HK40080157B - Anti-gprc5d monoclonal antibodies and uses thereof - Google Patents

Description

Anti-GPRC5D monoclonal antibodies and their uses

Background Technology

G protein-coupled receptor C family 5 member D (GPRC5D) is a member of the G protein-coupled receptor family. GPRC5D is a transmembrane protein. Overexpression of GPRC5D has been observed in patients with multiple myeloma. In particular, its high expression is significantly associated with poor disease and treatment outcomes.

Given its specific high expression on malignant cells, antibodies specific to GPRC5D have been proposed for the treatment of malignant tumors, for example, through bispecific T cell redirection antibodies or through antibody-dependent cytotoxicity (ADCC).

Summary of the Invention

This paper identifies anti-GPRC5D antibodies, including their humanized derivatives, with high affinity for the human GPRC5D protein. These antibodies, or fragments thereof, can target cancer cells expressing GPRC5D, and therefore have potential applications in cancer treatment, particularly hematologic malignancies.

One embodiment of this disclosure provides an antibody or antigen-binding fragment thereof with binding specificity to human G protein-coupled receptor C family 5 member D (GPRC5D) protein, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) comprising heavy chain complementarity-determining regions CDRH1, CDRH2, and CDRH3, and the light chain variable region (VL) comprising complementarity-determining regions CDRL1, CDRL2, and CDRL3. In some embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 each comprise: (a) the amino acid sequence of SEQ ID NO: 29-34; (b) the amino acid sequence of SEQ ID NO: 42-47; (c) the amino acid sequence of SEQ ID NO: 54-59; or (d) the amino acid sequence of SEQ ID NO: 68-73.

In some embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 each comprise the amino acid sequences of SEQ ID NO: 29-34. In some embodiments, VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and 35-37, and VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and 38-41. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 35, and VL comprises the amino acid sequence of SEQ ID NO: 38.

In some embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 each comprise the amino acid sequences of SEQ ID NO:42-47. In some embodiments, VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO:9 and 48-50, and VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO:10 and 51-53. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO:48, and VL comprises the amino acid sequence of SEQ ID NO:51.

In some embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 each comprise the amino acid sequences of SEQ ID NO: 54-59. In some embodiments, VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 61-64, and VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16 and 65-67. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 61, and VL comprises the amino acid sequence of SEQ ID NO: 65. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 63, and VL comprises the amino acid sequence of SEQ ID NO: 65.

In some embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 each comprise the amino acid sequences of SEQ ID NO: 68-73. In some embodiments, VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 74-79, and VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 80-86. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 76, and VL comprises the amino acid sequence of SEQ ID NO: 82. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 77, and VL comprises the amino acid sequence of SEQ ID NO: 82.

In some embodiments, the antibody or fragment thereof is humanized. In some embodiments, the antibody or fragment thereof has ADCC capability.

In some embodiments, an antibody or fragment thereof is also provided, the antibody or fragment thereof further comprising a cytotoxic drug conjugated to the antibody or fragment thereof.

A bispecific antibody is also provided, comprising the antigen-binding fragment of this disclosure and a second antigen-binding fragment specific to a second target protein. In some embodiments, the second target protein is selected from the group consisting of CD3, CD16, CD19, CD28, CD64, and 4-1BB. In some embodiments, the second target protein is CD3. In some embodiments, the second target protein is 4-1BB.

A method for treating cancer in a patient in need is also provided, comprising administering an antibody or fragment thereof disclosed herein to the patient. A method for treating cancer in a patient in need is also provided, comprising (a) treating T cells, natural killer (NK) cells, or macrophages in vitro with an antibody or fragment thereof disclosed herein, and (b) administering the treated cells to the patient.

In some implementations, the cancer is a blood cancer, such as B-cell cancer expressing GPRC5D (e.g., multiple myeloma).

Attached Figure Description

Figure 1 shows the binding of mouse antibodies collected from hybridoma supernatant to GPRC5D expressed on CHO-K1 cells.

Figure 2 shows the binding of the chimeric antibody to GPRC5D expressed on CHO-K1 cells.

Figure 3 shows antibody binding-induced endocytosis.

Figure 4 shows that the antibody has ADCC capability.

Figures 5-8 show the affinity test results of humanized antibodies derived from 6G10D9, 58F9G10, 34D3H1, and 37B9C4, respectively.

Figure 9 confirms the binding of the selected humanized antibody to GPRC5D expressed on CHO-K1 cells.

Figure 10 confirms the binding of the selected humanized antibody to GPRC5D expressed on NCI-H929 cells.

Figure 11 shows the ADCC efficacy of the tested humanized antibody.

Figure 12 shows the internalization-inducing activity of the tested humanized antibody.

Figure 13 shows the cell-killing activity of the tested antibody-drug conjugates.

Figure 14 shows the tumor-suppressive activity of the tested antibodies.

Detailed Implementation

definition

It should be noted that the terms "an" or "a type" refer to one or more of the entity; for example, "an antibody" should be understood to mean one or more antibodies. Thus, the terms "an" (or "a type"), "one or more", and "at least one" are used interchangeably herein.

As used herein, the term "polypeptide" is intended to encompass both the singular and plural forms of "polypeptide" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids and does not refer to a product of a specific length. Therefore, the definition of "polypeptide" includes peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to one or more chains of two or more amino acids, and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of a polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modifications by non-naturally occurring amino acids. Polypeptides may be derived from natural biological sources or produced through recombinant technologies, but are not necessarily translated from a specified nucleic acid sequence. They can be produced in any manner, including through chemical synthesis.

"Homology," "identity," or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence, which can be aligned for comparative purposes. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. "Unrelated" or "non-homologous" sequences share less than 40% identity with one of the sequences disclosed herein, but preferably less than 25%.

"Sequence identity" of a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) with another sequence is defined as the percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of the bases (or amino acids) that are the same when the two sequences are compared during alignment.

The term "equivalent nucleic acid or equivalent polynucleotide" refers to a nucleic acid having a nucleotide sequence that shares a degree of homology or sequence identity with the nucleotide sequence of a nucleic acid or its complement. Homologs of double-stranded nucleic acids are intended to include nucleic acids having a nucleotide sequence that shares a degree of homology with it or its complement. On the one hand, a homolog of a nucleic acid is capable of hybridizing with a nucleic acid or its complement. Similarly, "equivalent polypeptide" refers to a polypeptide that shares a degree of homology or sequence identity with 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 1, 2, 3, 4, or 5 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.

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 thereof. Therefore, the term "antibody" includes any protein or peptide containing a portion of at least an immunoglobulin molecule that has biological activity in binding to an antigen. Such 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 (FR) region or any portion thereof, or at least one portion of a binding protein.

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 dimeric forms. The term "antibody fragment" also includes any synthetic or genetically engineered protein that functions like an antibody by forming a complex with a specific antigen.

"Single-chain variable fragment" or "scFv" refers to a fusion protein of the variable region ( _VH ) of the heavy chain and the variable region ( _VL ) of the light chain of an immunoglobulin. In some aspects, the region is linked to a short linker peptide of 10 to about 25 amino acids. The linker may be enriched with glycine to improve flexibility and with serine or threonine to improve solubility, and may link the N-terminus of _VH to the C-terminus of _VL , and vice versa. Despite the removal of the constant region and the introduction of the linker, the protein retains the specificity of the original immunoglobulin. ScFv molecules are known in the art and are described, for example, in U.S. Patent 5,892,019.

The term antibody encompasses a wide range of polypeptides that can be distinguished biochemically. Those skilled in the art will understand that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with several subclasses (e.g., γ1-γ4). The properties of this chain determine the "class" of the antibody, namely IgG, IgM, IgA, IgG, or IgE. Immunoglobulin subclasses (isotypes), such as _IgG1 , _IgG2 , _IgG3 , _IgG4 , _IgG5 , etc., have been well characterized and are known to confer functional specificity. Given this disclosure, modified versions of each of these classes and isotypes are readily identifiable to those skilled in the art and are therefore within the scope of this disclosure. All immunoglobulin classes are obviously within the scope of this disclosure, and the following discussion will generally refer to the IgG class of 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 molecular weights of 53,000-70,000. The four chains are typically connected by disulfide bonds in a “Y” configuration, where the light chain encloses the heavy chain, starting from the opening of the “Y” and extending through the variable region.

The antibodies, antigen-binding peptides, variants, or derivatives disclosed herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primate-derived, or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as 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 from Fab expression libraries, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules disclosed herein can be 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.

Light chains are classified as kappa or lambda (κ, λ). Each heavy chain class can bind to either a κ or λ light chain. Typically, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the light and heavy chains are covalently bonded to each other, with the "tails" of the two heavy chains linked together by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus of the Y-configuration branch to the C-terminus at the base of each chain.

Both light and heavy chains are divided into structural homologous regions and functional homologous regions. The terms "constant" and "variable" are used functionally. In this regard, it should be understood that the variable domains (VK) of the light chain moiety and the variable domains (VH) of the heavy chain moiety determine antigen recognition and specificity. Conversely, the constant domains (CK) of the light chain and the constant domains (CH1, CH2, or CH3) of the heavy chain confer important biological properties, such as secretion, transplacental migration, Fc receptor binding, and complement binding. By convention, the numbering of constant domains increases as they move further away from the antibody's antigen-binding site or N-terminus. The N-terminal portion is the variable region, and the C-terminal portion is the constant region; the CH3 and CK domains actually contain the carboxyl termini of the heavy and light chains, respectively.

