma. Talquetamab (JNJ-64407564) (a bispecific IgG4 antibody) was used as the baseline antibody.

[0039] The results showed that many of these antibodies, despite being single-domain antibodies, exhibited excellent binding specificity and affinity comparable to talquetamab. Furthermore, unlike talquetamab, which does not cross-react with cynomolgus monkey GPRC5D protein, some of the newly identified antibodies demonstrated excellent cross-reactivity. These antibodies, A011, A018, A019, A020, A024, A026, A037, A038, A039, A042, A046, A049, A053, A055, A056, A057, and A059, are suitable for further clinical development.

[0040] Similar screening and testing were performed on the immunomodulatory nanobody library, from which superior binding agents were identified. The best antibodies included B006, B020, B028, B034, B036, B037, B043, B051, B054, B057, B059, B061, B069, B074, B079, B094, B104, B108, B116, B123, B128, B142, B149, and B161, which were also considered suitable for further clinical development. In particular, B051, B061, B104, B128, and B161 showed strong specific binding to GPRC5D and no nonspecific binding to GRPC5A and GPRC5C (Example 7). When constructed into chimeric antigen receptors (CARs), these antibodies exhibited potent antitumor activity (Example 8).

[0041] Therefore, according to one embodiment of this disclosure, a single-domain antibody with binding specificity to the human G protein-coupled receptor class C group 5 member D (GPRC5D) protein is provided. According to one embodiment of this disclosure, a single-domain antibody and a polypeptide comprising such a single-domain antibody are also provided.

[0042] In some embodiments, the single-domain antibody comprises CDR1, CDR2, and CDR3, which respectively have the CDR1, CDR2, and CDR3 sequences of any one of the identified antibodies A011, A018, A019, A020, A024, A026, A037, A038, A039, A042, A046, A049, A053, A055, A056, A057, and A059.

[0043] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody A057 (SEQ ID NO:1). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:18, CDR2 comprises the amino acid sequence of SEQ ID NO:19, and CDR3 comprises the amino acid sequence of SEQ ID NO:20.

[0044] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3, and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:1. In some embodiments, an antibody that binds to the same epitope on GPRC5D as A011 is also provided. In some embodiments, an antibody that competes with A011 for binding to GPRC5D is also provided.

[0045] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody A020 ​​(SEQ ID NO:2). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:21, CDR2 comprises the amino acid sequence of SEQ ID NO:22, and CDR3 comprises the amino acid sequence of SEQ ID NO:23.

[0046] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:2. In some embodiments, an antibody that binds to the same epitope on GPRC5D as A020 ​​is also provided. In some embodiments, an antibody that competes with A020 ​​for binding to GPRC5D is also provided.

[0047] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody A038 (SEQ ID NO:3). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:24, CDR2 comprises the amino acid sequence of SEQ ID NO:25, and CDR3 comprises the amino acid sequence of SEQ ID NO:26.

[0048] Some residues in CDR2 of A038 were found ( Post-translational modifications (PTMs) may occur at residues (SEQ ID NO: 25), and substitution of these residues can prevent or reduce this. Therefore, de-risked CDR (de-PTM) versions of this type are also provided. In one embodiment, de-risked CDR2 is SEQ ID NO: 69 (… In another implementation, the risk-free CDR2 is SEQ ID NO: 70 ( ).

[0049] Therefore, in one embodiment, CDR1 comprises the amino acid sequence of SEQ ID NO:24, CDR2 comprises the amino acid sequence of SEQ ID NO:69, and CDR3 comprises the amino acid sequence of SEQ ID NO:26. In another embodiment, CDR1 comprises the amino acid sequence of SEQ ID NO:24, CDR2 comprises the amino acid sequence of SEQ ID NO:70, and CDR3 comprises the amino acid sequence of SEQ ID NO:26.

[0050] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the antibody comprises CDR1, CDR2 (including SEQ ID NO: 25, 69, or 70) and CDR3, and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:3. In some embodiments, an antibody that binds to the same epitope on GPRC5D as A038 is also provided. In some embodiments, an antibody that competes with A038 for binding to GPRC5D is also provided.

[0051] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody A026 (SEQ ID NO:4). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:27, CDR2 comprises the amino acid sequence of SEQ ID NO:28, and CDR3 comprises the amino acid sequence of SEQ ID NO:29.

[0052] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:4. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:4. In some embodiments, an antibody that binds to the same epitope on GPRC5D as A026 is also provided. In some embodiments, an antibody that competes with A026 for binding to GPRC5D is also provided.

[0053] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody A055 (SEQ ID NO:5). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:30, CDR2 comprises the amino acid sequence of SEQ ID NO:31, and CDR3 comprises the amino acid sequence of SEQ ID NO:32.

[0054] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:5. In some embodiments, an antibody that binds to the same epitope on GPRC5D as A055 is also provided. In some embodiments, an antibody that competes with A055 for binding to GPRC5D is also provided.

[0055] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody A037 (SEQ ID NO:6). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:33, CDR2 comprises the amino acid sequence of SEQ ID NO:34, and CDR3 comprises the amino acid sequence of SEQ ID NO:35 (RFV).

[0056] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:6. In some embodiments, an antibody that binds to the same epitope on GPRC5D as A037 is also provided. In some embodiments, an antibody that competes with A037 for binding to GPRC5D is also provided.

[0057] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody A018 (SEQ ID NO:7). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:36, CDR2 comprises the amino acid sequence of SEQ ID NO:37, and CDR3 comprises the amino acid sequence of SEQ ID NO:38.

[0058] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:7. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:7. In some embodiments, an antibody that binds to the same epitope on GPRC5D as A018 is also provided. In some embodiments, an antibody that competes with A018 for binding to GPRC5D is also provided.

[0059] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody A049 (SEQ ID NO:8). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:39, CDR2 comprises the amino acid sequence of SEQ ID NO:40, and CDR3 comprises the amino acid sequence of SEQ ID NO:41.

[0060] Certain residues in CDR3 of A049 were found ( Post-translational modifications (PTMs) may occur at residues SEQ ID NO: 41, and substitution of these residues can prevent or reduce this. Therefore, de-risked CDR (de-PTM) versions of this type are also provided. In one embodiment, the de-risked CDR3 is SEQ ID NO: 71 (…). In another implementation, the risk-free CDR3 is SEQ ID NO: 72 ( In another implementation, the risk-free CDR3 is SEQ ID NO: 73 ( ).

[0061] Therefore, in one embodiment, CDR1 comprises the amino acid sequence of SEQ ID NO:39, CDR2 comprises the amino acid sequence of SEQ ID NO:40, and CDR3 comprises the amino acid sequence of SEQ ID NO:71. In one embodiment, CDR1 comprises the amino acid sequence of SEQ ID NO:39, CDR2 comprises the amino acid sequence of SEQ ID NO:40, and CDR3 comprises the amino acid sequence of SEQ ID NO:72. In one embodiment, CDR1 comprises the amino acid sequence of SEQ ID NO:39, CDR2 comprises the amino acid sequence of SEQ ID NO:40, and CDR3 comprises the amino acid sequence of SEQ ID NO:73.

[0062] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:8. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 (including SEQ ID NO: 41, 71, 72, or 73) and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:8. In some embodiments, an antibody that binds to the same epitope on GPRC5D as A049 is also provided. In some embodiments, an antibody that competes with A049 for binding to GPRC5D is also provided.

[0063] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody A053 (SEQ ID NO:9). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:42, CDR2 comprises the amino acid sequence of SEQ ID NO:43, and CDR3 comprises the amino acid sequence of SEQ ID NO:44.

[0064] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:9. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:9. In some embodiments, an antibody that binds to the same epitope on GPRC5D as A053 is also provided. In some embodiments, an antibody that competes with A053 for binding to GPRC5D is also provided.

[0065] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody A046 (SEQ ID NO:10). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:45, CDR2 comprises the amino acid sequence of SEQ ID NO:46, and CDR3 comprises the amino acid sequence of SEQ ID NO:47.

[0066] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:10. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:10. In some embodiments, an antibody that binds to the same epitope on GPRC5D as A046 is also provided. In some embodiments, an antibody that competes with A046 for binding to GPRC5D is also provided.

[0067] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody A039 (SEQ ID NO:11). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:48, CDR2 comprises the amino acid sequence of SEQ ID NO:49, and CDR3 comprises the amino acid sequence of SEQ ID NO:50.

[0068] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:11. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3, and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:11. In some embodiments, an antibody that binds to the same epitope on GPRC5D as A039 is also provided. In some embodiments, an antibody that competes with A039 for binding to GPRC5D is also provided.

[0069] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody A011 (SEQ ID NO:12). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:51, CDR2 comprises the amino acid sequence of SEQ ID NO:52, and CDR3 comprises the amino acid sequence of SEQ ID NO:53.

[0070] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:12. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:12. In some embodiments, an antibody that binds to the same epitope on GPRC5D as A011 is also provided. In some embodiments, an antibody that competes with A011 for binding to GPRC5D is also provided.

[0071] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody A042 (SEQ ID NO:13). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:54, CDR2 comprises the amino acid sequence of SEQ ID NO:55, and CDR3 comprises the amino acid sequence of SEQ ID NO:56.

[0072] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:13. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3, and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:13. In some embodiments, an antibody that binds to the same epitope on GPRC5D as A042 is also provided. In some embodiments, an antibody that competes with A042 for binding to GPRC5D is also provided.

