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NZ732567B2 - Antibodies targeting g-protein coupled receptor and methods of use - Google Patents

Antibodies targeting g-protein coupled receptor and methods of use Download PDF

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Publication number
NZ732567B2
NZ732567B2 NZ732567A NZ73256715A NZ732567B2 NZ 732567 B2 NZ732567 B2 NZ 732567B2 NZ 732567 A NZ732567 A NZ 732567A NZ 73256715 A NZ73256715 A NZ 73256715A NZ 732567 B2 NZ732567 B2 NZ 732567B2
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New Zealand
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seq
set forth
amino acids
sequence set
chain variable
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NZ732567A
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NZ732567A (en
Inventor
Renier J Brentjens
Cheng Liu
Eric L Smith
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Eureka Therapeutics Inc
Memorial Sloan Kettering Cancer Center
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Application filed by Eureka Therapeutics Inc, Memorial Sloan Kettering Cancer Center filed Critical Eureka Therapeutics Inc
Priority claimed from PCT/US2015/064122 external-priority patent/WO2016090329A2/en
Publication of NZ732567A publication Critical patent/NZ732567A/en
Publication of NZ732567B2 publication Critical patent/NZ732567B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels

Abstract

The presently disclosed subject matter provides antibodies that bind to GPRC5D and methods of using the same. The antibodies and fragments thereof are useful in treating cancer, particularly multiple myeloma. In a specific embodiment of the invention, the antibodies are defined by the HCDRs 1-3 defined by SEQ ID NOs: 220, 221, and 222, and the LCDRs 1-3 defined by SEQ ID NOs: 223, 224, and 225, respectively.

Description

ANTIBODIES TARGETING G-PROTEIN COUPLED RECEPTOR AND METHODS OF USE CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to United States Provisional Application Serial No. ,228, filed December 5, 2014, the contents of which is incorporated by nce in its entirety, and to which priority is claimed.
FIELD OF THE INVENTION The presently disclosed subject matter s to human antibodies that bind to a G—protein coupled or (e.g., a G—protein coupled receptor family C group 5 member D (GPRCSD)), and methods of using the same.
BACKGROUND G protein—coupled receptors, also known as seven—transmembrane domain receptors, 7TM receptors, heptahelical receptors, serpentine receptor, and G n— linked receptors, constitute a large protein family of receptors that sense molecules outside the cell and activate inside signal transduction ys and, ultimately, cellular ses. GPCRs can be categorized into six classes based on sequence homology and onal similarity: Class A (Rhodopsin—like), Class B (Secretin receptor family), Class C (Metabotropic glutamate/pheromone), Class D (Fungal mating pheromone receptors), Class E (Cyclic AMP receptors), and Class F (Frizzled/Smoothened).
G—protein coupled receptor family C group 5 member D (GPRCSD) is an orphan receptor with no known ligand or function in humans. It is a member of a family of retinoic acid—inducible G—protein—coupled receptors. It is overexpressed in multiple myeloma (MM) cells and is not expressed or expressed in a significantly lower level by any other cell type, benign or malignant, as shown in Figure 1. Several groups have identified this gene as highly differentially expressed by gene expression profiling of primary MM cells when compared to normal tissuel or other hematologic malignancies (Frigyesi, I., et al. Robust isolation of malignant plasma cells in multiple myeloma. Blood 123, 1336—1340 ; Cohen, Y., Gutwein, O., Garach—Jehoshua, Active 223858733 0., Bar—Haim, A. & Kornberg, A. GPRC5D is a ing marker for monitoring the tumor load and to target multiple myeloma cells. Hematology (Amsterdam, Netherlands) 18, 348—351 ; Bam, R., et al. GPRC5D Is a Cell e Plasma Cell Marker Whose Expression Is High In Myeloma Cells and d Following ure With Osteoclasts. Blood 122, 3099 ). It has been shown that higher mRNA expression correlates with worse l survival (Atamaniuk, J ., et al.
Overexpression of G protein—coupled receptor 5D in the bone marrow is associated with poor prognosis in patients with multiple myeloma. European journal of clinical investigation 42, 953—960 (2012)). Surface staining of Bone marrow aspirates from patients with MM demonstrate plasma cell specific staining (Bam, R., et al. GPRC5D Is a Cell Surface Plasma Cell Marker Whose Expression Is High In Myeloma Cells and Reduced Following Coculture With Osteoclasts. Blood 122, 3099 (2013)). Given the significant role for GPRC5D in multiple myeloma, antibodies that recognize GPRC5D, and methods of using such agents, are desired.
The presently disclosed subject matter provides human antibodies that bind to a G—protein coupled receptor (e.g., GPRC5D), and methods of using the same.
In n embodiments, the presently disclosed subject matter provides an isolated antibody, or an antigen—binding fragment f, comprising a heavy chain variable region sing an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 274, 286, 298, 310, 322, 334, 346 and 358, wherein the antibody or n—binding fragment thereof specifically binds to human GPRC5D.
In certain embodiments, the presently disclosed subject matter provides an isolated antibody, or an antigen—binding fragment thereof, comprising a light chain variable region comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 275, 287, 299, 311, 323, 335, 347 and 359, wherein the antibody or antigen—binding fragment thereof specifically binds to human GPRC5D.
In certain embodiments, the presently sed subject matter provides an isolated dy, or an antigen—binding fragment thereof, comprising (a) a heavy chain variable region comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 274, 286, 298, 310, 322, 334, 346 and 358; and (b) a light chain variable region sing an amino acid ce that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to an amino acid sequence selected from the group ting of SEQ ID NOS: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 275, 287, 299, 311, 323,335, 347 and 359, wherein the antibody or antigen—binding fragment thereof specifically binds to human GPRC5D.
In certain embodiments, the presently disclosed subject matter es an isolated antibody, or an antigen—binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are selected from the group consisting of: (i) a heavy chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% gous to the sequence set forth in SEQ ID NO:1, and a light chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:2; (ii) a heavy chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:5, and a light chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:6; (iii) a heavy chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:9, and a light chain le region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:lO; (iv) a heavy chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:l3, and a light chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:l4; (v) a heavy chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:l7, and a light chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:l8; (vi) a heavy chain variable region comprising amino acids having a ce that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:2l, and a light chain le region sing amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:22; (vii) a heavy chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% gous to the sequence set forth in SEQ ID NO:25, and a light chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:26; (viii) a heavy chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% gous to the sequence set forth in SEQ ID NO:29, and a light chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:30; (iX) a heavy chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:33, and a light chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:34; (X) a heavy chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% gous to the sequence set forth in SEQ ID NO:37, and a light chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:38; (Xi) a heavy chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:4l, and a light chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:42; (Xii) a heavy chain variable region sing amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:45, and a light chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:46; (xiii) a heavy chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the ce set forth in SEQ ID NO:49, and a light chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:50; (xiv) a heavy chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% gous to the sequence set forth in SEQ ID NO:53, and a light chain le region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:54; (xv) a heavy chain variable region sing amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:57, and a light chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:58; (xvi) a heavy chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:6l, and a light chain variable region sing amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:62; (xvii) a heavy chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:65, and a light chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:66; (xviii) a heavy chain variable region comprising amino acids having a ce that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:69, and a light chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:70; (xix) a heavy chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:73, and a light chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% gous to the sequence set forth in SEQ ID NO:74; (XX) a heavy chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:77, and a light chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:78; (XXi) a heavy chain variable region comprising amino acids having a ce that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:8l, and a light chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:82; (XXii) a heavy chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% gous to the sequence set forth in SEQ ID NO:85, and a light chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:86; ) a heavy chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:89, and a light chain variable region sing amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% gous to the sequence set forth in SEQ ID NO:90; (XXiv) a heavy chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:93, and a light chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:94; (XXV) a heavy chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:274, and a light chain le region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:275; (xxvi) a heavy chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% gous to the sequence set forth in SEQ ID NO:286, and a light chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the ce set forth in SEQ ID NO:287; (xxvii) a heavy chain le region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:298, and a light chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:299; (xxviii) a heavy chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:310, and a light chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:3ll; (XXiX) a heavy chain variable region comprising amino acids having a ce that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:322, and a light chain variable region sing amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:323; (XXX) a heavy chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:334, and a light chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:335; (XXXi) a heavy chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the ce set forth in SEQ ID NO:346, and a light chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:347; or (XXXii) a heavy chain variable region sing amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the ce set forth in SEQ ID NO:358, and a light chain variable region comprising amino acids having a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO:359, wherein the antibody or antigen—binding fragment f specifically binds to human GPRC5D.
In certain embodiments, the antibody or n—binding fragment comprises: (i) a heavy chain variable region comprising amino acids having a ce set forth in SEQ ID NO:1, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:2; (ii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:5, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:6; (iii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:9, and a light chain le region comprising amino acids having a sequence set forth in SEQ ID NO: 10; (iv) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:l3, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:l4; (v) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:l7, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO: 18; (vi) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:21, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:22; (vii) a heavy chain le region comprising amino acids having a ce set forth in SEQ ID NO:25, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:26; (viii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:29, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:30; (iX) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:33, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:34; (X) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:37, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:38; (Xi) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:4l, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:42; (Xii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:45, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:46; (Xiii) a heavy chain le region comprising amino acids having a ce set forth in SEQ ID NO:49, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:50; (xiv) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:53, and a light chain le region comprising amino acids having a sequence set forth in SEQ ID NO:54; (xv) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:57, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:58; (xvi) a heavy chain variable region sing amino acids having a sequence set forth in SEQ ID NO:6l, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:62; (xvii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:65, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:66; (xviii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:69, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:70; (xix) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:73, and a light chain le region comprising amino acids having a sequence set forth in SEQ ID NO:74; (xx) a heavy chain le region sing amino acids having a sequence set forth in SEQ ID NO:77, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:78; (xxi) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:81, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:82; (xxii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:85, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:86; (xxiii) a heavy chain variable region comprising amino acids having a ce set forth in SEQ ID NO:89, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:90; (xxiv) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:93, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:94; (xxv) a heavy chain variable region comprising amino acids having a ce set forth in SEQ ID NO:274, and a light chain variable region comprising amino acids having a set forth in SEQ ID NO:275; (xxvi) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:286, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:287; (xxvii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:298, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:299; (xxviii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:3lO, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:3ll; (XXiX) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:322, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:323; (XXX) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:334, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:335; (XXXi) a heavy chain variable region comprising amino acids having a ce set forth in SEQ ID NO:346, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:347; or ) a heavy chain variable region sing amino acids having a sequence set forth in SEQ ID NO:35 8, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:359.
In certain embodiments, the tly sed t matter provides an isolated antibody, or an antigen—binding nt thereof, comprising a heavy chain variable region comprising an amino acid ce selected from the group consisting of SEQ ID NOS: 1, 5, 9, l3, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 274, 286, 298, 310, 322, 334, 346 and 358, and conservative modifications thereof, wherein the antibody or antigen—binding nt f specifically binds to human GPRC5D.
In certain embodiments, the presently disclosed subject matter provides an isolated antibody, or an antigen—binding fragment thereof, comprising a light chain le region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 6, 10, l4, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 275, 287, 299, 311, 323, 335, 347 and 359, and vative modifications thereof, wherein the antibody or antigen—binding fragment thereof specifically binds to human GPRC5D.
In certain embodiments, the presently disclosed subject matter provides an isolated antibody, or an antigen—binding fragment thereof, comprising: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 274, 286, 298, 310, 322, 334, 346 and 358, and conservative modifications thereof; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 6, 10, 14, 18, 22, 26, , 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 275, 287, 299, 311, 323, 335, 347 and 359, and vative modifications thereof, wherein the antibody or antigen—binding fragment thereof specifically binds to human GPRC5D.
The presently disclosed subject matter also provides an isolated antibody or antigen—binding fragment thereof comprising a heavy chain variable region that comprises CDRl, CDR2, and CDR3 domains; and a light chain variable region that comprises CDRl, CDR2, and CDR3 domains, wherein the heavy chain variable region and light chain variable region CDR3 domains are selected from the group consisting of: (i) a heavy chain le region CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 126 and conservative modifications thereof; and a light chain variable region CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 129 and conservative modifications thereof; (ii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 132 and conservative modifications thereof; and a light chain le region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 135 and vative cations thereof; (iii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 138 and conservative modifications thereof; and a light chain variable region CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 141 and conservative modifications thereof; (iv) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 144 and conservative modifications thereof; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 147 and conservative modifications thereof; (v) a heavy chain variable region CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 150 and conservative modifications thereof; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 153 and conservative modifications thereof; (vi) a heavy chain variable region CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 156 and conservative modifications thereof; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 159 and conservative modifications thereof; (vii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 162 and conservative modifications thereof; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 165 and conservative modifications f; (viii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 168 and conservative modifications thereof; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 171 and conservative modifications thereof; (iX) a heavy chain variable region CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 174 and conservative modifications thereof; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 177 and conservative modifications thereof; (X) a heavy chain le region CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 180 and conservative modifications f; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 183 and conservative modifications thereof; (Xi) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 186 and conservative modifications thereof; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 189 and conservative modifications thereof; (Xii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 192 and conservative modifications thereof; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 195 and conservative modifications thereof; (Xiii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 198 and conservative modifications thereof; and a light chain le region CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 201 and conservative modifications thereof; (xiv) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 204 and vative modifications thereof; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 207 and conservative modifications thereof; (xv) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 210 and conservative modifications thereof; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 213 and conservative modifications thereof; (xvi) a heavy chain variable region CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 216 and conservative modifications thereof; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 219 and conservative modifications thereof; (xvii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:222 and conservative modifications thereof; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:225 and conservative modifications thereof; (xviii) a heavy chain le region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 228 and conservative modifications f; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 231 and conservative cations f; (xix) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 234 and conservative cations thereof; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:237 and conservative modifications thereof; (xx) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:240 and conservative cations thereof; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 243 and conservative modifications thereof; (xxi) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 246 and conservative modifications thereof; and a light chain variable region CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 249 and conservative modifications thereof; (XXii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 252 and conservative modifications thereof; and a light chain variable region CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 255 and conservative modifications thereof; (XXiii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 258 and vative modifications thereof; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 261 and conservative modifications thereof; (XXiv) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 264 and vative modifications f; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 267 and conservative modifications thereof; (XXV) a heavy chain le region CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 270 and conservative modifications thereof; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 273 and conservative modifications thereof; (xxvi) a heavy chain le region CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 282 and conservative modifications thereof; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 285 and conservative modifications thereof; (xxvii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 294 and conservative modifications f; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 297 and conservative modifications thereof; (xxviii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 305 and conservative modifications thereof; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 308 and conservative modifications thereof; (XXiX) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 318 and vative modifications thereof; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 321 and conservative modifications thereof; (XXX) a heavy chain variable region CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 330 and vative modifications thereof; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 333 and conservative modifications thereof; (XXXi) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 342 and conservative modifications thereof; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 345 and conservative modifications thereof; and (XXXii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 354 and conservative modifications thereof; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 357 and conservative modifications f, wherein the antibody or antigen—binding portion thereof specifically binds to GPRC5D.
In n embodiments, the heavy chain variable region and light chain le region CDR2 domains the antibody or antigen—binding portion thereof are ed from the group consisting of: (i) a heavy chain le region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 125 and conservative modifications thereof; and a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 128 and conservative modifications thereof; (ii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 131 and conservative modifications thereof; and a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 134 and conservative modifications thereof; (iii) a heavy chain le region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 137 and conservative modifications thereof; and a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 140 and conservative modifications thereof; (iv) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 143 and conservative modifications thereof; and a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 146 and conservative modifications thereof; (V) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 149 and conservative modifications thereof; and a light chain variable region CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 152 and conservative modifications thereof; (vi) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 155 and conservative modifications thereof; and a light chain le region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 158 and conservative modifications thereof; (vii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 161 and conservative modifications thereof; and a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 164 and conservative modifications thereof; (viii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 167 and conservative modifications thereof; and a light chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID NO: 170 and conservative modifications thereof; (iX) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 173 and conservative modifications thereof; and a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 176 and conservative modifications thereof; (X) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 179 and conservative modifications thereof; and a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 182 and conservative modifications thereof; (Xi) a heavy chain le region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 185 and conservative cations thereof; and a light chain variable region CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 188 and conservative modifications thereof; (Xii) a heavy chain variable region CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 191 and conservative modifications thereof; and a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 194 and conservative modifications thereof; (xiii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 197 and conservative modifications thereof; and a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:200 and conservative modifications thereof; (xiv) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 203 and conservative modifications thereof; and a light chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID NO: 206 and conservative modifications f; (xv) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 209 and conservative modifications thereof; and a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 212 and conservative modifications thereof; (xvi) a heavy chain le region CDR2 sing amino acids having the sequence set forth in SEQ ID NO: 215 and conservative modifications thereof; and a light chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID NO: 218 and conservative modifications thereof; (xvii) a heavy chain variable region CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 221 and conservative cations thereof; and a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 224 and conservative modifications thereof; (xviii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 227 and conservative modifications thereof; and a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:230 and conservative modifications thereof; (xix) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:233 and conservative modifications thereof; and a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:236 and conservative modifications thereof; (xx) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:239 and conservative modifications thereof; and a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:242 and conservative cations thereof; (XXi) a heavy chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID NO:245 and conservative modifications thereof; and a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:248 and conservative modifications thereof; (XXii) a heavy chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID NO:251 and conservative modifications thereof; and a light chain le region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:254 and vative modifications thereof; (XXiii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:257 and conservative modifications thereof; and a light chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID NO:260 and conservative modifications thereof; (XXiv) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:263 and conservative modifications thereof; and a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:266 and conservative modifications thereof; (XXV) a heavy chain variable region CDR2 comprising amino acids having the ce set forth in SEQ ID NO:269 and conservative modifications thereof; and a light chain le region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:272 and conservative modifications thereof; (xxvi) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:281 and conservative modifications thereof; and a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:284 and conservative modifications thereof; (xxvii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:293 and conservative modifications thereof; and a light chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID NO:296 and conservative modifications thereof; (xxviii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:304 and conservative modifications f; and a light chain le region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:307 and conservative modifications thereof; (XXiX) a heavy chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID NO:317 and conservative modifications thereof; and a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:320 and conservative modifications thereof; (XXX) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:329 and conservative modifications f; and a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:332 and conservative cations thereof; (XXXi) a heavy chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID NO:34l and conservative modifications thereof; and a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:344 and conservative modifications thereof; and (XXXii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:353 and conservative modifications f; and a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:356 and conservative modifications thereof, wherein the antibody or antigen—binding portion thereof specifically binds to GPRCSD.
In certain embodiments, the heavy chain le region and light chain variable region CDRl domains of the antibody or n—binding portion thereof are selected from the group consisting of: (i) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 124 and conservative modifications thereof; and a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 127 and conservative modifications thereof; (ii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 130 and conservative modifications thereof; and a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 133 and conservative modifications thereof; (iii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 136 and conservative modifications thereof; and a light chain le region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 139 and conservative cations thereof; (iv) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 142 and conservative modifications thereof; and a light chain le region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 145 and conservative modifications thereof; (v) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 148 and conservative modifications thereof; and a light chain le region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 151 and conservative modifications thereof; (vi) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 154 and conservative modifications thereof; and a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 157 and conservative modifications f; (vii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 160 and conservative modifications thereof; and a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 163 and conservative modifications thereof; (viii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 166 and conservative modifications thereof; and a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 169 and conservative modifications thereof; (iX) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 172 and conservative modifications thereof; and a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 175 and conservative modifications thereof; (X) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 178 and conservative modifications thereof; and a light chain variable region CDRl sing amino acids having the sequence set forth in SEQ ID NO: 181 and vative modifications thereof; (Xi) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 184 and conservative modifications thereof; and a light chain variable region CDRl sing amino acids having the sequence set forth in SEQ ID NO: 187 and conservative cations thereof; (xii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 190 and conservative modifications thereof; and a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 193 and conservative modifications thereof; (xiii) a heavy chain variable region CDRl sing amino acids having the sequence set forth in SEQ ID NO: 196 and conservative modifications f; and a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 199 and conservative modifications f; (xiv) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:202 and conservative modifications thereof; and a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:205 and conservative modifications thereof; (xv) a heavy chain le region CDRl sing amino acids having the sequence set forth in SEQ ID NO:208 and conservative modifications thereof; and a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:211 and conservative modifications thereof; (xvi) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:214 and conservative cations thereof; and a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:217 and conservative modifications thereof; (xvii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:220 and conservative modifications thereof; and a light chain le region CDRl sing amino acids having the sequence set forth in SEQ ID NO:223 and conservative modifications thereof; (xviii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:226 and vative modifications thereof; and a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:229 and conservative modifications thereof; (xix) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:232 and conservative modifications thereof; and a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:235 and conservative modifications thereof; (XX) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:238 and vative modifications thereof; and a light chain le region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:24l and conservative cations thereof; (XXi) a heavy chain variable region CDRl comprising amino acids having the ce set forth in SEQ ID NO:244 and conservative modifications thereof; and a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:247 and conservative modifications thereof; (XXii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:250 and vative modifications thereof; and a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:253 and conservative modifications thereof; (XXiii) a heavy chain variable region CDRl sing amino acids having the sequence set forth in SEQ ID NO:256 and conservative modifications thereof; and a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:259 and conservative modifications thereof; (XXiv) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:262 and vative modifications thereof; and a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:265 and conservative modifications thereof; (XXV) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:268 and conservative modifications thereof; and a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:27l and conservative modifications thereof; (XXVi) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:280 and conservative modifications thereof; and a light chain variable region CDRl comprising amino acids having the ce set forth in SEQ ID NO:283 and conservative modifications thereof; (xxvii) a heavy chain le region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:292 and conservative modifications thereof; and a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:295 and conservative modifications thereof; (xxviii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:303 and conservative modifications f; and a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:306 and vative modifications thereof; (XXiX) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:3l6 and conservative modifications thereof; and a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:3l9 and conservative modifications f; (XXX) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:328 and conservative modifications thereof; and a light chain le region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:331 and conservative modifications thereof; (XXXi) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:340 and conservative modifications thereof; and a light chain variable region CDRl comprising amino acids having the ce set forth in SEQ ID NO:343 and conservative modifications thereof; and (XXXii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:352 and conservative modifications f; and a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO:355 and vative modifications thereof, n the antibody or antigen—binding portion thereof ically binds to GPRCSD.
Furthermore, the presently disclosed subject matter provides an isolated antibody, or an antigen—binding portion thereof, comprising: (i) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 124; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 125; and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 126; (ii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 130; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 131; and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 132; (iii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 136; a heavy chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID NO: 137; and a heavy chain le region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 138; (iv) a heavy chain variable region CDRl sing amino acids having the sequence set forth in SEQ ID NO: 142; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 143; and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 144; (v) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 148; a heavy chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID NO: 149; and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 150; (vi) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 154; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 155; and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 156; (vii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 160; a heavy chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID NO: 161; and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 162; (viii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 166; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 167; and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 168; (iX) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 172; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 173; and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 174; (X) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 178; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 179; and a heavy chain le region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 180; (Xi) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 184; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 185; and a heavy chain le region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 186; (Xii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 190 a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 191; and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 192; (Xiii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 196; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 197; and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 198; (Xiv) a heavy chain variable region CDRl sing amino acids having the ce set forth in SEQ ID NO: 202; a heavy chain variable region CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 203; and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 204; (xv) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 208; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 209; and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 210; (xvi) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 214; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 215; and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 216; (xvii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 220; a heavy chain le region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 221; and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 222; (xviii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 226; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 227; and a heavy chain variable region CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 228; (xix) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 232; a heavy chain le region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 233; and a heavy chain variable region CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 234; (xx) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 238; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 239; and a heavy chain le region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 240; (xxi) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 244; a heavy chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID NO: 245; and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 246; (XXii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 250; a heavy chain variable region CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 251; and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 252; and (XXiii) a heavy chain le region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 256; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 257; and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 258; (XXiv) a heavy chain variable region CDRl sing amino acids having the sequence set forth in SEQ ID NO: 262; a heavy chain variable region CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 263; and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 264; (XXV) a heavy chain le region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 268; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 269; and a heavy chain le region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 270; (xxvi) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 280; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 281; and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 282; (xxvii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 292; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 293; and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 294; (xxviii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 303; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 304; and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 305; (XXiX) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 316; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 317; and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 318; (XXX) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 328; a heavy chain le region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 329; and a heavy chain le region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 330; (XXXi) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 340; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 341; and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 342; or (XXXii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 352; a heavy chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID NO: 353; and a heavy chain le region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 354; wherein the antibody or antigen—binding portion thereof specifically binds to GPRC5D.
Additionally, the tly disclosed subject matter provides an isolated antibody, or an antigen—binding portion thereof, comprising: (i) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 127; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:129; and a light chain variable region CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 130; (ii) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 133; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:134; and a light chain variable region CDR3 comprising SEQ ID NO: 135; (iii) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 139; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:140; and a light chain le region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 141; (iv) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 145; a light chain variable region CDR2 sing SEQ ID NO:146; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 147; (v) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 151; a light chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID NO:152; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 153; (vi) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 157; a light chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID NO:158; and a light chain variable region CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 159; (vii) a light chain variable region CDRl comprising amino acids having the ce set forth in SEQ ID NO: 163; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:164; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 165; (viii) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 169; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:170; and a light chain variable region CDR3 comprising SEQ ID NO: 171; (iX) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 175; a light chain variable region CDR2 comprising amino acids having the ce set forth in SEQ ID NO:176; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 177; (X) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 181; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:182; and a light chain le region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 183; (Xi) a light chain variable region CDRl sing amino acids having the sequence set forth in SEQ ID NO: 187; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID ; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 189; (Xii) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 193; a light chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID NO:194; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 195; (Xiii) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 199; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:200; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 201; (Xiv) a light chain le region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 205; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:206; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 207; (xv) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 211; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:212; and a light chain variable region CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 213; (xvi) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 217; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:218; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 219; (xvii) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 223; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:224; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 225; (xviii) a light chain variable region CDRl comprising amino acids having the ce set forth in SEQ ID NO: 229; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:230; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 231; (xix) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 235; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:236; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 237; (xx) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 241; a light chain le region CDR2 sing amino acids having the sequence set forth in SEQ ID ; and a light chain variable region CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 243; (xxi) a light chain variable region CDRl comprising amino acids having the ce set forth in SEQ ID NO: 247; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:248; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 249; (xxii) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 253; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:254; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 255; (XXiii) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 259; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:250; and a light chain variable region CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 261; or (XXiv) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 265; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:266; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 267; (XXV) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 271; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:272; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 273; (xxvi) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 283; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:284; and a light chain variable region CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 285; (xxvii) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 295; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:296; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 297; (xxviii) a light chain le region CDRl sing amino acids having the ce set forth in SEQ ID NO: 306; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:307; and a light chain variable region CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 308; (XXiX) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 319; a light chain le region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:320; and a light chain le region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 321; (XXX) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 331; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:332; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 333; (XXXi) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 343; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:344; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 345; or (XXXii) a light chain variable region CDRl comprising amino acids having the ce set forth in SEQ ID NO: 355; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:356; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 357, wherein the antibody or antigen—binding portion thereof specifically binds to GPRC5D.