As described above, the variable region allows antibodies to selectively recognize and specifically bind to epitopes on antigens. That is, a subset of the antibody's VK and VH domains, or complementarity-determining regions (CDRs), forms the variable region defining a three-dimensional antigen-binding site. This quaternary antibody structure forms antigen-binding sites located at the ends of the arms of the Y. More specifically, the antigen-binding site is defined by three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) on each of the VH and VK chains. In some cases, such as certain immunoglobulin molecules derived from camel species or engineered from camel immunoglobulins, the complete immunoglobulin molecule may consist only of the heavy chain, without the light chain. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993).

In naturally occurring antibodies, six "complementarity-determining regions" or "CDRs" present in each antigen-binding domain are short, discontinuous amino acid sequences specifically positioned to form the antigen-binding domain when the antibody exhibits its three-dimensional conformation in an aqueous environment. The remaining amino acids in the antigen-binding domain (called "framework" regions) exhibit less intermolecular variability. The framework regions primarily employ a β-sheet structure, and the CDRs form loops that connect to the β-sheet structure and, in some cases, form part of the β-sheet structure. Thus, the framework regions act as a scaffold that positions the CDRs in the correct orientation through interchain non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to an epitope on an immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to its homologous epitope. Those skilled in the art can readily identify the amino acids containing the CDR and framework regions, respectively, for any given heavy or light chain variable region, as they have been precisely defined (see "Sequences of Proteins of Immunological Interest", Kabat, E. et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. MoI. Biol., 196: 901-917 (1987)).

Where a term used and/or accepted in the art has two or more definitions, 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 a discontinuous antigen-binding site found within the variable region of both heavy and light chain polypeptides. Such a specific region has been described by Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and Chothia et al., J. MoI. Biol. 196: 901-917 (1987), which are incorporated herein by reference in their entirety. When compared with each other, the CDR definition according to Kabat and Chothia includes overlaps or subsets of amino acid residues. However, the application of any definition of a CDR used to refer to an antibody or a variant thereof is intended to be within the scope of the terms defined and used herein. For comparison, the table below lists the appropriate amino acid residues that cover the CDRs as defined in the references cited above. The exact residue numbers covering a particular CDR will vary depending on the sequence and size of the CDR. Given the amino acid sequence of the variable region of an antibody, those skilled in the art can routinely determine which residues contain a particular CDR.

Kabat Chothia CDR-H1 31-35 26-32 CDR-H2 50-65 52-58 CDR-H3 95-102 95-102 CDR-L1 24-34 26-32 CDR-L2 50-56 50-52 CDR-L3 89-97 91-96

Kabat et al. also defined a numbering system applicable to the variable domain sequence of 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 outside the sequence itself. As used herein, “Kabat numbering” refers to the numbering system shown by Kabat et al., U.S. Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).

In addition to the table above, the Kabat numbering system also describes the CDR regions as follows: CDR-H1 begins at approximately the 31st amino acid (i.e., approximately 9 residues after the first cysteine residue), consists of approximately 5–7 amino acids, and terminates at the next tryptophan residue. CDR-H2 begins at the 15th residue after the end of CDR-H1, consists of approximately 16–19 amino acids, and terminates at the next arginine or lysine residue. CDR-H3 begins at approximately the 33rd amino acid residue after the end of CDR-H2; consists of 3–25 amino acids; and terminates at the sequence W-G-X-G, where X is any amino acid. CDR-L1 begins at approximately the 24th residue (i.e., after the cysteine residue); consists of approximately 10–17 residues; and terminates at the next tryptophan residue. CDR-L2 begins at approximately the 16th residue after the end of CDR-L1, and consists of approximately 7 residues. CDR-L3 begins at approximately the 33rd residue after the end of CDR-L2 (i.e., after the cysteine residue); it contains approximately 7-11 residues and terminates at sequence F or W-G-X-G, where X is any amino acid.

The antibodies disclosed herein can be derived from any animal source, including birds and mammals. Preferably, the antibodies are from humans, mice, donkeys, rabbits, goats, guinea pigs, camels, llamas, horses, or chickens. In another embodiment, the variable region can be of condricthoid origin (e.g., from sharks).

As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from the immunoglobulin heavy chain. A polypeptide containing a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide for use in this disclosure may comprise a polypeptide chain containing a CH1 domain; a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, and a CH2 domain; a polypeptide chain containing both a CH1 domain and a CH3 domain; a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, and a CH3 domain; or a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of this disclosure comprises a polypeptide chain containing a CH3 domain. Further, an antibody for use in this disclosure may lack at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). As described above, those skilled in the art will understand that the heavy chain constant regions can be modified so that they differ in amino acid sequence from naturally occurring immunoglobulin molecules.

The heavy chain constant region of the antibody disclosed herein can be derived from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide can include a CH1 domain derived from an _IgG1 molecule and a hinge region derived from an _IgG3 molecule. In another example, the heavy chain constant region can include a hinge region partially derived from an _IgG1 molecule and partially derived from an _IgG3 molecule. In yet another example, the heavy chain portion can include a chimeric hinge partially derived from an _IgG1 molecule and partially derived from an _IgG4 molecule.

As used herein, the term "light chain constant region" includes an amino acid sequence derived from the antibody light chain. Preferably, the light chain constant region comprises at least one of a constant κ domain or a constant λ domain.

A "light chain-heavy chain pair" refers to a combination of light and heavy chains that can form a dimer through disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain.

As previously mentioned, the subunit structures and three-dimensional conformations of constant regions of various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the amino-terminal variable domain of the immunoglobulin heavy chain, and the term "CH1 domain" includes the first (most amino-terminal) constant region domain of the immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is located at the amino terminus of the hinge region of the immunoglobulin heavy chain molecule.

As used herein, the term "CH2 domain" includes, for example, the portion of a heavy chain molecule extending from approximately residue 244 to residue 360 of an antibody (using the conventional numbering scheme) (residues 244 to 360, Kabat numbering system; and residues 231–340, EU numbering system; see Kabat et al., U.S. Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983)). The CH2 domain is unique in that it does not pair tightly with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the intact native IgG molecule. It has also been well demonstrated that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues.

As used herein, the term "hinge region" encompasses the portion of the heavy chain molecule that connects the CH1 and CH2 domains. This hinge region contains approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be further subdivided into three distinct domains: the upper, middle, and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).

As used herein, the term "disulfide bond" refers to a covalent bond formed between two sulfur atoms. The amino acid cysteine contains a thiol group, which can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CK regions are linked by disulfide bonds, and the two heavy chains are linked by two disulfide bonds at positions 239 and 242 (using the Kabat numbering system) (position 226 or 229, EU numbering system).

As used herein, the term "chimeric antibody" will be considered to mean any antibody in which the immunoreactive region or immunoreactive site is derived from or originates from a first species and the constant region (which, according to this disclosure, may be whole, partial, or modified) is derived from a second species. In some embodiments, the target binding region or target binding site will be derived from a non-human source (e.g., mouse or primate), while the constant region is human.

As used in this paper, “percentage humanization” is calculated by determining the number of framework amino acid differences (i.e., non-CDR differences) between humanized and germline domains, subtracting that number from the total number of amino acids, dividing by the total number of amino acids, and then multiplying by 100.

"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 readily than to a random, unrelated epitope. The term "specific" is used herein 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" can be said to bind to epitope "C" with higher specificity than to related epitope "D."

As used herein, the term “treatment” refers to therapeutic treatment and preventative or defensive measures aimed at preventing or slowing (reducing) undesirable physiological changes or disorders, such as the progression of cancer. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, whether detectable or undetectable; reduction of disease severity; stabilization of disease status (i.e., no worsening); delay or slowing of disease progression; improvement or reduction of disease status; and remission (whether partial or complete). “Treatment” can also mean extended survival compared to the expected survival without treatment. Subjects requiring treatment include those who already have a condition or disorder, those who are susceptible to a condition or disorder, or those who wish to prevent a condition or disorder.

"Subject," "individual," "animal," "patient," or "mammal" refers to any subject requiring diagnosis, prognosis, or treatment, particularly a mammalian subject. Mammal subjects include humans, livestock, farm animals and zoo animals, sporting animals, or pets such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, domestic cattle, and dairy cows.

As used herein, phrases such as “patient in need of treatment” or “subject in need of treatment” include subjects who will benefit from the administration of antibodies or compositions used in this disclosure, such as those for detection, diagnostic procedures and/or treatment, such as mammalian subjects.

Anti-GPRC5D antibody

Using hybridoma technology, the attached experimental examples demonstrate the acquisition of a variety of mouse antibodies. Fourteen mouse antibodies were sequenced, and humanized antibodies were prepared. These humanized antibodies exhibited strong GPRC5D binding activity and were able to induce receptor-mediated endocytosis. In vivo experiments showed that these antibodies possessed activity in inducing ADCC and inhibiting tumor development.

Four mouse antibodies, 34D3H1, 37B9C4, 58F9G10, and 6G10D9, have undergone humanization. Some humanized antibodies, including 6-H3L3, 6-H4L3 (both derived from 6G10D9), 58-H1L1, 58-H3L1 (both derived from 58F9G10), 34-H1L1 (derived from 34D3H1), and 37-H1L1 (both derived from 37B9C4), further demonstrate promise for continued clinical development.