[0073] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody A024 (SEQ ID NO:14). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:57, CDR2 comprises the amino acid sequence of SEQ ID NO:58, and CDR3 comprises the amino acid sequence of SEQ ID NO:59.

[0074] Some residues in CDR3 of A024 were found ( Post-translational modifications (PTMs) may occur at residues (SEQ ID NO: 59), and substitution of these residues can prevent or reduce this. Therefore, de-risked CDR (de-PTM) versions of this type are also provided. In one embodiment, the de-risked CDR3 is SEQ ID NO: 74 (…). In another implementation, the de-risking CDR3 is SEQ ID NO: 75 ( ).

[0075] Therefore, in one embodiment, CDR1 comprises the amino acid sequence of SEQ ID NO:57, CDR2 comprises the amino acid sequence of SEQ ID NO:58, and CDR3 comprises the amino acid sequence of SEQ ID NO:74. In one embodiment, CDR1 comprises the amino acid sequence of SEQ ID NO:57, CDR2 comprises the amino acid sequence of SEQ ID NO:58, and CDR3 comprises the amino acid sequence of SEQ ID NO:75.

[0076] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:14. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 (including SEQ ID NO: 59, 74, or 75) and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:14. In some embodiments, an antibody that binds to the same epitope on GPRC5D as A024 is also provided. In some embodiments, an antibody that competes with A024 for binding to GPRC5D is also provided.

[0077] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody A059 (SEQ ID NO:15). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:60, CDR2 comprises the amino acid sequence of SEQ ID NO:61, and CDR3 comprises the amino acid sequence of SEQ ID NO:62.

[0078] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:15. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:15. In some embodiments, an antibody that binds to the same epitope on GPRC5D as A059 is also provided. In some embodiments, an antibody that competes with A059 for binding to GPRC5D is also provided.

[0079] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody A019 (SEQ ID NO:16). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:63, CDR2 comprises the amino acid sequence of SEQ ID NO:64, and CDR3 comprises the amino acid sequence of SEQ ID NO:65.

[0080] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:16. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:16. In some embodiments, an antibody that binds to the same epitope on GPRC5D as A016 is also provided. In some embodiments, an antibody that competes with A016 for binding to GPRC5D is also provided.

[0081] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody A056 (SEQ ID NO:17). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:66, CDR2 comprises the amino acid sequence of SEQ ID NO:67, and CDR3 comprises the amino acid sequence of SEQ ID NO:68.

[0082] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:17. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:17. In some embodiments, an antibody that binds to the same epitope on GPRC5D as A056 is also provided. In some embodiments, an antibody that competes with A056 for binding to GPRC5D is also provided.

[0083] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody B006 (SEQ ID NO:76). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:100, CDR2 comprises the amino acid sequence of SEQ ID NO:101, and CDR3 comprises the amino acid sequence of SEQ ID NO:102.

[0084] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:76. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3, and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:76. In some embodiments, an antibody that binds to the same epitope on GPRC5D as B006 is also provided. In some embodiments, an antibody that competes with B006 for binding to GPRC5D is also provided.

[0085] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody B020 (SEQ ID NO:77). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:103, CDR2 comprises the amino acid sequence of SEQ ID NO:104, and CDR3 comprises the amino acid sequence of SEQ ID NO:105.

[0086] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:77. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3, and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:77. In some embodiments, an antibody that binds to the same epitope on GPRC5D as BO20 is also provided. In some embodiments, an antibody that competes with BO20 for binding to GPRC5D is also provided.

[0087] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody B028 (SEQ ID NO:78). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:106, CDR2 comprises the amino acid sequence of SEQ ID NO:107, and CDR3 comprises the amino acid sequence of SEQ ID NO:108.

[0088] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:78. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:78. In some embodiments, an antibody that binds to the same epitope on GPRC5D as B028 is also provided. In some embodiments, an antibody that competes with B028 for binding to GPRC5D is also provided.

[0089] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody B034 (SEQ ID NO:79). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:109, CDR2 comprises the amino acid sequence of SEQ ID NO:110, and CDR3 comprises the amino acid sequence of SEQ ID NO:111.

[0090] Some residues in CDR2 of B034 were found ( Post-translational modifications (PTMs) may occur at residues (SEQ ID NO: 110), and substitution of these residues can prevent or reduce this. Therefore, de-risked CDR (de-PTM) versions of this type are also provided. In one embodiment, the de-risked CDR2 is SEQ ID NO: 172 (…). In another implementation, the risk-free CDR2 is SEQ ID NO: 173 ( In another implementation, the risk-free CDR2 is SEQ ID NO: 174 ( ).

[0091] Therefore, in one embodiment, CDR1 comprises the amino acid sequence of SEQ ID NO:109, CDR2 comprises the amino acid sequence of SEQ ID NO:172, and CDR3 comprises the amino acid sequence of SEQ ID NO:111. In one embodiment, CDR1 comprises the amino acid sequence of SEQ ID NO:109, CDR2 comprises the amino acid sequence of SEQ ID NO:173, and CDR3 comprises the amino acid sequence of SEQ ID NO:111. In one embodiment, CDR1 comprises the amino acid sequence of SEQ ID NO:109, CDR2 comprises the amino acid sequence of SEQ ID NO:174, and CDR3 comprises the amino acid sequence of SEQ ID NO:111.

[0092] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:79. In some embodiments, the antibody comprises CDR1, CDR2 (including SEQ ID NO: 172, 173, or 174) and CDR3, and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:79. In some embodiments, an antibody that binds to the same epitope on GPRC5D as B034 is also provided. In some embodiments, an antibody that competes with B034 for binding to GPRC5D is also provided.

[0093] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody B036 (SEQ ID NO: 80). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 112, CDR2 comprises the amino acid sequence of SEQ ID NO: 113, and CDR3 comprises the amino acid sequence of SEQ ID NO: 114.

[0094] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:80. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:80. In some embodiments, an antibody that binds to the same epitope on GPRC5D as B036 is also provided. In some embodiments, an antibody that competes with B036 for binding to GPRC5D is also provided.

[0095] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody B037 (SEQ ID NO: 81). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 115, CDR2 comprises the amino acid sequence of SEQ ID NO: 116, and CDR3 comprises the amino acid sequence of SEQ ID NO: 117.

[0096] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:81. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3, and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:81. In some embodiments, an antibody that binds to the same epitope on GPRC5D as B037 is also provided. In some embodiments, an antibody that competes with B037 for binding to GPRC5D is also provided.

[0097] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody B043 (SEQ ID NO: 82). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 118, CDR2 comprises the amino acid sequence of SEQ ID NO: 119, and CDR3 comprises the amino acid sequence of SEQ ID NO: 120.

[0098] Some residues in CDR2 of B043 were found ( The residues (SEQ ID NO: 119) may undergo post-translational modifications (PTMs), and if these residues are substituted, this can be prevented or reduced. Therefore, a de-risked CDR (de-PTM) version of this type is also provided. In one embodiment, the de-risked CDR2 is SEQ ID NO: 175 (…). In another implementation, the de-risking CDR2 is SEQ ID NO: 176 ( In another implementation, the risk-free CDR2 is SEQ ID NO: 177 ( ).

[0099] Therefore, in one embodiment, CDR1 comprises the amino acid sequence of SEQ ID NO:118, CDR2 comprises the amino acid sequence of SEQ ID NO:175, and CDR3 comprises the amino acid sequence of SEQ ID NO:120. In one embodiment, CDR1 comprises the amino acid sequence of SEQ ID NO:118, CDR2 comprises the amino acid sequence of SEQ ID NO:176, and CDR3 comprises the amino acid sequence of SEQ ID NO:120. In one embodiment, CDR1 comprises the amino acid sequence of SEQ ID NO:118, CDR2 comprises the amino acid sequence of SEQ ID NO:177, and CDR3 comprises the amino acid sequence of SEQ ID NO:120.

[0100] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO: 82. In some embodiments, the antibody comprises CDR1, CDR2 (including SEQ ID NO: 175, 176, or 177) and CDR3, and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 82. In some embodiments, an antibody that binds to the same epitope on GPRC5D as B043 is also provided. In some embodiments, an antibody that competes with B043 for binding to GPRC5D is also provided.

[0101] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody B051 (SEQ ID NO: 83). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 121, CDR2 comprises the amino acid sequence of SEQ ID NO: 122, and CDR3 comprises the amino acid sequence of SEQ ID NO: 123.

[0102] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:83. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:83. In some embodiments, an antibody that binds to the same epitope on GPRC5D as B051 is also provided. In some embodiments, an antibody that competes with B051 for binding to GPRC5D is also provided.

[0103] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody B054 (SEQ ID NO:84). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:124, CDR2 comprises the amino acid sequence of SEQ ID NO:125, and CDR3 comprises the amino acid sequence of SEQ ID NO:126.

[0104] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:84. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:84. In some embodiments, an antibody that binds to the same epitope on GPRC5D as B054 is also provided. In some embodiments, an antibody that competes with B054 for binding to GPRC5D is also provided.

[0105] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody B057 (SEQ ID NO:85). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:127, CDR2 comprises the amino acid sequence of SEQ ID NO:128, and CDR3 comprises the amino acid sequence of SEQ ID NO:129.