The tly disclosed subject matter also provides an isolated dy, or an antigen—binding portion thereof, comprising: (i) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 124; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 125; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 126; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 127; a light chain le region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:128; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 129; (ii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 130; a heavy chain le region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 131; a heavy chain le region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 132; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 133; a light chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID NO:l34; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 135; (iii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 136; a heavy chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID NO: 137; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 138; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 139; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:l40; and a light chain le region CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 141; (iv) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 142; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 143; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 144; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 145; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:l46; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 147; (v) a heavy chain le region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 148; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 149; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 150; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 151; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID ; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 153; (vi) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 154; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 155; a heavy chain le region CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 156; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 157; a light chain le region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:158; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 159; (vii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 160; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 161; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 162; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 163; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:164; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 165; (viii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 166; a heavy chain variable region CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 167; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 168; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 169; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:170; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 171; (ix) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 172; a heavy chain le region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 173; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 174; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 175; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:l76; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 177; (X) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 178; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 179; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 180; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 181; a light chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID NO:l82; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 183; (Xi) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 184; a heavy chain variable region CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 185; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 186; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 187; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:l88; and a light chain variable region CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 189; (xii) a heavy chain le region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 190; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 191; a heavy chain variable region CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 192; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 193; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID ; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 195; (xiii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 196; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 197; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 198; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 199; a light chain le region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:200; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 201; (xiv) a heavy chain variable region CDRl comprising amino acids having the ce set forth in SEQ ID NO: 202; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 203; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 204; a light chain variable region CDRl comprising amino acids having the ce set forth in SEQ ID NO: 205; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:206; and a light chain le region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 207; (xv) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 208; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 209; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 210; a light chain variable region CDRl comprising amino acids having the ce set forth in SEQ ID NO: 211; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:212; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 213; (xvi) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 214; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 215; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 216; a light chain variable region CDRl sing amino acids having the ce set forth in SEQ ID NO: 217; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:218; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 219; (xvii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 220; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 221; a heavy chain variable region CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 222; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 223; a light chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID ; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 225; (xviii) a heavy chain le region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 226; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 227; a heavy chain le region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 228; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 229; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:230; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 231; (xix) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 232; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 233; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 234; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 235; a light chain le region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:236; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 237; (xx) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 238; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 239; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 240; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 241; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:242; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 243; (XXi) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 244; a heavy chain le region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 245; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 246; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 247; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:248; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 249; (XXii) a heavy chain le region CDRl comprising amino acids having the ce set forth in SEQ ID NO: 250; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 251; a heavy chain variable region CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 252; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 253; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID ; and a light chain variable region CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 255; (XXiii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 256; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 257; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 258; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 259; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:260; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 261; (XXiv) a heavy chain variable region CDRl comprising amino acids having the ce set forth in SEQ ID NO: 262; a heavy chain variable region CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 263; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 264; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 265; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID ; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 267; (XXV) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 268; a heavy chain variable region CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 269; a heavy chain le region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 270; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 271; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:272; and (vi) a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 273; (xxvi) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 280; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 281; a heavy chain variable region CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 282; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 283; a light chain variable region CDR2 comprising amino acids having the ce set forth in SEQ ID NO:284; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 285; (xxvii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 292; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 293; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 294; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 295; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:296; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 297; (xxviii) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 303; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 304; a heavy chain le region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 305; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 306; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 307; and a light chain le region CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 308; (XXiX) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 316; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 317; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 318; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 319; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 320; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 321; (XXX) a heavy chain le region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 328; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 329; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 330; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 331; a light chain le region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 332; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 333; (XXXi) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 340; a heavy chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID NO: 341; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 342; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 343; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 344; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 345; or (XXXii) a heavy chain le region CDRl sing amino acids having the sequence set forth in SEQ ID NO: 352; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 353; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 354; a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 355; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 356; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 357. rmore, the presently disclosed subject matter provides an isolated antibody, or an antigen—binding portion f, which cross—competes for binding to human GPRC5D with any of the disclosed antibodies. In certain embodiments, the presently disclosed t matter es an isolated antibody, or an antigen—binding portion thereof, which binds to the same epitope on human GPRC5D with an isolated antibody, or an antigen—binding portion thereof of any of the antibodies disclosed herein.
In certain embodiments, the tly disclosed subject matter provides an isolated antibody, or an antigen—binding portion thereof, which cross—competes for binding to human GPRC5D with a reference antibody or reference antigen—binding portion thereof comprising: (i) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:1, and a light chain le region comprising amino acids having a sequence set forth in SEQ ID NO:2; (ii) a heavy chain variable region sing amino acids having a sequence set forth in SEQ ID NO:5, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:6; (iii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:9, and a light chain variable region sing amino acids having a sequence set forth in SEQ ID NO:lO; (iv) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:13, and a light chain le region comprising amino acids having a sequence set forth in SEQ ID NO:l4; (v) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:l7, and a light chain le region comprising amino acids having a sequence set forth in SEQ ID NO:lS; (vi) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:21, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:22; (vii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:25, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:26; (viii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:29, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:30; (ix) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:33, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:34; (x) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:37, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:38; (xi) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:4l, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:42; (xii) a heavy chain le region comprising amino acids having a sequence set forth in SEQ ID NO:45, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:46; (xiii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:49, and a light chain variable region comprising amino acids having a ce set forth in SEQ ID NO:50; (xiv) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:53, and a light chain variable region comprising amino acids having a ce set forth in SEQ ID NO:54; (xv) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:57, and a light chain variable region sing amino acids having a sequence set forth in SEQ ID NO:58; (xvi) a heavy chain le region comprising amino acids having a sequence set forth in SEQ ID NO:6l, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:62; (xvii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:65, and a light chain variable region comprising amino acids having a ce set forth in SEQ ID NO:66; (xviii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:69, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:70; (xix) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:73, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:74; (XX) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:77, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:78; (XXi) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:81, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:82; (XXii) a heavy chain variable region sing amino acids having a ce set forth in SEQ ID NO:85, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:86; (XXiii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:89, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:90; (XXiv) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:93, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:94; (XXV) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID , and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:275; (xxvi) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:286, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:287; (xxvii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:298, and a light chain le region comprising amino acids having a sequence set forth in SEQ ID NO:299; (xviii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:3lO, and a light chain le region comprising amino acids having a sequence set forth in SEQ ID NO:3ll; (XXi) a heavy chain variable region sing amino acids having a sequence set forth in SEQ ID NO:322, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:323; (XX) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:334, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:335; (XXi) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:346, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:347; or (XXii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:35 8, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:359.
In addition, the presently sed subject matter provides an isolated antibody, or an antigen—binding portion thereof, which binds to the same epitope on human GPRCSD as a reference antibody or reference antigen—binding portion f sing: (i) a heavy chain variable region sing amino acids having a sequence set forth in SEQ ID NO:1, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:2; (ii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:5, and a light chain le region comprising amino acids having a sequence set forth in SEQ ID NO:6; (iii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:9, and a light chain le region comprising amino acids having a sequence set forth in SEQ ID NO: 10; (iv) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:l3, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:l4; (v) a heavy chain le region comprising amino acids having a sequence set forth in SEQ ID NO:l7, and a light chain variable region sing amino acids having a sequence set forth in SEQ ID NO: 18; (vi) a heavy chain variable region comprising amino acids having a ce set forth in SEQ ID NO:21, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:22; (vii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:25, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:26; (viii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:29, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:30; (iX) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:33, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:34; (X) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:37, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:38; (xi) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:4l, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:42; (xii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:45, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:46; (xiii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:49, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:50; (xiv) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:53, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:54; (xv) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:57, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:58; (xvi) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:6l, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:62; (xvii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:65, and a light chain variable region comprising amino acids having a ce set forth in SEQ ID NO:66; (xviii) a heavy chain variable region sing amino acids having a sequence set forth in SEQ ID NO:69, and a light chain variable region sing amino acids having a sequence set forth in SEQ ID NO:70; (xix) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:73, and a light chain le region comprising amino acids having a sequence set forth in SEQ ID NO:74; (xx) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:77, and a light chain le region comprising amino acids having a sequence set forth in SEQ ID NO:78; (xxi) a heavy chain le region comprising amino acids having a sequence set forth in SEQ ID NO:81, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:82; (xxii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:85, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:86; ) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:89, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:90; (XXiv) a heavy chain le region comprising amino acids having a sequence set forth in SEQ ID NO:93, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:94; (XXV) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:274, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:275; (xxvi) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:286, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:287; (xxvii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:298, and a light chain variable region sing amino acids having a sequence set forth in SEQ ID NO:299; (xviii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:3lO, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:3ll; (XXi) a heavy chain variable region comprising amino acids having a ce set forth in SEQ ID NO:322, and a light chain le region comprising amino acids having a sequence set forth in SEQ ID NO:323; (XX) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:334, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:335; (XXi) a heavy chain variable region sing amino acids having a sequence set forth in SEQ ID NO:346, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:347; or (XXii) a heavy chain variable region comprising amino acids having a ce set forth in SEQ ID NO:35 8, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID .
In certain embodiments, the antibodies of the present sure bind to GPRCSD comprising the amino acid sequence set forth in SEQ ID NO:97. In n embodiments, the antibodies of the t disclosure binds to human GPRCSD with a binding affinity (Kd) of from about 1 x 10'9 M to about 1 x 10'8 M.
In certain embodiments, the antibodies of the present disclosure binds to one, two, three or four epitope region selected from the group consisting of an epitope region in N—terminal region comprising amino acids 1—27 of SEQ ID NO:97, an epitope region in ECLl region comprising amino acids 85—93 of SEQ ID NO:97, an epitope region in ECL2 region comprising amino acids 145—167 of SEQ ID NO:97, and an epitope region in ECL3 region comprising amino acids 226—239 of SEQ ID NO:97. In certain embodiments, the antibodies of the present sure bind to an epitope region comprising amino acids 16—23 of SEQ ID NO:97. In certain embodiments, the antibodies of the present disclosure bind to an epitope region comprising amino acids 15—23 of SEQ ID NO:97. In certain embodiments, the antibodies of the present sure bind to an epitope region comprising amino acids 16—25 of SEQ ID NO:97. In certain embodiments, the antibodies of the present disclosure bind to an epitope region sing amino acids 10—17 of SEQ ID NO:97.
In certain embodiments, the antibodies of the present disclosure bind to an epitope region comprising amino acids 5—17 of SEQ ID NO:97. In certain embodiments, the antibodies of the present disclosure bind to an epitope region sing amino acids 85-95 of SEQ ID NO:97. In certain embodiments, the antibodies of the present disclosure bind to an e region comprising amino acids 157—164 of SEQ ID NO:97. In certain embodiments, the antibodies of the present sure bind to an epitope region comprising amino acids 157—167 of SEQ ID NO:97. In certain embodiments, the antibodies of the present disclosure bind to an epitope region comprising amino acids 230—237 of SEQ ID NO:97. In certain embodiments, the antibodies of the present disclosure bind to an e region comprising amino acids 229—237 of SEQ ID NO:97. In certain embodiments, the antibodies of the present disclosure bind to an epitope region comprising amino acids 230—243 of SEQ ID NO:97. In certain embodiments, the antibodies of the present disclosure bind to an epitope region comprising amino acids 227—237 of SEQ ID NO:97.
The presently sed subject matter also provides an isolated antibody, or n—binding nt thereof, comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 100—123, 276, 288, 300, 312, 324, 336, 348 and 360.
In certain embodiments, the antibody or antigen—binding fragment thereof comprises a human variable region framework region. In certain embodiments, the antibody or antigen—binding fragment thereof is fully human or an antigen—binding fragment thereof. In certain embodiments, the antibody or antigen—binding fragment thereof is a chimeric antibody or an n—binding fragment thereof. In n embodiments, the antibody or antigen—binding portion thereof is a humanized dy or an antigen—binding fragment thereof. In certain ments, the antigen— binding fragment of the antibody is an Fab, Fab', F(ab')2, Fv or single chain Fv (scFv).
The presently disclosed subject matter also provides a composition comprising the antibody or antigen—binding fragment thereof disclosed herein, and a pharmaceutically acceptable carrier.
In addition, the presently disclosed subject matter es an immunoconjugate comprising the antibody or antigen—binding fragment thereof sed herein, linked to a therapeutic agent. In certain embodiments, the therapeutic agent is a drug, xin, or a radioactive isotope. The presently disclosed subject matter also provides a composition comprising such immunoconjugate and a pharmaceutically acceptable carrier.
Furthermore, the presently disclosed subject matter provides a bispecific molecule comprising the antibody or antigen—binding fragment thereof disclosed herein, linked to a second functional moiety. In certain embodiments, the second onal moiety has a different binding specificity than the antibody or antigen g fragment thereof. In certain embodiments, the second onal moiety has a binding specificity for an immune cell. In certain embodiments, the second functional moiety has a binding specificity for CD3.
The presently disclosed subject matter also provides a composition comprising such bispecific molecule and a pharmaceutically acceptable carrier.
In addition, the presently sed subject matter provides an isolated nucleic acid that encodes the antibody or antigen—binding fragment thereof sed , an expression vector comprising such nucleic acid molecule, and a host cell comprising gsuch sion vector. rmore, the presently disclosed subject matter provides a method for detecting GPRCSD in a whole cell or tissue. In certain ments, the method comprises: contacting a cell or tissue with the antibody or antigen—binding fragment thereof disclosed herein, wherein said antibody or antigen—binding fragment thereof ses a detectable label; and determining the amount of the labeled antibody or antigen—binding fragment thereof bound to said cell or tissue by measuring the amount of detectable label associated with said cell or tissue, wherein the amount of bound antibody or antigen—binding fragment thereof tes the amount of GPRCSD in said cell or tissue.
Furthermore, the presently sed subject matter provides a method of treating a tumor in a subject. In certain embodiments, the method comprises: administering an effective amount of the antibody or antigen—binding fragment thereof disclosed herein to the subject, thereby inducing death of a tumor cell in the subject.
In certain ments, the method reduces the number of the tumor cells. In certain embodiments, the method reduces the tumor size. In certain embodiments, the method eradicates the tumor in the subject. In certain embodiments, the subject is a human.
In on, the presently disclosed subject matter provides use of the antibody or antigen—binding fragment disclosed herein for the treatment of a tumor, and the antibody or antigen—binding fragment thereof disclosed herein for use in treating a tumor in a subject.
Furthermore, the presently sed subject matter provides a kit for treating a tumor, comprising the antibody or antigen—binding fragment thereof disclosed . In certain ments, the kit further comprises written instructions for using the antibody or antigen—binding fragment thereof for treating a subject having a tumor.
In n embodiments, the tumor is multiple myeloma or Waldenstrom’s Macroglobulinemia. In certain embodiments, the tumor is multiple myeloma.
BRIEF DESCRIPTION OF THE FIGURES The following ed Description, given by way of example, but not intended to limit the invention to specific embodiments described, may be understood in conjunction with the accompanying drawings.
Figure 1 s the human GPRCSD expression in various tissues.
Figure 2 illustrates the CLIPS technology. The CLIPS reaction takes place between bromo groups of the CLIPS scaffold and thiol sidechains of cysteines.
The reaction is fast and specific under mild conditions. Using this elegant chemistry, native protein sequences are transformed into CLIPS constructs with a range of ures. From left to right: two different single T2 loops, T3 double loop, conjugated T2+T3 loops, stabilized beta sheet, and stabilized alpha heliX rman et al., J. Mol. Recognit. 2007; 20: 283—29).
Figure 3 illustrates combinatorial clips library screening. The target protein (left) containing a tinuous conformational epitope is converted into a matrix library (middle). Combinatorial peptides are synthesized on a etary minicard and chemically converted into spatially defined CLIPS constructs (right).
Figure 4 depicts T3 looped M construct.
Figures 5A—5D illustrates heat map technology. (i) Table of combined peptides, with two sub—sequences indicated as "Loop 1" and "Loop 2". (ii) Data from A displayed as a matrix. (iii) Color bar indication of the heat map representation. (iv) Heat map visualization of data from A.
Figure 6 shows intensity profiles recorded for 2. Lines are drawn from the starting residue to the ending e of a single peptide on the height at which the signal for that peptide is recorded.
Figure 7 shows heatmap analysis of data recorded for ETlSO—S under high stringency conditions.
Figure 8 shows ity profiles recorded for ETlSO—lS.
Figure 9 shows intensity profiles recorded for ETlSO—S.
Figure 10 depicts schematic drawing of a GPCR containing seven transmembrane helices (TM) and 3 extracellular regions (ECLs). Colored arrows binding sites for each antibody is depicted.
Figure ll depicts scatterplot analysis of all data recorded for each sample.
On the al is the statistical data distribution.
Figure 12 s FACS analysis of PRCSD dies.
Figure 13 depicts FACS analysis of anti—GPRCSD antibodies.
Figure 14 depicts the FACS analysis of anti—GPRCSD/CD3 bispecific antibodies.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION All publications, s and other references cited herein are incorporated by reference in their entirety into the present disclosure.
In practicing the presently disclosed subject , many conventional techniques in molecular biology, microbiology, cell biology, biochemistry, and immunology are used, which are within the skill of the art. These techniques are described in greater detail in, for example, Molecular Cloning: a Laboratory Manual 3rd edition, J .F. Sambrook and D.W. Russell, ed. Cold Spring Harbor Laboratory Press 2001 ; Recombinant Antibodies for Immunotherapy, Melvyn Little, ed.
Cambridge University Press 2009; Oligonucleotide Synthesis" (M. J. Gait, ed., 1984); "Animal Cell Culture" (R. I. ey, ed., 1987); "Methods in logy" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (F. M. Ausubel et al., eds., 1987, and periodic s); "PCR: The Polymerase Chain Reaction", (Mullis et al., ed., 1994); "A cal Guide to Molecular Cloning" l Bernard V., 1988); "Phage Display: A Laboratory Manual" (Barbas et al., 2001 ). The contents of these references and other references containing standard protocols, widely known to and relied upon by those of skill in the art, including manufacturers' instructions are hereby incorporated by reference as part of the present disclosure.
Definitions In the description that follows, certain conventions will be followed as regards the usage of terminology. Generally, terms used herein are intended to be interpreted consistently with the meaning of those terms as they are known to those of skill in the art.
An "antigen—binding protein" is a protein or polypeptide that comprises an antigen—binding region or antigen—binding portion, that is, has a strong affinity to another le to which it binds. Antigen—binding ns encompass antibodies, chimeric antigen receptors (CARs) and fusion proteins.
"Antibody" and "antibodies" as those terms are known in the art refer to antigen binding proteins of the immune system. The term "antibody" as referred to herein includes whole, full length antibodies having an antigen—binding region, and any fragment thereof in which the "antigen—binding portion" or "antigen—binding " is retained, or single , for example, single chain variable nt (scFv), thereof. A naturally ing "antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter—connected by disulfide bonds.
Each heavy chain is comprised of a heavy chain le region (abbreviated herein as VH) and a heavy chain constant (CH) region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant CL region. The light chain constant region is sed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino—terminus to carboxy—terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4. The variable s of the heavy and light chains contain a g domain that interacts with an antigen.
The nt regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., or cells) and the first component (Cl q) of the classical complement .
The term "human antibody", as used herein, is intended to e antibodies having variable s in which both the framework and CDR regions are d from human germline globulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the presently disclosed t matter may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site— specific mutagenesis in vitro or by somatic mutation in viva).
The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies sing the tion are identical and/or bind the same epitope, except for possible t antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal dy preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being ed from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the presently disclosed subject matter may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage—display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
The term "recombinant human antibody", as used herein, includes all human antibodies that are prepared, sed, created or isolated by inant means, such as (a) antibodies isolated from an animal (e. g., a mouse) that is enic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies ed from a host cell transformed to express the human dy, e.g., from a transfectoma, (c) antibodies isolated from a inant, atorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that e splicing of human immunoglobulin gene ces to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL s of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL ces, may not naturally exist within the human antibody germline repertoire in vivo.
The term "humanized antibody" is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been d onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.
The term "chimeric antibody" is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.
As used , an antibody that "specifically binds to human GPRCSD" is intended to refer to an antibody that binds to human GPRCSD with a KD of 5 x 10'7 M or less, 1 x 10'7 M or less, 5 x 10'8 M or less, 1 x 10'8 M or less, 5 x 10'9 M or less, lx 10'9 M or less, 5 x 10'10 M or less, or 1 x 10'10 M or less.
An "antibody that competes for g" or "antibody that cross—competes for binding" with a reference antibody for binding to an n, e. g., GRPCSD, refers to an antibody that blocks g of the reference antibody to the antigen (e.g., GRPCSD) in a competition assay by 50% or more, and sely, the reference antibody blocks binding of the antibody to the n (e.g., GRPCSD) in a competition assay by 50% or more. An exemplary competition assay is described in "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY).
As used herein, "isotype" refers to the antibody class (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes.
The phrases "an antibody recognizing an antigen" and "an antibody specific for an n" are used interchangeably herein with the term "an antibody which binds specifically to an antigen (e.g., a GPRCSD polypeptide)." The term "antigen—binding portion" or "antigen—binding region" of an antibody, as used herein, refers to that region or portion of the antibody that binds to the antigen and which confers antigen specificity to the antibody; fragments of antigen—binding proteins, for example, dies includes one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a GPRCSD polypeptide). It has been shown that the antigen—binding function of an antibody can be med by fragments of a ength antibody. Examples of antigen—binding fragments encompassed within the term "antibody fragments" of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., 1989 Nature 341 :544—546), which ts of a VH domain; and an isolated complementarity determining region (CDR).
Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be , using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules. These are known as single chain Fv (scFv); see e.g., Bird et al., 1988 e 242:423—426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879—5883. These antibody nts are obtained using tional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
An "isolated antibody" or "isolated antigen—binding protein" is one which has been identified and separated and/or recovered from a component of its natural environment. "Synthetic antibodies" or "recombinant antibodies" are generally generated using recombinant technology or using peptide synthetic techniques known to those of skill in the art.
The terms "GPRCSD" and "G—protein d receptor family C group 5 member D" are used interchangeably, and include variants, isoforms, species homologs of human GPRCSD, and analogs having at least one common epitope with GPRCSD (e. g., human GPRCSD). An exemplary human GPRCSD sequence can be found under GenBank n Accession No: NP_06l 124. 1.
As used herein, the term e—chain variable fragment" or "scFv" is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin (e. g., mouse or human) covalently linked to form a VszVL dimer. The heavy (VH) and light chains (VL) are either joined directly or joined by a peptide—encoding linker (e. g., 10, 15, 20, 25 amino acids), which connects the N— terminus of the VH with the inus of the VL, or the C—terminus of the VH with the inus of the VL. The linker is usually rich in glycine for ?exibility, as well as serine or threonine for solubility. The linker can link the heavy chain variable region and the light chain variable region of the antibody or an antigen—binding fragment thereof. Non—limiting examples of s are disclosed in Shen et al., Anal.
Chem. 80(6):l9lO-l9l7 (2008) and hereby incorporated by reference in their entireties. In certain embodiments, the linker is a G4S linker.
In certain embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:309 as provided below: GGGGSGGGGSGGGGS [SEQ ID NO:309].
In certain ments, the nucleic acid ce encoding the amino acid sequence of SEQ ID NO:309 is set forth in SEQ ID NO:364, which is provided below: GGTGGAGGTGGATCAGGTGGAGGTGGATCTGGTGGAGGTGGAT CT [SEQ ID NO:364].
In one non—limiting example, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98 as provided below.
SRGGGGSGGGGSGGGGSLEMA [SEQ ID NO:98] In certain embodiments, the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:98 is set forth in SEQ ID NO:99, which is provided below: tctagaggtggtggtggtagcggcggcggcggctctggtggtggtggatccctcgagatggcc [SEQ ID NO:99] In certain embodiments, the linker ses amino acids having the following sequence GGGGS [SEQ ID NO:365].
In certain embodiments, the linker ses amino acids having the ing sequence S [SEQ ID NO:366].
In certain embodiments, the linker comprises amino acids having the following sequence GGGGSGGGS [SEQ ID NO:367].
In certain embodiments, the linker comprises amino acids having the following sequence GGGGSGGGGS [SEQ ID NO:368].
In certain embodiments, the linker ses amino acids having the following sequence GGGGSGGGGSGGGGGGGS [SEQ ID NO:369].
In certain embodiments, the linker comprises amino acids having the following sequence GGGGSGGGGSGGGGSGGGGS [SEQ ID NO:370].
In certain embodiments, the linker comprises amino acids having the following sequence GGGGSGGGGSGGGGSGGGGSGGGGS [SEQ ID NO:37l].
In certain embodiments, the linker comprises amino acids having the following sequence GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS [SEQ ID ].
In certain embodiments, the linker comprises amino acids having the following sequence GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS [SEQ ID NO:373].
In certain embodiments, the linker comprises amino acids having the following sequence KTHTCPPCP [SEQ ID ].
In certain embodiments, the linker comprises amino acids having the following sequence GGGGSGGGSEPKSCDKTHTCPPCP [SEQ ID NO:375].
In n embodiments, the linker comprises amino acids having the following sequence ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTP P PCPRCP [SEQ ID NO:376].
In n embodiments, the linker comprises amino acids having the following sequence GSGSGS [SEQ ID NO:377].
In certain embodiments, the linker comprises amino acids having the ing sequence AAA [SEQ ID ].
Despite removal of the constant regions and the introduction of a linker, scFv proteins retain the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be expressed from a nucleic acid comprising VH — and VL —encoding sequences as described by , et al. (Proc. Nat. Acad. Sci. USA, 9—5883, 1988). See, also, US. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and US. Patent Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs having inhibitory activity have been described (see, e.g., Zhao et al., Hyrbidoma mt) 2008 27(6):455—51; Peter et al., J CacheXia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol2009 183(4):2277—85; Giomarelli et al., Thromb Haemost 2007 97(6):955—63; Fife eta., J Clin Invst 2006 116(8):2252—61; Brocks et al., Immunotechnology 1997 3(3):173—84; Moosmayer et al., Ther Immunol 1995 2(10:31—40). Agonistic scFvs having stimulatory activity have been described (see, e.g., Peter et al., J Bioi Chem 2003 25278(38):36740—7; Xie et al., Nat Biotech 1997 768—71; Ledbetter et al., Crit Rev Immunoll997 17(5—6):427—55; Ho et al., BioChim Biophys Acta 2003 1638(3):257—66).
As used herein, "F(ab)" refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have a Fc portion, for example, an antibody digested by the enzyme papain yields two F(ab) nts and an EC nt (e. g., a heavy (H) chain constant region; Fc region that does not bind to an antigen).
As used herein, "F(ab')2" refers to an antibody fragment generated by pepsin digestion of whole IgG antibodies, wherein this fragment has two antigen binding (ab') (bivalent) regions, wherein each (ab') region comprises two separate amino acid chains, a part of a H chain and a light (L) chain linked by an S—S bond for binding an n and where the remaining H chain ns are linked together. A "F(ab')2" fragment can be split into two individual Fab' fragments.
As used herein, the term "vector" refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when ated with the proper control elements and which can transfer gene ces into cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors and plasmid vectors.