Therefore, according to one embodiment of this disclosure, an antibody or antigen-binding fragment thereof is provided that has binding specificity to human G protein-coupled receptor C family 5 member D (GPRC5D) protein. The antibody or fragment thereof includes a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising heavy chain complementarity-determining regions CDRH1, CDRH2, and CDRH3, and the light chain variable region comprising complementarity-determining regions CDRL1, CDRL2, and CDRL3.

In some embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 each comprise (a) the amino acid sequence of SEQ ID NO:29-34; (b) the amino acid sequence of SEQ ID NO:42-47; (c) the amino acid sequence of SEQ ID NO:54-59 or SEQ ID NO:54, 60 and 56-59; or (d) the amino acid sequence of SEQ ID NO:68-73.

In one embodiment, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 each comprise the amino acid sequences of SEQ ID NO: 29-34. Example sequences of VH include SEQ ID NO: 7 and 35-37 or sequences having at least 85%, 90%, or 95% sequence identity with any one of SEQ ID NO: 7 and 35-37. Example sequences of VL include SEQ ID NO: 8 and 38-41 or sequences having at least 85%, 90%, or 95% sequence identity with any one of SEQ ID NO: 8 and 38-41.

In one embodiment, VH comprises the amino acid sequence of SEQ ID NO:35 or a sequence having at least 85%, 90%, or 95% sequence identity with SEQ ID NO:35. In one embodiment, VL comprises the amino acid sequence of SEQ ID NO:38 or a sequence having at least 85%, 90%, or 95% sequence identity with SEQ ID NO:38.

In some embodiments, an antibody and its antigen-binding fragment that bind to the same epitope on FAPα as any of these antibodies are also provided. In some embodiments, an antibody and its antigen-binding fragment that compete with any of these antibodies for binding to FAPα are also provided, such as an antibody and its antigen-binding fragment having a VH comprising the amino acid sequence of SEQ ID NO:35 and a VL comprising the amino acid sequence of SEQ ID NO:38.

In one embodiment, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 each comprise the amino acid sequences of SEQ ID NO:42-47. Instance sequences of VH include SEQ ID NO:9 and 48-50 or sequences having at least 85%, 90%, or 95% sequence identity with any one of SEQ ID NO:9 and 48-50. Instance sequences of VL include SEQ ID NO:10 and 51-53 or sequences having at least 85%, 90%, or 95% sequence identity with any one of SEQ ID NO:10 and 51-53.

In one embodiment, VH comprises the amino acid sequence of SEQ ID NO:48 or a sequence having at least 85%, 90%, or 95% sequence identity with SEQ ID NO:48. In one embodiment, VL comprises the amino acid sequence of SEQ ID NO:51 or a sequence having at least 85%, 90%, or 95% sequence identity with SEQ ID NO:51.

In some embodiments, an antibody and its antigen-binding fragment that bind to the same epitope on FAPα as any of these antibodies are also provided. In some embodiments, an antibody and its antigen-binding fragment that compete with any of these antibodies for binding to FAPα are also provided, such as an antibody and its antigen-binding fragment having a VH comprising the amino acid sequence of SEQ ID NO:48 and a VL comprising the amino acid sequence of SEQ ID NO:51.

In one embodiment, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 each comprise the amino acid sequences of SEQ ID NO: 54-59. Instance sequences of VH include SEQ ID NO: 61-64 or sequences having at least 85%, 90%, or 95% sequence identity with any one of SEQ ID NO: 61-64. Instance sequences of VL include SEQ ID NO: 16 and 65-67 or sequences having at least 85%, 90%, or 95% sequence identity with any one of SEQ ID NO: 16 and 65-67.

In one embodiment, VH comprises the amino acid sequence of SEQ ID NO:61 or a sequence having at least 85%, 90%, or 95% sequence identity with SEQ ID NO:61. In one embodiment, VL comprises the amino acid sequence of SEQ ID NO:65 or a sequence having at least 85%, 90%, or 95% sequence identity with SEQ ID NO:65.

In some embodiments, an antibody and its antigen-binding fragment that bind to the same epitope on FAPα as any of these antibodies are also provided. In some embodiments, an antibody and its antigen-binding fragment that compete with any of these antibodies for binding to FAPα are also provided, such as an antibody and its antigen-binding fragment having a VH comprising the amino acid sequence of SEQ ID NO:61 and a VL comprising the amino acid sequence of SEQ ID NO:65.

In one embodiment, VH comprises the amino acid sequence of SEQ ID NO:63 or a sequence having at least 85%, 90%, or 95% sequence identity with SEQ ID NO:63. In one embodiment, VL comprises the amino acid sequence of SEQ ID NO:65 or a sequence having at least 85%, 90%, or 95% sequence identity with SEQ ID NO:65.

In some embodiments, an antibody and its antigen-binding fragment that bind to the same epitope on FAPα as any of these antibodies are also provided. In some embodiments, an antibody and its antigen-binding fragment that compete with any of these antibodies for binding to FAPα are also provided, such as an antibody and its antigen-binding fragment having a VH comprising the amino acid sequence of SEQ ID NO:63 and a VL comprising the amino acid sequence of SEQ ID NO:65.

In one embodiment, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences of SEQ ID NO:54, 60, and 56-59, respectively. Instance sequences of VH include SEQ ID NO:15 or sequences having at least 85%, 90%, or 95% sequence identity with any one of SEQ ID NO:15. Instance sequences of VL include SEQ ID NO:16 and 65-67 or sequences having at least 85%, 90%, or 95% sequence identity with any one of SEQ ID NO:16 and 65-67.

In one embodiment, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 each comprise the amino acid sequences of SEQ ID NO: 68-73. Example sequences of VH include SEQ ID NO: 1 and 74-79, or sequences having at least 85%, 90%, or 95% sequence identity with any one of SEQ ID NO: 1 and 74-79. Example sequences of VL include SEQ ID NO: 2 and 80-86, or sequences having at least 85%, 90%, or 95% sequence identity with any one of SEQ ID NO: 2 and 80-86.

In one embodiment, VH comprises the amino acid sequence of SEQ ID NO:76 or a sequence having at least 85%, 90%, or 95% sequence identity with SEQ ID NO:76. In one embodiment, VL comprises the amino acid sequence of SEQ ID NO:82 or a sequence having at least 85%, 90%, or 95% sequence identity with SEQ ID NO:82.

In some embodiments, an antibody and its antigen-binding fragment that bind to the same epitope on FAPα as any of these antibodies are also provided. In some embodiments, an antibody and its antigen-binding fragment that compete with any of these antibodies for binding to FAPα are also provided, such as an antibody and its antigen-binding fragment having a VH comprising the amino acid sequence of SEQ ID NO:76 and a VL comprising the amino acid sequence of SEQ ID NO:82.

In one embodiment, VH comprises the amino acid sequence of SEQ ID NO:77 or a sequence having at least 85%, 90%, or 95% sequence identity with SEQ ID NO:77. In one embodiment, VL comprises the amino acid sequence of SEQ ID NO:82 or a sequence having at least 85%, 90%, or 95% sequence identity with SEQ ID NO:82.

In some embodiments, an antibody and its antigen-binding fragment that bind to the same epitope on FAPα as any of these antibodies are also provided. In some embodiments, an antibody and its antigen-binding fragment that compete with any of these antibodies for binding to FAPα are also provided, such as an antibody and its antigen-binding fragment having a VH comprising the amino acid sequence of SEQ ID NO:77 and a VL comprising the amino acid sequence of SEQ ID NO:82.

In some embodiments, the antibody or a fragment thereof has ADCC capability. Methods and materials suitable for manufacturing antibodies with ADCC capability are known in the art, such as by using a suitable Fc fragment or by reducing/removing fucosylation.

In some embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 each comprise (a) the amino acid sequence of SEQ ID NO: 29-34; (b) the amino acid sequence of SEQ ID NO: 42-47; (c) the amino acid sequence of SEQ ID NO: 54-59 or SEQ ID NO: 54, 60, and 56-59; or (d) the amino acid sequence of SEQ ID NO: 68-73, wherein each of the CDR sequences comprises one, two, or three conserved amino acid substitutions.

"Conservative amino acid substitution" refers to the substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged 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 peptides 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 members of the side chain family that differ in sequence and/or composition but are structurally similar.

The table below provides non-limiting examples of conserved amino acid substitutions, where a similarity score of 0 or higher indicates a conserved substitution between two amino acids.

Table A. Amino acid similarity matrix

C G P S A T D E N Q H K R V M I L F Y W W -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17 Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10 F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 0 1 2 9 L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6 I -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5 M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6 V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4 R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6 K -5 -2 -1 0 -1 0 0 0 1 1 0 5 H -3 -2 0 -1 -1 -1 1 1 2 3 6 Q -5 -1 0 -1 0 -1 2 2 1 4 N -4 0 -1 1 0 0 2 1 2 E -5 0 -1 0 0 0 3 4 D -5 1 -1 0 0 0 4 T -2 0 0 1 1 3 A -2 1 1 1 2 S 0 1 1 1 P -3 -1 6 G -3 5 C 12  

Table B. Conserved Amino Acid Substitutions

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

In some embodiments, the anti-CCR8 antibody is a modified mAb containing a modified heavy chain constant region, such as a non-fucosylated heavy chain, which binds to the activated Fcγ receptor mediating enhanced ADCC with a higher affinity compared to an unmodified mAb. In some embodiments, the anti-CCR8 antibody contains the heavy chain of a human IgG1 variant, including single or combined L234Y, L235Q, G236W, S239D/M, F243L, H268D, D270E, R292P, S298A, Y300L, V305I, K326D, A330L/M, I332E, K334A/E, and P396L (which enhances ADCC function) (all EU designations).