[0106] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:85. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:85. In some embodiments, an antibody that binds to the same epitope on GPRC5D as B057 is also provided. In some embodiments, an antibody that competes with B057 for binding to GPRC5D is also provided.

[0107] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody B059 (SEQ ID NO: 86). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 130, CDR2 comprises the amino acid sequence of SEQ ID NO: 131, and CDR3 comprises the amino acid sequence of SEQ ID NO: 132.

[0108] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:86. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:86. In some embodiments, an antibody that binds to the same epitope on GPRC5D as B059 is also provided. In some embodiments, an antibody that competes with B059 for binding to GPRC5D is also provided.

[0109] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody B061 (SEQ ID NO: 87). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 133, CDR2 comprises the amino acid sequence of SEQ ID NO: 134, and CDR3 comprises the amino acid sequence of SEQ ID NO: 135.

[0110] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:87. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:87. In some embodiments, an antibody that binds to the same epitope on GPRC5D as B061 is also provided. In some embodiments, an antibody that competes with B061 for binding to GPRC5D is also provided.

[0111] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody B069 (SEQ ID NO:88). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:136, CDR2 comprises the amino acid sequence of SEQ ID NO:137, and CDR3 comprises the amino acid sequence of SEQ ID NO:138.

[0112] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:88. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:88. In some embodiments, an antibody that binds to the same epitope on GPRC5D as B069 is also provided. In some embodiments, an antibody that competes with B069 for binding to GPRC5D is also provided.

[0113] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody B074 (SEQ ID NO: 89). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 139, CDR2 comprises the amino acid sequence of SEQ ID NO: 140, and CDR3 comprises the amino acid sequence of SEQ ID NO: 141.

[0114] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:89. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:89. In some embodiments, an antibody that binds to the same epitope on GPRC5D as B074 is also provided. In some embodiments, an antibody that competes with B074 for binding to GPRC5D is also provided.

[0115] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody B079 (SEQ ID NO: 90). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 142, CDR2 comprises the amino acid sequence of SEQ ID NO: 143, and CDR3 comprises the amino acid sequence of SEQ ID NO: 144.

[0116] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:90. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:90. In some embodiments, an antibody that binds to the same epitope on GPRC5D as B079 is also provided. In some embodiments, an antibody that competes with B079 for binding to GPRC5D is also provided.

[0117] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody B094 (SEQ ID NO: 91). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 145, CDR2 comprises the amino acid sequence of SEQ ID NO: 146, and CDR3 comprises the amino acid sequence of SEQ ID NO: 147.

[0118] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:91. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:91. In some embodiments, an antibody that binds to the same epitope on GPRC5D as B094 is also provided. In some embodiments, an antibody that competes with B094 for binding to GPRC5D is also provided.

[0119] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody B104 (SEQ ID NO: 92). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 148, CDR2 comprises the amino acid sequence of SEQ ID NO: 149, and CDR3 comprises the amino acid sequence of SEQ ID NO: 150.

[0120] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:92. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:92. In some embodiments, an antibody that binds to the same epitope on GPRC5D as B104 is also provided. In some embodiments, an antibody that competes with B104 for binding to GPRC5D is also provided.

[0121] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody B108 (SEQ ID NO: 93). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 151, CDR2 comprises the amino acid sequence of SEQ ID NO: 152, and CDR3 comprises the amino acid sequence of SEQ ID NO: 153.

[0122] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:93. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3, and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:93. In some embodiments, an antibody that binds to the same epitope on GPRC5D as B108 is also provided. In some embodiments, an antibody that competes with B108 for binding to GPRC5D is also provided.

[0123] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody B116 (SEQ ID NO: 94). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 154, CDR2 comprises the amino acid sequence of SEQ ID NO: 155, and CDR3 comprises the amino acid sequence of SEQ ID NO: 156.

[0124] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:94. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3, and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:94. In some embodiments, an antibody that binds to the same epitope on GPRC5D as B116 is also provided. In some embodiments, an antibody that competes with B116 for binding to GPRC5D is also provided.

[0125] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody B123 (SEQ ID NO:95). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:157, CDR2 comprises the amino acid sequence of SEQ ID NO:158, and CDR3 comprises the amino acid sequence of SEQ ID NO:159.

[0126] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:95. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:95. In some embodiments, an antibody that binds to the same epitope on GPRC5D as B123 is also provided. In some embodiments, an antibody that competes with B123 for binding to GPRC5D is also provided.

[0127] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody B128 (SEQ ID NO: 96). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 160, CDR2 comprises the amino acid sequence of SEQ ID NO: 161, and CDR3 comprises the amino acid sequence of SEQ ID NO: 162.

[0128] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:96. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:96. In some embodiments, an antibody that binds to the same epitope on GPRC5D as B128 is also provided. In some embodiments, an antibody that competes with B128 for binding to GPRC5D is also provided.

[0129] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody B142 (SEQ ID NO: 97). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 163, CDR2 comprises the amino acid sequence of SEQ ID NO: 164, and CDR3 comprises the amino acid sequence of SEQ ID NO: 165.

[0130] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:97. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:97. In some embodiments, an antibody that binds to the same epitope on GPRC5D as B142 is also provided. In some embodiments, an antibody that competes with B142 for binding to GPRC5D is also provided.

[0131] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody B149 (SEQ ID NO: 98). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 166, CDR2 comprises the amino acid sequence of SEQ ID NO: 167, and CDR3 comprises the amino acid sequence of SEQ ID NO: 168.

[0132] Certain residues in CDR2 of B149 were found ( The residues (SEQ ID NO: 167) may undergo post-translational modifications (PTMs), and substitution of these residues can prevent or reduce this. Therefore, a de-risked CDR (de-PTM) version of this type is also provided. In one embodiment, the de-risked CDR2 is SEQ ID NO: 178 (…). In another implementation, the risk-free CDR2 is SEQ ID NO: 179 ( ).

[0133] Therefore, in one embodiment, CDR1 comprises the amino acid sequence of SEQ ID NO:166, CDR2 comprises the amino acid sequence of SEQ ID NO:178, and CDR3 comprises the amino acid sequence of SEQ ID NO:168. In another embodiment, CDR1 comprises the amino acid sequence of SEQ ID NO:166, CDR2 comprises the amino acid sequence of SEQ ID NO:179, and CDR3 comprises the amino acid sequence of SEQ ID NO:168.

[0134] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO: 98. In some embodiments, the antibody comprises CDR1, CDR2 (including SEQ ID NO: 178 or 179) and CDR3, and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 98. In some embodiments, an antibody that binds to the same epitope on GPRC5D as B149 is also provided. In some embodiments, an antibody that competes with B149 for binding to GPRC5D is also provided.

[0135] In one embodiment, the single-domain antibody comprises CDR1, CDR2, and CDR3 of antibody B161 (SEQ ID NO: 99). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 169, CDR2 comprises the amino acid sequence of SEQ ID NO: 170, and CDR3 comprises the amino acid sequence of SEQ ID NO: 171.

[0136] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:99. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3, and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:99. In some embodiments, an antibody that binds to the same epitope on GPRC5D as B161 is also provided. In some embodiments, an antibody that competes with B161 for binding to GPRC5D is also provided.

[0137] "Conservative amino acid substitution" refers to replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the prior art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, non-essential amino acid residues in immunoglobulin polypeptides are preferably replaced by another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced by a string of amino acids that are structurally similar but differ in the order and / or composition of the side chain family members.

[0138] Those skilled in the art will also understand that the antibodies disclosed herein can be modified so that their amino acid sequences differ from those of the naturally occurring binding polypeptides from which they are derived. For example, a polypeptide or amino acid sequence derived from a specified protein may be similar to the starting sequence, for example, having a certain percentage of identity with the starting sequence, such as being 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the starting sequence.

[0139] The antibodies, variants, or derivatives disclosed herein include modified derivatives, i.e., modified by covalent linking any type of molecule to the antibody such that the covalent link does not prevent the antibody from binding to the epitope. For example, but not limited to, antibodies can be modified by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or linking to cellular ligands or other proteins. Any combination of chemical modifications can be performed using known techniques, including but not limited to: specific chemical cleavage, acetylation, formylation, and the metabolic synthesis of tunicamycin. Additionally, antibodies may contain one or more non-classical amino acids. Antibody-drug conjugates

[0140] In some implementations, the antibody may be conjugated with a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.

[0141] In one embodiment, the antibody of this disclosure is covalently linked to a pharmaceutical moiety. The pharmaceutical moiety may be, or may be modified to include, a group that reacts with a coupling site on the antibody. For example, the pharmaceutical moiety may be linked via alkylation (e.g., at the ε-amino group of lysine or the N-terminus of the antibody), reductive amination of oxidized carbohydrates, transesterification between hydroxyl and carboxyl groups, amidation of an amino or carboxyl group, and coupling with a thiol group.

[0142] In some embodiments, the average range of p for the number of drug moieties conjugated to each antibody molecule is 1 to 8; 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, the average range of p is 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In other embodiments, the average range of p is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the average range of p is about 1 to about 20, about 1 to about 10, about 2 to about 10, about 2 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, or about 1 to about 2. In some embodiments, the range of p is about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, or about 2 to about 3.