"CDRs" are defined as the complementarity determining region amino acid sequences of an antibody which are the hypervariable regions of immunoglobulin heavy and light chains. See, e. g., Kabat et al., Sequences of ns of Immunological Interest, 4th U. S. Department of Health and Human Services, National Institutes of Health . The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain which are ariable in sequence ("complementarity determining regions" or "CDRs") and/or form structurally defined loops ("hypervariable loops") and/or n the antigen— ting residues ("antigen contacts"). Generally, antibodies comprise three heavy chain and three light chain CDRs or CDR regions in the variable region. CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope.
An "isolated dy" is one which has been ted from a component of its l environment. In certain embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS— PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see, e. g., Flatman et al., J. Chromatogr. B 848:79—87 (2007).
An "isolated nucleic acid" refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is t extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
An "isolated nucleic acid encoding an antibody" (including references to a ic antibody, e.g. an anti—KLB antibody) refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector separate vectors, and such c acid molecule(s) t at one or more locations in a host cell.
The term 4 ‘vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self—replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors." An "immunoconjugate" is an dy conjugated to one or more heterologous molecule(s), including, but not limited to, a cytotoxic agent.
An "effective amount" of an agent, e. g., an anti—GPRCSD antibody or an antigen—binding nt thereof, a pharmaceutical comprision comprising thereof, refers to an amount effective, at s and for periods of time necessary, to achieve the desired therapeutic or prophylactic result, e.g., treating a tumor (e.g., multiple myeloma).
An "individual" or ct" is a mammal. Mammals include, but are not limited to, icated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non—human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.
As used herein, "treatment" (and grammatical variations thereof such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of al pathology. Desirable s of treatment include, but are not limited to, preventing occurrence or recurrence of disease, ation of symptoms, shment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of e progression, ration or palliation of the e state, and remission or improved prognosis. In certain embodiments, antibodies of the presently disclosed subject matter are used to delay development of a disease or to slow the progression of a disease, e.g., a tumor (multiple myeloma).
As used , the term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the tions of the measurement system. For example, "about" can mean within 3 or more than 3 rd deviations, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. atively, particularly with respect to biological systems or ses, the term can mean within an order of magnitude, preferably within 5—fold, and more preferably within 2—fold, of a value.
As described herein, any concentration range, percentage range, ratio range or r range is to be understood to include the value of any integer within the recited range and, when riate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
Anti-GPRCSD Antibodies The antibodies of the presently disclosed subject matter are characterized by particular functional features or properties of the antibodies. For example, the antibodies bind specifically to GPRCSD (e.g., bind to human GPRCSD and may cross—react with GPRCSD from other species, such as mouse). In certain embodiments, an antibody of the presently disclosed subject matter binds to GPRCSD with high affinity, for example with a Kd of 1 x 10'7 M or less, e.g., about about 1 x '8 M or less, about 1 x 10'9 M or less, or about 1 x 10'10 M or less. In certain embodiments, a presently disclosed anti—GPRCSD antibody binds to GPRCSD (e.g., human GPRCSD) with a Kd of from about 1 x 10'10 M to about 1 x 10'7 M, e.g., about from about 1 x 10'10 M to about 1 x 10'9 M, from 1 x 10'9 M to about 1 x 10'8 M, or from about 1 x 10'8 M to about 1 x 10'7 M. In certain embodiments, a presently disclosed anti—GPRCSD antibody binds to GPRCSD (e.g., human GPRCSD) with a Kd of about 1 x 10'8 M or less. In certain embodiments, a presently disclosed anti— GPRCSD dy binds to GPRCSD (e.g., human GPRCSD) with a Kd of from about 1 x 10'9 M to about 1 x 10'8 M. In certain embodiments, a presently disclosed anti— GPRCSD dy binds to GPRCSD (e.g., human GPRCSD) with a Kd of from about 1 x 10'9 M to about 1.5 x 10'9 M. In certain embodiments, a presently disclosed anti— GPRCSD dy binds to GPRCSD (e.g., human GPRCSD) with a Kd of about 1.2 x 10'9 M. In certain embodiments, a presently disclosed anti—GPRCSD antibody binds to GPRCSD (e.g., human GPRCSD) with a Kd of from about 4 x 10'9 M to about 5 x '9 M. In certain embodiments, a tly disclosed PRCSD antibody binds to GPRCSD (e.g., human GPRCSD) with a Kd of about 5 x 10'9 M. In certain ments, a presently disclosed anti—GPRCSD antibody binds to GPRCSD (e.g., human GPRCSD) with a Kd of about 4.8 X 10'9 M. In certain embodiments, a presently disclosed anti—GPRCSD antibody binds to GPRCSD (e. g., human GPRCSD) with a Kd of from about 8 X 10'9 M to about 9 X 10'9 M. In certain embodiments, a presently sed anti—GPRCSD antibody binds to GPRCSD (e. g., human GPRCSD) with a Kd of about 8 X 10'9 M. In certain embodiments, a presently sed anti— GPRCSD antibody binds to GPRCSD (e. g., human GPRCSD) with a Kd of about 8.1 X '9 M.
The heavy and light chains of an antibody of the presently disclosed subject matter can be full—length (e.g, an antibody can include at least one (e.g., one or two) complete heavy , and at least one (e.g., one or two) complete light chains) or can include an antigen—binding portion (a Fab, F(ab')2, Fv or a single chain Fv fragment ("scFv")). In certain ments, the antibody heavy chain nt region is chosen from, e. g and IgE, ., IgGl , IgG2, IgG3, IgG4, IgM, IgAl , IgA2, IgD, particularly chosen from, e.g, IgGl and IgG4, more particularly, IgGl , IgG2, IgG3, (e. g., human IgG1 ). In another embodiment, the antibody light chain constant region is chosen from, e.g., kappa or lambda, particularly kappa.
I . Single-chain variable fragments (scFvsl In certain ments, the presently sed t matter includes antibodies that have the scFv sequence fused to one or more constant domains to form an antibody with an Fc region of a human immunoglobulin to yield a bivalent protein, increasing the overall avidity and stability of the antibody. In addition, the Fc portion allows the direct conjugation of other molecules, including but not limited to ?uorescent dyes, cytot0Xins, radioisotopes etc. to the antibody for eXample, for use in antigen quantitation studies, to immobilize the antibody for affinity measurements, for targeted delivery of a therapeutic agent, to test for iated cytot0Xicity using immune effector cells and many other applications.
The s presented here highlight the specificity, sensitivity and utility of the antibodies of the invention in targeting a GRPCSD polypeptide.
The molecules of the invention are based on the identification and ion of single chain variable fragments (scFvs) using phage display, the amino acid sequence of which confers the molecules' specificity for a GRPCSD polypeptide of interest and forms the basis of all antigen binding ns of the disclosure. The scFv, therefore, can be used to design a diverse array of "antibody" molecules, including, for example, full length antibodies, fragments thereof, such as Fab and F(ab')2, minibodies, fusion proteins, including scFv—Fc fusions, multivalent dies, that is, antibodies that have more than one icity for the same antigen or different antigens, for example, bispecific antibodies, tribodies, etc. (see Cuesta et al., alent dies: when design surpasses ion. Trends in Biotechnology 28:355—362 2010).
In certain embodiments, the antigen—binding protein is a full length antibody, the heavy and light chains of an antibody of the presently disclosed subject matter can be ength (e.g., an antibody can include at least one, and ably two, te heavy chains, and at least one, and preferably two, complete light chains) or can include an antigen—binding portion (a Fab, F(ab')2, Fv or a single chain Fv fragment ("scFv")). In certain embodiments, the dy heavy chain constant region is chosen from, e. g., IgGl and IgE.
, IgG2, IgG3, IgG4, IgM, IgAl , IgA2, IgD, In certain embodiments, the immunoglobulin isotype is selected from IgGl , IgG2, IgG3, and IgG4, more ularly, IgGl (e.g., human IgGl). The choice of antibody isotype can depend on the immune effector function that the antibody is designed to elicit.
In constructing a recombinant immunoglobulin, appropriate amino acid sequences for constant regions of various immunoglobulin isotypes and methods for the production of a wide array of antibodies are known to those of skill in the art.
In certain embodiments, the antibody or other antigen binding protein is an anti—GPRCSD scFv or an antigen—binding fragment thereof having an antigen— binding region that ses the amino acid sequence of SEQ ID NO: 100 and specifically binds to a GPRCSD polypeptide (e.g., a GPRCSD polypeptide having the amino acid sequence SEQ ID NO:97 which is provided below, or fragments thereof).
MYKDCIESTGDYFLLCDAEGPWGIILESLAILGIVVTILLLLAFLFLMRKIQDCS QWNVLPTQLLFLLSVLGLFGLAFAFIIELNQQTAPVRYFLFGVLFALCFSCLLA HASNLVKLVRGCVSFSWTTILCIAIGCSLLQIIIATEYVTLIMTRGMMFVNMTP CQLNVDFVVLLVYVLFLMALTFFVSKATFCGPCENWKQHGRLIFITVLFSIlIW VVWISMLLRGNPQFQRQPQWDDPVVCIALVTNAWVFLLLYIVPELCILYRSCR QECPLQGNACPVTAYQHSFQVENQELSRARDSDGAEEDVALTSYGTPIQPQT VDPTQECFIPQAKLSPQQDAGGV [SEQ ID NO:97] The N—terminal region of human GPRCSD has amino acids 1—27 of SEQ ID NO:97.
The extracellular loop I (ECLl) region of human GPRCSD has amino acids 85—93 of SEQ ID NO:97. The extracellular loop 2 (ECL2) region of human GPRCSD has amino acids 145—167 of SEQ ID NO:97. The extracellular loop 3 (ECL3) region of human GPRCSD has amino acids 226—239 of SEQ ID NO:97.
In certain embodiments, the antibody or other antigen binding protein is an anti—GPRCSD scFv or an antigen—binding fragment thereof having an antigen— g region that comprises the amino acid sequence of SEQ ID NO:lOO and specifically binds to a GPRCSD polypeptide (e.g., a GPRCSD polypeptide having the amino acid sequence SEQ ID NO:97, or fragments thereof), which is designated as ET150—153 scFv (also referred to as —3 scFv").
In certain embodiments, the anti—GPRCSD scFv antibody ses a heavy chain variable region sing amino acids having the sequence set forth in SEQ ID NO:1 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:2, optionally with (iii) a linker sequence, for example a linker peptide, between the heavy chain variable region and the light chain variable region. In certain embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the anti—GPRCSD scFv antibody is an scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table I. In n embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:1, as shown in Table I. In certain embodiments, the anti—GPRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:2, as shown in Table I. In n embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the ce set forth in SEQ ID NO:1 and a VL comprising amino acids having the sequence set forth in SEQ ID NO:2, as shown in Table I. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:124 or vative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:125 or conservative modifications thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:126 or conservative cations thereof, as shown in Table I. In certain embodiments, the anti—GPRCSD scFv comprises a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:127 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:128 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:129 or conservative cations thereof, as shown in Table 1. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the ce set forth in SEQ ID NO:124 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:125 or conservative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:126 or conservative modifications thereof, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:127 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:128 or conservative cations thereof, and a VL CDR3 comprising amino acids having the ce set forth in SEQ ID NO:129 or conservative modifications f, as shown in Table 1. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 124, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 125, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:126, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:127, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:128, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 129.
Antigen A GPRCSD polypeptide having the amino acid ce of SEQ ID NO:97 GYTFTSYY [SEQ ID GYTFTSYY [SEQ ID ARGMYRSLLFYDP NO:124] ] [SEQ ID ] RSNVGNYY [SEQ DNN [SEQ ID NO:128] GTWDGSLSAHV [SEQ ID NO:127] ID NO:129] QVQLVQSGSELKKPGASVRVSCTASGYTFTSYYMHWVRQAPGQGLEW MGVINPNAGSTRYAQKFQGRVTMSTDTSTSTAYMDLSSLRSEDTAVYY CARGMYRSLLFYDPWGQGTLVTVSS [SEQ ID NO: 1] Caggtgcagctggtgcagtctgggtctgagttgaagaagcctggggcctcagtcagagtctcctgcacggcttctg gatacaccttcaccagttactatatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggagtaat caaccctaatgctggcagcacaagatacgcacagaaattccagggcagagtcaccatgagcactgacacgtcca cgagcacagcctacatggacctgagcagtctgagatctgaggacacggccgtgtattactgtgcgcgcggtatgta ccgttctctgctgttctacgatccgtggggtcaaggtactctggtgaccgtctcctca [SEQ ID NO:3] QSVLTQPPSVSAAPGQKVTIPCSGSRSNVGNYYVSWYQQLPGTAPKLLI YDNNKRPSGIPDRFSGSKSGTSATLGlTGLQTGDEADYFCGTWDGSLSA HVFGTGTKVTVLG [SEQ ID NO:2] Cagtctgtgttgacgcagccgccctcagtgtctgcggccccaggacagaaggtcaccatcccctgctctggaagc cgttccaacgttgggaattattatgtgtcctggtaccagcaactcccaggaacagcccccaaactcctcatttatgac aagcgaccctcagggattcctgaccgattctctggctccaagtctggcacgtcagccaccctgggcatcac ccagactggggacgaggccgattatttctgcggaacatgggatggcagcctgagtgcccatgtcttcgga actgggaccaaggtcaccgtcctaggt [SEQ ID NO:4] QSVLTQPPSVSAAPGQKVTIPCSGSRSNVGNYYVSWYQQLPGTAPKLLI PSGIPDRFSGSKSGTSATLGlTGLQTGDEADYFCGTWDGSLSA HVFGTGTKVTVLGSRGGGGSGGGGSGGGGSLEMAQVQLVQSGSELKK VSCTASGYTFTSYYMHWVRQAPGQGLEWMGVINPNAGSTRY AQKFQGRVTMSTDTSTSTAYMDLSSLRSEDTAVYYCARGMYRSLLFYD PWGQGTLVTVSS [SEQ ID ] In certain embodiments, the antibody or other antigen binding protein is an anti—GPRCSD scFv or an antigen—binding fragment thereof having an antigen— binding region that comprises the amino acid sequence of SEQ ID NO: 101 and specifically binds to a GPRCSD polypeptide (e.g., a GPRCSD polypeptide having the amino acid sequence SEQ ID NO:97, or fragments thereof), which is designated as ET150—l66 scFv (also referred to as "ET150—l6 scFv").
In certain embodiments, the anti—GPRCSD scFv antibody comprises a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:5 and a light chain le region comprising amino acids having the sequence set forth in SEQ ID NO:6, optionally with (iii) a linker ce, for example a linker peptide, between the heavy chain variable region and the light chain variable region. In certain embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the anti—GPRCSD scFv antibody is an scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 2. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:5, as shown in Table 2. In certain ments, the anti—GPRCSD scFv comprises a VL comprising amino acids having the ce set forth in SEQ ID NO:6, as shown in Table 2. In n embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:5 and a VL comprising amino acids having the sequence set forth in SEQ ID NO:6, as shown in Table 2. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:l30 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:l3l or conservative modifications thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:l32 or conservative modifications thereof, as shown in Table 2. In n embodiments, the anti—GPRCSD scFv comprises a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 133 or conservative modifications f, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:l34 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the ce set forth in SEQ ID NO:l35 or conservative modifications thereof, as shown in Table 2. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:l30 or vative modifications thereof, a VH CDR2 sing amino acids having the sequence set forth in SEQ ID NO:l3l or conservative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:l32 or conservative modifications thereof, a VL CDRl sing amino acids having the sequence set forth in SEQ ID NO:l33 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:l34 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:l35 or vative modifications thereof, as shown in Table 2. In certain ments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 130, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 131, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID , a VL CDRI comprising amino acids having the sequence set forth in SEQ ID NO: 133, a VL CDR2 comprising amino acids haVing the sequence set forth in SEQ ID NO:134, and a VL CDR3 comprising amino acids haVing the sequence set forth in SEQ ID NO:135.
Table 2 Antigen A GPRCSD polypeptide haVing the amino acid sequence of SEQ ID NO:97 GFTFSNYA [SEQ ID T [SEQ ID ARGSVRYTDI [SEQ ID NO:130] NO:131] NO:132] SGAIAGAY [SEQ DDN [SEQ ID QSYDYDSSNVL [SEQ ID NO:133] NO:134] ID NO:135] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLE GSGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY YCARGSVRYTDIWGQGTLVTVSS [SEQ ID NO:5] Gaggtgcagctggtggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagc ctctggattcacctttagcaactatgccatgagttgggtccgccaggctccagggaagggactggagtgggtct cagctattagtggtagtggtaacacatactacgcagactccgtgaagggccggttcaccatctccagagacaat tccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtatattactgtgcgcgcg gttctgttcgttacactgatatctggggtcaaggtactctggtgaccgtctcctca [SEQ ID NO:7] NFMLTQPHSVSESPGKTVSISCTRTSGAIAGAYVQWFQQRPGSAPTTV IYDDNKRPSGVPDRFSGSIDKSSNSASLTISGLKTEDEADYYCQSYDY DSSNVLFGGGTKLTVLG [SEQ ID NO:6] Aattttatgctgactcagccccactcagtgtcggagtctccggggaagacggtaagcatctcctgcacccgca ccagtggcgccattgccggcgcctatgtgcagtggttccagcagcgcccgggcagtgcccccaccactgtga tctatgacgataacaaaagaccctctggggtccctgatcggttctctgggtccatcgacaagtcctccaactctg cctccctcaccatctctggactgaagactgaggacgaggctgactattattgtcagtcttatgattatgatagcag caatgtgctattcggcggagggaccaagctgaccgtcctaggt [SEQ ID NO:8] NFMLTQPHSVSESPGKTVSISCTRTSGAIAGAYVQWFQQRPGSAPTTV IYDDNKRPSGVPDRFSGSIDKSSNSASLTISGLKTEDEADYYCQSYDY DSSNVLFGGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAEVQLVESG GGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSAISGS GNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSV RYTDIVVGQGTLVTVSS [SEQ ID NO: 101] In certain ments, the antibody or other antigen binding n is an anti—GPRCSD scFv or an antigen—binding fragment thereof having an n— binding region that comprises the amino acid sequence of SEQ ID NO:102 and specifically binds to a GPRCSD polypeptide (e. g., a GPRCSD polypeptide having the amino acid sequence SEQ ID NO:97, or fragments f), which is designated as ETlSO—l70 scFv (also referred to as "ET150—20 scFv").
In certain embodiments, the anti—GPRCSD scFv antibody comprises a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:9 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:lO, optionally with (iii) a linker sequence, for example a linker peptide, between the heavy chain variable region and the light chain variable region. In certain ments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the anti—GPRCSD scFv antibody is an c fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 3. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:9, as shown in Table 3. In certain embodiments, the anti—GPRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:lO, as shown in Table 3. In certain ments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:9 and a VL sing amino acids having the sequence set forth in SEQ ID NO:lO, as shown in Table 3. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:l36 or conservative modifications f, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:l37 or conservative cations thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:l38 or conservative modifications thereof, as shown in Table 3. In certain embodiments, the anti—GPRCSD scFv comprises a VL CDRl comprising amino acids having the ce set forth in SEQ ID NO:l39 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:l40 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:l4l or conservative modifications thereof, as shown in Table 3. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:136 or conservative modifications f, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:137 or conservative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:l38 or conservative modifications thereof, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:139 or conservative cations thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:l40 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the ce set forth in SEQ ID NO:l4l or conservative modifications thereof, as shown in Table 3. In certain ments, the PRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:136, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:137, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:138, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:139, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:l40, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 141.
Table 3 Antigen A GPRCSD ptide having the amino acid sequence of SEQ ID NO:97 GFTFNNYW [SEQ IKQDGSEK [SEQ ID ARSMSTAV [SEQ ID ID NO:136] NO:137] NO:l38] QSISSY [SEQ ID AAS [SEQ ID NO: 140] QQSYSVPYT [SEQ ID NO:139] NO:l4l] Full VH EVQLVQSGGGLVQPGGSLRLSCATSGFTFNNYWMSWVRQAPGKGLE WVANIKQDGSEKYYADSVRGRFTISRDNAKNSLSLQLNNLRAEDTAV MSTAWGYDEWGQGTLVTVSS [SEQ ID NO:9] DNA Gaggtgcagctggtgcagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcaacct ctggattcacctttaataactattggatgagttgggtccgccaggctccagggaaggggctggagtgggtggcc aacataaagcaagatggaagtgagaaatactacgcggactctgtgaggggccgattcaccatctccagagaca acgccaagaactcactgtctctgcaattgaacaacctgagagccgaggacacggccgtgtattactgtgcgcgc tctatgtctactgcttggggttacgatgaatggggtcaaggtactctggtgaccgtctcctca [SEQ ID NO:1 1] Full VL DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY AASSLQSGVPSRFSGSGSGTDFTLTISSLQPADFATYYCQQSYSVPYTF EIKR [SEQ ID NO: 10] DNA Gacatccagttgacccagtctccatcctccctgtctgcatctgtcggagacagagtcaccatcacttgccgggca agtcagagcattagcagctatttaaattggtatcaacagaaaccagggaaagcccctaagctcctgatctatgctg catccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcag cagtctgcaacctgcagattttgcaacttactactgtcaacagagttacagtgtcccgtacacttttggccagggga ccaa__ct __aatcaaac _t [SEQ ID NO: 12] scFv DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY AASSLQSGVPSRFSGSGSGTDFTLTISSLQPADFATYYCQQSYSVPYTF GQGTKLEIKRSRGGGGSGGGGSGGGGSLEMAEVQLVQSGGGLVQPG CATSGFTFNNYWMSWVRQAPGKGLEWVANIKQDGSEKYYA FTISRDNAKNSLSLQLNNLRAEDTAVYYCARSMSTAWGYDE WGQGTLVTVSS [SEQ ID NO:lOZ] In certain embodiments, the antibody or other antigen binding protein is an anti—GPRCSD scFv or an antigen—binding fragment thereof having an antigen— binding region that ses the amino acid sequence of SEQ ID NO: 103 and specifically binds to a GPRCSD polypeptide (e.g., a GPRCSD polypeptide having the amino acid ce SEQ ID NO:97, or fragments thereof), which is designated as ETlSO—l7l scFv (also referred to as "ET150—21 scFv").
In certain embodiments, the anti—GPRCSD scFv antibody comprises a heavy chain variable region sing amino acids having the sequence set forth in SEQ ID NO:l3 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:l4, optionally with (iii) a linker sequence, for example a linker peptide, between the heavy chain variable region and the light chain variable region. In certain embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the PRCSD scFv antibody is an scFv—Fc fusion n or full length human IgG with VH and VL regions or CDRs selected from Table 4. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:l3, as shown in Table 4. In certain embodiments, the anti—GPRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:14, as shown in Table 4. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:l3 and a VL comprising amino acids having the sequence set forth in SEQ ID NO:14, as shown in Table 4. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the ce set forth in SEQ ID NO:l42 or vative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:143 or conservative modifications thereof, and a VH CDR3 sing amino acids having the sequence set forth in SEQ ID NO:144 or conservative modifications thereof, as shown in Table 4. In certain embodiments, the anti—GPRCSD scFv comprises a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:l45 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:146 or vative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:147 or conservative modifications f, as shown in Table 4. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:l42 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:143 or conservative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:144 or conservative cations thereof, a VL CDRl sing amino acids having the sequence set forth in SEQ ID NO:l45 or conservative modifications thereof, a VL CDR2 comprising amino acids having the ce set forth in SEQ ID NO:146 or conservative modifications thereof, and a VL CDR3 sing amino acids having the sequence set forth in SEQ ID NO:147 or conservative modifications thereof, as shown in Table 4. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:142, a VH CDR2 sing amino acids having the sequence set forth in SEQ ID NO:143, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:144, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:145, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:146, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:147.
Antigen A GPRCSD polypeptide having the amino acid sequence of SEQ ID NO:97 GYTFTSYY [SEQ INPSGGST [SEQ ID ARGSSRWGGWTGDY ID NO:l42] NO:l43] [SEQ ID NO:l44] SSDVGGYNF [SEQ DVS [SEQ ID NO:l46] SSYTSTRTVIFAGGTKVTV ID NO:l45] L [SEQ ID NO:l47] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEW MGIINPSGGSTRYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYC ARGSSRWGGWTGDYWGQGTLVTVSS [SEQ ID NO: 13] Caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtttcctgcaaggcatct ggatacaccttcaccagctactatatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggaataat caaccctagtggtggtagcacaaggtacgcacagaagttccagggcagagtcaccatgaccagggacacgtcaac gagcacagtctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgcgcggttcttctc gctggggtggttggactggtgattactggggtcaaggtactctggtgaccgtctcctca [SEQ ID NO:15] QSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKVMI YDVSKRPSGISNRFSGSKSGNTASLTISGLQVEDEAEYYCSSYTSTRTVIF AGGTKVTVLG [SEQ ID NO: 14] gccctgactcagcctgcctccgtgtctgggtctcctggacagtcgatcaccatctcctgcactggaaccagc agtgacgttggtggttataactttgtctcctggtaccaacagcacccaggcaaagcccccaaagtcatgatttatgatg tcagtaagcggccctcagggatttctaatcgcttctctggctccaagtctggcaacacggcctccctgaccatctctgg gctccaggttgaggacgaggctgaatattactgcagctcatatacaagcactagaactgtgatattcgccggaggga ccaaggtcaccgtcctaggt [SEQ ID NO: 16] QSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKVMI YDVSKRPSGISNRFSGSKSGNTASLTISGLQVEDEAEYYCSSYTSTRTVIF SRGGGGSGGGGSGGGGSLEMAQVQLVQSGAEVKKPGASVKVSCKASG YMHWVRQAPGQGLEWMGIINPSGGSTRYAQKFQGRVTMTRDT STSTVYMELSSLRSEDTAVYYCARGSSRWGGWTGDYWGQGTLVTVSS [SEQ ID NO: 103] In n embodiments, the antibody or other antigen binding protein is an anti—GPRCSD scFV or an antigen—binding fragment thereof haVing an antigen— binding region that ses the amino acid sequence of SEQ ID NO: 104 and specifically binds to a GPRCSD polypeptide (e.g., a GPRCSD polypeptide haVing the amino acid sequence SEQ ID NO:97, or fragments thereof), which is designated as ETlSO—l75 scFV (also referred to as —25 scFV").
In certain embodiments, the anti—GPRCSD scFv antibody comprises a heavy chain le region comprising amino acids having the sequence set forth in SEQ ID NO:l7 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:lS, optionally with (iii) a linker ce, for example a linker peptide, between the heavy chain variable region and the light chain variable region. In certain embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the anti—GPRCSD scFv antibody is an scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 5. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:l7, as shown in Table 5. In certain embodiments, the anti—GPRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:lS, as shown in Table 5. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:l7 and a VL comprising amino acids having the sequence set forth in SEQ ID NO:lS, as shown in Table 5. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:l48 or conservative cations f, a VH CDR2 sing amino acids having the sequence set forth in SEQ ID NO:l49 or conservative modifications thereof, and a VH CDR3 comprising amino acids having the sequence set forth in0 SEQ ID NO:lSO or conservative modifications f, as shown in Table 5. In certain embodiments, the PRCSD scFv comprises a VL CDRl sing amino acids having the sequence set forth in SEQ ID NO:lSl or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:152 or conservative cations thereof, and a VL CDR3 comprising amino acids having the ce set forth in SEQ ID NO:lS3 or vative modifications thereof, as shown in Table 5. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:l48 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:l49 or conservative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:lSO or conservative modifications thereof, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:lSl or conservative modifications thereof, a VL CDR2 sing amino acids having the sequence set forth in SEQ ID NO: 152 or conservative modifications f, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:lS3 or conservative modifications thereof, as shown in Table 5. In certain embodiments, the PRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 148, a VH CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 149, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 150, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 15 l, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:lSZ, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 153.