In some embodiments, the antibody comprises an amino acid sequence or one or more portions that are not typically associated with the antibody. Exemplary modifications will be described in more detail below. For example, the antibodies of this disclosure may comprise a flexible linker sequence or may be modified to add a functional portion (e.g., a PEG, a drug, a toxin, or a marker).

The antibodies, variants, or derivatives thereof disclosed herein include modified derivatives, i.e., modified by covalent linking the antibody to any type of molecule 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, linking to cellular ligands or other proteins, etc. Any of a number 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

GPRC5D is overexpressed on certain malignant hematologic cells, such as multiple myeloma cells. Therefore, the antibodies and fragments disclosed herein can be used to target those malignant cells to inhibit, inactivate, or destroy them. In one instance, the antibody or fragment is conjugated to an agent that helps to inhibit, inactivate, or destroy malignant cells. This document shows that the antibody can induce GPRC5D-mediated endocytosis.

In some embodiments, the antibody or fragment may be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG. In some embodiments, the conjugator may be a short interfering RNA (siRNA) or an intrinsic regulator, such as an interferon gene stimulating factor (STING) agonist or a TLR7/8 agonist.

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

In some embodiments, the number p of pharmaceutical moieties conjugated to each antibody molecule ranges from 1 to 8 on average; 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p ranges from 2 to 8 on average, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In other embodiments, p averages 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, p averages from about 1 to about 20, from about 1 to about 10, from about 2 to about 10, from about 2 to about 9, from about 1 to about 8, from about 1 to about 7, from about 1 to about 6, from about 1 to about 5, from about 1 to about 4, from about 1 to about 3, or from about 1 to about 2. In some embodiments, p ranges from about 2 to about 8, from about 2 to about 7, from about 2 to about 6, from about 2 to about 5, from about 2 to about 4, or from about 2 to about 3.

For example, when the chemical activation of a protein leads to the formation of a free thiol group, the protein can conjugate with a thiol-reactive agent. On the one hand, this agent is essentially specific to free thiol groups. On the other hand, it is essentially 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., iodides, bromides, or chlorides), vinyl sulfones, and pyridine thiols.

Drugs can be linked to antibodies or fragments via adapters. Suitable adapters include, for example, cleavable and non-cleavable adapters. Cleavable adapters are generally readily cleavable under intracellular conditions. Suitable cleavable adapters include, for example, peptide adapters that can be cleaved by intracellular proteases, such as lysosomal proteases or endosomal proteases. In exemplary embodiments, the adapter can be a dipeptide adapter, such as a valine-citrulline (val-cit), a phenylalanine-lysine (phe-lys) adapter, or a maleimide hexanoate-valine-citrulline-p-aminobenzyloxycarbonyl (mc-Val-Cit-PABA) adapter. Another adapter is sulfosuccinimide-4-[N-maleimidemethyl]cyclohexane-1-carboxylic acid ester (smcc). Sulfo-smcc conjugation occurs via a maleimide group that reacts with a thiol (thiol, -SH), while its Sulfo-NHS ester is reactive to primary amines (such as those found at the N-terminus of lysine and proteins or peptides). Another type of linker is maleimide hexanoyl (mc). Other suitable linkers include those that are hydrolyzable at a specific pH or within a pH range, such as hydrazone linkers. Other suitable cleavable linkers include disulfide linkers. Linkers 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 linkers.

The linker may include a group for binding to the antibody. For example, the linker may include 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., iodide, bromide, or chloride), vinyl sulfone, and pyridine thiol).

In some embodiments, the drug component is a cytotoxic agent 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 in tumor or cancer cells, or treat cancer in patients. The conjugate can be used accordingly in various environments to treat cancer in animals. 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 GPRC5D, and the conjugate and/or drug can be taken up into tumor cells or cancer cells via receptor-mediated endocytosis.

Once inside the cell, one or more specific peptide sequences within the conjugate (e.g., in the connector) are hydrolyzed and 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 inhibition, or other activities. In some embodiments, the drug is cleaved from the antibody outside the tumor cell or cancer cell, and the drug subsequently penetrates the cell or acts on the cell surface.

Examples of drug portions or payloads are selected from the group consisting of: DM1 (matansine, N2'-deacetylated-N2'-(3-mercapto-1-oxopropyl)- or N2'-deacetylated-N2'-(3-mercapto-1-oxopropyl)-matansine), mc-MMAD (6-maleimide hexanoyl-monomethylaurestatin-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 auratestatin F or N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrolidin-1-yl)-1-oxohexyl]-N-methyl-L-valine-L-valine-(3R,4) S,5S)-3-methoxy-5-methyl-4-(methylamino)heptanyl-(αR,βR,2S)-β-methoxy-α-methyl-2-pyrrolidinepropionyl-L-phenylalanine) and mc-Val-Cit-PABA-MMAE(6-maleimide hexanoyl-ValcCit-(p-aminobenzyloxycarbonyl)-monomethylauratestatin E or N-[[[4-[[N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrolo-1-yl)-1-oxohexyl]-L- Valyl-N5-(aminocarbonyl)-L-ornithine]amino]phenyl]methoxy]carbonyl]-N-methyl-L-valyl-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-valylamide). DM1 is a derivative of the microtubule inhibitor maytansine, while MMAD, MMAE, and MMAF are derivatives of auristatin. In some embodiments, the pharmaceutical part is selected from the group consisting of mc-MMAF and mc-Val-Cit-PABA-MMAE. In some implementations, the drug component is maytansinoid or aurestatin.

Antibodies or fragments may be conjugated or fused with therapeutic agents, which may include detectable markers (such as radiolabels), immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutics 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.

Antibodies can be detectably labeled by conjugating them to chemiluminescent compounds. The presence of the chemiluminescently labeled antigen-binding peptide can then be determined by detecting the presence of light emitted during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds include luminol, isoluminol, theromatic acridine ester, imidazole, acridine salts, and oxalates.

Antibodies can also be detectably labeled using fluorescently emitting metals (such as ^Eu152 or other lanthanides). These metals can be linked to antibodies using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). The technique of conjugating different parts to antibodies is well known; see, for example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” *Monoclonal Antibodies and Cancer Therapy*, Reisfeld et al. (eds.), pp. 243–56 (Alan R. Liss, Inc. (1985)); Hellstrom et al., “Antibodies For Drug Delivery,” *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,” *Monoclonal Antibodies '84: Biological and Clinical Applications*, Pinchera et al. (eds.), pp. 475–506 (1985); “Analysis, Results, and Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer "Therapy", Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press, pp. 303-16 (1985), and Thorpe et al. "The Preparation and Cytotoxic Properties of Antibody-Toxin Conjugates", Immunol. Rev. (52:119-58 (1982)).

Bifunctional molecules and combination therapies

GPRC5D is overexpressed on certain malignant hematologic cells, such as multiple myeloma cells. Therefore, the antibodies and fragments disclosed herein can be used to target those malignant cells to inhibit, inactivate, or destroy them. In some embodiments, bifunctional or bispecific molecules/antibodies targeting both the GPRC5D protein and immune cells are provided.

In some implementations, immune cells are selected from the group consisting of T cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, phagocytes, natural killer cells, eosinophils, basophils, and mast cells. Molecules that can be targeted on immune cells include, for example, CD3, CD16, CD19, CD28, CD64, and 4-1BB (also known as CD137). Other examples include PD-1, CTLA-4, LAG-3 (also known as CD223), CD28, CD122, TIM3, OX-40 or OX40L, CD40 or CD40L, LIGHT, ICOS/ICOSL, GITR/GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM or BTLA (also known as CD272), killer cell immunoglobulin-like receptor (KIR), and CD47. Specific examples of bispecificity include, but are not limited to, GPRC5D/CD3.

Different forms of bispecific antibodies are also provided. In some embodiments, each of the anti-PD-L1 fragment and the second fragment is independently selected from Fab fragments, single-chain variable fragments (scFv), or single-domain antibodies. In some embodiments, the bispecific antibody further includes an Fc fragment.

Polynucleotides encoding antibodies and methods for preparing said antibodies

This disclosure also provides isolated polynucleotide or nucleic acid molecules encoding antibodies, variants, or derivatives thereof. The polynucleotides of this disclosure may encode the entire heavy chain variable region and light chain variable region of an antigen-binding polypeptide, variant, or derivative thereof, either on the same polynucleotide molecule or on different polynucleotide molecules. Additionally, the polynucleotides of this disclosure may encode portions of the heavy chain variable region and light chain variable region of an antigen-binding polypeptide, variant, or derivative thereof, either on the same polynucleotide molecule or on different polynucleotide molecules.

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 as described herein. For example, a fully human antibody against a specific antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such an antibody in response to an antigen attack, but whose endogenous loci have been disabled. Exemplary techniques that can be used to prepare such antibodies are described in U.S. Patents: 6,150,584, 6,458,592, and 6,420,140, which are incorporated herein by reference in their entirety.