[0143] For example, when the chemical activation of a protein leads to the formation of a free thiol group, the protein can be coupled with a thiol-reacting agent. In one aspect, the agent is a reagent that is substantially specific to free thiol groups. Such agents include, for example, maleimides, haloacetamides (e.g., iodinated, brominated, or chlorinated), haloesters (e.g., iodinated, brominated, or chlorinated), halomethyl ketones (e.g., iodinated, brominated, or chlorinated), benzyl halides (e.g., iodinated, brominated, or chlorinated), vinyl sulfones, and pyridyl thioides.

[0144] The drug can be linked to an antibody or fragment via a linker. Suitable linkers include, for example, cleavable and non-cleavable linkers. Cleavable linkers are generally readily cleavable under intracellular conditions. Suitable cleavable linkers include, for example, peptide linkers that can be cleaved by intracellular proteases, such as lysosomal proteases or endosomal proteases. In an exemplary embodiment, the linker may be a dipeptide linker, such as a valine-citrulline linker, a phenylalanine-lysine linker, or a maleimide hexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl linker (mc-Val-Cit-PABA). Another linker is sulfosuccinimide-4-[N-maleimidemethyl]cyclohexane-1-carboxylate (smcc). Sulfonyl-smcc coupling occurs via a maleimide group that reacts with a thiol group (-SH), while its sulfonyl-NHS ester reacts with a primary amine, such as lysine and a primary amine at the N-terminus of a protein or peptide. Another linker is maleimide hexanoyl (mc). Other suitable adapters include those that are hydrolyzable at a specific pH or within a pH range, such as hydrazone adapters. Other suitable cleavable adapters include disulfide adapters. These adapters can be covalently bound to antibodies to such an extent that the antibody must be degraded within the cell to release the drug, such as MC adapters.

[0145] The linker may contain groups for linking to the antibody. For example, the linker may contain amino, hydroxy, carboxyl, or thiol reactive groups (e.g., maleimide, haloacetamide (e.g., iodo, bromide, or chloride), haloester (e.g., iodo, bromide, or chloride), halomethyl ketone (e.g., iodo, bromide, or chloride), benzyl halide (e.g., iodo, bromide, or chloride), vinyl sulfone, and pyridyl thio).

[0146] In some embodiments, the drug component is a cytotoxic or cell growth inhibitor, immunosuppressant, radioactive isotope, toxin, etc. The conjugate can be used to inhibit the proliferation of tumor cells or cancer cells, induce apoptosis of tumor cells or cancer cells, or treat cancer in patients. Therefore, the conjugate can be used to treat animal cancers in a variety of settings. The conjugate can be used to deliver drugs to tumor cells or cancer cells. Not bound by theory, in some embodiments, the conjugate binds to or is associated with cancer cells expressing GPC3, and the conjugate and / or drug can be taken up into tumor cells or cancer cells via receptor-mediated endocytosis.

[0147] Once inside the cell, one or more specific peptide sequences within the conjugate (e.g., in the connector) are cleaved by one or more tumor cell or cancer cell-associated proteases, resulting in drug release. The released drug then migrates freely within the cell and induces cytotoxicity, cell growth inhibition, or other activities. In some embodiments, the drug is cleaved from the antibody outside the tumor cell or cancer cell, subsequently penetrating the cell or acting on the cell surface.

[0148] Examples of drug portions or payloads are selected from: DM1 (matansine, N2'-deacetylated-N2'-(3-mercapto-1-oxopropyl)- or N2'-deacetylated-N2'-(3-mercapto-1-oxopropyl)-matansine), mc-MMAD (6-maleimide hexanoyl-monomethyl olritatin-D) Or N-methyl-L-valine-N-[(1S,2R)-2-methoxy-4-[(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[[(1S)-2-phenyl-1-(2-thiazolyl)ethyl]amino]propyl]-1-pyrrolidinyl]-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-(9Cl)-L-valineamide), mc-MMAF (maleimide hexanoyl-monomethyl guanetazine F) Or N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrolo-1-yl)-1-oxohexyl]-N-methyl-L-valine-L-valine-(3R,4S,5S)-3-methoxy-5-methyl-4-(methylamino)heptanoyl-(αR,βR,2S)-β-methoxy-α-methyl-2-pyrrolidinepropionyl-L-phenylalanine) and mc-Val-Cit-PABA-MMAE (6-maleimide hexanoyl-ValcCit-(p-aminobenzyloxycarbonyl)-monomethyloritidine E) Or N-[[[4-[[N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrolo-1-yl)-1-oxohexyl]-L-valine-N5-(aminocarbonyl)-L-guanine]amino]phenyl]methoxy]carbonyl]-N-methyl-L-valine-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-1-pyrrolidinyl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-valineamide). DM1 is a derivative of the microtubule inhibitor maytansine, while MMAD, MMAE, and MMAF are derivatives of olistatin. In some embodiments, the pharmaceutical fraction is selected from mc-MMAF and mc-Val-Cit-PABA-MMAE. In some embodiments, the pharmaceutical fraction is a maytansine compound or olistatin.

[0149] The antibody may be conjugated to or fused with a therapeutic agent, which may include detectable markers such as radioactive markers, immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutic or diagnostic agents, cytotoxic agents (which may be drugs or toxins), ultrasound enhancers, non-radioactive markers, combinations thereof, and other such agents known in the art.

[0150] The antibody can be detectably labeled by conjugation to a chemiluminescent compound. The presence of the chemiluminescently labeled antigen-binding peptide is then determined by detecting the luminescence produced during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridine ester, imidazole, acridine salts, and oxalates.

[0151] The antibody can also use a fluorescent metal (such as...) 152Detectable labeling can be achieved using Eu or other lanthanide metals. These metals can be linked to antibodies using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). The technique of conjugating various components to antibodies is well known; see, for example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al., (eds.), Marcel Dekker, Inc., pp. 623-53 (1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506(1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use OfRadiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For CancerDetection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16(1985) and Thorpe et al., "The Preparation And Cytotoxic Properties OfAntibody-Toxin Conjugates”, Immunol. Rev. (52:119-58 (1982)). Multifunctional molecules

[0152] Multifunctional molecules are provided that contain antibodies specific to GPRC5D, such as those disclosed herein, and one or more antibody or antigen-binding fragments specific to a second antigen or a second epitope on GPRC5D.

[0153] In some implementations, the second antigen is a protein expressed on immune cells, such as T cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, phagocytes, natural killer cells, eosinophils, basophils, and mast cells.

[0154] In some embodiments, the second antigen is BCMA, CD123, CS1, CD20, CD19, CD22, CD3, CD47, PD1, PD-L1, LAG3, TIM3, CTLA4, VISTA, CSFR1, A2AR, CD73, CD39, CD40, 4-1BB, OX40, SIRPA, CD16, CD28, ICOS, CTLA4, BTLA, TIGIT, HVEM, CD27, VEGFR, or VEGF.

[0155] In some embodiments, the second specificity is for a different epitope on GPRC5D, or for amino acid residues on GPRC5D that are different from or overlap with the amino acid residues bound to the antibodies disclosed herein.

[0156] Different forms of bispecific antibodies are also provided. In some embodiments, the second fragment may be selected from a Fab fragment, a single-chain variable fragment (scFv), or a single-domain antibody. In some embodiments, the bispecific antibody also comprises an Fc fragment.

[0157] Bifunctional molecules that not only contain antibodies or antigen-binding fragments are also provided. As tumor antigen-targeting molecules, antibodies or antigen-binding fragments specific to GPRC5D (such as those described herein) can optionally be combined with immune cytokines or ligands via peptide linkers. The linked immune cytokines or ligands include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, GM-CSF, TNF-α, CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT, and GITRL. Such bifunctional molecules can combine immune checkpoint blockade effects with local immunomodulation at the tumor site.

[0158] A specific type of bispecific or bifunctional antibody, called a T-cell connector, is also available. T-cell connectors that are specific to T cells (such as CD3 bispecific antibodies) provide a targeted immuno-oncology platform that connects the patient's own T cells to malignant cells. These bispecific T-cell connectors ensure a direct connection between T cells and cancer cells, thereby enabling T-cell activation and resulting in cytotoxic activity against cancer cells.

[0159] Therefore, in one embodiment, this disclosure provides a T-cell connective comprising an antibody or peptide of the present disclosure. The T-cell connective can have different molecular designs and is particularly useful for treating liquid or solid tumors. In some embodiments, the T-cell connective is bispecific, targeting both GPRC5D and CD3. In some embodiments, the T-cell connective is trispecific, targeting both GPRC5D and CD3, as well as another epitope on GPRC5D or another tumor antigen. Chimeric antigen receptor

[0160] In one embodiment, a chimeric antigen receptor (CAR) is also provided, comprising an antibody of the present disclosure as a targeting unit. In some embodiments, the CAR comprises one or more antibodies of the present disclosure or fragments thereof, a transmembrane domain, a co-stimulatory domain, and a CD3ζ intracellular domain.

[0161] The transmembrane domain can be designed to fuse with an extracellular domain containing the antibody or fragment, optionally via a hinge domain. It can also fuse with an intracellular domain, such as a co-stimulatory domain. In some embodiments, the transmembrane domain may comprise a native transmembrane region of a co-stimulatory domain (e.g., the TM region of CD28T or 4-1BB used as a co-stimulatory domain) or a native transmembrane region of a hinge domain (e.g., the TM region of CD8α or CD28T used as a hinge domain).