Table 5 n A GPRCSD polypeptide having the amino acid sequence of SEQ ID NO:97 GSTFSSYA [SEQ ID ISGRGRST [SEQ ID ARYYKSKDH [SEQ NO:l48] NO:l49] ID NO:lSO] RSNIGTNY [SEQ ID RNH [SEQ ID NO: 152] AAWDDNLSGVV NO:lSl] [SEQ ID NO:lS3] Full VH EVQLVETGGGLVQPGGSLRLSCAASGSTFSSYAMSWVRQAPGKGLE WVSAISGRGRSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV YYCARYYKSSKDHWGQGTLVTVSS [SEQ ID NO: 17] DNA Gaggtgcagctggtggagactgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcc tctggatccacctttagcagctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctc agctattagtggtcgtggtcgtagcacatactacgcagactccgtgaagggccggttcaccatctccagagaca attccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtatattactgtgcgcgc tactacaaatcttctaaagatcattggggtcaaggtactctggtgaccgtctcctca [SEQ ID NO: 19] Full VL QSVLTQPPSLSGAPGQRVTISCSGSRSNIGTNYVSWXQQLPGTAPKLLI YRNHQWPSGVPDRFTGSKSGTSASLAISGLRSEDEADYYCAAWDDNL SGVVFGGGTKLTVLG [SEQ ID NO: 18] DNA Cagtctgtgttgacgcagccgccctcactgtctggggccccagggcagagggtcaccatctcttgttccggaag caggtccaacatcggaactaattatgtatcctggnaccagcaactcccaggaacggcccccaaactcctcatcta taggaatcatcagtggccctcaggggtccctgaccgattcactggctccaagtctggcacctcagcctccctggc catcagtgggctccggtccgaggatgaggctgattactactgtgcagcatgggatgacaatttgagtggtgtggt gttcggcggagggaccaagctgaccgtcctaggt [SEQ ID NO:20] scFv QSVLTQPPSLSGAPGQRVTISCSGSRSNIGTNYVSWXQQLPGTAPKLLI YRNHQWPSGVPDRFTGSKSGTSASLAISGLRSEDEADYYCAAWDDNL SGVVFGGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAEVQLVETGGG LVQPGGSLRLSCAASGSTFSSYAMSWVRQAPGKGLEWVSAISGRGRS TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYKSSK TLVTVSS [SEQ ID NO:104] In certain embodiments, the antibody or other n binding protein is an anti—GPRCSD scFv or an antigen—binding fragment thereof having an antigen— binding region that comprises the amino acid sequence of SEQ ID NO: 105 and specifically binds to a GPRCSD polypeptide (e.g., a GPRCSD polypeptide having the amino acid sequence SEQ ID NO:97, or fragments thereof), which is designated as ET150—154 scFv (also referred to as "ET150—4 scFv").
In certain embodiments, the anti—GPRCSD scFv antibody comprises a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:21 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:22, optionally with (iii) a linker sequence, for example a linker e, between the heavy chain variable region and the light chain le region. In n embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the anti—GPRCSD scFv antibody is an scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 6. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:21, as shown in Table 6. In n embodiments, the PRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:22, as shown in Table 6. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:21 and a VL comprising amino acids having the sequence set forth in SEQ ID NO:22, as shown in Table 6. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:154 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:155 or vative modifications thereof, and a VH CDR3 sing amino acids having the sequence set forth in SEQ ID NO:156 or conservative modifications thereof, as shown in Table 6. In certain embodiments, the anti—GPRCSD scFv ses a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:157 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:158 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:159 or conservative modifications thereof, as shown in Table 6. In certain ments, the anti—GPRC5D scFv comprises a VH CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:154 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:155 or conservative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:156 or conservative cations thereof, a VL CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:157 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:158 or conservative cations f, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:159 or conservative modifications thereof, as shown in Table 6. In certain ments, the anti—GPRC5D scFv comprises a VH CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 154, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 155, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:156, a VL CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 157, a VL CDR2 sing amino acids having the sequence set forth in SEQ ID NO:158, and a VL CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 159.
Antigen AYTFTDYY [SEQ INPKSGRT [SEQ ID ARVYGYSRWSGFDL ID NO:154] NO:155] [SEQ ID NO:156] SSNIGSNY [SEQ ID RNN [SEQ ID AAWDDSLSGYV [SEQ NO:157] NO:158] ID NO:159] QVQLVQSGAEVQRPGASVRVSCKAIAYTFTDYYIHWVRQAPGQGP EWMGWINPKSGRTQYAPKFQDRVTLARETPISTASMELRGLTSDDT AVYYCARVYGYSRWSGFDLWGQGTLVTVSS [SEQ ID NO:21] DNA Caggtccagctggtgcagtctggggctgaggtgcagaggcctggggcctcagtgagggtctcctgcaag gctattgcgtacaccttcaccgactactatatccactgggtgcgacaggcccctggacaagggcctgagtgg atggggtggatcaaccctaaaagtggtcgcacacagtatgcaccgaagtttcaagacagggtcaccctggc gacgcccatcagcacagcctccatggagctgcgcggactgacatctgacgacacggccgtgtat tactgtgcgcgcgtttacggttactctcgttggtctggtttcgatctgtggggtcaaggtactctggtgaccgtc tcctca [SEQ ID NO:23] Full VL QAVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKL QRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWD DSLSGYVFGTGTKVTVLG [SEQ ID NO:22] DNA Caggctgtgctgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgttctgg aagcagctccaacatcggaagtaattatgtatactggtaccagcagctcccaggaacggcccccaaactcct catctataggaataatcagcggccctcaggggtccctgaccgattctctggctccaagtctggcacctcagc ctccctggccatcagtgggctccggtccgaggatgaggctgattattactgtgcagcatgggatgacagcct ;at;; __ttattcttc ; _aact; ; _accaa; _tcacc _tccta; _t [SEQ ID NO:24] scFv QAVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKL LIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWD VFGTGTKVTVLGSRGGGGSGGGGSGGGGSLEMAQVQLV QSGAEVQRPGASVRVSCKAIAYTFTDYYIHWVRQAPGQGPEWMG WINPKSGRTQYAPKFQDRVTLARETPISTASMELRGLTSDDTAVYY CARVYGYSRWSGFDLWGQGTLVTVSS [SEQ ID NO: 105] In certain embodiments, the antibody or other antigen binding protein is an anti—GPRCSD scFv or an antigen—binding fragment thereof having an antigen— binding region that ses the amino acid ce of SEQ ID NO: 106 and specifically binds to a GPRCSD polypeptide (e.g., a GPRCSD polypeptide having the amino acid sequence SEQ ID NO:97, or fragments thereof), which is designated as 156 scFv (also referred to as "ET150—6 .
In certain embodiments, the anti—GPRCSD scFv antibody comprises a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:25 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:26, optionally with (iii) a linker sequence, for example a linker peptide, between the heavy chain variable region and the light chain variable region. In certain embodiments, the linker ses amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the anti—GPRCSD scFv antibody is an scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 7. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:25, as shown in Table 7. In certain embodiments, the anti—GPRCSD scFv comprises a VL comprising amino acids having the ce set forth in SEQ ID NO:26, as shown in Table 7. In certain embodiments, the anti—GPRCSD scFv comprises a VH sing amino acids having the sequence set forth in SEQ ID NO:25 and a VL comprising amino acids having the sequence set forth in SEQ ID NO:26, as shown in Table 7. In certain embodiments, the PRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:l60 or conservative modifications thereof, a VH CDR2 comprising amino acids having the ce set forth in SEQ ID NO:l6l or conservative modifications thereof, and a VH CDR3 sing amino acids having the sequence set forth in SEQ ID NO:l62 or conservative modifications thereof, as shown in Table 7. In n embodiments, the PRCSD scFv comprises a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:163 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:164 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:l65 or conservative modifications thereof, as shown in Table 7. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:l60 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:l6l or conservative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:l62 or conservative modifications thereof, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:163 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:164 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:l65 or conservative modifications thereof, as shown in Table 7. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 160, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 161, a VH CDR3 comprising amino acids having the ce set forth in SEQ ID NO:l62, a VL CDRl sing amino acids having the sequence set forth in SEQ ID NO:163, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:164, and a VL CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 165.
Antigen A GPRCSD polypeptide having the amino acid sequence of SEQ ID NO:97 YY [SEQ INPNGGGT [SEQ ID ARGHKVYKSHPTGGYDR ID NO:l60] NO: 161] [SEQ ID NO:l62] SRDVGGYNY [SEQ EVS [SEQ ID NO: 164] SSYTSSSTLD [SEQ ID ID NO:l63] NO:l65] QVQLVQSGAEVKQPGASVKVSCQASGYTFTTYYMHWVRQAPGQGLE WMGIINPNGGGTFYAQKFQDRVTMTRDTSTGTVYMELSSLRSDDTAVY YCARGHKVYKSHPTGGYDRWGQGTLVTVSS [SEQ ID NO:25] Caggtgcagctggtgcaatctggggctgaggtgaagcagcctggggcctcagtgaaggtttcctgccaggcatct ggatacaccttcaccacttattatatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggaataat caaccctaatggtggtggcacattctacgcacagaagttccaggacagagtcaccatgaccagggacacgtccac gggcacagtctacatggaactgagcagcctgagatctgacgacactgccgtgtattactgtgcgcgcggtcataaa gtttacaaatctcatccgactggtggttacgatcgttggggtcaaggtactctggtgaccgtctcctca [SEQ ID NO:27] QSALTQPASVSGSPGQSITISCTGTSRDVGGYNYVSWYQQYPGKAPKLM IYEVSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLD VTVLG [SEQ ID NO:26] Caatctgccctgactcagcctgcctccgtgtctgggtctcctggacagtcgatcaccatctcctgcactggaaccag ccgtgacgttggtggttataactatgtctcctggtaccaacagtacccaggcaaagcccccaaactcatgatttatga ggtcagtaagcggccctcaggggtttctaatcgcttctctggctccaagtctggcaacacggcctccctgaccatct ctgggctccaggctgaggacgaggctgattattactgcagctcatataccagtagcagcactttagacttcggaact tccta; ;t [SEQ ID NO:28] QSALTQPASVSGSPGQSITISCTGTSRDVGGYNYVSWYQQYPGKAPKLM IYEVSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLD FGTGTKVTVLGSRGGGGSGGGGSGGGGSLEMAQVQLVQSGAEVKQPG ASVKVSCQASGYTFTTYYMHWVRQAPGQGLEWMGIINPNGGGTFYAQ KFQDRVTMTRDTSTGTVYMELSSLRSDDTAVYYCARGHKVYKSHPTG GYDRWGQGTLVTVSS [SEQ ID NO: 106] In certain ments, the antibody or other antigen g protein is an anti—GPRCSD scFV or an antigen—binding nt thereof haVing an antigen— binding region that comprises the amino acid sequence of SEQ ID NO: 107 and specifically binds to a GPRCSD polypeptide (e.g., a GPRCSD polypeptide haVing the amino acid sequence SEQ ID NO:97, or fragments thereof), which is designated as ET150—157 scFv (also ed to as "ETlSO—7 scFv").
In certain ments, the anti—GPRCSD scFv antibody comprises a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:29 and a light chain le region comprising amino acids having the sequence set forth in SEQ ID NO:30, optionally with (iii) a linker sequence, for example a linker peptide, between the heavy chain le region and the light chain variable region. In certain ments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In n embodiments, the anti—GPRCSD scFv antibody is an scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 8. In n embodiments, the anti—GPRCSD scFv ses a VH comprising amino acids having the sequence set forth in SEQ ID NO:29, as shown in Table 8. In certain embodiments, the PRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:30, as shown in Table 8. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:29 and a VL comprising amino acids having the sequence set forth in SEQ ID NO:30, as shown in Table 8. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:l66 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:l67 or conservative modifications thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:l68 or conservative modifications thereof, as shown in Table 8. In certain embodiments, the anti—GPRCSD scFv comprises a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:l69 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:l70 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:l7l or conservative modifications thereof, as shown in Table 8. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:l66 or conservative cations thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:l67 or vative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:168 or conservative modifications thereof, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:169 or conservative modifications f, a VL CDR2 sing amino acids having the sequence set forth in SEQ ID NO:170 or conservative modifications f, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:171 or conservative modifications thereof, as shown in Table 8. In n embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 166, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 167, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:168, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 169, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:170, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 171.
Table 8 Antigen A GPRCSD polypeptide having the amino acid sequence of SEQ ID NO:97 CDRs 1 2 3 GGTFSSYA [SEQ ID HPIFGTA [SEQ ID ARSHVAW NO:166] NO: 167] SLLDY [SEQ ID NO: NY [SEQ ID RNN [SEQ ID NO: AAWDDSL NO: 169] 170] SGVV [SEQ ID NO: 171] Full VH EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVR QAPGQGLEWMGGIIPIFGTAKYAQKFQGRVTITADESTS TAYMELSSLRSEDTAVYYCARSHVAWSLLDYWGQGTL VTVSS [SEQ ID NO:29] DNA Gaggtccagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaagg tctcctgcaaggcttctggaggcaccttcagcagctatgctatcagctgggtgcgacagg cccctggacaagggcttgagtggatgggagggattatccctatctttggtacagcaaaata tgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcc tacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgcgctct catgttgcttggtctctgctggattactggggtcaaggtactctggtgaccgtctcctca [SEQ ID NO:31] Full VL SYELTQPPSASGTPGQRVTISCSGSSSNIGSNYVSWYQQL PGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLR SEDEADYYCAAWDDSLSGVVFGGGTKLTVLG [SEQ ID NO:30] DNA chtatgagctgactcagccaccctcagcgtctgggacccccgggcagagggtcacca tctcttgttctggaagcagctccaacatcggaagtaattatgtatcctggtaccagcagctc ccaggaacggcccccaaactcctcatctataggaataatcagcggccctcaggggtccc tgaccgattctctggctccaagtctggcacctcagcctccctggccatcagtgggctccgg gatgaggctgattattactgtgcagcatgggatgacagcctgagtggtgtggtat tc -c- -a- - - accaa_____ctacctcctat [SEQ ID NO:32] scFv SYELTQPPSASGTPGQRVTISCSGSSSNIGSNYVSWYQQL PGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLR SEDEADYYCAAWDDSLSGVVFGGGTKLTVLGSRGGGG SGGGGSGGGGSLEMAEVQLVQSGAEVKKPGSSVKVSC KASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTAKYA QKFQGRVTlTADESTSTAYMELSSLRSEDTAVYYCARSH VAWSLLDYWGQGTLVTVSS [SEQ ID NO: 107] In certain embodiments, the antibody or other n binding protein is an anti—GPRCSD scFv or an antigen—binding fragment thereof having an antigen— binding region that comprises the amino acid sequence of SEQ ID NO: 108 and specifically binds to a GPRCSD polypeptide (e. g., a GPRCSD polypeptide having the amino acid sequence SEQ ID NO:97, or fragments thereof), which is designated as ET150—159 scFv (also ed to as —9 scFv").
In certain embodiments, the anti—GPRCSD scFv antibody comprises a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:33 and a light chain variable region sing amino acids having the sequence set forth in SEQ ID NO:34, optionally with (iii) a linker sequence, for example a linker peptide, between the heavy chain variable region and the light chain variable . In certain embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In n embodiments, the PRCSD scFv antibody is an scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 9. In certain embodiments, the PRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:33, as shown in Table 9. In certain embodiments, the anti—GPRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:34, as shown in Table 9. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:33 and a VL comprising amino acids having the sequence set forth in SEQ ID NO:34, as shown in Table 9. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:172 or conservative modifications thereof, a VH CDR2 comprising amino acids having the ce set forth in SEQ ID NO:173 or conservative modifications thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:174 or conservative modifications thereof, as shown in Table 9. In certain embodiments, the anti—GPRCSD scFv comprises a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:175 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:176 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:177 or conservative modifications thereof, as shown in Table 9. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:172 or conservative modifications thereof, a VH CDR2 sing amino acids having the sequence set forth in SEQ ID NO:173 or conservative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:174 or conservative modifications thereof, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:175 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:176 or vative modifications f, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:177 or conservative modifications thereof, as shown in Table 9. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID , a VH CDR2 comprising amino acids having the ce set forth in SEQ ID , a VH CDR3 sing amino acids having the sequence set forth in SEQ ID NO:174, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:175, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:176, and a VL CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 177.
Table 9 A GPRCSD polypeptide having the amino acid sequence of SEQ ID GGTFSSYA [SEQ ID MNPNSGNT [SEQ ID ARYQSYKGSQSD No:172] NO:173] S [SEQ ID NO:174] SSNIGSNY[SEQ ID RNN [SEQ ID NO: 176] AAWDDSLSGWV No:175] [SEQ ID NO:177] Full VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGL EWMGWMNPNSGNTGYAQKFQGRVTMTRNTSISTAYMELSSLRSE DTAVYYCARYQSYKGSQSDSWGQGTLVTVSS [SEQ ID NO:33] DNA cagctggtgcagtctggggctgaggtgaagaagcctgggtcctcagtgaaggtctcctgcaag gcttctggaggcaccttcagcagctatgctatcagctgggtgcgacaggcccctggacaagggcttgagt ggatgggatggatgaaccctaacagtggtaacacaggctatgcacagaagttccagggcagagtcaccat gaacacctccataagcacagcctacatggagctgagcagcctgagatctgaggacacggccgt gtattactgtgcgcgctaccagtcttacaaaggttctcagtctgattcttggggtcaaggtactctggtgaccg tctcctca [SEQ ID NO:35] Full VL QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPK NQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAW DDSLSGWVFGGGTKLTVLG [SEQ ID NO:34] DNA Cagtctgtgttgacgcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgttctg gaagcagctccaacatcggaagtaattatgtatactggtaccagcagctcccaggaacggcccccaaact cctcatctataggaataatcagcggccctcaggggtccctgaccgattctctggctccaagtctggcacctc agcctccctggccatcagtgggctccggtccgaggatgaggctgattattactgtgcagcatgggatgaca __--cctat Htt - H-tttc -c- -a- - accaa_____ctacctcctat [SEQ ID NO:36] scFv QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPK LLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAW DDSLSGWVFGGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAQVQL VQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWM GWMNPNSGNTGYAQKFQGRVTMTRNTSISTAYMELSSLRSEDTA VYYCARYQSYKGSQSDSWGQGTLVTVSS [SEQ ID NO:108] In certain embodiments, the antibody or other antigen binding protein is an anti—GPRCSD scFv or an antigen—binding fragment f having an antigen— binding region that comprises the amino acid sequence of SEQ ID NO: 109 and specifically binds to a GPRCSD polypeptide (e.g., a GPRCSD polypeptide having the amino acid sequence SEQ ID NO:97, or fragments thereof), which is designated as as l60 scFv (also referred to as —lO .
In certain embodiments, the anti—GPRCSD scFv antibody comprises a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:37 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:38, optionally with (iii) a linker sequence, for example a linker peptide, between the heavy chain le region and the light chain variable region. In certain embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the anti—GPRCSD scFv antibody is an scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 10. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:37, as shown in Table 10. In certain embodiments, the anti—GPRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:38, as shown in Table 10. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the ce set forth in SEQ ID NO:37 and a VL sing amino acids having the sequence set forth in SEQ ID NO:38, as shown in Table 10. In n embodiments, the anti—GPRCSD scFv ses a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:l78 or vative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:l79 or conservative cations thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:lSO or conservative cations f, as shown in Table 10. In certain embodiments, the PRCSD scFv comprises a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:lSl or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:182 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:lS3 or conservative modifications thereof, as shown in Table 10. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:l78 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:l79 or conservative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:lSO or conservative modifications thereof, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:lSl or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:182 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:lS3 or conservative modifications thereof, as shown in Table 10. In certain embodiments, the anti—GPRCSD scFV comprises a VH CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 178, a VH CDR2 sing amino acids haVing the sequence set forth in SEQ ID NO: 179, a VH CDR3 comprising amino acids haVing the sequence set forth in SEQ ID NO:180, a VL CDR1 comprising amino acids haVing the sequence set forth in SEQ ID NO: 181, a VL CDR2 comprising amino acids haVing the sequence set forth in SEQ ID NO:182, and a VL CDR3 comprising amino acids haVing the ce set forth in SEQ ID NO: 183.
Table 10 Antigen A GPRCSD polypeptide haVing the amino acid sequence of SEQ ID NO:97 CDRs 1 2 3 VH GYTFTSYY[SEQ ID INPSGGST [SEQ ID ARGGSKKWSGEKW NO:178] NO:179] RRENFDY [SEQ ID NO: 180] VL SSDVGGYNY [SEQ DVS [SEQ ID NO:182] SSYTRSSTEV [SEQ 81] IDNO:183] Full VH EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQG LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED TAVYYCARGGSKKWSGEKWRRENFDYWGQGTLVTVSS [SEQ ID NO:37] DNA Gaggtccagctggtacagtctggggctgaggtgaagaagcctggggcctcagtgaaggtttcctgcaagg gatacaccttcaccagctactatatgcactgggtgcgacaggcccctggacaagggcttgagtgga tgggaataatcaaccctagtggtggtagcacaagctacgcacagaagttccagggcagagtcaccatgacc agggacacgtccacgagcacagtctacatggagctgagcagcctgagatctgaggacacggccgtgtatt actgtgcgcgcggtggttctaaaaaatggtctggtgaaaaatggcgtcgtgaaaacttcgattactggggtca aggtactctggtgaccgtctcctca [SEQ ID NO:39] Full VL QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAP KLMIYDVSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSY TRSSTEVFGGGTKLTVLG [SEQ ID NO:38] DNA Caatctgccctgactcagcctgcctccgtgtctgggtctcctggacagtcgatcaccatctcctgcactggaa ccagcagtgacgttggtggttataactatgtctcctggtaccaacagcacccaggcaaagcccccaaactca tgatttatgatgtcagtaagcggccctcaggggtttctaatcgcttctctggctccaagtctggcaacacggcc tccctgaccatctctgggctccaggctgaggacgaggctgattattactgcagctcatatacaagaagcagc actgaggtattcggcggagggaccaagctgaccgtcctaggt [SEQ ID NO:40] scFV QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAP KLMIYDVSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSY TRSSTEVFGGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAEVQLVQ SGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGI INPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYC ARGGSKKWSGEKWRRENFDYWGQGTLVTVSS [SEQ ID NO: 109] In certain embodiments, the antibody or other antigen g protein is an anti—GPRCSD scFv or an antigen—binding nt thereof having an antigen— binding region that ses the amino acid sequence of SEQ ID NO: 110 and specifically binds to a GPRCSD polypeptide (e.g., a GPRCSD polypeptide having the amino acid sequence SEQ ID NO:97, or fragments thereof), which is ated as ET150—161 scFv (also referred to as "ET150—11 scFv").
In certain embodiments, the anti—GPRCSD scFv antibody comprises a heavy chain variable region sing amino acids having the sequence set forth in SEQ ID NO:4l and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:42, optionally with (iii) a linker sequence, for example a linker peptide, between the heavy chain variable region and the light chain variable region. In certain embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the PRCSD scFv dy is an scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 11. In certain ments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:4l, as shown in Table 11. In certain embodiments, the anti—GPRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:42, as shown in Table 11. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:4l and a VL comprising amino acids having the ce set forth in SEQ ID NO:42, as shown in Table 11. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:184 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 185 or conservative modifications thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 186 or conservative modifications f, as shown in Table 11. In certain embodiments, the anti—GPRCSD scFv comprises a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 187 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 188 or vative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 189 or conservative modifications thereof, as shown in Table 11. In certain embodiments, the anti—GPRCSD scFv ses a VH CDRl sing amino acids having the sequence set forth in SEQ ID NO: 184 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 185 or conservative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 186 or conservative modifications thereof, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 187 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 188 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 189 or conservative modifications thereof, as shown in Table 11. In certain ments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 184, a VH CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 185, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 186, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 187, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 188, and a VL CDR3 sing amino acids having the ce set forth in SEQ ID NO: 189.
Table 11 Antigen A GPRCSD polypeptide having the amino acid sequence of SEQ ID NO:97 NO: 184] NO: 186] SSNIGSNT [SEQ ID RNN [SEQ ID NO: 188] AAWDDSLSGL NO: 187] [SEQ ID NO:l89] QMQLVQSGAEVKKPGASVKVSCKASEYTFTRHILHWVRQAPGQSL EWMGWINPGNGNTKYSQKFQVRVTFTRDTSASTVYMELSSLRSED TAVYYCARLPDQWGQGTLVTVSS [SEQ ID NO:41] DNA Cagatgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtttcctgcaagg cttctgaatacaccttcactaggcatattctacattgggtgcgccaggctcccggacaaagccttgagtggat gggatggatcaacccaggcaatggtaatacaaaatattcacagaagttccaggtcagagtcacctttaccag ggacacatccgcgagcacagtctatatggagctgagcagcctgagatctgaagacacggccgtgtattact mtcc__cctcc ; at ; ; tcaa; tactct; tacc _tctcctca [SEQ ID NO:43] Full VL SYVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKL LIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWD DSLSGLFGTGTKVTVLG [SEQ ID NO:42] DNA chtatgtgctgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgttctgga agcagctccaacatcggaagtaatactgtaaactggtaccagcagctcccaggaacggcccccaaactcct catctataggaataatcagcggccctcaggggtccctgaccgattctctggctccaagtctggcacctcagc ctccctggccatcagtgggctccggtccgaggatgaggctgattattactgtgcagcatgggatgacagcct _a - t - - tctcttc ; _aact; ; _accaa; _tcacc _tccta; _t [SEQ ID NO:44] scFv SYVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKL LIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWD DSLSGLFGTGTKVTVLGSRGGGGSGGGGSGGGGSLEMAQMQLVQ SGAEVKKPGASVKVSCKASEYTFTRHILHWVRQAPGQSLEWMGWI NPGNGNTKYSQKFQVRVTFTRDTSASTVYMELSSLRSEDTAVYYC ARLPDQWGQGTLVTVSS [SEQ ID NO: l 10] In certain embodiments, the antibody or other antigen binding protein is an anti—GPRCSD scFv or an n—binding nt thereof having an antigen— binding region that ses the amino acid sequence of SEQ ID NO: 111 and specifically binds to a GPRCSD polypeptide (e.g., a GPRCSD polypeptide having the amino acid sequence SEQ ID NO:97, or fragments thereof), which is designated as ETlSO—l62 scFv (also referred to as "ET150—12 scFv").