Treatment

As described herein, the antibodies, variants, or derivatives disclosed herein can be used in certain therapeutic and diagnostic methods.

This disclosure further relates to antibody-based therapies involving the administration of the antibodies of this disclosure to patients, such as animals, mammals, and humans, to treat one or more disorders or conditions described herein. Therapeutic compounds of this disclosure include, but are not limited to, the antibodies of this disclosure (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding the antibodies of this disclosure (including variants and derivatives thereof as described herein).

The antibodies disclosed herein can also be used to treat or inhibit cancer. As mentioned above, GPRC5D can be overexpressed in cancer cells, particularly multiple myeloma. Inhibition of GPRC5D has been shown to be useful in cancer treatment.

Therefore, in some embodiments, methods for treating cancer in patients in need are provided. In one embodiment, the method involves administering an effective amount of the antibody disclosed herein to the patient. In some embodiments, at least one cancer cell in the patient overexpresses GPRC5D. In some embodiments, the antibody or fragment has ADCC capability. In some embodiments, the antibody or fragment further comprises a cytotoxic agent. In some embodiments, the antibody is bispecific, further targeting immune cells, such as cytotoxic T cells.

This disclosure also provides cell therapies, such as chimeric antigen receptor (CAR) T-cell (or NK cell, macrophage) therapy. Suitable cells can be used, which are contacted with the anti-GPRC5D antibody of this disclosure (or alternatively engineered to express the anti-GPRC5D antibody of this disclosure). After such contact or engineering, the cells can then be introduced into a cancer patient in need of treatment. The cancer patient can have any type of cancer as disclosed herein. The cells (e.g., T cells) can be, for example, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, natural killer (NK) cells, macrophages, or combinations thereof, but are not limited thereto.

In some implementations, the cells are isolated from the cancer patient themselves. In other implementations, the cells are provided by a donor or cell bank. When cells are isolated from a cancer patient, unwanted immune responses can be minimized.

Non-limiting examples of cancer include blood cancers, such as multiple myeloma. Other examples include leukemia (including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)) and lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease) and multiple myeloma.

The specific dosage and treatment regimen for any given patient will depend on a number of factors, including the specific antibody used, its variants or derivatives, the patient's age, weight, general health condition, sex, diet, as well as the timing of administration, excretion rate, concomitant medications, and the severity of the specific disease being treated. The healthcare professional's judgment regarding these factors is within the realm of ordinary skill in the art. The 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.

Methods of administration for antibodies or variants include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Antigen-binding peptides or compositions can be administered via any convenient route, such as by infusion or bolus injection, by absorption through the epithelial or mucosal inner layers of the skin (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 (e.g., via powder, ointment, drops, or transdermal patch), orally, or as oral or nasal sprays.

As used in this article, the term "parenteral" refers to the route of administration, including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

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

It may be necessary to apply the antigen-binding peptides or compositions of this disclosure topically to the area requiring treatment; this can be achieved, for example, but not limited to, local infusion during surgery, topical application (e.g., postoperatively in combination with wound dressings), by injection, via catheter, via suppository, or via implantation, said implant being a porous, non-porous, or gel-like material, including membranes, such as sialic acid membranes or fibers. Preferably, when administering the proteins (including antibodies) of this disclosure, care must be taken to use materials that do not absorb the proteins.

The amount of the disclosed antibody that can effectively treat, inhibit, and prevent inflammatory, immune, or malignant diseases, disorders, or conditions can be determined using standard clinical techniques. Alternatively, in vitro analysis can be used to help determine the optimal dose range. The precise dose used in the formulation also depends on the route of administration and the severity of the disease, disorder, or condition, and should be determined based on the practitioner's judgment and the individual patient's situation. The effective dose can be deduced from dose-response curves from in vitro or animal model testing systems.

As a general recommendation, the dose of the antigen-binding peptide of this disclosure administered to patients is typically 0.1 mg/kg to 100 mg/kg of patient body weight, 0.1 mg/kg to 20 mg/kg of patient body weight, or 1 mg/kg to 10 mg/kg of patient body weight. Generally, human antibodies have a longer half-life in the human body than antibodies from other species due to the immune response to exogenous peptides. Therefore, lower doses and lower frequencies of administration of human antibodies are generally possible. Furthermore, the dosage and frequency of administration of the antibodies of this disclosure can be reduced by modification, such as lipolysis, to enhance antibody uptake and tissue penetration (e.g., into the brain).

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.

In another embodiment, the compositions disclosed herein are administered in combination with other treatment or preventative measures (e.g., radiotherapy).

Diagnostic methods

Overexpression of GPRC5D has been observed in certain tumor samples, and patients with GPRC5D-overexpressing cells may respond to treatment with the anti-GPRC5D antibody disclosed herein. Therefore, the antibody disclosed herein can also be used for diagnostic and prognostic purposes.

Samples, preferably comprising cells, can be obtained from a patient, who may be a cancer patient or a patient requiring diagnosis. The cells are cells from tumor tissue or tumor masses, blood samples, urine samples, or any sample from the patient. After optional pretreatment of the sample, the sample can be incubated with the antibody of this disclosure under conditions that allow the antibody to interact with the potentially present GPRC5D protein in the sample. The presence of the GPRC5D protein in the sample can be detected using methods such as ELISA, utilizing the anti-GPRC5D antibody.

The presence of GPRC5D protein in a sample (optionally in a certain amount or concentration) can be used to diagnose cancer, as an indicator of a patient's suitability for antibody therapy, or as an indicator of whether a patient has (or has not) responded to cancer treatment. For prognostic methods, one, two, or more tests can be performed at certain stages after the initiation of cancer treatment to indicate treatment progress.

Composition

This disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of antibody and an acceptable carrier. In some embodiments, the composition further comprises a second anticancer agent (e.g., an immune checkpoint inhibitor).

In one specific implementation, the term "pharmaceuticalally acceptable" means a substance approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other recognized pharmacopoeia for use in animals, and more particularly for humans. Further, "pharmaceuticalally acceptable carrier" will generally be any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation adjuvant.

The term "carrier" refers to a diluent, adjuvant, excipient, or excipient that is administered with 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 the preferred carrier when the drug composition is administered intravenously. Saline solutions, as well as aqueous solutions of glucose and glycerol, can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers, such as acetates, citrates, or phosphates. Antimicrobial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and agents for tonicity adjustment, such as sodium chloride or dextrose. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The composition can be formulated into suppositories with conventional binders and carriers (e.g., 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 drug carriers are described in E.W. 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 purified form, together with an appropriate amount of carrier, to provide a form suitable for appropriate administration to the patient. The formulation should be suitable for the route of administration. Parenteral preparations can be packaged in glass or plastic ampoules, disposable syringes, or multi-dose vials.

In one embodiment, the composition is formulated according to standard 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 relieve pain at the injection site. Typically, the components are provided individually or in combination in unit dosage forms, for example, as a dry lyophilized powder or anhydrous concentrate in a closed container, such as an ampoule or capsule indicating the amount of active agent. When the composition is to be administered by infusion, it can be dispensed using an infusion bottle filled with sterile pharmaceutical-grade water or saline. When the composition is to be administered by injection, a single ampoule of sterile water for injection or saline can be provided so that the components can be mixed prior to administration.

The compounds disclosed herein can be formulated into neutral or salt forms. 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, iron hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

Example

Example 1: Generation of mouse monoclonal antibody against human GPRC5D

Mice of different strains were immunized with human GPRC5D protein, resulting in the generation of hybridomas. More than twenty hybridoma clones were collected for further analysis.

The binding of antibodies harvested from hybridoma supernatant to human GPRC5D protein expressed on CHO K1 cells was tested. CHO-K1 cells stably expressing human GPRC5D were harvested from flasks. 100 μl of cells (1 × ^10⁶ cells/ml) were incubated with serially diluted 3-fold mouse antibody on ice for 30 min. After washing twice with 200 μl of FACS buffer, the cells were incubated with secondary antibody on ice for 30 min. The cells were then washed twice with 200 μl of FACS buffer and transferred to BD Falcon 5 ml tubes for FACS analysis. The results showed that the mouse antibody could bind to human GPRC5D with a high EC50 (Figure 1). The results are shown in the table below. All antibodies exhibited good binding activity.

Fourteen hybridomas were subcloned and the VH/VL sequences were determined (see Table 1).

Table 1. VH/VL sequences of the lead mouse antibody

Example 2. Binding of chimeric antibody to GPRC5D

The mouse VH and VK genes were synthesized and then cloned into vectors containing the human γ1 constant domain and the human κ constant domain, respectively. Purified chimeric antibodies were produced from transfected CHO cells.

CHO-K1 cells stably expressing human GPRC5D were harvested from flasks. 100 μl of cells (1 × ^10⁶ cells/ml) were incubated on ice for 30 min with a primary chimeric antibody serially diluted 3-fold from 300 nM to 0.001 nM. After washing twice with 200 μl of FACS buffer, the cells were incubated on ice with the secondary antibody for 30 min. The cells were then washed twice with 200 μl of FACS buffer and transferred to BD Falcon 5 ml tubes for FACS analysis. The results showed that the chimeric antibody can bind to human GPRC5D with a high EC50 (Figure 2).

The results are shown in the table below.