[0162] In some embodiments, the transmembrane domain may contain sequences that cross the cell membrane but extend into the cytoplasm and / or extracellular space. For example, a transmembrane region may contain a membrane-crossing sequence, which itself may further contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids that extend into the cytoplasm and / or extracellular space. Thus, a transmembrane domain contains a membrane-crossing region but may further contain amino acids that extend beyond the membrane interior or outer surface; such sequences can still be considered "transmembrane domains".

[0163] In some embodiments, the transmembrane domain is fused to the cytoplasmic domain via a short linker. Optionally, a short peptide or polypeptide linker (preferably 2 to 10 amino acids in length) can form a connection between the transmembrane domain and the proximal cytoplasmic signaling domain of the chimeric receptor. Glycine-serine duplexes (GS), glycine-serine-glycine triplets (GSG), or alanine-alanine-alanine triplets (AAA) provide suitable linkers.

[0164] In some embodiments, the CAR further includes a co-stimulatory domain. In some embodiments, the co-stimulatory domain is located between a transmembrane domain and an activation domain. Exemplary co-stimulatory domains include, but are not limited to: CD2, CD3δ, CD3ε, CD3γ, CD4, CD7, CD8a, CD8, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), and CD49d (ITGA4). CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B cell antigen receptor complex-associated α chain), CD79B (B cell antigen receptor complex-associated β chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD1 50 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD1 58F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-ζ), CD2 58 (LIGHT), CD268 (BAFFR), CD270 (TNFRSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 (SLAMF7), CD335 (NK-p46), CD336 (NK-p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), inducible T cell co-stimulatory factor (ICOS), LFA-1 (CD11a / CD12)18) KG2C, DAP-10, ICAM-1, Kp80 (KLRF1), IL-2Rβ, IL-2Rγ, IL-7Rα, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fcγ receptor, MHC class I molecules, MHC class II molecules, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, activated NK cell receptor, Toll-like ligand receptor, and fragments or combinations thereof.

[0165] In some embodiments, the cytoplasmic portion of the CAR also includes a signaling / activation domain. In one embodiment, the signaling / activation domain is a CD3ζ domain, or an amino acid sequence having at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with a CD3ζ domain.

[0166] Bispecific and bicistronic CARs are also provided. For bispecific CARs, the CAR protein further comprises a second antigen-binding fragment (e.g., scFv) that binds to a second target. Non-limiting examples of the second target include BCMA, CD123, CS1, CD20, CD19, CD22, CD3, CD47, PD1, PD-L1, LAG3, TIM3, CTLA4, VISTA, CSFR1, A2AR, CD73, CD39, CD40, 4-1BB, OX40, SIRPA, CD16, CD28, ICOS, CTLA4, BTLA, TIGIT, HVEM, CD27, VEGFR, and VEGF. Bicistronic CARs comprise two CAR molecules that can be expressed by the same vector encoding both. Both CAR molecules can be expressed in target cells (e.g., T cells).

[0167] This disclosure also provides cells comprising the CAR or encoding nucleotides. Suitable cells may be used that are transduced with or contacted with a vector encoding a CAR comprising the anti-GPRC5D antibody of this disclosure (or alternatively engineered to express the anti-GPRC5D antibody of this disclosure). The cells (e.g., T cells, NK cells, monocytes, macrophages) may be, for example, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, γδ T cells, or combinations thereof, without limitation. Polynucleotides, mRNA, and methods for expressing or preparing antibodies

[0168] This disclosure also provides polynucleotide or nucleic acid molecules encoding antibodies, variants or derivatives thereof, or said CARs. The polynucleotides of this disclosure may encode the entire heavy and light chain variable regions of an antigen-binding polypeptide, its variants or derivatives, either on the same polynucleotide molecule or on separate polynucleotide molecules. Additionally, the polynucleotides of this disclosure may encode portions of the heavy and light chain variable regions of an antigen-binding polypeptide, its variants or derivatives, either on the same polynucleotide molecule or on separate polynucleotide molecules.

[0169] In some embodiments, the polynucleotide is an mRNA molecule. In some embodiments, the mRNA can be introduced into target cells to express the antibody or a fragment thereof.

[0170] mRNA can be synthesized using any of a variety of known methods. For example, the mRNA can be synthesized via in vitro transcription (IVT). In short, IVT typically uses a linear or circular DNA template containing a promoter, a ribonucleoside triphosphate pool, a buffer system that may contain DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNase inhibitors. The exact conditions will vary depending on the specific application.

[0171] In some implementations, a DNA template is transcribed in vitro to prepare mRNA encoding an antibody. A suitable DNA template typically has a promoter for in vitro transcription (e.g., a T3, T7, or SP6 promoter), followed by the desired nucleotide sequence encoding the desired antibody (e.g., heavy or light chain encoding) and a termination signal.

[0172] The desired antibody-encoding (e.g., heavy or light chain-encoding) mRNA sequence can be determined using standard methods and incorporated into a DNA template. For example, virtual reverse translation based on the degenerate genetic code can be performed, starting with the desired amino acid sequence (e.g., the desired heavy or light chain sequence). Optimization algorithms can then be used to select appropriate codons. Typically, the G / C ratio can be optimized to achieve the highest possible G / C ratio while taking into account tRNA frequency (depending on codon usage). The optimized RNA sequence can be constructed and displayed, for example, using appropriate display equipment, and compared with the original (wild-type) sequence. Secondary structure can also be analyzed to calculate the RNA's stabilizing and destabilizing properties or regions.

[0173] The mRNA can be synthesized as unmodified or modified mRNA. Typically, the mRNA is modified to enhance stability. Modifications to the mRNA can include, for example, modifications to RNA nucleotides. Therefore, modified mRNA can include, for example, backbone modifications, sugar modifications, or base modifications. In some embodiments, the mRNA encoding the antibody (e.g., heavy and light chain-encoded mRNA) can be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including but not limited to purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and analogs or derivatives of purines and pyrimidines as modified nucleotides, such as 1-methyladenine, 2-methyladenine, etc. -Adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil) ), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio Uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, methyl uracil-5-oxyacetate, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, 13-D-mannosyl-queosine, wybutoxosine, as well as phosphoramide, thiophosphate, peptide nucleotide, methylphosphonate, 7-dezoguanosine, 5-methylcytosine, and inosine. Preparation of such analogues is known to those skilled in the art, for example from U.S. Patent Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530, and 5,700,642, the disclosures of which are incorporated herein by reference in their entirety.

[0174] In some embodiments, the mRNA (e.g., the heavy and light chains encoding mRNA) may contain RNA backbone modifications. Typically, backbone modifications are chemical modifications to the backbone phosphate groups of the nucleotides contained in the RNA. Exemplary backbone modifications typically include, but are not limited to, modifications selected from: methylphosphonates, methylphosphamides, phosphoramides, thiophosphates (e.g., cytidine 5'-O-(1-thiophosphate)), boron phosphates, positively charged guanidine groups, etc., meaning by replacing the phosphodiester bonds with other anionic, cationic, or neutral groups.

[0175] In some embodiments, the mRNA (e.g., the heavy and light chains encoding mRNA) may contain sugar modifications. Typical sugar modifications involve chemically modifying the sugars in the contained nucleotides, including but not limited to sugar modifications selected from: 2'-deoxy-2'-fluoro-oligonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamino-oligonucleotides (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O -alkyl-oligonucleotides, 2'-deoxy-2'-C-alkyl-oligonucleotides (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyl-oligonucleotides and their isomers (2'-cytarabine 5'-triphosphate, 2'-cytarabine 5'-triphosphate), or azidotriphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0176] In some implementations, the mRNA (e.g., the heavy and light chains encoding mRNA) may contain modifications of nucleotide bases (base modifications). Modified nucleotides containing base modifications are also called base-modified nucleotides. Examples of such base-modified nucleotides include, but are not limited to: 2-amino-6-chloropurine riboside 5'-triphosphate, 2-aminoadenosine 5'-triphosphate, 2-thiocytidine 5'-triphosphate, 2-thiouridine 5'-triphosphate, 4-thiouridine 5'-triphosphate, 5-aminoallylcytidine 5'-triphosphate, 5-aminoallyluridine 5'-triphosphate, 5-bromocytidine 5'-triphosphate, 5-bromouridine 5'-triphosphate, 5-iodocytidine 5'-triphosphate, 5-iodouridine 5'-triphosphate, 5-methylcytidine 5'-triphosphate, 5-methyluridine 5'-triphosphate. Acids, 6-azacytidine 5'-triphosphate, 6-azauridine 5'-triphosphate, 6-chloropurine riboside 5'-triphosphate, 7-deadenine 5'-triphosphate, 7-deadenine guanosine 5'-triphosphate, 8-azaadenine 5'-triphosphate, 8-azidoadenine 5'-triphosphate, benzimidazole riboside 5'-triphosphate, N1-methyladenosine 5'-triphosphate, N1-methylguanosine 5'-triphosphate, N6-methyladenosine 5'-triphosphate, O6-methylguanosine 5'-triphosphate, pseudouridine 5'-triphosphate, puromycin 5'-triphosphate, or flavin 5'-triphosphate.

[0177] Typically, mRNA synthesis involves adding a "cap" at the N-terminus (5') and a "tail" at the C-terminus (3'). The presence of the cap is important for providing resistance to nucleases found in most eukaryotic cells. The presence of the "tail" is used to protect the mRNA from degradation by exonucleases.