In certain embodiments, the anti—GPRCSD scFv antibody comprises a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:45 and a light chain le region comprising amino acids having the sequence set forth in SEQ ID NO:46, ally with (iii) a linker sequence, for example a linker peptide, between the heavy chain variable region and the light chain variable region. In certain embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the anti—GPRCSD scFv antibody is an c fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 12. In certain embodiments, the PRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:45, as shown in Table 12. In certain embodiments, the anti—GPRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:46, as shown in Table 12. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:45 and a VL comprising amino acids having the sequence set forth in SEQ ID NO:46, as shown in Table 12. In certain embodiments, the anti—GPRCSD scFv ses a VH CDRl comprising amino acids having the ce set forth in SEQ ID NO:l90 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 191 or conservative modifications f, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 192 or conservative modifications thereof, as shown in Table 12. In certain embodiments, the anti—GPRCSD scFv comprises a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 193 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 194 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 195 or vative modifications thereof, as shown in Table 12. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 190 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 191 or vative cations thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 192 or conservative modifications thereof, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 193 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 194 or conservative cations thereof, and a VL CDR3 sing amino acids having the ce set forth in SEQ ID NO: 195 or conservative modifications thereof, as shown in Table 12. In certain ments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 190, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 191, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 192, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 193, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 194, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 195.
Table 12 n A GPRCSD polypeptide having the amino acid sequence of SEQ ID NO:97 GFTFGDYG [SEQ INWNGGST [SEQ ID ARSKQDY [SEQ ID ID NO: 190] NO: 191] NO: 192] SRDAGGYNY [SEQ EVT [SEQ ID NO: 194] SSYGGSNNFRV ID NO: 193] [SEQ ID NO:l95] Full VH EVQLVESGGGVVRPGGSLRLSCAASGFTFGDYGMSWVRQAPGKG LEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARSKQDYWGQGTLVTVSS [SEQ ID NO:45] DNA Gaggtgcagctggtggagtctgggggaggtgtggtacggcctggggggtccctgagactctcctgtgca gcctctggattcacctttggtgattatggcatgagctgggtccgccaagctccagggaaggggctggagtg ggtctctggtattaattggaatggtggtagcacaggttatgcagactctgtgaagggccgattcaccatctcc aacgccaagaactccctgtatctgcaaatgaacagtctgagagccgaggacacggccgtatatt actgtgcgcgctctaaacaggattactggggtcaaggtactctggtgaccgtctcctca [SEQ ID NO:47] Full VL QSALTQPPSASGSPGQSVTISCTGTSRDAGGYNYFSWYQQHPGKA PKLLIYEVTKRPSGVPDRFSGSKSGKTASLTVSGLQADDEAVYYCS SYGGSNNFRVFGGGTKLTVLG [SEQ ID NO:46] DNA Cagtctgccctgactcagcctccctccgcgtccgggtctcctggacagtcagtcaccatctcctgcactgg aaccagcagggacgctggtggttataattatttctcctggtaccaacaacacccaggcaaagcccccaaac tcctgatttatgaggtcactaagcggccctcaggggtccctgatcgcttctctggctccaagtctggcaaga ccctgaccgtctctgggctccaggctgacgatgaggctgtatattactgcagctcatatggaggc agcaacaactttcgggtgttcggcggagggaccaagctgaccgtcctaggt [SEQ ID NO:48] scFV QSALTQPPSASGSPGQSVTISCTGTSRDAGGYNYFSWYQQHPGKA PKLLIYEVTKRPSGVPDRFSGSKSGKTASLTVSGLQADDEAVYYCS SYGGSNNFRVFGGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAEV QLVESGGGVVRPGGSLRLSCAASGFTFGDYGMSWVRQAPGKGLE WVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARSKQDYWGQGTLVTVSS [SEQ ID NO:111] In certain embodiments, the antibody or other antigen binding protein is an anti—GPRCSD scFV or an antigen—binding fragment thereof haVing an antigen— binding region that comprises the amino acid sequence of SEQ ID NO: 112 and specifically binds to a GPRCSD polypeptide (e.g., a GPRCSD polypeptide haVing the amino acid ce SEQ ID NO:97, or nts thereof), which is designated as ET150—163 scFV (also referred to as "ET150—13 scFV").
In n embodiments, the anti—GPRCSD scFv antibody comprises a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:49 and a light chain variable region comprising amino acids having the ce set forth in SEQ ID NO:50, optionally with (iii) a linker sequence, for example a linker peptide, between the heavy chain variable region and the light chain le region. In certain embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the anti—GPRCSD scFv antibody is an c fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 13. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:49, as shown in Table 13. In certain embodiments, the anti—GPRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:50, as shown in Table 13. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the ce set forth in SEQ ID NO:49 and a VL comprising amino acids having the ce set forth in SEQ ID NO:50, as shown in Table 13. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:l96 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 197 or conservative modifications thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 198 or conservative modifications thereof, as shown in Table 13. In certain embodiments, the PRCSD scFv comprises a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 199 or conservative modifications thereof, a VL CDR2 sing amino acids having the sequence set forth in SEQ ID NO:200 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:201 or conservative cations thereof, as shown in Table 13. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 196 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 197 or conservative modifications thereof, a VH CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 198 or vative modifications thereof, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 199 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:200 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the ce set forth in SEQ ID NO:201 or conservative modifications thereof, as shown in Table 13. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRI comprising amino acids having the ce set forth in SEQ ID NO: 196, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 197, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 198, a VL CDRI comprising amino acids having the sequence set forth in SEQ ID NO: 199, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:200, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:201.
Table 13 GFSFSGTA[SEQ ID ISSTGRST [SEQ ID NO: ARVSFDY [SEQ ID NO: 196] NO: 198] SSNIGAGYD [SEQ ID GNS [SEQ ID NO:200] QSYDSSLSGSYV NO: 199] [SEQ ID NO:201] EVQLVETGGNLVQPGASLRLSCAASGFSFSGTAMHWVRQAPGKGLE WVSTISSTGRSTYYRDSVKGRFTISRDNSKNTLYLQMNSLRGEDTAV YYCARVSFDYWGQGTLVTVSS [SEQ ID NO:49] Gaggtgcagctggtggagactgggggaaacttggtacagccgggggcgtccctgagactctcctgtgcagc ctctggattcagctttagtggcactgccatgcactgggtccgccaggctccagggaaggggctggaatgggtc tcgactattagtagtactgggcgtagcacatactacagagactccgtgaagggccggttcaccatctccagaga caattccaagaacacgctgtatctgcaaatgaacagcctgagaggcgaggacacggccgtatattactgtgcg cgcgtttctttcgattactggggtcaaggtactctggtgaccgtctcctca [SEQ ID NO:51] QSVVTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPK LLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDS SLSGSYVFGTGTKLTVLG [SEQ ID NO:50] Cagtctgtcgtgacgcagccgccctcagtgtctggggccccagggcagagggtcaccatctcctgcactggg tccaacatcggggcaggttatgatgtacactggtaccagcagcttccaggaacagcccccaaactcc tcatctatggtaacagcaatcggccctcaggggtccctgaccgattctctggctccaagtctggcacctcagcct ccctggccatcactgggctccaggctgaggatgaggctgattattactgccagtcctatgacagcagcctgagt ggctcctacgtcttcggaactgggaccaagctgaccgtcctaggt [SEQ ID NO:52] QSVVTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPK LLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDS SLSGSYVFGTGTKLTVLGSRGGGGSGGGGSGGGGSLEMAEVQLVET PGASLRLSCAASGFSFSGTAMHWVRQAPGKGLEWVSTISST RDSVKGRFTISRDNSKNTLYLQMNSLRGEDTAVYYCARVSF DYWGQGTLVTVSS [SEQ ID NO:112] In certain embodiments, the antibody or other antigen binding protein is an anti—GPRCSD scFv or an antigen—binding fragment thereof having an antigen— binding region that ses the amino acid sequence of SEQ ID NO: 113 and specifically binds to a GPRCSD polypeptide (e.g., a GPRCSD polypeptide having the amino acid ce SEQ ID NO:97, or fragments thereof), which is designated as ET150—151 scFv (also referred to as "ET150—1 scFv").
In n embodiments, the anti—GPRCSD scFv antibody comprises a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:53 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:54, ally with (iii) a linker sequence, for example a linker peptide, between the heavy chain variable region and the light chain variable region. In certain embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the anti—GPRCSD scFv antibody is an scFv—Fc fusion protein or full length human IgG with VH and VL s or CDRs selected from Table 14. In n embodiments, the anti—GPRCSD scFv ses a VH comprising amino acids having the sequence set forth in SEQ ID NO:53, as shown in Table 14. In certain embodiments, the anti—GPRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:54, as shown in Table 14. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:53 and a VL comprising amino acids having the sequence set forth in SEQ ID NO:54, as shown in Table 14. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:202 or conservative modifications thereof, a VH CDR2 sing amino acids having the sequence set forth in SEQ ID NO:203 or conservative modifications thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 204 or conservative modifications thereof, as shown in Table 14. In certain embodiments, the anti—GPRCSD scFv comprises a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 205 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 206 or conservative modifications thereof, and a VL CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 207 or conservative modifications thereof, as shown in Table 14. In certain embodiments, the anti—GPRC5D scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 202 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 203 or conservative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 204 or conservative modifications f, a VL CDRl comprising amino acids having the ce set forth in SEQ ID NO: 205 or conservative cations thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 206 or vative modifications f, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 207 or conservative modifications thereof, as shown in Table 14. In certain embodiments, the anti—GPRC5D scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 202, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 203, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 204, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 205, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 206, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 207.
Table 14 Antigen A GPRC5D polypeptide having the amino acid sequence of SEQ ID NO:97 GFTFSSYA [SEQ ID ISGRGRST [SEQ ID GAFDL NO: 202] NO: 203] [SEQ ID NO: 204] SSDVGGYNY [SEQ ID DVS [SEQ ID NO: SSYTSSSTLV [SEQ NO: 205] 206] ID NO: 207] EVQLVESGGAFVQPGGSLRLSCAASGFTFSSYAMTWVRQAPGKGL EWVSTISGRGRSTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT AVYYCARYYHAGAFDLWGQGTLVTVSS [SEQ ID NO:53] DNA Gaggtgcagctggtggagtctgggggagcctttgtacagcctggggggtccctgagactctcctgtgcag gattcacctttagcagctatgccatgacctgggtccgccaggctccagggaagggcctggaatg ggtctcgactattagtggtcgtggtcgtagcacattctacgcagactccgtgaagggccggtttaccatctcc agagacaattccaagaacacgctatatctgcaaatgaacagtctgagagccgaggacacggccgtatatt actgtgcgcgctactaccatgctggtgctttcgatctgtggggtcaaggtactctggtgaccgtctcctca [SEQ ID NO:55] Full VL QSVVTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKA PKLMIYDVSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCS SYTSSSTLVFGGGTKLTVLG [SEQ ID NO:54] DNA Cagtctgtcgtgacgcagcctgcctccgtgtctgggtctcctggacagtcgatcaccatctcctgcactgg aaccagcagtgacgttggtggttataactatgtctcctggtaccaacagcacccaggcaaagcccccaaac tcatgatttatgatgtcagtaagcggccctcaggggtttctaatcgcttctctggctccaagtctggcaacac ggcctccctgaccatctctgggctccaggctgaggacgaggctgattattactgcagctcatatacaagca _____cacacttttattc -c- -a- - - accaactacctcctat [SEQ ID NO:56] scFv QSVVTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKA PKLMIYDVSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCS SYTSSSTLVFGGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAEVQL FVQPGGSLRLSCAASGFTFSSYAMTWVRQAPGKGLEWV STISGRGRSTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY YCARYYHAGAFDLWGQGTLVTVSS [SEQ ID NO: 1 l3] In certain embodiments, the antibody or other antigen binding protein is an anti—GPRCSD scFv or an antigen—binding fragment thereof having an antigen— binding region that comprises the amino acid sequence of SEQ ID NO: 114 and specifically binds to a GPRCSD polypeptide (e.g., a GPRCSD polypeptide having the amino acid sequence SEQ ID NO:97, or fragments thereof), which is designated as ET150—152 scFv (also referred to as "ET150—2 .
In certain embodiments, the anti—GPRCSD scFv antibody comprises a heavy chain le region comprising amino acids having the sequence set forth in SEQ ID NO:57 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:58, ally with (iii) a linker sequence, for e a linker peptide, between the heavy chain variable region and the light chain variable region. In certain embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain ments, the anti—GPRCSD scFv antibody is an scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 15. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:57, as shown in Table 15. In certain embodiments, the PRC5D scFv comprises a VL sing amino acids having the sequence set forth in SEQ ID NO:58, as shown in Table 15. In certain embodiments, the anti—GPRC5D scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:57 and a VL comprising amino acids having the sequence set forth in SEQ ID NO:58, as shown in Table 15. In certain embodiments, the anti—GPRC5D scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:208 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 209 or conservative modifications thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 210 or conservative modifications f, as shown in Table 15. In certain embodiments, the anti—GPRC5D scFv ses a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 211 or conservative modifications f, a VL CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 212 or vative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 213 or conservative modifications thereof, as shown in Table 15. In certain embodiments, the anti—GPRC5D scFv comprises a VH CDRl comprising amino acids having the ce set forth in SEQ ID NO: 208 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 209 or conservative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 210 or conservative cations thereof, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 211 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 212 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 213 or conservative modifications thereof, as shown in Table 15. In certain embodiments, the anti—GPRC5D scFv comprises a VH CDRl comprising amino acids having the ce set forth in SEQ ID NO: 208, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 209, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 210, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 211, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 212, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 213.
Table 15 Antigen A GPRCSD polypeptide having the amino acid sequence of SEQ ID NO:97 GYTFNRYA [SEQ ISAYNGNS [SEQ ID ARMAYDS [SEQ ID ID NO: 208] NO: 209] NO: 210] SNDVGAYKY [SEQ DVF [SEQ ID NO: 212] FSLTSSNTYV [SEQ IDNO: 211] IDNO: 213] Full VH QMQLVQSGAEVKKPGASVKVSCKASGYTFNRYAITWVRQAPGQG LEWMGWISAYNGNSHYAQKLQGRVTMTTDTSTGTAYMELRRLRS DDTAVYYCARMAYDSWGQGTLVTVSS [SEQ ID NO:57] DNA Cagatgcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaagg cttctggttacacctttaacagatatgctatcacctgggtgcgacaggcccctggacaaggccttgagtggat gggatggatcagcgcttacaatggtaattcacactatgcacagaagctccagggcagagtcaccatgacca cagacacatccacgggcacagcctatatggagctgaggaggctgagatctgacgacacggccgtgtatta gcgcatggcttacgattcttggggtcaaggtactctggtgaccgtctcctca [SEQ ID NO:59] Full VL PASVSGSPGQSLTISCTGTSNDVGAYKYVSWYQQYPGKAP KLILYDVFKRPSGVSNRFSGSKSDNTASLTISGLQAEDEADYYCFSL TSSNTYVFGTGTKVTVLG [SEQ ID NO:58] DNA Cagtctgtgttgacgcagcctgcctccgtgtctgggtctcctggacagtcgctcaccatctcctgcactggaa ccagcaatgacgttggtgcttataagtatgtctcctggtatcaacagtacccaggcaaagcccccaaactcat actttatgatgtctttaagcggccctcaggggtctctaatcgcttctctggctccaagtctgacaacacggcctc cctgaccatctctgggctccaggctgaggacgaggctgattattactgcttctcacttacaagcagtaacactt at_tcttc ; __aact ; ; _tcacc _tccta; _t [SEQ ID NO:60] scFV QSVLTQPASVSGSPGQSLTISCTGTSNDVGAYKYVSWYQQYPGKAP KLILYDVFKRPSGVSNRFSGSKSDNTASLTISGLQAEDEADYYCFSL TSSNTYVFGTGTKVTVLGSRGGGGSGGGGSGGGGSLEMAQMQLV QSGAEVKKPGASVKVSCKASGYTFNRYAITWVRQAPGQGLEWMG WISAYNGNSHYAQKLQGRVTMTTDTSTGTAYMELRRLRSDDTAV YYCARMAYDSWGQGTLVTVSS [SEQ ID NO:114] In certain ments, the antibody or other antigen binding protein is an anti—GPRCSD scFV or an antigen—binding fragment thereof haVing an antigen— binding region that comprises the amino acid sequence of SEQ ID NO: 115 and specifically binds to a GPRCSD polypeptide (e.g., a GPRCSD polypeptide haVing the amino acid sequence SEQ ID NO:97, or fragments thereof), which is designated as ET150—155 scFv (also referred to as "ET150—5 scFv").
In certain embodiments, the anti—GPRCSD scFv antibody comprises a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:6l and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:62, optionally with (iii) a linker sequence, for e a linker peptide, n the heavy chain variable region and the light chain variable region. In certain embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the anti—GPRCSD scFv antibody is an scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 16. In certain ments, the anti—GPRCSD scFv ses a VH comprising amino acids having the sequence set forth in SEQ ID NO:6l, as shown in Table 16. In certain embodiments, the anti—GPRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:62, as shown in Table 16. In certain embodiments, the PRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:6l and a VL comprising amino acids having the sequence set forth in SEQ ID NO:62, as shown in Table 16. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:214 or conservative cations thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 215 or conservative modifications thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 216 or vative modifications thereof, as shown in Table 16. In certain embodiments, the anti—GPRCSD scFv comprises a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 217 or vative cations thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 218 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 219 or conservative modifications thereof, as shown in Table 16. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 214 or vative modifications thereof, a VH CDR2 sing amino acids having the sequence set forth in SEQ ID NO: 215 or conservative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 216 or conservative modifications thereof, a VL CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 217 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 218 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 219 or conservative modifications thereof, as shown in Table 16. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 214, a VH CDR2 sing amino acids having the sequence set forth in SEQ ID NO: 215, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 216, a VL CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 217, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 218, and a VL CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 219.
Table 16 Antigen A GPRCSD polypeptide having the amino acid sequence of SEQ ID NO:97 GFTFSDYY [SEQ ID ISSSGSTI [SEQ ID NO: ARGYGKAYDQ NO: 214] [SEQ ID NO: 216] RSNVGGNY [SEQ RSN [SEQ ID NO: 218] ATWDDSLSGFV ID NO: 217] [SEQ ID NO: 219] Full VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGL EWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDT RGYGKAYDQWGQGTLVTVSS [SEQ ID NO:61] DNA Gaggtgcagctggtggagtctgggggaggcttggtcaagcctggagggtccctgagactctcctgtgca ggattcaccttcagtgactactacatgagctggatccgccaggctccagggaaggggctggagt gggtttcatacattagtagtagtggtagtaccatatactacgcagactctgtgaagggccgattcaccatctc cagggacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggccgtata ttactgtgcgcgcggttacggtaaagcttacgatcagtggggtcaaggtactctggtgaccgtctcctca [SEQ ID NO:63] Full VL QSVLTQPPSASGTPGQRVTISCSGSRSNVGGNYVFWYQQVPGATP KLLIYRSNQRPSGVPDRFAGSKSGSSASLAISGLRSEDEADYYCAT WDDSLSGFVFGTGTKVTVLG [SEQ ID NO:62] DNA Cagtctgtgttgactcagccaccctcagcgtctgggacccccggacagagggtcaccatctcttgttctgg aagcaggtccaacgtaggaggtaattatgtattttggtaccagcaagtccccggagcgacccccaaactcc tcatctata;ataatcac ; _ccctc ; ; ; ; cc _attc ct; tct; _ctcctca gcctccctggccatcagtggactccggtccgaggatgaggctgattattactgtgcaacatgggatgacag cct at tttt tcttc aact accaa tcacc tccta t[SEQ ID NO:64] scFv QSVLTQPPSASGTPGQRVTISCSGSRSNVGGNYVFWYQQVPGATP KLLIYRSNQRPSGVPDRFAGSKSGSSASLAISGLRSEDEADYYCAT WDDSLSGFVFGTGTKVTVLGSRGGGGSGGGGSGGGGSLEMAEVQ LVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWV GSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY YCARGYGKAYDQWGQGTLVTVSS [SEQ ID ] In certain embodiments, the dy or other antigen binding protein is an anti—GPRCSD scFv or an antigen—binding fragment thereof having an antigen— binding region that comprises the amino acid sequence of SEQ ID NO: 116 and specifically binds to a GPRCSD polypeptide (e.g., a GPRCSD polypeptide having the amino acid sequence SEQ ID NO:97, or fragments thereof), which is designated as ET150—158 scFv (also referred to as "ET150—8 scFv").
In certain embodiments, the anti—GPRCSD scFv antibody comprises a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:65 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:66, optionally with (iii) a linker sequence, for example a linker peptide, between the heavy chain variable region and the light chain variable region. In certain embodiments, the linker comprises amino acids having the ce set forth in SEQ ID NO:98. In n embodiments, the anti—GPRCSD scFv antibody is an scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 17. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:65, as shown in Table 17. In certain embodiments, the anti—GPRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:66, as shown in Table 17. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:65 and a VL comprising amino acids having the ce set forth in SEQ ID NO:66, as shown in Table 17. In certain ments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:220 or conservative modifications f, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 221 or conservative modifications thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 222 or conservative modifications thereof, as shown in Table 17. In n embodiments, the anti—GPRC5D scFv comprises a VL CDRl comprising amino acids having the ce set forth in SEQ ID NO: 223 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 224 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 225 or conservative modifications thereof, as shown in Table 17. In certain embodiments, the anti—GPRC5D scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 220 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 221 or conservative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 222 or conservative modifications thereof, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 223 or vative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 224 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 225 or conservative modifications thereof, as shown in Table 17. In certain ments, the PRC5D scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 220, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 221, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 222, a VL CDRl comprising amino acids having the ce set forth in SEQ ID NO: 223, a VL CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 224, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 225.
Table 17 A GPRC5D polypeptide having the amino acid sequence of SEQ ID NO:97 VH-———GFTFRSHS[SEQID ISSDSTYT [SEQ ID ARSGGQWKYYDY NO: 220] NO: 221] [SEQ ID NO: 222] SLRSYY [SEQ ID GKN [SEQ ID NO: 224] NSRDSSGNPPVV SGGGLVHPGGSLRLSCAASGFTFRSHSMNWVRQAPGKGL EWVSSISSDSTYTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTA VYYCARSGGQWKYYDYWGQGTLVTVSS [SEQ ID NO:65] Caggtgcagctggtggagtctgggggaggcctggtccaccctggggggtccctgagactctcctgtgcagc ctctggattcaccttcagaagccatagcatgaactgggtccgccaggctccagggaaggggctggagtggg tctcatccattagtagtgatagtacttacacatactacgcagactcagtgaagggccgattcaccatctccagag acaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggccgtatattactgtg cgcgctctggtggtcagtggaaatactacgattactggggtcaaggtactctggtgaccgtctcctca [SEQ ID NO:67] SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLV IYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSG NPPVVFGGGTKLTVLG [SEQ ID NO:66] Tcttctgagctgactcaggaccctgctgtgtctgtggccttgggacagacagtcaggatcacatgccaagga gacagcctcagaagctattatgcaagctggtaccagcagaagccaggacaggcccctgtacttgtcatctatg gtaaaaacaaccggccctcagggatcccagaccgattctctggctccagctcaggaaacacagcttccttga ccatcactggggctcaggcggaagatgaggctgactattactgtaactcccgggacagcagtggtaaccccc ctgtggtattcggcggagggaccaagctgaccgtcctaggt [SEQ ID NO:68] DPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLV IYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSG NPPVVFGGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAQVQLVESG GGLVHPGGSLRLSCAASGFTFRSHSMNWVRQAPGKGLEWVSSISSD STYTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSG GQWKYYDYWGQGTLVTVSS [SEQ ID NO: 1 l6] In certain embodiments, the antibody or other antigen binding protein is an anti—GPRCSD scFv or an antigen—binding nt thereof having an antigen— binding region that comprises the amino acid sequence of SEQ ID NO: 117 and ically binds to a GPRCSD polypeptide (e.g., a GPRCSD polypeptide having the amino acid sequence SEQ ID NO:97, or fragments thereof), which is designated as l68 scFv (also ed to as "ETlSO—lS scFv").
In certain embodiments, the anti—GPRCSD scFv antibody comprises a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:69 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:70, optionally with (iii) a linker sequence, for example a linker e, between the heavy chain variable region and the light chain variable region. In certain embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the anti—GPRCSD scFv antibody is an scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 18. In certain embodiments, the anti—GPRCSD scFv comprises a VH sing amino acids having the sequence set forth in SEQ ID NO:69, as shown in Table 18. In certain embodiments, the anti—GPRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:70, as shown in Table 18. In certain embodiments, the anti—GPRCSD scFv comprises a VH sing amino acids having the sequence set forth in SEQ ID NO:69 and a VL comprising amino acids having the sequence set forth in SEQ ID NO:70, as shown in Table 18. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:226 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 227 or conservative modifications thereof, and a VH CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 228 or conservative modifications thereof, as shown in Table 18. In certain embodiments, the anti—GPRCSD scFv comprises a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 229 or vative modifications f, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 230 or conservative modifications thereof, and a VL CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 231 or conservative modifications thereof, as shown in Table 18. In n embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 226 or conservative modifications thereof, a VH CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 227 or conservative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 228 or conservative modifications thereof, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 229 or conservative cations f, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 230 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 231 or conservative modifications thereof, as shown in Table 18. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 226, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 227, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 228, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 229, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 230, and a VL CDR3 comprising amino acids haVing the sequence set forth in SEQ ID NO: 231.
Table 18 Antigen A GPRCSD polypeptide haVing the amino acid sequence of SEQ ID NO:97 GFTFSNYA [SEQ ID INGRGSST [SEQ ID NO: ARYISRGLGDS NO: 226] [SEQ ID NO: 228] NSNIERNY[SEQ ID DND [SEQ ID NO: 230] GTWDSSLRGWV NO: 229] [SEQ ID NO: 231] Full VH EVQLVESGGGLIQPGGSLRLSCAASGFTFSNYAMNWVRQAPGKGL EWVSTINGRGSSTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT RYISRGLGDSWGQGTLVTV [SEQ ID NO:69] DNA Gaggtgcagctggtggagtccgggggaggcttgatacagcctggggggtccctgagactctcctgtgca gcctctggattcacctttagcaactatgccatgaactgggtccgccaggctccagggaaggggctggagt gggtctcaactattaatggtcgtggtagtagtacaatctacgcagactccgtgaagggccggttcaccatct ccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacagccacgt attactgtgcgcgctacatctctcgtggtctgggtgattcttggggtcaaggtactctggtgaccgtctcctca [SEQ ID NO:71] Full VL QSVVTQPPSMSAAPGQQVTISCSGGNSNIERNYVSWYLQLPGTAP KLVIFDNDRRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGT WDSSLRGWVFGGGTKLTVLG [SEQ ID NO:70] DNA Cagtctgtcgtgacgcagccgccctcaatgtctgcggccccaggacagcaagtcaccatctcctgctctg gaggcaactccaacattgagagaaattatgtatcctggtacctccagctccctggaacagcccccaaactc gtcatttttgacaatgataggcgaccctcagggattcctgaccgattctctggctccaagtctggcacgtcag ccaccctgggcatcaccggactccagactggggacgaggccgattattactgcggaacatgggatagca taa Htt - H-tttc -c- -a- - accaa_____ctacctcctat [SEQ ID NO:72] scFV QSVVTQPPSMSAAPGQQVTISCSGGNSNIERNYVSWYLQLPGTAP KLVIFDNDRRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGT GWVFGGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAEV QLVESGGGLIQPGGSLRLSCAASGFTFSNYAMNWVRQAPGKGLE WVSTINGRGSSTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA TYYCARYISRGLGDSWGQGTLVTV [SEQ ID NO:117] In certain embodiments, the antibody or other antigen binding protein is an anti—GPRCSD scFv or an antigen—binding fragment thereof having an antigen— binding region that comprises the amino acid sequence of SEQ ID NO: 118 and specifically binds to a GPRCSD polypeptide (e.g., a GPRCSD ptide having the amino acid sequence SEQ ID NO:97, or nts thereof), which is designated as 164 scFv (also referred to as "ET150—14 scFv").