Minimum Response Maximum response LogEC50 Curve slope EC50 span 58F9G10 -521.8 414579 0.3596 0.8258 2.289 415101 40A5B2 1487 440269 0.3436 0.8189 2.206 438781 38A9E10A11 3016 386984 0.2453 1.037 1.759 383968 37B9C4 2078 405062 0.249 1.045 1.774 402985 34D3H1 3448 507844 0.5214 1.012 3.322 504396 29B2C10 4326 430443 0.342 1.041 2.198 426116 21C3C11B3 5774 403938 0.2293 1.286 1.696 398164 6G10D9 6886 458124 0.1012 1.343 1.262 451238

Example 3. Internalization of chimeric GPRC5D antibody into EC50 cells on CHO-GPRC5D cells

pHAb dyes are pH sensor dyes that exhibit very low fluorescence at pH > 7, and their fluorescence increases significantly as the solution becomes more acidic. The excitation maximum (Ex) of pHAb dyes is at 532 nm, and the emission maximum (Em) is at 560 nm. pHAb dye-conjugated antibodies can be used to monitor receptor-mediated antibody internalization. When an antibody-pHAb dye conjugate binds to its receptor on the cell membrane, it exhibits minimal fluorescence. However, after receptor-mediated internalization, the antibody-pHAb dye conjugate is transported to acidic endosomes and lysosomal vesicles, causing the pHAb dye to fluoresce. This fluorescence can be detected using various techniques, including cell imaging, flow cytometry, and fluorescent plate-based readers with appropriate filters.

Stably transfected human GPRC5D CHO cells were harvested using 0.05% trypsin/EDTA (Gibco, 25300-054) and seeded in 96-well black plates (Thermo Scientific #165305) at a density of 10K/90μl/well. The plates were incubated for 20–24 hours before treatment with pHAb-labeled antibody.

For internalization, add the pHAb-conjugated chimeric GPRC5D antibody to the cells at varying concentrations and gently mix on a plate mixer for 1–2 minutes, then incubate overnight to allow internalization (which can be detected within a few hours). Read the plate using a fluorescent plate reader (Tecan Infinity M1000 Pro at Ex/Em: 532nm/560nm). For higher sensitivity, replace the culture medium with PBS before reading the plate.

The results of DAR normalization are shown in Figure 3. As shown in Figure 3, the tested chimeric antibody exhibits strong internalization activity.

Example 4. Antibody-dependent cytotoxicity (ADCC) assay

The ADCC Reporter Bioassay uses an alternative reading early in ADCC MOA pathway activation: gene transcriptional activation via the NFAT (nuclear factor for activating T cells) pathway in effector cells. Additionally, the ADCC Reporter Bioassay uses engineered Jurkat cells as effector cells, stably expressing the FcγRIIIa receptor, the V158 (high-affinity) variant, and the NFAT-responsive element that drives firefly luciferase expression. Antibody bioactivity in ADCC MOA is quantified by luciferase activity generated by NFAT pathway activation; luciferase activity in effector cells is quantified by luminescent readings. High signal intensity and low background are observed.

With or without ADCC Bioassay target cells (GPRC5D), serially diluted GPRC5D chimeric monoclonal antibodies were incubated with engineered Jurkat effector cells (ADCC Bioassay effector cells) at 37°C for 6 hours to induce ADCC. Luciferase activity was quantified using Bio-Glo ^™ reagents (Figure 4). All tested antibodies exhibited strong ADCC-inducing capabilities.

Example 5: Humanization of mouse mAb

Humanized mAbs were generated using the variable region gene of mouse antibodies. In the first step of this process, the amino acid sequences of the VH and VL of the mAb were compared with available databases of human Ig gene sequences to find the best-matching human Ig gene sequence overall.

The amino acid sequence of the humanized antibody is provided below.

Humanized sequence

A.34D3H1

Table 2A. Humanization of 34D3H1–VH

Table 2B. CDR Sequences

CDR sequence SEQ ID NO: CDR-H1 SYGMS 29 CDR-H2 TISSGGSYTYYPDSVKG 30 CDR-H3 QGGDAMDY 31

Table 2C.34D3H1–VL Humanization

Table 2D.CDR Sequence

CDR sequence SEQ ID NO: CDR-L1 RASQSINNNLH 32 CDR-L2 YASQSIS 33 CDR-L3 QQSNSRLT 34

Table 2E. Humanized Antibodies

VL VL v1 VL v2 VL v3 VL v4 VH 34-XI VH v1 34-H1L1 34-H1L2 34-H1L3 34-H1L4 VH v2 34-H2L1 34-H2L2 34-H2L3 34-H2L4 VH v3 34-H3L1 34-H3L2 34-H3L3 34-H3L4

B.37B9C4

Table 3A.37B9C4–VH Humanization

Table 3B. CDR Sequences

CDR sequence SEQ ID NO: CDR-H1 DYWMN 42 CDR-H2 EIRLKSNNYATHYAESVKG 43 CDR-H3 PLLWFRRYYAMDY 44

Humanization of Table 3C.37B9C4–VL

Table 3D.CDR Sequence

CDR sequence SEQ ID NO: CDR-L1 SASQGISNYLN 45 CDR-L2 YTSSLHS 46 CDR-L3 QQYSKLPFT 47

Table 3E. Humanized Antibodies

VL VL v1 VL v2 VL v3 VH 37B9C4-XI VH v1 37-H1L1 37-H1L2 37-H1L3 VH v2 37-H2L1 37-H2L2 37-H2L3 VH v3 37-H3L1 37-H3L2 37-H3L3

C.58F9G10

Table 4A. Humanization of 58F9G10–VH

Table 4B. CDR Sequences

Table 4C.58F9G10–VL Humanization

Table 4D.CDR Sequence

Table 3E. Humanized Antibodies

VL VL v1 VL v2 VL V3 VH 58F9G10-XI VH v1 58-H1L1 58-H1L2 58-H1L3 VH v2 58-H2L1 58-H2L2 1) 58-H2L3: VH v3 58-H3L1 58-H3L2 58-H3L3 VH v4 58-H4L1 58-H4L2 58-H4L3

D.6G10D9

Table 5A. Humanization of 6G10D9–VH

Table 5B. CDR Sequences

Table 5C.6G10D9–VL Humanization

Table 5D.CDR Sequence

CDR sequence SEQ ID NO: CDR-L1 KASQNVGTAVV 71 CDR-L2 SASNRYT 72 CDR-L3 QQYSSYPYT 73

Table 5E. Humanized Antibodies

Example 6. Testing of humanized antibodies

This embodiment tested the ability of some humanized antibodies to bind to GPRC5D expressed on CHO-K1 cells.

Among the humanized antibodies derived from 6G10D9 tested, 6-H2L1 and 6-H3L3 outperformed the other antibodies (Figure 5, Table 6).

Table 6.6 Activity of humanized antibodies against G10D9 binding to GPRC5D

Minimum Response Maximum response LogEC50 Curve slope EC50 span 6G10D9-XI 6858 121725 0.2992 1.525 1.991 114867 6-H1L1 192.6 ~6397 ~3.102 1.198 ~1264 ~6205 6-H1L2 191.9 898894 5.224 1.19 167639 898702 6-H1L3 226.2 16478302 5.389 1.156 244790 16478076 6-H2L1 189.1 661560 5.039 1.232 109294 661371 6-H2L2 192.5 1271084 5.37 1.191 234364 1270892 6-H2L3 215.7 10592380 5.039 1.23 109279 10592164 6-H3L1 -205.1 74293 2.352 0.8392 225.1 74498 6-H3L2 68.94 47463 2.244 0.9532 175.3 47394 6-H3L3 3994 119304 0.9553 1.13 9.022 115310 6-H4L1 61.5 50293 1.654 0.9277 45.05 50231 6-H4L2 9.56 39449 1.976 0.911 94.63 39440 6-H4L3 2344 126413 0.9049 1.052 8.033 124069

As shown in Figure 6 and Table 7, all humanized versions of 58F9G10 appear to have good performance.

Table 7. Activity of humanized antibody against GPRC5D of 58F9G10

Similarly, as shown in Figure 7 and Table 8, all humanized versions of 34D3H1 appear to have good performance comparable to chimeric antibodies.

Table 8.34 Activity of humanized antibody against GPRC5D of D3H1

Minimum Response Maximum response LogEC50 Curve slope EC50 span 34-XI 16525 192054 0.4947 1.454 3.124 175530 34-H1L1 10921 196570 0.5908 1.314 3.898 185650 34-H1L2 13856 182030 0.5836 1.436 3.834 168174 34-H1L3 10620 185840 0.5732 1.167 3.743 175220 34-H1L4 10299 188925 0.5999 1.195 3.98 178626 34-H2L1 7854 187153 0.6188 1.173 4.157 179298 34-H2L2 8736 178873 0.6254 1.118 4.221 170137 34-H2L3 7063 185842 0.5832 1.11 3.83 178779 34-H2L4 4393 192405 0.6092 1.041 4.066 188012 34-H3L1 11139 178248 0.5207 1.228 3.317 167109 34-H3L2 9036 179809 0.6464 1.146 4.43 170773 34-H3L3 9118 192976 0.6508 1.1 4.475 183858 34-H3L4 12751 192533 0.5981 1.263 3.963 179782

Furthermore, as shown in Figure 8 and Table 9, all humanized versions of 37B9C4 appear to have good performance comparable to chimeric counterparts.