[0178] Therefore, in some embodiments, the mRNA (e.g., heavy and light chain encoding mRNA) includes a 5' cap structure. The 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one terminal phosphate group from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphates via guanylate transferase, creating a 5'5'5 triphosphate linker; then, the 7-nitrogen of guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp(5'(A,G(5')ppp(5)A and G(5)ppp(5')G.

[0179] In some embodiments, the mRNA (e.g., heavy and light chain-encoded mRNA) comprises a 3' poly(A) tail. The poly-A tail at the 3' end of the mRNA typically comprises about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 175 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 125 adenosine nucleotides, 10 to 100 adenosine nucleotides, about 10 to 75 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides). In some embodiments, the mRNA encoding an antibody (e.g., heavy and light chain-encoded mRNA) comprises a 3' poly(C) tail. A suitable poly-C tail at the 3' end of mRNA typically contains about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). The poly-C tail can be added to or can replace the poly-A tail.

[0180] In some embodiments, the mRNA (e.g., the heavy and light chains encoding mRNA) includes a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region includes one or more elements that affect mRNA stability or translation, such as an iron-responsive element. In some embodiments, the length of the 5' untranslated region can be between about 50 and 500 nucleotides (e.g., about 50 to 400 nucleotides, about 50 to 300 nucleotides, about 50 to 200 nucleotides, or about 50 to 100 nucleotides).

[0181] In some embodiments, the 5' region of the mRNA (e.g., heavy and light chain-encoded mRNAs) contains a sequence encoding a signal peptide, such as those described herein. In a particular embodiment, a signal peptide derived from human growth hormone (hGH) is incorporated into the 5' region. Typically, the signal peptide-encoded sequence is directly or indirectly linked to the heavy or light chain-encoded sequence at the N-terminus.

[0182] This technology can be used to deliver any antibody known in the art, as well as antibodies that can be generated against a desired antigen using standard methods. This invention can be used to deliver monoclonal antibodies, polyclonal antibodies, antibody mixtures or combinations, human or humanized antibodies, chimeric antibodies, or bispecific antibodies.

[0183] Methods for preparing antibodies are well known in the art and are described herein. In some embodiments, both the variable and constant regions of the antigen-binding polypeptide of this disclosure are fully human. Fully human antibodies can be prepared using techniques described in the art and herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal modified to produce such antibodies in response to antigen challenge, but whose endogenous loci have been inactivated. Exemplary techniques that can be used to prepare such antibodies are described in U.S. Patents: 6,150,584; 6,458,592; 6,420,140, ​​the entire contents of which are incorporated herein by reference. Treatment and Uses

[0184] As described herein, the antibodies, variants thereof, antibody-drug conjugates, chimeric antigen receptors (CARs) and CAR cells, encoding polynucleotides or derivatives may be used in certain therapeutic and diagnostic approaches.

[0185] This disclosure also relates to antibody-based therapies comprising administering antibodies or fragments of this disclosure, antibody-drug conjugates, chimeric antigen receptors (CARs) and CAR cells, encoding polynucleotides or derivatives thereof to patients, such as animals, mammals, and humans, to treat one or more of the conditions or illnesses described herein. Therapeutic molecules or cells of this disclosure include, but are not limited to: antibodies of this disclosure (including variants and derivatives thereof described herein), antibody-drug conjugates, chimeric antigen receptors (CARs) and CAR cells, and nucleic acids or polynucleotides encoding antibodies of this disclosure (including variants and derivatives thereof described herein).

[0186] The molecules or cells disclosed herein can also be used to treat or suppress cancer. As mentioned above, GPRC5D can be overexpressed in tumor cells, particularly in hematologic malignancies. In some embodiments, the cancer is a B-cell cancer expressing GPRC5D, such as multiple myeloma.

[0187] Therefore, in some embodiments, methods for treating cancer in patients in need are provided. In one embodiment, the method includes administering an effective amount of the molecules or cells disclosed herein to the patient. In some embodiments, at least one cancer cell (e.g., stromal cells) in the patient overexpresses GPRC5D.

[0188] This disclosure also provides cell therapies, such as chimeric antigen receptor (CAR) T-cell therapy. Suitable cells can be used, which are transduced or contacted with a vector encoding a CAR containing the anti-GPRC5D antibody of this disclosure (or alternatively engineered to express the anti-GPRC5D antibody of this disclosure). After contact or engineering, the cells can be introduced into a cancer patient in need of treatment. The cancer patient can have any type of cancer disclosed herein. The cells (e.g., T cells, NK cells, monocytes, macrophages) can be, for example, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, γδ T cells, or combinations thereof, without limitation.

[0189] In some embodiments, the cells are isolated from the cancer patient themselves. In other embodiments, the cells are provided by a donor or from a cell bank. When cells are isolated from a cancer patient, adverse immune responses can be minimized.

[0190] Non-limiting examples of cancer include bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer. In some embodiments, the cancer is one or more of stomach cancer, pancreatic cancer, esophageal cancer, ovarian cancer, and lung cancer.

[0191] The antibodies or variants or derivatives thereof disclosed herein can be used to treat, prevent, diagnose, and / or predict other diseases or conditions associated with increased cell survival, including but not limited to the progression and / or metastasis of malignancies and related conditions such as leukemia (including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia (including medulloblastic, promyelocytic, granulocytic, monocytic, and erythroleukemia)) and chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors, including but not limited to sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, and liposarcoma. Chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. The specific dosage and treatment regimen for any given patient will depend on a number of factors, including the specific antibody used, its variant or derivative, the patient's age, weight, general health condition, sex, and diet, as well as the timing of administration, excretion rate, drug combination, and the severity of the specific disease being treated. The judgment of these factors by healthcare professionals is within the realm of common skill. Dosage will also depend on the individual patient being treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The dosage used can be determined using principles of pharmacology and pharmacokinetics well known in the art.

[0192] The methods of administration of the antibodies or fragments include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antigen-binding peptides or compositions can be administered via any convenient route, such as by infusion or bolus injection, absorption through epithelial or mucosal linings (e.g., oral mucosa, rectal and intestinal mucosa), and can be administered together with other bioactive agents. Therefore, pharmaceutical compositions containing the antigen-binding peptides of this disclosure can be administered orally, rectally, parenterally, intracerebrospinal, intravaginally, intraperitoneally, topically (by powder, ointment, drops, or transdermal patch), buccally, or as oral or nasal sprays.

[0193] The term "parenteral" as used in this article refers to administration methods including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

[0194] Administration can be systemic or local. Additionally, it may be desirable to introduce the antibodies of this disclosure into the central nervous system via any suitable route, including intraventricular and intrathecal injection; intraventricular injection can be facilitated by an intraventricular catheter (e.g., connected to a reservoir, such as the Ommaya reservoir). Lung administration may also be employed, for example, by using an inhaler or nebulizer, and in combination with a nebulizer formulation.

[0195] It may be desirable to apply the antigen-binding peptides or compositions of this disclosure topically to areas requiring treatment; this can be achieved, for example (non-limitingly), by local infusion during surgery, by topical application (e.g., postoperatively in combination with wound dressings), by injection, by catheter, by suppository, or by implantation of a porous, non-porous, or gel-like material, including membranes such as silicone rubber membranes, or fibers. Preferably, when administering the proteins (including antibodies) of this disclosure, care must be taken to use materials that do not absorb proteins.

[0196] The effective dose of the antibody disclosed herein in treating, inhibiting, and preventing inflammatory, immune, or malignant diseases, symptoms, or conditions can be determined using standard clinical techniques. Alternatively, in vitro assays may be used to help determine the optimal dose range. The precise dose to be used in the formulation also depends on the route of administration and the severity of the disease, symptom, or condition, and should be determined based on the physician's judgment and the individual patient's situation. The effective dose can be extrapolated from dose-response curves derived from in vitro or animal model testing systems.

[0197] As a general recommendation, the dosage of the disclosed antibody administered to patients is typically from 0.001 mg / kg to 100 mg / kg, 0.01 mg / kg to 20 mg / kg, or 0.5 mg / kg to 10 mg / kg of the patient's body weight. Generally, due to the immune response to exogenous peptides, human antibodies have a longer half-life in the human body than antibodies from other species. Therefore, it is generally possible to reduce the dosage of human antibodies and decrease the frequency of administration. Furthermore, the dosage and frequency of administration of the disclosed antibody can be reduced by modifying (e.g., lipidation) to enhance antibody uptake and tissue penetration (e.g., into the brain).

[0198] In another embodiment, the compositions of this disclosure are administered in combination with cytokines. Cytokines that can be administered with the compositions of this disclosure include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α.

[0199] In other embodiments, the compositions disclosed herein are administered in combination with other treatment or preventative measures, such as radiotherapy. Composition

[0200] This disclosure also provides pharmaceutical compositions. Such compositions comprise effective amounts of antibodies, antibody-drug conjugates, chimeric antigen receptors (CARs) and CAR cells, encoding polynucleotides or derivatives, and acceptable carriers. In some embodiments, the compositions further comprise a second anticancer agent (e.g., an immune checkpoint inhibitor).

[0201] In the specific implementation plan, the term "pharmaceutical acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other recognized animal (especially human) pharmacopoeias. Furthermore, "pharmaceutical acceptable carriers" will typically be non-toxic solid, semi-solid, or liquid fillers, diluents, encapsulating materials, or any type of formulation excipient.