In certain embodiments, the anti—GPRCSD scFv antibody comprises a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:73 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:74, optionally with (iii) a linker sequence, for example a linker peptide, between the heavy chain variable region and the light chain variable region. In certain embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the anti—GPRCSD scFv antibody is an scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 19. In certain ments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the ce set forth in SEQ ID NO:73, as shown in Table 19. In certain embodiments, the anti—GPRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:74, as shown in Table 19. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:73 and a VL comprising amino acids having the sequence set forth in SEQ ID NO:74, as shown in Table 19. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:232 or conservative cations thereof, a VH CDR2 sing amino acids having the sequence set forth in SEQ ID NO: 233 or conservative modifications thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 234 or conservative modifications thereof, as shown in Table 19. In certain embodiments, the anti—GPRCSD scFv comprises a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 235 or conservative modifications thereof, a VL CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 236 or conservative cations thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 237 or conservative modifications thereof, as shown in Table 19. In n embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 232 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 233 or conservative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 234 or conservative modifications thereof, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 235 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 236 or conservative modifications thereof, and a VL CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 237 or conservative modifications thereof, as shown in Table 19. In certain embodiments, the anti—GPRC5D scFv ses a VH CDRl sing amino acids having the sequence set forth in SEQ ID NO: 232, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 233, a VH CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 234, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 235, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 236, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 237.
Table 19 Antigen A GPRC5D polypeptide having the amino acid ce of SEQ ID NO:97 YY [SEQ ID INPSGGST [SEQ ID ARAGMGMDT NO: 232] NO: 233 [SEQ ID NO: 234] SSDVGGYNY [SEQ EVS [SEQ ID NO: 236] SSYAGSNTLV ID NO: 235] [SEQ ID NO: 237] Full VH QMQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQ GLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSE DTAVYYCARAGMGMDTWGQGTLVTVSS [SEQ ID NO:73] DNA Cagatgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtttcctgcaag gcatctggatacaccttcaccagctactatatgcactgggtgcgacaggcccctggacaagggcttgagtg gatgggaataatcaaccctagtggtggtagcacaagctacgcacagaagttccagggcagagtcaccatg accagggacacgtccacgagcacagtctacatggagctgagcagcctgagatctgaggacacggccgt gtattactgtgcgcgcgctggtatgggtatggatacttggggtcaaggtactctggtgaccgtctcctca [SEQ ID NO:75] Full VL QSALTQPPSASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKA PKLMIYEVSKRPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCS SYAGSNTLVFGGGTKLTVLG [SEQ ID NO:74] DNA Cagtctgccctgactcagcctccctccgcgtccgggtctcctggacagtcagtcaccatctcctgcactgg aaccagcagtgacgttggtggttataactatgtctcctggtaccaacagcacccaggcaaagcccccaaac tcatgatttatgaggtcagtaagcggccctcaggggtccctgatcgcttctctggctccaagtctggcaaca cggcctccctgaccgtctctgggctccaggctgaggatgaggctgattattactgcagctcatatgcaggc aacacctttttc -c- -a- - - accaactacctcctat [SEQ ID NO:76] scFv QSALTQPPSASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKA PKLMIYEVSKRPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCS SYAGSNTLVFGGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAQMQ LVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE WMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDT AVYYCARAGMGMDTWGQGTLVTVSS [SEQ ID NO: 1 18] In certain embodiments, the antibody or other antigen g protein is an anti—GPRCSD scFv or an antigen—binding fragment f having an antigen— binding region that ses the amino acid sequence of SEQ ID NO: 119 and specifically binds to a GPRCSD polypeptide (e.g., a GPRCSD polypeptide having the amino acid sequence SEQ ID NO:97, or fragments thereof), which is designated as ETlSO—l65 scFv (also referred to as "ETlSO—lS scFv").
In certain embodiments, the anti—GPRCSD scFv antibody comprises a heavy chain le region comprising amino acids having the sequence set forth in SEQ ID NO:77 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:78, optionally with (iii) a linker sequence, for example a linker peptide, between the heavy chain variable region and the light chain variable region. In certain embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the PRCSD scFv antibody is an scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 20. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:77, as shown in Table 20. In n embodiments, the PRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:78, as shown in Table 20. In certain embodiments, the PRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:77 and a VL comprising amino acids having the sequence set forth in SEQ ID NO:78, as shown in Table 20. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:238 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 239 or conservative modifications thereof, and a VH CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 240 or conservative modifications f, as shown in Table 20. In certain embodiments, the anti—GPRCSD scFv comprises a VL CDRl sing amino acids having the ce set forth in SEQ ID NO: 241 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 242 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 243 or conservative modifications thereof, as shown in Table 20. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 238 or conservative cations thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 239 or conservative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 240 or conservative modifications thereof, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 241 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 242 or conservative cations thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 243 or conservative modifications thereof, as shown in Table 20. In certain ments, the PRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 238, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 239, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 240, a VL CDRl comprising amino acids having the ce set forth in SEQ ID NO: 241, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 242, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 243.
Table 20 A GPRCSD polypeptide having the amino acid sequence of SEQ ID NO:97 GYTFTAYS [SEQ ID INPSSGGA [SEQ ID ARNVGGQADD NO: 238] NO: 239] [SEQ ID NO: 240] SSDIGGYNY [SEQ ID EVN [SEQ ID NO: 242] ASFAGRKTLV NO: 241] [SEQ ID NO: 243] Full VH QVQLVQSGAEVKKPGASVKVSCRASGYTFTAYSLHWVRQAPGQG LEWMGWINPSSGGAVYAQKFQGRVTMTRDTSISTAYMELSGLRSD DTAVYYCARNVGGQADDWGQGTLVTVSS [SEQ ID NO:77] DNA cagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcagg gcttctggatacaccttcaccgcctactctttacactgggtgcgacaggcccctggacaagggcttgagtgg atgggatggatcaaccctagcagtggtggcgcagtttatgcacagaaatttcagggtagggtcaccatgacc agggacacgtccatcagcacagcctacatggagctgagtggcctgagatctgacgacacggccgtgtatta ctgtgcgcgcaacgttggtggtcaggctgatgactggggtcaaggtactctggtgaccgtctcctca [SEQ ID NO:79] Full VL PPSASGSPGQSVTISCTGTSSDIGGYNYVSWYQQHPGKAP VNKRPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCAS FAGRKTLVFGGGTKLTVLG [SEQ ID NO:78] DNA Caatctgccctgactcagcctccctccgcgtccgggtctcctggacagtcagtcaccatctcctgcactgga accagcagtgacattggtggttataactatgtctcctggtaccaacagcacccaggcaaagcccccaaactc atgatttatgaggtcaataagcggccctcaggggtccctgatcgcttctcgggctccaagtctggcaacacg gcctccctgaccgtctctgggctccaggctgaggatgaggctgattattactgcgcctcatttgcgggcagg aagacattggtcttcggcggagggaccaagctgaccgtcctaggt [SEQ ID NO:80] scFv QSALTQPPSASGSPGQSVTISCTGTSSDIGGYNYVSWYQQHPGKAP KLMIYEVNKRPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCAS FAGRKTLVFGGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAQVQL VQSGAEVKKPGASVKVSCRASGYTFTAYSLHWVRQAPGQGLEWM GWINPSSGGAVYAQKFQGRVTMTRDTSISTAYMELSGLRSDDTAV YYCARNVGGQADDWGQGTLVTVSS [SEQ ID NO: 1 l9] In certain embodiments, the antibody or other antigen binding protein is an PRCSD scFv or an antigen—binding fragment thereof having an antigen— binding region that comprises the amino acid ce of SEQ ID NO: 120 and specifically binds to a GPRCSD polypeptide (e.g., a GPRCSD polypeptide having the amino acid sequence SEQ ID NO:97, or fragments thereof), which is designated as ETlSO—l67 scFv (also referred to as "ETlSO—l7 scFv").
In certain embodiments, the anti—GPRCSD scFv antibody ses a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:81 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:82, optionally with (iii) a linker sequence, for example a linker peptide, between the heavy chain variable region and the light chain variable region. In certain embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the PRCSD scFv antibody is an scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 21. In n ments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:81, as shown in Table 21. In certain embodiments, the anti—GPRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:82, as shown in Table 21. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the ce set forth in SEQ ID NO:81 and a VL comprising amino acids having the sequence set forth in SEQ ID NO:82, as shown in Table 21. In certain ments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:244 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 245 or conservative modifications thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 246 or conservative modifications thereof, as shown in Table 21. In certain embodiments, the anti—GPRCSD scFv comprises a VL CDRl comprising amino acids having the ce set forth in SEQ ID NO: 247 or conservative modifications thereof, a VL CDR2 sing amino acids having the sequence set forth in SEQ ID NO: 248 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 249 or conservative modifications f, as shown in Table 21. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 244 or conservative modifications thereof, a VH CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 245 or conservative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 246 or conservative modifications thereof, a VL CDRl sing amino acids having the sequence set forth in SEQ ID NO: 247 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 248 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 249 or conservative modifications thereof, as shown in Table 21. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 244, a VH CDR2 comprising amino acids haVing the sequence set forth in SEQ ID NO: 245, a VH CDR3 sing amino acids haVing the sequence set forth in SEQ ID NO: 246, a VL CDRI comprising amino acids haVing the sequence set forth in SEQ ID NO: 247, a VL CDR2 comprising amino acids haVing the sequence set forth in SEQ ID NO: 248, and a VL CDR3 comprising amino acids haVing the sequence set forth in SEQ ID NO: 249.
Table 21 Antigen A GPRC5D polypeptide haVing the amino acid sequence of SEQ ID NO:97 VH GYTFTAYS [SEQ ID INPSSGGA [SEQ ID ARNVGGHADD NO: 244] NO: 245] [SEQ ID NO: 246] STDIGGYNY [SEQ EVN [SEQ ID NO: 248] ASFAGRKTLV [SEQ ID NO: 247] ID NO: 249] QVQLVQSGAEVKKPGASVKVSCRASGYTFTAYSLHWVRQAPGQGL EWMGWINPSSGGAVYAQKFQGRVTMTRDTSISTAYMELSGLRSDDT RNVGGHADDWGQGTLVTVSS [SEQ ID NO:81] Caggtgcagctggtgcagtctggggctgaggtgaaaaagcctggggcctcagtgaaagtctcctgcagggc ttctggatacaccttcaccgcctactctttacactgggtgcgacaggcccctggacaagggcttgagtggatgg gatggatcaaccctagcagtggtggcgcagtttatgcacagaaatttcagggtagggtcaccatgaccaggga cacgtccatcagcacagcctacatggagctgagtggcctgagatctgacgacacggccgtgtattactgtgcg cgcaacgttggtggtcacgctgatgactggggtcaaggtactctggtgaccgtctcctca [SEQ ID NO:83] QSALTQPPSASGSPGQSVTISCTGTSTDIGGYNYVSWYQHHPSKAPKL KRPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCASFAG RKTLVFGGGTKLTVLG [SEQ ID NO:82] Caatctgccctgactcagcctccctccgcgtccgggtctcctggacagtcagtcaccatctcctgcactggaac cagcactgacattggtggttataactatgtctcctggtaccaacaccacccaagcaaagcccccaaactcatgat ttatgaggtcaataagcggccctcaggggtccctgatcgcttctcgggctccaagtctggcaacacggcctccc tgaccgtctctgggctccaggctgaggatgaggctgattattactgcgcctcatttgcgggcaggaagacattg gtcttcggcggagggaccaagctgaccgtcctaggt [SEQ ID NO:84] QSALTQPPSASGSPGQSVTISCTGTSTDIGGYNYVSWYQHHPSKAPKL MIYEVNKRPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCASFAG RKTLVFGGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAQVQLVQSG AEVKKPGASVKVSCRASGYTFTAYSLHWVRQAPGQGLEWMGWINP SSGGAVYAQKFQGRVTMTRDTSISTAYMELSGLRSDDTAVYYCARN VGGHADDWGQGTLVTVSS [SEQ ID NO:120] In n embodiments, the antibody or other antigen g protein is an anti—GPRC5D scFv or an antigen—binding nt thereof having an antigen— g region that comprises the amino acid sequence of SEQ ID NO: 121 and specifically binds to a GPRC5D polypeptide (e.g., a GPRC5D polypeptide having the amino acid sequence SEQ ID NO:97, or fragments thereof), which is designated as ETl50—l69 scFv (also referred to as "ETl50—l9 scFv").
In certain embodiments, the anti—GPRC5D scFv antibody comprises a heavy chain le region comprising amino acids having the sequence set forth in SEQ ID NO:85 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:86, optionally with (iii) a linker sequence, for example a linker peptide, between the heavy chain variable region and the light chain variable region. In certain embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the PRC5D scFv antibody is an scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 22. In certain embodiments, the PRC5D scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:85, as shown in Table 22. In certain embodiments, the anti—GPRC5D scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:86, as shown in Table 22. In certain embodiments, the anti—GPRC5D scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:85 and a VL comprising amino acids having the sequence set forth in SEQ ID NO:86, as shown in Table 22. In certain embodiments, the anti—GPRC5D scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:250 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 251 or conservative modifications thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 252 or conservative modifications thereof, as shown in Table 22. In n ments, the anti—GPRC5D scFv comprises a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 253 or vative modifications thereof, a VL CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 254 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 255 or conservative modifications thereof, as shown in Table 22. In certain embodiments, the anti—GPRC5D scFv comprises a VH CDRl sing amino acids having the sequence set forth in SEQ ID NO: 250 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 251 or conservative cations thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 252 or conservative modifications thereof, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 253 or vative modifications f, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 254 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 255 or conservative modifications thereof, as shown in Table 22. In certain embodiments, the anti—GPRC5D scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 250, a VH CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 251, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 252, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 253, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 254, and a VL CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 255.
Table 22 Antigen A GPRC5D polypeptide having the amino acid sequence of SEQ ID NO:97 GFTFNTYG [SEQ ID ISANNGHT [SEQ ID ARGGYHHQMQRYY NO: 250] NO: 251] KATSVYSDY [SEQ ID NO: 252] SSNIGNNY [SEQ ID DNN [SEQ ID NO: GTWDSSLSGVV [SEQ NO: 253] 254] ID NO: 255] QVQLVQSGGEVKKPGASVKVSCKASGFTFNTYGISWVRQAPGQGLE WMGWISANNGHTKSAQRFQDRVAMATDTSTSTAYMELRSLKFDDT AVYYCARGGYHHQMQRYYKATSVYSDYWGQGTLVTVSS [SEQ ID NO:85] Caggtccagctggtgcagtctggaggtgaggtgaagaagcctggggcctcagtgaaggtctcctgcaagg cttctggtttcacctttaacacctatggcatcagttgggtgcgacaggcccctggacaagggcttgagtggatg ggatggatcagcgctaacaatggtcacacaaagtctgcacagaggttccaggacagagtcgccatggccac agacacatccacgagcacggcctacatggagctgaggagcctgaaatttgacgacacggccgtgtattactg tgcgcgcggtggttaccatcatcagatgcagcggtactacaaagctacttctgtttactctgattactggggtca QSVVTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKL LIYDNNKRPSGIPDRFSGSKSGTSATLGlTGLQTGDEADYYCGTWDS SLSGVVFGGGTKLTVLG [SEQ ID NO:86] gtcgtgacgcagccgccctcagtgtctgcggccccaggacagaaggtcaccatctcctgctctgga agcagctccaacattgggaataattatgtatcctggtaccagcaactcccaggaacagcccccaaactcctca tttatgacaataataagcgaccctcagggattcctgaccgattctctggctccaagtctggcacgtctgccacc ctgggcatcaccggactccagactggggacgaggccgattattactgcggaacatgggatagcagcctgag KRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDS SLSGVVFGGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAQVQLVQS GGEVKKPGASVKVSCKASGFTFNTYGISWVRQAPGQGLEWMGWIS ANNGHTKSAQRFQDRVAMATDTSTSTAYMELRSLKFDDTAVYYCA QMQRYYKATSVYSDYWGQGTLVTVSS [SEQ ID NO: 121] In certain embodiments, the antibody or other antigen binding protein is an anti—GPRCSD scFv or an antigen—binding fragment thereof having an antigen— binding region that comprises the amino acid sequence of SEQ ID NO: 122 and specifically binds to a GPRCSD polypeptide (e. g., a GPRCSD polypeptide having the amino acid sequence SEQ ID NO:97, or fragments thereof), which is designated as ET150—172 scFv (also referred to as "ET150—22 scFv").
In certain embodiments, the PRCSD scFv antibody ses a heavy chain variable region sing amino acids having the sequence set forth in SEQ ID NO:89 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:90, optionally with (iii) a linker sequence, for example a linker peptide, between the heavy chain variable region and the light chain variable region. In certain embodiments, the linker comprises amino acids having the ce set forth in SEQ ID NO:98. In certain embodiments, the anti—GPRCSD scFv antibody is an scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 23. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:89, as shown in Table 23. In certain embodiments, the anti—GPRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:90, as shown in Table 23. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:89 and a VL sing amino acids having the sequence set forth in SEQ ID NO:90, as shown in Table 23. In certain embodiments, the anti—GPRC5D scFv comprises a VH CDRl sing amino acids having the sequence set forth in SEQ ID NO:256 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 257 or conservative modifications thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 258 or conservative modifications thereof, as shown in Table 23. In certain embodiments, the anti—GPRC5D scFv comprises a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 259 or vative modifications thereof, a VL CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 260 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 261 or conservative modifications thereof, as shown in Table 23. In n embodiments, the anti—GPRC5D scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 256 or conservative modifications f, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 257 or conservative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 258 or conservative modifications thereof, a VL CDRl comprising amino acids having the ce set forth in SEQ ID NO: 259 or conservative modifications thereof, a VL CDR2 sing amino acids having the sequence set forth in SEQ ID NO: 260 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 261 or conservative modifications thereof, as shown in Table 23. In certain embodiments, the anti—GPRC5D scFv comprises a VH CDRl sing amino acids having the sequence set forth in SEQ ID NO: 256, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 257, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 258, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 259, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 260, and a VL CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 261.
Table 23 A GPRC5D polypeptide having the amino acid sequence of SEQ ID NO:97 GYTFTSYY [SEQ ID INPSGGSS [SEQ ID ARAGMGMDT [SEQ NO: 256] NO: 257] ID NO: 258] SSDVGGYNY [SEQ ID EVS [SEQ ID NO: 260] SSYAGSNTLV [SEQ NO: 259] ID NO: 261] Full VH QMQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGL EWMGIINPSGGSSSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAV YYCARAGMGMDTWGQGTLVTVSS [SEQ ID NO:89] DNA Cagatgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtttcctgcaaggcat ctggatacaccttcaccagctactatatgcactgggtgcgacaggcccctggacaagggcttgagtggatggga aaccctagtggtggtagctcaagctacgcacagaagttccagggcagagtcaccatgaccagggaca cgtccacgagcacagtctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgcg cgctggtatgggtatggatacttggggtcaaggtactctggtgaccgtctcctca [SEQ ID NO:9l] Full VL QSALTQPPSASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKL MIYEVSKRPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCSSYAGS NTLVFGGGTKLTVLG [SEQ ID NO:90] DNA gccctgactcagcctccctccgcgtccgggtctcctggacagtcagtcaccatctcctgcactggaac cagcagtgacgttggtggttataactatgtctcctggtaccaacagcacccaggcaaagcccccaaactcatgat ttatgaggtcagtaagcggccctcaggggtccctgatcgcttctctggctccaagtctggcaacacggcctccct ctctgggctccaggctgaggatgaggctgattattactgcagctcatatgcaggcagcaacaccttggt gttcggcggagggaccaagctgaccgtcctaggt [SEQ ID NO:92] scFv QSALTQPPSASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKL MIYEVSKRPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCSSYAGS NTLVFGGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAQMQLVQSGAE VKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGG SSSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARAGMG MDTWGQGTLVTVSS [SEQ ID NO: 122] In certain embodiments, the antibody or other antigen binding protein is an anti—GPRC5D scFv or an antigen—binding fragment thereof having an n— binding region that comprises the amino acid sequence of SEQ ID NO: 123 and specifically binds to a GPRC5D polypeptide (e.g., a GPRC5D polypeptide having the amino acid sequence SEQ ID NO:97, or nts thereof), which is designated as ETl50—l73 scFv (also referred to as "ETl50—23 scFv").
In certain embodiments, the anti—GPRC5D scFv antibody comprises a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:93 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:94, optionally with (iii) a linker sequence, for e a linker peptide, between the heavy chain variable region and the light chain variable region. In certain embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the anti—GPRCSD scFv antibody is an scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 24. In certain embodiments, the PRCSD scFv comprises a VH sing amino acids having the ce set forth in SEQ ID NO:93, as shown in Table 24. In n embodiments, the anti—GPRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:94, as shown in Table 24. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:93 and a VL comprising amino acids having the sequence set forth in SEQ ID NO:94, as shown in Table 24. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:262 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 263 or conservative modifications thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 264 or conservative modifications thereof, as shown in Table 24. In n embodiments, the anti—GPRCSD scFv ses a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 265 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 266 or vative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 267 or conservative modifications f, as shown in Table 24. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 262 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 263 or conservative modifications thereof, a VH CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 264 or conservative cations thereof, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 265 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 266 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 267 or conservative modifications thereof, as shown in Table 24. In certain embodiments, the anti—GPRCSD scFv ses a VH CDRl comprising amino acids having the ce set forth in SEQ ID NO: 262, a VH CDR2 comprising amino acids haVing the ce set forth in SEQ ID NO: 263, a VH CDR3 comprising amino acids haVing the sequence set forth in SEQ ID NO: 264, a VL CDRI comprising amino acids haVing the sequence set forth in SEQ ID NO: 265, a VL CDR2 comprising amino acids haVing the sequence set forth in SEQ ID NO: 266, and a VL CDR3 comprising amino acids haVing the sequence set forth in SEQ ID NO: 267.
Table 24 n A GPRCSD polypeptide haVing the amino acid sequence of SEQ ID NO:97 GYTFTSYY [SEQ ID INPSGGST [SEQ ID NO: ARDVISGFDS NO: 262] 263] [SEQ ID NO: SSDVGGYNY [SEQ ID GVS [SEQ ID NO: 266] SSYAGVNNLM NO: 265] [SEQ ID NO: Full VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQG LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED TAVYYCARDVISGFDSWGQGTLVTVSS [SEQ ID NO:93] DNA Caggtgcagctggtgcaatctggggctgaggtgaagaagcctggggcctcagtgaaggtttcctgcaagg catctggatacaccttcaccagctactatatgcactgggtgcgacaggcccctggacaagggcttgagtgga tgggaataatcaaccctagtggtggtagcacaagctacgcacagaagttccagggcagagtcaccatgacc agggacacgtccacgagcacagtctacatggagctgagcagcctgagatctgaggacactgccgtgtatta ctgtgcgcgcgacgttatctctggtttcgattcttggggtcaaggtactctggtgaccgtctcctca [SEQ ID NO:95] Full VL QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQSPGKAP RLMIYGVSKRPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCSS YAGVNNLMFGGGTKLTVLG [SEQ ID NO:94] DNA Cagtctgccctgactcagcctgcctccgtgtctgggtctcctggacagtcgatcaccatctcctgcactgga accagcagtgacgttggtggttataactatgtctcctggtaccaacaatccccaggcaaagcccccagactc atgatttatggggtcagtaagcggccctctggggtccctgatcgcttctctggctccaagtctggcaacacgg cctccctgaccgtctctgggctccaggctgaagatgaggctgattattactgcagctcatatgcaggcgtcaa caatttaatgttcggcggagggaccaagctgaccgtcctaggt [SEQ ID NO:96] scFV QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQSPGKAP RLMIYGVSKRPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCSS YAGVNNLMFGGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAQVQL VQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEW MGlINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAV YYCARDVISGFDSWGQGTLVTVSS [SEQ ID NO: 123] In certain embodiments, the antibody or other antigen binding protein is an PRC5D scFv or an antigen—binding fragment thereof having an antigen— binding region that comprises the amino acid sequence of SEQ ID NO:276 and specifically binds to a GPRC5D polypeptide (e. g., a GPRC5D polypeptide having the amino acid ce SEQ ID NO:97, or fragments thereof), which is designated as 024 scFv (also referred to as "ETl50—l74 scFv").
In n embodiments, the anti—GPRC5D scFv antibody comprises a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:274 and a light chain variable region sing amino acids having the sequence set forth in SEQ ID NO:275, optionally with (iii) a linker sequence, for example a linker peptide, between the heavy chain variable region and the light chain variable region. In certain ments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the anti—GPRC5D scFv antibody is an scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 25.
In certain embodiments, the anti—GPRC5D scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:274, as shown in Table 25. In certain embodiments, the anti—GPRC5D scFv comprises a VL comprising amino acids having the ce set forth in SEQ ID NO:275, as shown in Table 25. In certain embodiments, the anti—GPRC5D scFv comprises a VH comprising amino acids having the ce set forth in SEQ ID NO:274 and a VL comprising amino acids having the sequence set forth in SEQ ID NO:275, as shown in Table 25. In certain embodiments, the anti—GPRC5D scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:268 or conservative cations f, a VH CDR2 sing amino acids having the sequence set forth in SEQ ID NO:269 or conservative modifications thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:270 or conservative modifications thereof, as shown in Table 8. In certain embodiments, the anti—GPRC5D scFv comprises a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:27l or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:272 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:273 or conservative modifications f, as shown in Table 25. In certain embodiments, the anti—GPRCSD scFv ses a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:268 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:269 or conservative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:270 or conservative modifications f, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:27l or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:272 or conservative modifications thereof, and a VL CDR3 sing amino acids having the sequence set forth in SEQ ID NO:273 or conservative modifications thereof, as shown in Table 25. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the ce set forth in SEQ ID NO:268, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:269, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:270, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:27l, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:272, and a VL CDR3 comprising amino acids having the ce set forth in SEQ ID .