Table 9.37 Activity of humanized antibody against GPRC5D for 37B9C4

Based on the above data, humanized antibodies 6-H3L3, 6-H4L3, 58-H1L1, 58-H3L1, 34-H1L1, and 37-H1L1 were selected for further confirmatory testing.

Example 7. Confirmatory testing of selected humanized antibodies

This embodiment tested the ability of several humanized antibodies (6-H3L3, 6-H4L3, 58-H1L1, 58-H3L1, 34-H1L1, and 37-H1L1) to bind to human GPRC5D protein expressed on CHO-K1 and NCI-H929 cells.

The binding data with CHO-K1 are plotted in Figure 9 and summarized in Table 10 below.

Table 10 shows the binding of GPRC5D on CHO-K1 cells.

Minimum Response Maximum response LogEC50 Curve slope EC50 span 6-XI 523.9 104077 0.4723 1.095 2.967 103553 58-XI 52.25 107000 0.5562 0.9481 3.599 106948 6-H3L3 172 134045 1.412 0.8045 25.82 133873 6-H4L3 109.4 145833 1.421 0.7392 26.34 145724 58-H1L1 275.4 99454 0.579 0.972 3.793 99178 58-H3L1 75.37 104402 0.612 0.9135 4.093 104326 34-XI 688.6 117714 0.5776 1.054 3.781 117025 37-XI 792.6 113379 0.3877 1.175 2.442 112586 34-H1L1 170.8 119038 0.781 0.8745 6.04 118867 37-H1L1 523.2 102496 0.511 1.042 3.244 101973

Table 11. Binding of GPRC5D on NCI-H929 cells

Minimum Response Maximum response LogEC50 Curve slope EC50 span 6-XI 269.4 13785 0.05146 1.313 1.126 13515 58-XI 261.4 14738 0.1596 1.269 1.444 14476 6-H3L3 129.8 21859 1.465 0.8063 29.18 21730 6-H4L3 80.05 28987 1.606 0.7142 40.33 28907 58-H1L1 281 13474 0.1648 1.319 1.462 13193 58-H3L1 275.5 13934 0.3 1.297 1.995 13658 34-XI 181.1 18146 0.7587 0.9911 5.738 17965 34-H1L1 199.3 17911 0.8048 1.011 6.379 17712 37-XI 337.6 13807 -0.05621 1.41 0.8786 13470 37-H1L1 341.8 13534 -0.1991 1.419 0.6323 13192

Therefore, these data confirm that the selected humanized antibodies are suitable for further clinical development.

Example 8. ADCC of humanized antibodies

The ADCC Reporter Bioassay uses an alternative reading early in ADCC MOA pathway activation: gene transcriptional activation via the NFAT (nuclear factor for activating T cells) pathway in effector cells. Additionally, the ADCC Reporter Bioassay uses engineered Jurkat cells as effector cells, stably expressing the FcγRIIIa receptor, the V158 (high-affinity) variant, and the NFAT-responsive element that drives firefly luciferase expression. Antibody bioactivity in ADCC MOA is quantified by luciferase activity generated by NFAT pathway activation; luciferase activity in effector cells is quantified by luminescent readings. High signal intensity and low background are observed.

In the presence of ADCC Bioassay target cells (expressing GPRC5D), serially diluted human antibody containing GPRC5D was incubated with engineered Jurkat effector cells (ADCC Bioassay effector cells) at 37°C for 6 hours to induce luciferase activity. The activity was quantified using Bio-Glo ^™ reagents.

The results are shown in Figure 11, which demonstrate that these humanized antibodies induce strong ADCC activity in cell lines overexpressing GPRC5D and endogenous MM (multiple myeloma) cell lines.

Example 9. Internalization of humanized antibodies

This embodiment tested the internalization-inducing activity of humanized antibodies. A novel hydrophilic and bright pH sensor dye (pHAb dye) was used, which does not fluoresce at neutral pH but becomes highly fluorescent at acidic pH as internalization occurs. It can be used to detect the internalization process. NCI-H929 and MM.1R cells endogenously expressing human GPRC5D were used as target cells, and pHAb dye-labeled detection antibodies were added to evaluate the internalization of GPRC5D human antibodies in vitro.

Serially diluted human GPRC5D antibodies were incubated at 37°C for 24 hours. Luciferase activity was then measured. The results in Figure 12 show that these humanized antibodies exhibit very strong internalization activity against both GPRC5D-overexpressing cell lines and endogenous MM cell lines.

Example 10. Killing activity of GPRC5D ADC

This embodiment tested the cytotoxic activity of two antibody-drug conjugates (ADCs) against target cells. These ADCs included 37B9C4 and 58F9G10, respectively, conjugated to the toxic drug monomethylaurestatin E (MMAE).

HEK293 and NCI-H929 engineered cells expressing human GPRC5D (HEK293/H_GPRC5D and NCI-H929/H_GPRC5D), as well as NCI-H929 and MM.1R cells endogenously expressing GPRC5D, were seeded at 3000–4000 cells per well in 96-well plates. Cells were treated with various concentrations of 37B9C4-MMAE and 58F9G10-MMAE for 5 days. Cell viability was measured using CellTiter-Glo reagent at 3000–4000 cells per well in 96-well plates. Luciferase activity was detected by Envison assay. The results in Figure 13 and Table 12 indicate that these humanized antibodies possess strong cytotoxic activity.

Table 12 ADC Killing Activity

Example 11. In vivo antitumor activity

In this embodiment, the antitumor activity of antibody 37B9C4(H1L1) was tested using a CDX animal model.

In this study, 6-8 week old female NCG mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were used. Each mouse was subcutaneously inoculated with 2 × ^10⁶ MM.1R tumor cells in 0.1 ml of PBS containing matrix gel (V:V = 1:1) in the right axilla (lateral) for tumor development. When the tumor volume reached approximately 60 ^mm³ , the animals were randomly assigned to groups and treatment was initiated for efficacy studies. 37B9C4 was administered intravenously (iv) at doses of 1 mg/kg, 3 mg/kg, and 10 mg/kg on days 0, 7, and 14, respectively. The experiment was terminated on day 19, at which point the mean tumor volume in the excipient groups exceeded 2000 ^mm³ . The mean tumor volumes in the PBS group, 37B9C4 (1 mg/kg) group, 37B9C4 (3 mg/kg) group, and 37B9C4 (10 mg/kg) group were 2498.58 ^mm³ , 1196.15 ^mm³ , 0.00 ^mm³ (6/6 CR), and 0.00 ^mm³ (6/6 CR), respectively. Tumor size was measured three times weekly in two dimensions using calipers, and volume was expressed in ^mm³ using the following formula: V = 0.5a × ^b² , where a and b are the major and minor diameters of the tumor, respectively. Data points represent the mean of the groups (n = 6), and error bars represent the standard error of the mean (SEM).

The body weight of MM.1R tumor-bearing mice was monitored periodically as an indirect measure of toxicity. Figure 14A shows the tumor growth curves of MM.1R tumor-bearing mice after administration of 37B9C4. The antibody dose-dependently inhibited tumor growth in this tumor model and achieved complete tumor inhibition at 3 mg/kg or higher.

Detailed changes in body weight and relative body weight in MM.1R tumor-bearing mice after drug administration are shown in Figure 14B. During the drug administration period, there was no significant weight loss in any group of mice, and the mice in the drug administration group tolerated the treatment well, demonstrating the safety of the treatment.

***

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 that such modifications and variations fall within the scope of the appended claims and their equivalents.

All publications and patent applications mentioned in this specification are incorporated herein by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

sequence list

<110> Shanghai Lixin Pharmaceutical R&D Co., Ltd.

<120> Anti-GPRC5D Monoclonal Antibody and Its Uses

<130> FSP1V222255JW

<150> PCT/CN2021/070314

<151> 2021-01-05

<160> 86

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Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Glu Ile Arg Leu Lys Ser Asn Asn Tyr Ala Thr His Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr

85 90 95

Tyr Cys Thr Arg Pro Leu Leu Trp Phe Arg Arg Tyr Tyr Ala Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 10

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 10

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Ile Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro

65 70 75 80

Ala Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 11

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 11

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ala Gly Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Leu Arg Ser Arg Gly Tyr Phe Asp Tyr Trp Gly Arg Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 12

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 12

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Leu Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Met Gln Ser

65 70 75 80

Glu Asp Leu Ala Gly Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 13

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 13

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Asn

20 25 30

Ile Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Ala Gly Ser Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Ile Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Glu Val Tyr Tyr Arg Tyr Gly Ala Trp Phe Ala Tyr Trp

100 105 110

Gly His Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 14

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 14

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr

20 25 30

Ser Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln His Ser Trp

85 90 95

Glu Ile Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 15

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 15

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Thr Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Tyr Ile His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Ser Cys Tyr Asn Gly Ala Thr Ser Phe Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Phe Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Thr Glu Leu Arg Gly Pro Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 16

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 16

Gln Thr Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Asn Asn Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 17

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 17

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ile Asn Tyr

20 25 30

Leu Ile Glu Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Asn Trp Asp Val Trp Gly Gln Gly Thr Thr Leu Thr Val Ser

100 105 110

Ser

<210> 18

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 18

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Val Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Thr Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 19

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 19

Gln Val His Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Asn Ser Ala Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Val Leu Leu Leu Arg Val Leu Asp Phe Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 20