[0202] The term "carrier" refers to a diluent, adjuvant, excipient, or medium used to administer a therapeutic agent. Such drug carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the drug composition is administered parenterally. Saline and glucose solutions, as well as glycerol solutions, can also be used as liquid carriers, particularly for injectable solutions. Suitable drug excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting or emulsifying agents, or pH buffers, such as acetates, citrates, or phosphates. Antimicrobial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and agents for regulating tension, such as sodium chloride or glucose, are also envisioned. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The compositions can be formulated into suppositories using conventional binders and carriers, such as triglycerides. Oral formulations may include standard carriers, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in EW Martin's Remington's Pharmaceutical Sciences, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of an antigen-binding polypeptide, preferably in a purified form, along with a suitable amount of carrier to provide a dosage form suitable for administration to a patient. The formulation should be suitable for the route of administration. Parenteral formulations can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0203] In one embodiment, the composition is formulated according to conventional procedures to be a pharmaceutical composition suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in a sterile isotonic buffer solution. If necessary, the composition may also include a solubilizer and a local anesthetic, such as lidocaine, to reduce pain at the injection site. Typically, the components are provided individually or mixed together in unit dosage forms, for example, as a dry lyophilized powder or an anhydrous concentrate in an airtight container (such as an ampoule or sachet) indicating the active dose. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, sterile water for injection or saline can be provided in ampoules so that the components can be mixed prior to administration.

[0204] The compounds disclosed herein can be formulated in a neutral or salt form. Pharmaceutically acceptable salts include those that form with anions, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those that form with cations, such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. Example Example 1: Screening of anti-GPRC5D single-domain antibodies

[0205] This embodiment demonstrates the screening of positive clones by panning a synthesized humanized single-domain antibody library and primary screening, wherein Hu-GPRC5D-VLP, MM1s, HEK293, Hu-GPRC5D-HEK293 and NCI-H929 are used as antigens (or controls). Materials and Methods Panning of humanized single-domain antibody libraries (liquid chromatography) and Kingfisher coating.

[0206] Consumption of nonspecific phages: The phage suspension of the prepared humanized single-domain antibody library was diluted with 2.5% BSA solution and blocked, then incubated with Dynabeads. The consumed phages that did not bind nonspecifically after incubation were collected.

[0207] Dynabeads blocking: Freshly prepared magnetic beads were blocked with 2.5% BSA.

[0208] Positive screening: The coated magnetic beads are incubated with consumed phages and then further washed according to the liquid phase magnetic bead screening system method.

[0209] Elution: Elute the magnetic beads with trypsin and collect the eluent containing bacteriophages.

[0210] Infection and culture: The collected phages were mixed with logarithmic-phase SS320 cells and incubated at 37°C for 30 minutes. After incubation, the infected cells were spread on 2YT-Car+-Tet+ plates and incubated overnight at 37°C.

[0211] Titer test: The eluted phage solution was serially diluted 10-fold and incubated with SS320 cells in log phase. Then, 2 μL of the mixture was spread on a plate and incubated overnight at 37°C.

[0212] Statistics: Three rounds of panning were conducted, with antigen concentrations decreasing in a 3-fold gradient, yielding antibody clones with high affinity. The enrichment of positive clones was assessed based on input and output titers. Screening of humanized single-domain antibody libraries (immunotube method)

[0213] Blocking: Block the prepared humanized single-domain antibody library phage suspension with 5% PBSM.

[0214] Immunotube coating and blocking: Diluted antigen was added to the immunotubes, coated overnight at 4°C, then washed and blocked with 5% PBSM.

[0215] Positive panning: Phage suspensions are incubated in antigen-coated immunotubes. Binding and subsequent washing are performed according to the immunotube screening system method.

[0216] Elution: Elute the immunosorbent tubes with trypsin and collect the eluent containing bacteriophages.

[0217] Infection and culture: The collected phages were mixed with logarithmic-phase SS320 cells and incubated at 37°C for 30 minutes. After incubation, the infected cells were spread on 2YT-Car+-Tet+ plates and incubated overnight at 37°C.

[0218] Titer test: The eluted phage solution was serially diluted 10-fold and incubated with SS320 cells in log phase. Then, 2 μL of the mixture was spread on a plate and incubated overnight at 37°C.

[0219] Statistics: Three rounds of panning were conducted, with antigen concentrations decreasing in a 3-fold gradient, yielding antibody clones with high affinity. The enrichment of positive clones was assessed based on input and output titers. Screening of humanized single-domain antibody libraries (cell-based screening)

[0220] Phage blocking: The prepared phage library was blocked at room temperature on a four-dimensional rotator with 5% PBSM for 1 hour.

[0221] Incubation

[0222] 1) Negative selection: Add the blocked phage suspension to the negative cell solution, then place the mixture on a track shaker (room temperature, 80 rpm, 1 hour). Note: Add the phage suspension carefully to avoid washing out the cells.

[0223] 2) Positive selection: Use a disposable pipette to add the negatively selected phages to the corresponding positive cell bottles, and incubate the mixture on a track shaker (room temperature, 80 rpm, 2 hours).

[0224] Washing: Discard the supernatant and add 5% FBS-PBS solution to the flask. To ensure all cells are covered by the liquid, briefly mix on a track shaker at 80 rpm for 1 minute. Repeat the washing process several times, the exact number depending on the panning round.

[0225] Elution: Add 1 mL of 100 mM glycine-HCl solution (pH 2.2) to the bottle, mix the sample, and incubate on a shaker at room temperature (80 rpm) for 10 minutes. Then add 70 μL of Tris-base solution (1 M) for neutralization and adjust the pH to 7.0–7.2. Collect the eluent containing positive phages into a 15 mL centrifuge tube for later use.

[0226] Infection and culture: The collected phages were mixed with logarithmic-phase SS320 cells and incubated at 37°C for 30 minutes. After incubation, the infected cells were spread on 2YT-Car+-Tet+ plates and incubated overnight at 37°C.

[0227] Titer test: The eluted phage solution was serially diluted 10-fold and incubated with SS320 cells in log phase. Then, 2 μL of the mixture was spread on a plate and incubated overnight at 37°C.

[0228] Statistics: Multiple rounds of screening were conducted, with antigen concentrations decreasing in a 3-fold gradient, yielding antibody clones with high affinity. The enrichment of positive clones was assessed based on input and output titers. Positive antibody signal verification

[0229] The expression supernatant of the phage library after three rounds of screening was serially diluted 5-fold with 5% PBSM and then subjected to ELISA.

[0230] ELISA method

[0231] 1) Coating: 2 μg / mL antigen Hu-GPRC5D-VLP, 30 μL / well, incubate overnight at 4°C, wash 3 times with PBST.

[0232] 2) Blocking: Block with 5% PBSM for 1 hour at room temperature, and wash with PBST 3 times.

[0233] 3) Primary antibody: Add the serially diluted VHH or phage expression supernatant as described above, 30 μL / well, incubate at room temperature for 1 hour, and then wash the plate 3 times with PBST.

[0234] 4) Secondary antibody: Add 1:5000 diluted secondary antibody Anti-Flag-HRP to the VHH supernatant, dilute with PBS, 30 μL / well, incubate at room temperature for 1 hour, and wash the plate 6 times with PBST; add 1:20000 diluted secondary antibody Anti-M13-HRP to the phage, dilute with 5% PBSM, 30 μL / well, incubate at room temperature for 1 hour, and then wash the plate 6 times with PBST.

[0235] 5) Termination: Add TMB, 30 μL / well, incubate at room temperature for 5 to 10 minutes, then add 2 M stop solution, 30 μL / well to terminate the reaction. Read the OD value of each well at 450 nm using a microplate reader.

[0236] Quantitative ELISA method

[0237] 1) Coating: Dilute Anti-Flag to 1 μg / mL with PBS, 30 μL / well, incubate at room temperature for 16 hours, and wash the plate 3 times with PBST.

[0238] 2) Blocking: Block with 5% PBSM for 1 hour at room temperature, and wash with PBST 3 times.

[0239] 3) Primary antibody: Add 30 μL of serially diluted VHH supernatant per well, incubate at room temperature for 1 hour, and then wash the plate 3 times with PBST.

[0240] 4) Secondary antibody: Add 30 μL of diluted secondary antibody Anti-VHH-HRP (1:7000), incubate at room temperature for 1 hour, and then wash the plate 6 times with PBST.

[0241] 5) Termination: Add 30 μL of TMB per well, incubate at room temperature for 5-20 minutes, then add 30 μL of 2 M stop solution per well to terminate the reaction. Read the OD value of each well at 450 nm using a microplate reader.

[0242] Monoclonal primary screening

[0243] Centrifugation was used to separate the supernatant of monoclonal phages for primary screening ELISA.

[0244] ELISA method

[0245] 1) Coating: 2 μg / mL Hu-GPRC5D-VLP, 30 μL / well, overnight at 4°C, wash 3 times with PBST.

[0246] 2) Blocking: Block with 5% PBSM for 1 hour at room temperature, then wash with PBST 3 times.

[0247] 3) Primary antibody: Add 30 μL of serially diluted phage supernatant per well, incubate at room temperature for 1 hour, and wash the plate 3 times with PBST.

[0248] 4) Secondary antibody: Add 30 μL of diluted secondary antibody Anti-M13-HRP (1:7000), incubate at room temperature for 1 hour, and then wash the plate 6 times with PBST.