Table 25 Antigen A GPRCSD polypeptide having the amino acid ce of SEQ ID NO:97 GFTFGDYG [SEQ ID NO: 268] ID NO: 270] SRDAGGYNY [SEQ ID EVT [SEQ ID NO: 272] SSYGGSNNFRV NO: 271] [SEQ ID NO: 273] EVQLVESGGGVVRPGGSLRLSCAASGFTFGDYGMSWVRQAPGKGL EWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARSKQDYWGQGTLVTVSS [SEQ ID NO:274] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGG GGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGG TGATTATGGCATGAGCTGGGTCCGCCAAGCTCCAGGGAAGGGGC TGGAGTGGGTCTCTGGTATTAATTGGAATGGTGGTAGCACAGGTT ATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAAC GCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCCGA GGACACGGCCGTATATTACTGTGCGCGCTCTAAACAGGATTACTG GGGTCAAGGTACTCTGGTGACCGTCTCCTCA [SEQ ID NO:277] MKKTAIAIAVALAGFATVAQAAELQSALTQPPSASGSPGQSVTISCT GTSRDAGGYNYFSWYQQHPGKAPKLLIYEVTKRPSGVPDRFSGSKS GKTASLTVSGLQADDEAVYYCSSYGGSNNFRVFGGGTKLTVLG [SEQ ID NO:275] ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTT CGCTACCGTGGCCCAGGCGGCCGAGCTCCAGTCTGCCCTGACTCA GCCTCCCTCCGCGTCCGGGTCTCCTGGACAGTCAGTCACCATCTC CTGCACTGGAACCAGCAGGGACGCTGGTGGTTATAATTATTTCTC CTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCCTGATTT ATGAGGTCACTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTG GCTCCAAGTCTGGCAAGACGGCCTCCCTGACCGTCTCTGGGCTCC AGGCTGACGATGAGGCTGTATATTACTGCAGCTCATATGGAGGC AGCAACAACTTTCGGGTGTTCGGCGGAGGGACCAAGCTGACCGT CCTAGGT [SEQ ID NO:278] MKKTAIAIAVALAGFATVAQAAELQSALTQPPSASGSPGQSVTISCT GTSRDAGGYNYFSWYQQHPGKAPKLLIYEVTKRPSGVPDRFSGSKS GKTASLTVSGLQADDEAVYYCSSYGGSNNFRVFGGGTKLTVLGw GGGSGGGGSGGGGSLEMAEVQLVESGGGVVRPGGSLRLSCAASGF MSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISR DNAKNSLYLQMNSLRAEDTAVYYCARSKQDYWGQGTLVTVSS [SEQ ID NO:276] ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTT CGCTACCGTGGCCCAGGCGGCCGAGCTCCAGTCTGCCCTGACTCA GCCTCCCTCCGCGTCCGGGTCTCCTGGACAGTCAGTCACCATCTC CTGCACTGGAACCAGCAGGGACGCTGGTGGTTATAATTATTTCTC CTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCCTGATTT ATGAGGTCACTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTG GCTCCAAGTCTGGCAAGACGGCCTCCCTGACCGTCTCTGGGCTCC AGGCTGACGATGAGGCTGTATATTACTGCAGCTCATATGGAGGC AGCAACAACTTTCGGGTGTTCGGCGGAGGGACCAAGCTGACCGT CCTAGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTG GTGGTGGTGGATCCCTCGAGATGGCCGAGGTGCAGCTGGTGGAG TCTGGGGGAGGTGTGGTACGGCCTGGGGGGTCCCTGAGACTCTC CTGTGCAGCCTCTGGATTCACCTTTGGTGATTATGGCATGAGCTG GGTCCGCCAAGCTCCAGGGAAGGGGCTGGAGTGGGTCTCTGGTA TTAATTGGAATGGTGGTAGCACAGGTTATGCAGACTCTGTGAAG GGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTA TCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCGTATATT CGCGCTCTAAACAGGATTACTGGGGTCAAGGTACTCTG GTGACCGTCTCCTCA [SEQ ID NO:279] In certain embodiments, the antibody or other antigen binding protein is an anti—GPRCSD scFV or an antigen—binding fragment thereof haVing an n— binding region that ses the amino acid sequence of SEQ ID NO: 288 and specifically binds to a GPRCSD polypeptide (e. g., a GPRCSD polypeptide having the amino acid sequence SEQ ID NO:97, or fragments thereof), which is designated as ET150—026 scFv (also referred to as "ETlSO—l76 scFv").
In certain embodiments, the anti—GPRCSD scFv antibody comprises a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:286 and a light chain variable region sing amino acids having the ce set forth in SEQ ID NO:287, optionally with (iii) a linker sequence, for e a linker peptide, between the heavy chain variable region and the light chain variable region. In certain embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the anti—GPRCSD scFv dy is an scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 26. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID , as shown in Table 26. In certain embodiments, the anti—GPRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:287, as shown in Table 26. In certain ments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:286 and a VL comprising amino acids having the sequence set forth in SEQ ID NO:287, as shown in Table 26. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:280 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:281 or conservative modifications thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:282 or conservative modifications thereof, as shown in Table 26. In certain embodiments, the anti—GPRCSD scFv comprises a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:283 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:284 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the ce set forth in SEQ ID NO:285 or conservative cations thereof, as shown in Table 26. In certain embodiments, the PRCSD scFv ses a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:280 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:281 or conservative modifications thereof, a VH CDR3 sing amino acids having the sequence set forth in SEQ ID NO:282 or conservative cations thereof, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:283 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:284 or conservative modifications f, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:285 or conservative modifications thereof, as shown in Table 26. In n embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:280, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:28l, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:282, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:283, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:284, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:285.
Table 26 Antigen A GPRCSD polypeptide having the amino acid sequence of SEQ ID NO:97 CDRs l 2 3 GFTFSNYA [SEQ ID lTNSGRST [SEQ ID NO: ARVTHRRYGSTF NO: 280] 281] DS [SEQ ID NO: SSNIGSNT [SEQ ID SNN [SEQ ID NO: 284] AAWDDSVNGYV NO: 283] [SEQ ID NO: 285] QLQLQESGGGSVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLE WVSAlTNSGRSTYYADSVKGRFTISRDNSKNTLSLQMSSLRAEDTAV YYCARVTHRRYGSTFDSRGQGTLVTVSS [SEQ ID ] CAGCTGCAGGAGTCGGGGGGAGGCTCGGTACAGCCGGG GGGGTCTCTGAGACTGTCCTGTGCAGCCTCTGGATTCACCTTTAG CAACTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGC TGGAGTGGGTCTCAGCTATCACTAATAGTGGTCGTAGTACATACT ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAAT TCCAAGAACACGCTGTCTTTGCAAATGAGCAGCCTGAGAGCCGA AGACACGGCCGTGTATTACTGTGCGCGCGTTACTCATCGTCGTTA CGGTTCTACTTTCGATTCTCGGGGTCAAGGTACTCTGGTGACCGT CTCCTCA ACTAGTGGCCAGGCCGGCCAGC [SEQ ID NO:289] MKKTAIAIAVALAGFATVAQAAELSYELTQPPSASGTPGQRVSISCS GSSSNIGSNTVNWYQQFPGTAPKLLIHSNNQRPSGVPDRFSGSKSGT SASLAISGPQSEDEADYYCAAWDDSVNGYVFGTGTKVTVLG [SEQ ID NO:287] ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTT CGCTACCGTGGCCCAGGCGGCCGAGCTCTCCTATGAGCTGACTCA GCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAGCATCT CTTGTTCTGGAAGCAGCTCCAACATCGGGAGTAATACTGTAAACT GGTACCAACAGTTCCCCGGAACGGCCCCCAAACTCCTCATCCATA GTAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCT CCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCCCCAGT ATGAGGCTGATTATTACTGTGCAGCTTGGGATGACAGTG TGAATGGTTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTAG GT [SEQ ID NO:290] AIAVALAGFATVAQAAELSYELTQPPSASGTPGQRVSISCS GSSSNIGSNTVNWYQQFPGTAPKLLIHSNNQRPSGVPDRFSGSKSGT SASLAISGPQSEDEADYYCAAWDDSVNGYVFGTGTKVTVLGSRGG GGSGGGGSGGGGSLEMAQLQLQESGGGSVQPGGSLRLSCAASGFTF SNYAMSWVRQAPGKGLEWVSAITNSGRSTYYADSVKGRFTISRDNS QMSSLRAEDTAVYYCARVTHRRYGSTFDSRGQGTLVTVSS [SEQ ID NO:288] ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTT CGCTACCGTGGCCCAGGCGGCCGAGCTCTCCTATGAGCTGACTCA GCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAGCATCT CTTGTTCTGGAAGCAGCTCCAACATCGGGAGTAATACTGTAAACT GGTACCAACAGTTCCCCGGAACGGCCCCCAAACTCCTCATCCATA GTAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCT CCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCCCCAGT CTGAGGATGAGGCTGATTATTACTGTGCAGCTTGGGATGACAGTG TGAATGGTTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTAG GTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGT GGTGGATCCCTCGAGATGGCCCAGCTGCAGCTGCAGGAGTCGGG GGGAGGCTCGGTACAGCCGGGGGGGTCTCTGAGACTGTCCTGTG CTGGATTCACCTTTAGCAACTATGCCATGAGCTGGGTCC GCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATCACT AATAGTGGTCGTAGTACATACTACGCAGACTCCGTGAAGGGCCG GTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTCTTTGCA CAGCCTGAGAGCCGAAGACACGGCCGTGTATTACTGTG CGCGCGTTACTCATCGTCGTTACGGTTCTACTTTCGATTCTCGGG GTCAAGGTACTCTGGTGACCGTCTCCTCA [SEQ ID NO:29l] In certain embodiments, the antibody or other antigen binding protein is an anti—GPRCSD scFV or an antigen—binding fragment thereof haVing an antigen— binding region that comprises the amino acid sequence of SEQ ID NO: 300 and specifically binds to a GPRCSD polypeptide (e.g., a GPRCSD polypeptide haVing the amino acid sequence SEQ ID NO:97, or fragments thereof), which is ated as ET150—028 scFv (also referred to as "ETlSO—l78 scFv").
In certain embodiments, the anti—GPRCSD scFv antibody comprises a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:298 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:299, ally with (iii) a linker sequence, for e a linker peptide, between the heavy chain variable region and the light chain variable region. In n embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the anti—GPRCSD scFv dy is an scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 27. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:298, as shown in Table 27. In certain ments, the anti—GPRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:299, as shown in Table 27. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:298 and a VL comprising amino acids having the sequence set forth in SEQ ID NO:299, as shown in Table 27. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:292 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:293 or conservative modifications thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:294 or conservative modifications thereof, as shown in Table 27. In certain embodiments, the anti—GPRCSD scFv ses a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:295 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:296 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:297 or conservative modifications thereof, as shown in Table 27. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the ce set forth in SEQ ID NO:292 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:293 or conservative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:294 or conservative modifications thereof, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:295 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:296 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:297 or conservative cations thereof, as shown in Table 27. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:292, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:293, a VH CDR3 comprising amino acids having the ce set forth in SEQ ID NO:294, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:295, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:296, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:297.
Table 27 Antigen A GPRCSD ptide having the amino acid sequence of SEQ ID NO:97 CDRs l 2 3 GGTFRSYA [SEQ ID lIPMLDIT [SEQ ID NO: ARTYSRSPFHME NO: 292] 293] DF [SEQ ID NO: NT [SEQ ID RNN [SEQ ID NO: 296] AAWDASRQGV NO: 295] [SEQ ID NO: 297] Full VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFRSYAITWVRQAPGQGL EWMGRIIPMLDITNYAQKFQGRVTlTADKSTSTAYMELSSLRSEDTA VYYCARTYSRSPFHMEDFWGQGTLVTVSS [SEQ ID NO:298] DNA CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTG GGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTC CGCAGCTATGCTATCACCTGGGTGCGACAGGCCCCTGGACAAGG GCTTGAGTGGATGGGAAGGATCATCCCTATGCTTGATATAACAA ACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGA CAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGA TCTGAGGACACGGCCGTGTATTACTGTGCGCGCACTTACTCTCG TTCTCCGTTCCATATGGAAGATTTCTGGGGTCAAGGTACTCTGGT GACCGTCTCCTCA [SEQ ID NO:300] Full VL MKKTAIAIAVALAGFATVAQAAELQPVLTQPPSASGTPGQRVTISCS GSSSNIGGNTVSWYQQVPGTAPRLLIFRNNQRPPGVPDRFSGSKSGT SGLRSEDEADYYCAAWDASRQGVFGGGTKLTVLG [SEQ ID NO:299] DNA ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTT CGCTACCGTGGCCCAGGCGGCCGAGCTCCAGCCTGTGCTGACTC AGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATC TCTGGAAGCAGCTCCAATATCGGAGGTAACACTGTCAG CTGGTACCAGCAGGTCCCAGGAACGGCCCCCAGACTCCTCATTT TTAGGAATAATCAACGGCCCCCAGGGGTCCCTGACCGATTCTCT GGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCT CCGGTCTGAGGATGAGGCTGATTATTACTGTGCAGCATGGGACG CCAGTCGACAAGGGGTGTTCGGCGGAGGGACCAAGCTGACCGT CCTAGGT [SEQ ID NO:301] scFV MKKTAIAIAVALAGFATVAQAAELQPVLTQPPSASGTPGQRVTISCS GSSSNIGGNTVSWYQQVPGTAPRLLIFRNNQRPPGVPDRFSGSKSGT SASLAISGLRSEDEADYYCAAWDASRQGVFGGGTKLTVLGSRGGG GSGGGGSGGGGSLEMAQVQLVQSGAEVKKPGSSVKVSCKASGGT FRSYAITWVRQAPGQGLEWMGRIIPMLDlTNYAQKFQGRVTITADK STSTAYMELSSLRSEDTAVYYCARTYSRSPFHMEDFWGQGTLVTV SS [SEQ ID NO:300] DNA ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTT CGCTACCGTGGCCCAGGCGGCCGAGCTCCAGCCTGTGCTGACTC AGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATC TCTTGTTCTGGAAGCAGCTCCAATATCGGAGGTAACACTGTCAG CTGGTACCAGCAGGTCCCAGGAACGGCCCCCAGACTCCTCATTT TTAGGAATAATCAACGGCCCCCAGGGGTCCCTGACCGATTCTCT GGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCT CCGGTCTGAGGATGAGGCTGATTATTACTGTGCAGCATGGGACG CCAGTCGACAAGGGGTGTTCGGCGGAGGGACCAAGCTGACCGT CCTAGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTG GTGGTGGTGGATCCCTCGAGATGGCCCAGGTGCAGCTGGTGCAG TCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTC CTGCAAGGCTTCTGGAGGCACCTTCCGCAGCTATGCTATCACCT GGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAAG GATCATCCCTATGCTTGATATAACAAACTACGCACAGAAGTTCC AGGGCAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGC CTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGT ATTACTGTGCGCGCACTTACTCTCGTTCTCCGTTCCATATGGAAG ATTTCTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA [SEQ ID NO:302] In n embodiments, the dy or other antigen binding protein is an anti—GPRCSD scFV or an antigen—binding fragment thereof haVing an antigen— binding region that comprises the amino acid ce of SEQ ID NO:312 and specifically binds to a GPRCSD polypeptide (e.g., a GPRCSD polypeptide haVing the amino acid ce SEQ ID NO:97, or fragments thereof), which is designated as ET150—029 scFV (also referred to as "ETlSO—l79 scFV").
In certain embodiments, the anti—GPRCSD scFv dy comprises a heavy chain le region comprising amino acids having the sequence set forth in SEQ ID NO:310 and a light chain variable region sing amino acids having the sequence set forth in SEQ ID NO:3ll, optionally with (iii) a linker sequence, for e a linker peptide, between the heavy chain variable region and the light chain variable region. In n embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the anti—GPRCSD scFv antibody is an scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 28. In certain ments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:310, as shown in Table 28. In certain embodiments, the anti—GPRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:3ll, as shown in Table 28. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:310 and a VL comprising amino acids having the sequence set forth in SEQ ID NO:3ll, as shown in Table 28. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl sing amino acids having the sequence set forth in SEQ ID NO:303 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:304 or conservative modifications thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:305 or conservative modifications thereof, as shown in Table 28. In certain ments, the anti—GPRCSD scFv comprises a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:306 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:307 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:308 or conservative modifications thereof, as shown in Table 28. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:303 or conservative modifications thereof, a VH CDR2 sing amino acids having the sequence set forth in SEQ ID NO:304 or vative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:305 or conservative cations thereof, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:306 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:307 or conservative cations thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:308 or conservative modifications thereof, as shown in Table 28. In certain embodiments, the PRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:303, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:304, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:305, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:306, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:307, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:308.
Table 28 n A GPRCSD polypeptide having the amino acid sequence of SEQ ID NO:97 VH GFTFSSYA[SEQ ID ISGSGGST [SEQ ID ARKYQDV [SEQ ID VL SSNIGSNT[SEQ ID RNN [SEQ ID NO: 307] LSGRV NO: 306] - [SEQID NO: 308] Full VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGL EWVSAISGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDT AVYYCARKYQDVWGQGTLVTVSS [SEQ ID NO:3lO] DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTG GGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTA GCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGG GCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGAC AATGCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAG ACACGGCCGTATATTACTGTGCGCGCAAATACCAGGAT GTTTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA [SEQ ID NO:3 13] Full VL MKKTAIAIAVALAGFATVAQAAELQSVLTQPPSASGTPGQRVTISCS GSSSNIGSNTVNWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGT SASLAISGLRSEDEADYYCAAWDDSLSGRVFGGGTKLTVLG [SEQ ID NO:3l 1] DNA ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTT CGCTACCGTGGCCCAGGCGGCCGAGCTCCAGTCTGTGCTGACGC AGCCGCCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATC TCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATACTGTAAA CTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCT ATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCT GGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCT CCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATG ACAGCCTGAGTGGTAGGGTGTTCGGCGGAGGGACCAAGCTGAC CGTCCTAGGT [SEQ ID NOz3l4] scFv MKKTAIAIAVALAGFATVAQAAELQSVLTQPPSASGTPGQRVTISCS GSSSNIGSNTVNWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGT SASLAISGLRSEDEADYYCAAWDDSLSGRVFGGGTKLTVLGSRGG GGSGGGGSGGGGSLEMAEVQLVESGGGLVQPGGSLRLSCAASGFT FSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRD NAKNTLYLQMNSLRAEDTAVYYCARKYQDVWGQGTLVTVSS [SEQ ID ] DNA ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTT CGCTACCGTGGCCCAGGCGGCCGAGCTCCAGTCTGTGCTGACGC AGCCGCCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATC TCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATACTGTAAA CTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCT ATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCT GGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCT CGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATG ACAGCCTGAGTGGTAGGGTGTTCGGCGGAGGGACCAAGCTGAC CGTCCTAGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCT CTGGTGGTGGTGGATCCCTCGAGATGGCCGAGGTGCAGCTGGTG GAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAG CTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCA GCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGT GAAGGGCCGGTTCACCATCTCCAGAGACAATGCCAAGAACACG CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCG TATATTACTGTGCGCGCAAATACCAGGATGTTTGGGGTCAAGGT ACTCTGGTGACCGTCTCCTCA [SEQ ID NOz3lS] In certain ments, the antibody or other antigen binding protein is an PRCSD scFv or an antigen—binding nt thereof having an antigen— binding region that comprises the amino acid sequence of SEQ ID NO:324 and specifically binds to a GPRCSD polypeptide (e.g., a GPRCSD polypeptide having the amino acid sequence SEQ ID NO:97, or fragments thereof), which is designated as ET150—030 scFv (also referred to as "ETlSO—lSO scFv").
In certain embodiments, the anti—GPRCSD scFv dy comprises a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:322 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:323, optionally with (iii) a linker sequence, for example a linker peptide, between the heavy chain variable region and the light chain variable region. In certain embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the PRCSD scFv antibody is an scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 29. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID , as shown in Table 29. In certain embodiments, the anti—GPRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:323, as shown in Table 29. In certain embodiments, the PRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:322 and a VL comprising amino acids having the sequence set forth in SEQ ID NO:323, as shown in Table 29. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:3l6 or conservative modifications thereof, a VH CDR2 sing amino acids having the sequence set forth in SEQ ID NO:3l7 or conservative modifications thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:318 or conservative modifications thereof, as shown in Table 29. In certain embodiments, the anti—GPRCSD scFv comprises a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:3l9 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:320 or vative modifications f, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:32l or conservative modifications thereof, as shown in Table 29. In n embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:3l6 or vative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:3l7 or conservative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:318 or vative modifications f, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:3l9 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:320 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:32l or conservative modifications thereof, as shown in Table 29. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:316, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:317, a VH CDR3 comprising amino acids haVing the sequence set forth in SEQ ID NO:318, a VL CDR1 comprising amino acids haVing the sequence set forth in SEQ ID NO:319, a VL CDR2 comprising amino acids haVing the ce set forth in SEQ ID NO:320, and a VL CDR3 comprising amino acids haVing the sequence set forth in SEQ ID NO:321.
Table 29 Antigen A GPRCSD1polypeptide haVing the amino acid sequence of SEQ ID NO:97 VH—_GFSFSGTA[SEQID ISSTGRST [SEQ ID NO: ARPVSSMTLSIQS NO: 316] 317] DG [SEQ ID NO: VL SSNIGAGYD [SEQ ID GNS [SEQ ID NO: 320] QSYDSSLRGYV NO: 319] [SEQ ID NO: 321] Full VH QVQLVQSGGGVVQPGRSLRLSCAASGFSFSGTAMHWVRQAPGKGL EWVSTISSTGRSTYYRDSVKGRFTISRDNSKNTLYLQMNSLRGEDTA VYYCARPVSSMTLSIQSDGWGQGTLVTVSS [SEQ ID NO:322] CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGG GAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCAGCTTTAG TGGCACTGCCATGCACTGGGTCCGCCAGGCTCCAGGGAAGGGGC TGGAATGGGTCTCGACTATTAGTAGTACTGGGCGTAGCACATACT ACAGAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAAT TCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGGCGA GGACACGGCCGTATATTACTGTGCGCGCCCGGTTTCTTCTATGAC TATCCAGTCTGATGGTTGGGGTCAAGGTACTCTGGTGAC CGTCTCCTCA [SEQ ID ] MKKTAIAIAVALAGFATVAQAAELQSVLTQPPSVSGAPGQRVTISCT GSSSNIGAGYDV TSASLAITGLQAEDEADYYCQSYDSSLRGYVFGTGTKVTVLG [SEQ ID NO:323] ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTT CGCTACCGTGGCCCAGGCGGCCGAGCTCCAGTCTGTGTTGACGC AGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATC TCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGT ACACTGGTACCAGCAGCTTCCAGGAAGAGCCCCCAAACTCCTCA TCTATGGTAACAGCAATCGGCCCTCAGGGGTCCCTGACCGATTCT CCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGC TCCAGGCTGAGGATGAGGCTGATTATTACTGCCAGTCCTATGACA GCAGCCTGAGAGGTTATGTCTTCGGAACTGGGACCAAGGTCACC GTCCTAGGT [SEQ ID NO:326] MKKTAIAIAVALAGFATVAQAAELQSVLTQPPSVSGAPGQRVTISCT GSSSNIGAGYDV TSASLAITGLQAEDEADYYCQSYDSSLRGYVFGTGTKVTVLGSRGG GGSGGGGSGGGGSLEMAQVQLVQSGGGVVQPGRSLRLSCAASGFS HWVRQAPGKGLEWVSTISSTGRSTYYRDSVKGRFTISRDN SKNTLYLQMNSLRGEDTAVYYCARPVSSMTLSIQSDGWGQGTLVT VSS [SEQ ID NO:324] ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTT CGCTACCGTGGCCCAGGCGGCCGAGCTCCAGTCTGTGTTGACGC AGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATC TCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGT ACACTGGTACCAGCAGCTTCCAGGAAGAGCCCCCAAACTCCTCA TCTATGGTAACAGCAATCGGCCCTCAGGGGTCCCTGACCGATTCT CTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGC TCCAGGCTGAGGATGAGGCTGATTATTACTGCCAGTCCTATGACA GCAGCCTGAGAGGTTATGTCTTCGGAACTGGGACCAAGGTCACC GTCCTAGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTC TGGTGGTGGTGGATCCCTCGAGATGGCCCAGGTGCAGCTGGTGC AGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTC TCCTGTGCAGCCTCTGGATTCAGCTTTAGTGGCACTGCCATGCAC TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGGTCTCGAC TATTAGTAGTACTGGGCGTAGCACATACTACAGAGACTCCGTGA AGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG TATCTGCAAATGAACAGCCTGAGAGGCGAGGACACGGCCGTATA TTACTGTGCGCGCCCGGTTTCTTCTATGACTCTGTCTATCCAGTCT GATGGTTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA [SEQ ID NO:327] In n embodiments, the antibody or other antigen binding protein is an PRCSD scFv or an n—binding fragment thereof having an antigen— binding region that comprises the amino acid sequence of SEQ ID NO:336 and specifically binds to a GPRCSD polypeptide (e.g., a GPRCSD polypeptide having the amino acid sequence SEQ ID NO:97, or fragments thereof), which is designated as ET150—03l scFv (also referred to as "ETlSO—lSl scFv").
In certain embodiments, the anti—GPRCSD scFv antibody comprises a heavy chain le region comprising amino acids having the sequence set forth in SEQ ID NO:334 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:335, optionally with (iii) a linker sequence, for example a linker peptide, n the heavy chain variable region and the light chain variable . In certain embodiments, the linker comprises amino acids having the ce set forth in SEQ ID NO:98. In certain embodiments, the anti—GPRCSD scFv antibody is an scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 30. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:334, as shown in Table 30. In certain embodiments, the anti—GPRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:335, as shown in Table 30. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:334 and a VL comprising amino acids having the sequence set forth in SEQ ID NO:335, as shown in Table 30. In certain embodiments, the PRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:328 or conservative modifications thereof, a VH CDR2 sing amino acids having the ce set forth in SEQ ID NO:329 or conservative cations thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:330 or conservative cations thereof, as shown in Table 30. In certain embodiments, the anti—GPRCSD scFv comprises a VL CDRl sing amino acids having the sequence set forth in SEQ ID NO:33l or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:332 or conservative cations thereof, and a VL CDR3 comprising amino acids having the ce set forth in SEQ ID NO:333 or conservative modifications thereof, as shown in Table 30. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl sing amino acids having the sequence set forth in SEQ ID NO:328 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:329 or conservative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:330 or conservative modifications thereof, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:33l or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:332 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:333 or conservative modifications thereof, as shown in Table 30. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the ce set forth in SEQ ID NO:328, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID , a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:330, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:331, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:332, and a VL CDR3 comprising amino acids haVing the sequence set forth in SEQ ID NO:333.