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 20

Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly

1 5 10 15

Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Asn Lys Tyr

20 25 30

Leu Thr Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile

35 40 45

Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Leu

85 90 95

Thr Phe Gly Thr Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 21

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 21

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Gly Gly Ile Arg Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Gly Leu Arg Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 22

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 22

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Ser Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 23

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 23

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Ala Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Leu Arg Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 24

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 24

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Ser Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Asn Ser Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 25

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 25

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met His Trp Val Lys Gln Ser His Gly Glu Asn Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Leu Arg Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 26

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 26

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Thr Ser Ile Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Ser Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Asn Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Tyr Asn Ser Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 27

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 27

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ile Ser Tyr

20 25 30

Leu Ile Glu Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Asn Trp Asp Val Trp Gly Gln Gly Thr Thr Leu Thr Val Ser

100 105 110

Ser

<210> 28

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 28

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Val Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Thr Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 29

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 29

Ser Tyr Gly Met Ser

1 5

<210> 30

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 30

Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys

1 5 10 15

Gly

<210> 31

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 31

Gln Gly Gly Asp Ala Met Asp Tyr

1 5

<210> 32

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 32

Arg Ala Ser Gln Ser Ile Asn Asn Asn Leu His

1 5 10

<210> 33

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 33

Tyr Ala Ser Gln Ser Ile Ser

1 5

<210> 34

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 34

Gln Gln Ser Asn Ser Arg Leu Thr

1 5

<210> 35

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 35

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Gly Gly Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 36

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 36

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Gly Gly Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 37

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 37

Glu Val His Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Thr Pro Gly Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Asn

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Gly Gly Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 38

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 38

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Asn Asn

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Ser Gln Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Arg Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 39

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 39

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Asn Asn

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ser Asn Ser Arg Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 40

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 40

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Asn Asn

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ser Asn Ser Arg Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 41

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 41

Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Val Gly

1 5 10 15

Asp Arg Val Thr Leu Thr Cys Arg Ala Ser Gln Ser Ile Asn Asn Asn

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ser Asn Ser Arg Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 42

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 42

Asp Tyr Trp Met Asn

1 5

<210> 43

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 43

Glu Ile Arg Leu Lys Ser Asn Asn Tyr Ala Thr His Tyr Ala Glu Ser

1 5 10 15

Val Lys Gly

<210> 44

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 44

Pro Leu Leu Trp Phe Arg Arg Tyr Tyr Ala Met Asp Tyr

1 5 10

<210> 45

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 45

Ser Ala Ser Gln Gly Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 46

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 46

Tyr Thr Ser Ser Leu His Ser

1 5

<210> 47

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 47

Gln Gln Tyr Ser Lys Leu Pro Phe Thr

1 5

<210> 48

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 48

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Glu Ile Arg Leu Lys Ser Asn Asn Tyr Ala Thr His Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Pro Leu Leu Trp Phe Arg Arg Tyr Tyr Ala Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 49

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 49

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Glu Ile Arg Leu Lys Ser Asn Asn Tyr Ala Thr His Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Arg Pro Leu Leu Trp Phe Arg Arg Tyr Tyr Ala Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 50

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 50

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Glu Ile Arg Leu Lys Ser Asn Asn Tyr Ala Thr His Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Arg Pro Leu Leu Trp Phe Arg Arg Tyr Tyr Ala Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 51

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 51

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 52

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 52

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 53

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 53

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Ile Thr Ile Thr Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 54

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 54

Gly Tyr Tyr Ile His

1 5

<210> 55

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 55

Tyr Ile Ser Ser Tyr Asn Ala Ala Thr Ser Phe Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 56

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 56

Thr Glu Leu Arg Gly Pro Trp Phe Ala Tyr

1 5 10

<210> 57

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 57

Ser Ala Ser Ser Ser Val Ser Tyr Met Asn

1 5 10

<210> 58

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 58

Asp Thr Ser Lys Leu Ala Ser

1 5

<210> 59

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 59

Gln Gln Trp Ser Asn Asn Pro Leu Thr

1 5

<210> 60

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 60

Tyr Ile Ser Cys Tyr Asn Gly Ala Thr Ser Phe Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 61

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 61

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Ser Ser Tyr Asn Ala Ala Thr Ser Phe Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Thr Glu Leu Arg Gly Pro Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 62

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 62

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Ser Ser Tyr Asn Ala Ala Thr Ser Phe Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Val Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Thr Glu Leu Arg Gly Pro Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 63

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 63

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Tyr Ile His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Ser Ser Tyr Asn Ala Ala Thr Ser Phe Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Val Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Phe Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Thr Glu Leu Arg Gly Pro Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 64

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 64

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Tyr Ile His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Ser Ser Tyr Asn Ala Ala Thr Ser Phe Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Phe Thr Val Asp Thr Ser Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Phe Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Thr Glu Leu Arg Gly Pro Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 65

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 65

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Asn Asn Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 66

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 66

Asp Thr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Asn Asn Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 67

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 67

Asp Thr Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Met Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Asn Asn Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 68

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 68

Thr Tyr Thr Met His

1 5

<210> 69

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 69

Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys

1 5 10 15

Asp

<210> 70

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 70

Leu Arg Ser Arg Gly Tyr Phe Asp Tyr

1 5

<210> 71

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 71

Lys Ala Ser Gln Asn Val Gly Thr Ala Val Val

1 5 10

<210> 72

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 72

Ser Ala Ser Asn Arg Tyr Thr

1 5

<210> 73

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 73

Gln Gln Tyr Ser Ser Tyr Pro Tyr Thr

1 5

<210> 74

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 74

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Leu Arg Ser Arg Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 75

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 75

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Leu

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Arg Ser Arg Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 76

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 76

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Ala Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Leu

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Leu Arg Ser Arg Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 77

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 77

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Ala Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Leu

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Leu Arg Ser Arg Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Leu Thr Val Ser Ser Ser

115

<210> 78

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 78

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Ala Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Leu

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Leu Arg Ser Arg Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Leu Thr Val Ser Ser Ser

115

<210> 79

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 79

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Ala Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Leu

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Ala Thr Leu Thr Ala Gly Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Leu Arg Ser Arg Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Leu Thr Val Ser Ser Ser

115

<210> 80

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 80

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Val Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 81

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 81

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Val Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Phe Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 82

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 82

Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Val Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Met Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Phe Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 83

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 83

Asp Ile Val Met Thr Gln Ser Pro Ser Phe Leu Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Val Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Met Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Phe Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 84

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 84

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Val Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 85

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 85

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Thr Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Val Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Phe Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 86

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 86

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Thr Ser Pro Gly

1 5 10 15

Glu Arg Val Thr Ile Ser Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Val Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Ile Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Met Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Phe Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

Claims (15)

1. An antibody or antigen-binding fragment thereof having binding specificity to human G protein-coupled receptor C family 5 member D (GPRC5D) protein, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises heavy chain complementarity-determining regions CDRH1, CDRH2 and CDRH3, and the light chain variable region (VL) comprises complementarity-determining regions CDRL1, CDRL2 and CDRL3, and wherein the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 are as shown in SEQ ID NO:42-47, respectively. 2. The antibody or its antigen-binding fragment according to claim 1, wherein the amino acid sequences of VH and VL are as follows: (a) As shown in SEQ ID NO:9 and SEQ ID NO:10; (b) As shown in SEQ ID NO:48 and SEQ ID NO:51; (c) As shown in SEQ ID NO:49 and SEQ ID NO:52; or (d) As shown in SEQ ID NO:50 and SEQ ID NO:53. 3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the amino acid sequence of VH is as shown in SEQ ID NO:48, and the amino acid sequence of VL is as shown in SEQ ID NO:51. 4. The antibody or antigen-binding fragment thereof having binding specificity to human G protein-coupled receptor C family 5 member D (GPRC5D) protein according to any one of claims 1-3, wherein the antibody is humanized. 5. The antibody or antigen-binding fragment thereof having binding specificity to human G protein-coupled receptor C family 5 member D (GPRC5D) protein according to any one of claims 1-3, which has ADCC capability. 6. The antibody or antigen-binding fragment thereof having binding specificity to human G protein-coupled receptor C family 5 member D (GPRC5D) protein according to any one of claims 1-3, further comprising a cytotoxic drug conjugated to said antibody or antigen-binding fragment thereof. 7. A bispecific antibody comprising the antigen-binding fragment according to any one of claims 1-3, and a second antigen-binding fragment specific to a second target protein. 8. The bispecific antibody according to claim 7, wherein the second target protein is selected from the group consisting of CD3, CD16, CD19, CD28, CD64 and 4-1BB. 9. The bispecific antibody according to claim 7, wherein the second target protein is CD3. 10. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-3 in the preparation of a medicament for treating cancer in patients in need; The cancer in question is a B-cell carcinoma expressing GPRC5D. 11. Use of T cells, natural killer (NK) cells, or macrophages in the preparation of a medicament for treating cancer in patients in need, wherein the T cells, the natural killer (NK) cells, or the macrophages are treated with an antibody or an antigen-binding fragment thereof as described in any one of claims 1-3; The cancer in question is a B-cell carcinoma expressing GPRC5D. 12. The use according to claim 11, wherein the T cells, the NK cells, or the macrophages are isolated from the individual prior to treatment with the antibody or its antigen-binding fragment. 13. The use according to claim 12, wherein the T cells, the NK cells, or the macrophages are isolated from the patient. 14. The use according to claim 11, wherein the T cell is a tumor-infiltrating T lymphocyte, a CD4+ T cell, a CD8+ T cell, or a combination thereof. 15. The use according to claim 10 or 11, wherein the B-cell carcinoma expressing GPRC5D is multiple myeloma.