[0249] 5) Termination: Add 30 μL of TMB per well, incubate at room temperature for 5 to 10 minutes, then add 30 μL of 2 M stop solution per well to terminate the reaction. Read the OD value of each well at 450 nm using a microplate reader. result

[0250] Seven single-domain antibodies that strongly bind to human GPRC5D protein were selected for further testing, including A003, A004, A007, A009, A011, A018, A019, A020, A022, A024, A026, A036, A037, A038, A039, A042, A046, A049, A053, A054, A055, A056, A057, A058, and A059. Example 2. Testing antibodies using FACS

[0251] This embodiment tested the binding affinity of the antibody to the human GPRC5D protein expressed on HEK293 cells, measured by flow cytometry (FACS). The bispecific IgG4 antibody Talquetamab (JNJ-64407564) was used as the baseline antibody.

[0252] The results are as follows Figure 1A -C and Table 1 are shown. As shown in the figure, most of the tested antibodies have affinity comparable to or better than talquetamab. Table 1. FACS Tests on Hu-GPRC5D-HEK293 * "Talquetamab-HC(IgG4SP)" is an anti-GPRC5D monoclonal antibody derived from talquetamab. Example 3. Combination of FACS testing with cynomolgus monkey GRPC5D

[0253] This embodiment tested the binding affinity of the antibody to the cynomolgus monkey GPRC5D protein expressed on HEK293 cells, measured by flow cytometry (FACS).

[0254] The results are as follows Figure 2A -C and Table 2 are shown. As shown in the figure, talquetamab does not cross-react with cynomolgus monkey GPRC5D, but most of the tested antibodies bind well to cynomolgus monkey proteins. Table 2. FACS testing on Cy-GPRC5D-HEK293 Example 4. Background Combination Test

[0255] This embodiment tested the binding affinity of the antibody to HEK293, which is negative for human GPRC5D protein.

[0256] As shown in Table 3, most of the test antibodies do not bind to GPRC5D negative cells. Table 3. FACS on HEK-293 Example 5. Binding with GPRC5D-positive MM.1s cells

[0257] This embodiment tested the binding affinity of the antibody to GPRC5D-positive human B lymphoblasts of multiple myeloma, which was measured by flow cytometry (FACS).

[0258] The results are as follows Figure 3A -C and Table 4 are shown. As shown in the figure, most of the tested antibodies have affinity comparable to or better than the benchmark antibody talquetamab. Table 4. FACS testing on MM.1s

[0259] Based on the test data above, the following antibodies were identified as suitable for further clinical development, including A011, A018, A019, A020, A024, A026, A037, A038, A039, A042, A046, A049, A053, A055, A056, A057, and A059. Their sequences are shown in Tables 5-6. Table 5. Selected antibody sequences

[0260] The CDR sequences of these antibodies are summarized in Table 6. Sequence examination revealed that some contain residues that are susceptible to post-translational modification (PTM), which could complicate production. Therefore, these residues can be modified to prevent PTM (hence the term "de-PTM" version). The de-PTM sequences are also included in the table. Table 6. CDR Sequences Example 6: Screening of the immune library

[0261] In this embodiment, an immune nanobody library was used to identify antibodies with satisfactory binding affinity and specificity. Candidate antibodies were evaluated as shown in Examples 2-5. The best candidates and their sequences are shown in Tables 7 and 8. Table 7. Optimal Antibodies from the Immune Library Table 8. CDR Sequences Example 7. Detection of binding activity by FACS

[0262] In this embodiment, the binding activity of selected antibodies (B051, B061, B104, B128, and B161) obtained from Example 6 was measured by FACS.

[0263] In short, human HEK293 cells expressing GPRC5D (Hu-GPRC5D-HEK293) were seeded at 1 x 10^5 cells / well. The cells were centrifuged and washed with FACS buffer. 100 μL of antibody solution was added to the cells, and the cells were incubated at 4°C for 60 minutes.

[0264] Centrifuge the cells and wash twice with FACS buffer. Add 100 μL of anti-human IgG, Fcγ (PE) (Jackson 109-115-098 1:200) to the cells and incubate at 4°C for 30 minutes. Then resuspend the cells in FACS buffer and measure binding activity by flow cytometry.

[0265] The results are as follows Figure 4 As shown in Table 9. Table 9. Antibody binding as measured by FACS

[0266] Using the same method, the potential nonspecific binding of the antibody to GPRC5A and GPRC5C was examined. Figure 5 As shown, B051, B061, B104, B128, and B161 all do not have nonspecific binding to HEK293 cells expressing GPRC5A or GPRC5C. Example 8. Construction and Testing of CAR

[0267] In this embodiment, the VHH antibodies B051, B061, B104, B128 and B161 selected in Example 6 were constructed into a chimeric antigen receptor, and their activity was then tested in the Jurkat NFAT-GFP reporter gene assay.

[0268] All 5 CARs showed ( Figure 6 The CAR-mediated signal showed strong interaction with wild-type H929 (H929-WT), but was lower when co-cultured with GPRC5D knockout H929 (H929-GPRC5D-KO). Here, the GPRC5D control CAR was used as a positive control, and the lysozyme control CAR was used as a negative control.

[0269] In vitro tumor re-challenge assays based on luciferase were performed to evaluate the killing efficacy of GPRC5D CARs. Primary T cells were transduced with OL CARs (B051, B061, B104, B128, and B161), GPRC5D control CARs (GPRC5D control #1 and GPRC5D control #2), and a negative lysozyme control CAR (lysozyme CAR), and in vitro killing assays were performed for two rounds and three days. Figure 7 As shown, the CAR of the present invention outperformed the GPRC5D control CAR in both rounds, demonstrating strong tumor control efficacy. * * *

[0270] The scope of this disclosure is not limited to the specific embodiments described, which are intended as a single illustration of various aspects of this disclosure, and any functionally equivalent compositions or methods are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of this disclosure without departing from the spirit or scope of this disclosure. Therefore, this disclosure is intended to cover modifications and variations thereof, provided they fall within the scope of the appended claims and their equivalents.

[0271] All publications and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication or patent application is specifically and individually indicated as incorporated by reference.

Claims

1. A single domain antibody or a polypeptide comprising the same, wherein the single domain antibody has binding specificity for human G protein-coupled receptor class C, group 5, member D (GPRC5D) protein and comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1, CDR2, and CDR3 comprise the following amino acid sequences, respectively: SEQ ID NO: 169, 170, and 171; SEQ ID NO: 160, 161, and 162; SEQ ID NO: 121, 122, and 123; SEQ ID NO: 133, 134, and 135; or SEQ ID NO: 148, 149, and 150.

2. The antibody or polypeptide of claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 99, 96, 83, 87, and 92, or an amino acid sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 99, 96, 83, 87, and 92 and retaining the CDRs of the corresponding sequence.

3. The antibody or polypeptide of claim 1, wherein the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO: 169, 170, and 171, respectively.

4. The antibody or polypeptide of claim 3, comprising the amino acid sequence of SEQ ID NO:

99.

5. The antibody or polypeptide of claim 1, wherein the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO: 160, 161, and 162, respectively.

6. The antibody or polypeptide of claim 5, comprising the amino acid sequence of SEQ ID NO:

96.

7. The antibody or polypeptide of claim 1, wherein the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO: 121, 122, and 123, respectively.

8. The antibody or polypeptide of claim 7, comprising the amino acid sequence of SEQ ID NO:

83.

9. The antibody or polypeptide of claim 1, wherein the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO: 133, 134, and 135, respectively.

10. The antibody or polypeptide of claim 9, comprising the amino acid sequence of SEQ ID NO:

87.

11. The antibody or polypeptide of claim 1, wherein the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO: 148, 149, and 150, respectively.

12. The antibody or polypeptide of claim 11, comprising the amino acid sequence of SEQ ID NO:

92.

13. A multispecific antibody comprising the antibody of any one of claims 1-12 and a second antibody or antigen binding fragment having binding specificity for a target antigen other than GPRC5D. ​ ​ ​ ​ ​ 14. The multispecific antibody of claim 13, wherein the second target antigen is selected from BCMA, CD123, CS1, CD20, CD19, CD22, CD3, CD47, PD1, PD-L1, LAG3, TIM3, CTLA4, VISTA, CSFR1, A2AR, CD73, CD39, CD40, 4-1BB, OX40, SIRPA, CD16, CD28, ICOS, CTLA4, BTLA, TIGIT, HVEM, CD27, VEGFR, or VEGF.

15. A chimeric antigen receptor (CAR) comprising the antibody of any one of claims 1-12, a transmembrane domain, a costimulatory domain, and a CD3 zeta intracellular domain.

16. A polynucleotide encoding the antibody or polypeptide of any one of claims 1-12, the multispecific antibody of claim 13 or 14, or the CAR of claim 15.

17. A cell comprising the polynucleotide of claim 16.

18. An antibody-drug conjugate comprising the antibody of any one of claims 1-12 conjugated to a drug moiety.

19. A method of treating cancer in a patient in need thereof, comprising administering to the patient the antibody or polypeptide of any one of claims 1-12, the multispecific antibody of claim 13 or 14, the CAR of claim 15, the polynucleotide of claim 16, the cell of claim 17, or the antibody-drug conjugate of claim 18.

20. The method of claim 19, wherein the cancer is a hematological cancer.

21. The method of claim 20, wherein the hematological cancer is a GPRC5D-expressing B-cell cancer.

22. The method of claim 21, wherein the GPRC5D-expressing B-cell cancer is multiple myeloma.

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