Table 30 Antigen A GPRCSD polypeptide haVing the amino acid ce of SEQ ID NO:97 VH GYTFTSYY [SEQ INPSGGST [SEQ ID ARGQKYHSQYSRGG ID NO: 328] NO: 329] TGGGMTQDM [SEQ ID NO: 330] SSNIGNNY [SEQ ID DNN [SEQ ID NO: 332] GTWDSSLRNWV NO: 331] [SEQ ID NO: 333] QMQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGL EWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTA VYYCARGQKYHSQYSRGGTGGGMTQDMWGQGTLVTVSS [SEQ ID CAGATGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGG GGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCAC CAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCT TGAGTGGATGGGAATAATCAACCCTAGTGGTGGTAGCACAAGCTA CGCACAAAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGT CCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGG ACACGGCCGTGTATTACTGTGCGCGCGGTCAGAAATACCATTCTC AGTACTCTCGTGGTGGTACTGGTGGTGGTATGACTCAGGATATGT GGGGTCAAGGTACTCTGGTGACCGTCTCCTCA [SEQ ID NO:337] MKKTAIAIAVALAGFATVAQAAELQSVVTQPPSVSAAPGQRVTISCS GGSSNIGNNYVSWFQQLPRTAPKLLIYDNNKRPSGIPDRFSGSKSGTS AALDITVLQTGDEADYYCGTWDSSLRNWVFGGGTKLTVLG [SEQ ID NO:335] ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTC GCTACCGTGGCCCAGGCGGCCGAGCTCCAGTCTGTCGTGACGCAG CCGCCCTCTGTGTCTGCGGCCCCAGGACAGAGGGTCACCATCTCC GGAGGTAGTTCCAACATTGGGAATAATTATGTTTCCTGGT TCCAACAACTCCCACGAACAGCCCCCAAACTCCTCATTTATGACA ATAATAAGCGACCCTCAGGGATTCCTGACCGATTCTCTGGCTCCA GCACGTCAGCCGCCCTGGACATCACCGTTCTCCAGACTG GGGACGAGGCCGATTATTACTGCGGAACTTGGGATAGCAGCCTGA GAAATTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT [SEQ ID NO:338] MKKTAIAIAVALAGFATVAQAAELQSVVTQPPSVSAAPGQRVTISCS GGSSNIGNNYVSWFQQLPRTAPKLLIYDNNKRPSGIPDRFSGSKSGTS AALDITVLQTGDEADYYCGTWDSSLRNWVFGGGTKLTVLGSRGGG WO 90329 2015/064122 GSGGGGSGGGGSLEMAQMQLVQSGAEVKKPGASVKVSCKASGYTF TSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTS TSTVYMELSSLRSEDTAVYYCARGQKYHSQYSRGGTGGGMTQDMW GQGTLVTVSS [SEQ ID NO:336] ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTC GCTACCGTGGCCCAGGCGGCCGAGCTCCAGTCTGTCGTGACGCAG CCGCCCTCTGTGTCTGCGGCCCCAGGACAGAGGGTCACCATCTCC TGCTCTGGAGGTAGTTCCAACATTGGGAATAATTATGTTTCCTGGT TCCAACAACTCCCACGAACAGCCCCCAAACTCCTCATTTATGACA ATAATAAGCGACCCTCAGGGATTCCTGACCGATTCTCTGGCTCCA AGTCTGGCACGTCAGCCGCCCTGGACATCACCGTTCTCCAGACTG GGGACGAGGCCGATTATTACTGCGGAACTTGGGATAGCAGCCTGA GAAATTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTT CTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGGTG GATCCCTCGAGATGGCCCAGATGCAGCTGGTGCAGTCTGGGGCTG AGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCAT CTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGG CCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTAGTG GTGGTAGCACAAGCTACGCACAAAAGTTCCAGGGCAGAGTCACC ATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAG CAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGCGCGG TCAGAAATACCATTCTCAGTACTCTCGTGGTGGTACTGGTGGTGGT ATGACTCAGGATATGTGGGGTCAAGGTACTCTGGTGACCGTCTCC TCA [SEQ ID NO:339] In certain embodiments, the antibody or other antigen binding protein is an PRCSD scFv or an antigen—binding fragment thereof having an antigen— binding region that comprises the amino acid sequence of SEQ ID NO:348 and ically binds to a GPRCSD polypeptide (e.g., a GPRCSD polypeptide having the amino acid ce SEQ ID NO:97, or fragments thereof), which is ated as ET150—032 scFv (also referred to as "ET150—182 scFv").
In certain embodiments, the PRCSD scFv antibody comprises a heavy chain variable region comprising amino acids having the ce set forth in SEQ ID NO:346 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:347, optionally with (iii) a linker sequence, for example a linker peptide, between the heavy chain variable region and the light chain variable region. In certain embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the anti—GPRCSD scFv antibody is an scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 31. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID WO 90329 2015/064122 NO:346, as shown in Table 31. In certain embodiments, the anti—GPRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:347, as shown in Table 31. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:346 and a VL comprising amino acids having the ce set forth in SEQ ID NO:347, as shown in Table 31. In certain ments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:340 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:34l or conservative modifications f, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:342 or conservative modifications thereof, as shown in Table 31. In certain embodiments, the anti—GPRCSD scFv comprises a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:343 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:344 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the ce set forth in SEQ ID NO:345 or conservative modifications thereof, as shown in Table 31. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:340 or conservative modifications thereof, a VH CDR2 sing amino acids having the sequence set forth in SEQ ID NO:34l or conservative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:342 or conservative modifications thereof, a VL CDRl comprising amino acids having the ce set forth in SEQ ID NO:343 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:344 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:345 or conservative modifications thereof, as shown in Table 31. In certain embodiments, the anti—GPRCSD scFv ses a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:340, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:34l, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:342, a VL CDRl comprising amino acids having the ce set forth in SEQ ID NO:343, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:344, and a VL CDR3 comprising amino acids having the ce set forth in SEQ ID NO:345.
Table 31 Antigen A GPRCSD polypeptide having the amino acid sequence of SEQ ID NO:97 GYTFSRYY [SEQ ID —[SEQMNPNSGNT ID ARGRYHVIDY SSDVGGYNH [SEQ EVT [SEQ ID NO: 344] SSYAGSAHWV Full VH EVQLVQSGAEVKKPGASVKVSCKASGYTFSRYYIHWVRQAPGQG LEWMGWMNPNSGNTGYAQKFQGRVTMTRNTSISTAYMELSSLRS YCARGRYHVIDYWGQGTLVTVSS [SEQ ID NO:346] DNA GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTG GGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTC AGCAGGTACTATATACACTGGGTGCGACAGGCCCCTGGACAAG GGCTTGAGTGGATGGGATGGATGAACCCTAACAGTGGTAACAC TGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGG AACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGA GATCTGAGGACACGGCCGTGTATTACTGTGCGCGCGGTCGTTAC CATGTTATCGATTACTGGGGTCAAGGTACTCTGGTGACCGTCTC CTCA [SEQ ID NO:349] Full VL MKKTAIAIAVALAGFATVAQAAELQSVLTQPPSASGSPGQSLTISC TGTSSDVGGYNHVSWYQQYPGKAPKLMIYEVTKRPSGVPDRFSG SKSGNTASLTVSGLQAEDEADYYCSSYAGSAHWVFGGGTKLTVL G [SEQ ID NO:347] DNA ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTT TCGCTACCGTGGCCCAGGCGGCCGAGCTCCAGTCTGTGTTGACT CAGCCACCCTCCGCGTCCGGGTCTCCTGGACAGTCACTCACCAT CACTGGAACCAGCAGTGACGTTGGTGGTTATAACCATG TCTCCTGGTACCAACAGTACCCAGGCAAAGCCCCCAAACTCAT GATTTATGAGGTCACTAAGCGGCCCTCAGGGGTCCCTGATCGCT TCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCGTCTCT GGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCAGCTCAT ATGCAGGCAGCGCCCATTGGGTGTTCGGCGGAGGGACCAAGCT GACCGTCCTAGGT [SEQ ID NO:350] scFV MKKTAIAIAVALAGFATVAQAAELQSVLTQPPSASGSPGQSLTISC TGTSSDVGGYNHVSWYQQYPGKAPKLMIYEVTKRPSGVPDRFSG SKSGNTASLTVSGLQAEDEADYYCSSYAGSAHWVFGGGTKLTVL GSGGGGSGGGGSLEMAEVQLVQSGAEVKKPGASVKVSC KASGYTFSRYYIHWVRQAPGQGLEWMGWMNPNSGNTGYAQKFQ GRVTMTRNTSISTAYMELSSLRSEDTAVYYCARGRYHVIDYWGQ GTLVTVSS [SEQ ID NO:348] DNA. ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTT TCGCTACCGTGGCCCAGGCGGCCGAGCTCCAGTCTGTGTTGACT CAGCCACCCTCCGCGTCCGGGTCTCCTGGACAGTCACTCACCAT CTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACCATG TCTCCTGGTACCAACAGTACCCAGGCAAAGCCCCCAAACTCAT GATTTATGAGGTCACTAAGCGGCCCTCAGGGGTCCCTGATCGCT TCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCGTCTCT GGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCAGCTCAT ATGCAGGCAGCGCCCATTGGGTGTTCGGCGGAGGGACCAAGCT GACCGTCCTAGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGC GGCTCTGGTGGTGGTGGATCCCTCGAGATGGCCGAGGTCCAGC TGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT GAAGGTTTCCTGCAAGGCATCTGGATACACCTTCAGCAGGTACT ATATACACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTG GATGGGATGGATGAACCCTAACAGTGGTAACACAGGCTATGCA CAGAAGTTCCAGGGCAGAGTCACCATGACCAGGAACACCTCCA TAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGA CACGGCCGTGTATTACTGTGCGCGCGGTCGTTACCATGTTATCG ATTACTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCAISEQID NO:351] In n embodiments, the dy or other antigen binding protein is an PRCSD scFv or an antigen—binding fragment thereof having an antigen— binding region that comprises the amino acid sequence of SEQ ID NO:360 and specifically binds to a GPRCSD polypeptide (e. g., a GPRCSD polypeptide having the amino acid sequence SEQ ID NO:97, or fragments thereof), which is ated as ET150—033 scFv (also ed to as "ETlSO—183 scFv").
In certain embodiments, the anti—GPRCSD scFv antibody comprises a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:358 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:359, optionally with (iii) a linker sequence, for example a linker peptide, between the heavy chain variable region and the light chain variable region. In certain ments, the linker comprises amino acids having the ce set forth in SEQ ID NO:98. In certain embodiments, the anti—GPRCSD scFv antibody is an c fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 32. In certain embodiments, the PRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:358, as shown in Table 32. In certain embodiments, the anti—GPRCSD scFv comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:359, as shown in Table 32. In certain embodiments, the anti—GPRCSD scFv comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:358 and a VL comprising amino acids having the ce set forth in SEQ ID NO:359, as shown in Table 32. In n embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:352 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:353 or conservative modifications thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:354 or conservative modifications thereof, as shown in Table 32. In certain embodiments, the anti—GPRCSD scFv comprises a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:355 or conservative cations thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:356 or conservative modifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:357 or conservative modifications thereof, as shown in Table 32. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:352 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:353 or conservative cations thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:354 or conservative modifications thereof, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO:355 or conservative cations thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:356 or conservative cations thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:357 or conservative modifications f, as shown in Table 32. In certain embodiments, the anti—GPRCSD scFv comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:352, a VH CDR2 comprising amino acids having the ce set forth in SEQ ID NO:353, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:354, a VL CDRl comprising amino acids having the ce set forth in SEQ ID NO:355, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:356, and a VL CDR3 sing amino acids having the sequence set forth in SEQ ID NO:357.
Table 32 n A GPRC5D polypeptide having the amino acid sequence of SEQ ID NO:97 VH YY [SEQ INPNNGGT [SEQ ID ARSYDY [SEQ ID NO: ID NO: 352] NO: 353] 354] VL SSNIGSNY [SEQ RNN [SEQ ID NO: 356] AAWDDSLSGRV ID NO: 355] [SEQ ID NO: 357] Full VH SGAEVKKPGSSVKVSCKASGYTFNTYYLHWVRQAPGQG LEWMGRINPNNGGTNYAQKFQGRVTMTRDTSINTAYMELSRLRSD DTAVYYCARSYDYWGQGTLVTVSS [SEQ ID NO:358] DNA CAGCTGCAGCTGGTGCAATCTGGGGCTGAGGTGAAGAAGCCTG GGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTC AACACCTACTATCTGCACTGGGTACGACAGGCCCCTGGACAAGG GCTTGAGTGGATGGGACGGATCAACCCTAACAATGGTGGCACA AACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGG ACACGTCCATCAACACAGCCTACATGGAGCTGAGCAGGCTGAG ATCTGACGACACGGCCGTGTATTACTGTGCGCGCTCTTACGATT ACTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA [SEQ ID NO:36 1] Full VL MKKTAIAIAVALAGFATVAQAAELQAVLTQPPSASGTPGQRVTISC SGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKS GTSASLAISGLRSEDEADYYCAAWDDSLSGRVFGTGTKVTVLG [SEQ ID NO:359] DNA ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTT CGCTACCGTGGCCCAGGCGGCCGAGCTCCAGGCTGTGCTGACTC AGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATC TCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATTATGTATA CTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCT ATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCT GGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCT CCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATG ACAGCCTGAGTGGTCGGGTCTTCGGAACTGGGACCAAGGTCACC GTCCTAGGT [SEQ ID NO:362] scFV MKKTAIAIAVALAGFATVAQAAELQAVLTQPPSASGTPGQRVTISC SGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKS GTSASLAISGLRSEDEADYYCAAWDDSLSGRVFGTGTKVTVLGSRG GGGSGGGGSGGGGSLEMAQLQLVQSGAEVKKPGSSVKVSCKASG YTFNTYYLHWVRQAPGQGLEWMGRINPNNGGTNYAQKFQGRVT MTRDTSINTAYMELSRLRSDDTAVYYCARSYDYWGQGTLVTVSS [SEQ ID ] DNA ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTT CGCTACCGTGGCCCAGGCGGCCGAGCTCCAGGCTGTGCTGACTC AGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATC TCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATTATGTATA CTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCT ATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCT GGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCT CCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATG TGAGTGGTCGGGTCTTCGGAACTGGGACCAAGGTCACC GTCCTAGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTC TGGTGGTGGTGGATCCCTCGAGATGGCCCAGCTGCAGCTGGTGC GGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTC AAGGCTTCTGGATACACCTTCAACACCTACTATCTGCAC TGGGTACGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAC GGATCAACCCTAACAATGGTGGCACAAACTATGCACAGAAGTTT CAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAACACAG CCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGT GTATTACTGTGCGCGCTCTTACGATTACTGGGGTCAAGGTACTCT GGTGACCGTCTCCTCAISEQH)N036? The presently disclosed subject matter further provides anti—GPRCSD scFv antibodies comprising a heavy chain le region, a light chain variable , a linker peptide between the heavy chain variable region and the light chain variable region, and an His—tag and an HA—tag. In n embodiments, the amino acid sequence of the His—tag and HA—tag comprises the amino acid sequence of SEQ ID NO:379,which is provided below: TSGQAGQHHHHHHGAYPYDVPDYAS [SEQ ID NO: 379] The tide sequence encoding SEQ ID NO: 379 is SEQ ID NO: 380, which is provided below: ACTAGTGGCCAGGCCGGCCAGCACCATCACCATCACCATGGCGCATACCC GTACGACGTTCCGGACTACGCTTCT[SEQIDPKI38m 2. Monoclonal Antibodies The presently disclosed subject matter provides human antibodies (e.g., human monoclonal antibodies) that specifically bind to GPRCSD (e.g., human GPRCSD) and were isolated and structurally characterized as described in Example 2.
The VH amino acid sequences of human anti—GPRCSD antibodies ET150—153, ETlSO-l66, ETlSO-l70, ETlSO-l7l, ETlSO-l75, ET150-154, 156, ETlSO- 157, 159, ET150-160, ET150-16l, ET150-162, ET150-163, ETTSO-lSL ET150-152, ETlSO-lSS, 158, ETlSO-l68, ETlSO-l65, ETlSO-l67, ETlSO— 169, ET150-172, ET150-173, ET150-024, ET150-026, ET150—028, ET150—029, ET150-030, ET150-03l, ET150-032 and ET150-033 are shown in SEQ ID NOs: l, 5, 9,l3,l7,2l,25,29,33,37,41,45,49,53,57,6l,65,69,73,77,8l,85,89,93,274, 286, 298, 310, 322, 334, 346 and 358, respectively. The VL amino acid sequences of ET150-153, ET150-166, ET150-170, ET150-171, ET150-175, ET150-154, ET150- 156, ET150-157, ET150-159, ET150-160, ET150-161, ET150-162, ET150-163, ET150-151, ET150-152, ET150-155, ET150-158, ET150-168, ET150—165, ET150— 167, ET150-169, ET150-172, ET150-173, ET150-024, ET150-026, ET150—028, ET150—029, ET150—030, ET150-031, ET150-032 and 033 are shown in SEQ ID NOs: 2, 6, 10, 14, 18, 22,26, 30, 34, 38, 42, 46, 50, 54,58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 275, 287, 299, 311, 323, 335, 347 and 359, respectively.
Given that each of ET150-153, ET150-166, ET150-170, ET150-171, 175, 154, 156, ET150-157, 159, ET150—160, ET150— 161, ET150-162, ET150-163, ET150-151, ET150-152, ET150-155, ET150—158, ET150-168, ET150-165, ET150-167, ET150-169, ET150-172, ET150-173, ET150- 024, ET150-026, ET150-028, ET150-029, ET150-030, ET150-031, ET150-032 and ET150—033 antibodies can bind to , the VH and VL sequences can be "mixed and d" to create other anti—GPRC5D binding molecules. GPRC5D binding of such "mixed and matched" dies can be tested using the binding assays known in the art, including for example, ELISAs, Western blots, RIAs, Biacore analysis.
Preferably, when VH and VL chains are mixed and matched, a VH sequence from a particular VH/VL pairing is ed with a structurally similar VH ce. se, a VL sequence from a particular VH/VL pairing is replaced with a structurally similar VL sequence.
In certain embodiments, the presently disclosed t matter provides an isolated antibody, or antigen—binding portion thereof comprising: (i) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 274, 286, 298, 310, 322, 334, 346 and 358; and (ii) a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42,46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 275, 287, 299, 311, 323, 335, 347 and 359; wherein the antibody specifically binds GPRC5D, e.g., human GPRC5D.
Preferred heavy and light chain combinations include: (i) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:1, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:2; or (ii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:5, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:6; (iii) a heavy chain variable region comprising amino acids having a ce set forth in SEQ ID NO:9, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO: 10; (iv) a heavy chain variable region sing amino acids having a sequence set forth in SEQ ID NO:l3, and a light chain variable region sing amino acids having a sequence set forth in SEQ ID NO: 14; (v) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:l7, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO: 18; (vi) a heavy chain le region comprising amino acids having a sequence set forth in SEQ ID NO:21, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:22; (vii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:25, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:26; (viii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:29, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:30; (iX) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:33, and a light chain le region comprising amino acids having a sequence set forth in SEQ ID NO:34; (X) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:37, and a light chain variable region comprising amino acids having a ce set forth in SEQ ID NO:38; (Xi) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:4l, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:42; (Xii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:45, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:46; (xiii) a heavy chain le region comprising amino acids having a sequence set forth in SEQ ID NO:49, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:50; (xiv) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:53, and a light chain le region comprising amino acids having a sequence set forth in SEQ ID NO:54; (xv) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:57, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:58; (xvi) a heavy chain variable region sing amino acids having a ce set forth in SEQ ID NO:6l, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:62; (xvii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:65, and a light chain le region comprising amino acids having a sequence set forth in SEQ ID NO:66; (xviii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:69, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:70; (xix) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:73, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:74; (xx) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:77, and a light chain variable region comprising amino acids having a ce set forth in SEQ ID NO:78; (xxi) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:81, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:82; (xxii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:85, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:86; (xxiii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:89, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:90; (X) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:93, and a light chain variable region sing amino acids having a sequence set forth in SEQ ID NO:94. (xvi) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:274, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:275; (xvii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:286, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:287; (xviii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:298, and a light chain variable region comprising amino acids having a ce set forth in SEQ ID NO:299; (XiX) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:3lO, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:3l 1; (xx) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:322, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:323; (XXi) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:334, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:335; (XXii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:346, and a light chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:347; or (XXiii) a heavy chain variable region comprising amino acids having a sequence set forth in SEQ ID NO:358, and a light chain le region comprising amino acids having a sequence set forth in SEQ ID NO:359.
In certain embodiments, the presently disclosed subject matter provides antibodies that se the heavy chain and light chain CDRls, CDR2s and CDR3s ofET150-153,ETlSO-l66,ETlSO-l70,ETlSO-l7l,ET150-l75,ET150-154,ET150- 156, ET150-157, 159, l60, ETlSO-l6l, ETlSO-l62, l63, lSl, ET150-152, ETlSO-lSS, ET150-158, ETlSO-l68, ETlSO—l65, ETlSO— 167, l69, l72, ETlSO-l73, ET150-024, ET150-026, ET150—028, ET150—029, ET150—030, ET150-031, ET150-032 and ET150-033 antibodies. The amino acid sequences of the VH CDRls of ET150—153, ET150—166, ET150—l70, ET150-171, ET150-175, ET150-154, ET150-156, ET150-157, ET150-159, ET150— 160, ET150-161, ET150-162, ET150-163, 151, ET150-152, ET150-155, ET150-158, ET150-168, ET150-165, 167, ET150-169, ET150-172, ET150- 173, ET150-024, ET150-026, ET150-028, ET150—029, 030, ET150—031, ET150-032 and ET150-033 are shown in SEQ ID NOs: 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196, 202, 208, 214, 220, 226, 232, 238, 244, 250, 256, 262, 268, 280, 292, 303, 316, 328, 340 and 352, tively. The amino acid sequences of the VH CDR2s of 153, ET150—166, ET150—170, ET150—171, ET150-175, ET150-154, ET150-156, 157, ET150-159, ET150—160, ET150— 161, ET150-162, ET150-163, ET150-151, ET150-152, ET150-155, ET150—158, ET150-168, 165, ET150-167, ET150-169, ET150-172, 173, ET150- 024, ET150-026, ET150-028, ET150-029, ET150-030, ET150-031, ET150-032 and ET150—033 antibodies are shown in SEQ ID NOs: 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197, 203, 209, 215, 221, 227, 233, 239, 245, 251, 257, 263, 269, 281, 293, 304, 317, 329, 341 and 353, respectively. The amino acid sequences of the VH CDR3s of ET150-153, ET150-166, ET150-170, ET150-171, ET150-175, ET150-154, ET150-156, ET150-157, ET150-159, 160, 161, ET150- 162, ET150-163, ET150-151, ET150-152, ET150-155, ET150—158, ET150—168, ET150-165, ET150-167, ET150-169, ET150-172, ET150-173, ET150-024, ET150- 026, ET150—028, ET150—029, ET150—030, 031, ET150—032 and ET150—033 are shown in SEQ ID NOs: 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, 240, 246, 252, 258, 264, 270, 282, 294, 305, 318, 330, 342 and 354, respectively.
The amino acid sequences of the VL CDRls of 1 ET150—153, ET150—l66, ET150-170, ET150-171, ET150-175, ET150-154, ET150-156, ET150-157, ET150- 159, 160, ET150-161, ET150-162, ET150-163, ET150-151, ET150-152, ET150-155, ET150-158, ET150-168, ET150-165, ET150-167, ET150-169, ET150- 172, ET150-173, ET150-024, ET150-026, ET150—028, ET150—029, ET150—030, ET150-031, ET150-032 and ET150-033 are shown in SEQ ID NOs: 127, 133, 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, 211, 217, 223, 229, 235, 241, 247, 253, 259, 265, 271, 283, 295, 306, 319, 331, 343 and 355, respectively. The amino acid sequences of the VL CDR2s of ET150—153, ET150—166, ET150—l70, ET150-171, ET150-175, ET150-154, ET150-156, ET150-157, ET150-159, ET150— 160, ET150-161, 162, ET150-163, ET150-151, ET150-152, ET150-155, ET150-158, ET150-168, ET150-165, ET150-167, ET150-169, 172, ET150- 173, ET150-024, ET150-026, ET150-028, ET150—029, ET150—030, ET150—031, ET150-032 and ET150-033 are shown in SEQ ID NOs: 128, 134, 140, 146, 152, 158, 164, 170, 176, 182, 188, 194, 200, 206, 212, 218, 224, 230, 236, 242, 248, 254, 260, 266, 272, 284, 296, 307, 320, 332, 344 and 356, respectively. The amino acid sequences of the VL CDR3s of ET150—153, ET150—166, ET150—170, ET150—171, 175, ET150-154, ET150-156, ET150-157, 159, ET150—160, ET150— 161, ET150-162, ET150-163, ET150-151, ET150-152, ET150-155, ET150—158, ET150-168, ET150-165, ET150-167, ET150-169, ET150-172, ET150-173, ET150- 024, ET150-026, ET150-028, ET150-029, ET150-030, ET150-031, ET150-032 and ET150-033 are shown in SEQ ID NOs: 129, 135, 141, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, 213, 219, 225, 231, 237, 243, 249, 255, 261, 267, 273, 285, 297, 308, 321, 333, 345 and 357, respectively. The CDR regions are delineated using the Kabat system , E. A., et al. (1991) Sequences of Proteins of logical Interest, Fifth Edition, US. Department of Health and Human Services, NIH Publication No. 91—3242).
Given that each of these antibodies can bind to GPRC5D and that antigen— binding specificity is provided primarily by the CDRl, CDR2, and CDR3 regions, the VH CDRl, CDR2, and CDR3 sequences and VL CDRl, CDR2, and CDR3 sequences can be "mixed and matched" (i.e., CDRs from different antibodies can be mixed and match, although each antibody must n a VH CDRl, CDR2, and CDR3 and a V L CDRl, CDR2, and CDR3) to create other PRC5D binding molecules.
GPRC5D binding of such "mixed and matched" antibodies can be tested using the binding assays described above. When VH CDR sequences are mixed and matched, the CDRl, CDR2 and/or CDR3 sequence from a particular VH sequence is ed with a structurally similar CDR sequence(s). Likewise, when VL CDR ces are mixed and matched, the CDRl, CDR2 and/or CDR3 sequence from a particular VL sequence preferably is ed with a structurally similar CDR sequence(s). It will be readily apparent to the ordinarily skilled artisan that novel VH and VL ces can be created by substituting one or more VH and/or VL CDR region sequences with structurally similar sequences from the CDR sequences of the antibodies disclosed herein ET150-153, ET150-166, 170, ET150-171, ET150-175, ET150-154, ET150-156, ET150-157, 159, ET150-160, ET150-161, ET150-162, ET150- 163, ET150-151, ET150-152, ET150-155, ET150-158, ET150—168, ET150—165, ET150-167, ET150-169, ET150-172, ET150-173, ET150-024, ET150-026, ET150- 028, ET150—029, ET150—030, ET150—031, ET150—032 and ET150—033.
In certain embodiments, the presently disclosed t matter provides an isolated antibody, or antigen—binding n f comprising: (iX) a heavy chain variable region CDRl sing an amino acid sequence selected from the group consisting of SEQ ID NOs: 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196, 202, 208, 214, 220, 226, 232, 238, 244, 250, 256, 262, 268, 280, 292, 303, 316, 328, 340 and 352; (ii)a heavy chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197, 203, 209, 215, 221, 227, 233, 239, 245, 251, 257, 263, 269, 281, 293, 304, 317, 329, 341 and 353; (iii) a heavy chain variable region CDR3 sing an amino acid sequence selected from the group consisting of SEQ ID NOs: 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, 240, 246, 252, 258, 264, 270, 282, 294, 305, 318, 330, 342 and 354; (iv) a light chain variable region CDRl comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 127, 133, 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, 211, 217, 223, 229, 235, 241, 247, 253, 259, 265, 271, 283, 295, 306, 319, 331, 343 and 355; (v) a light chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 128, 134, 140, 146, 152, 158, 164, 170, 176, 182, 188, 194, 200, 206, 212, 218, 224, 230, 236, 242, 248, 254, 260, 266, 272, 284, 296, 307, 320, 332, 344 and 356; and (f) a light chain variable region CDR3 comprising an amino acid sequence selected from the group ting of SEQ ID NOs: 129, 135, 141, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, 213, 219, 225, 231, 237, 243, 249, 255, 261, 267, 273, 285, 297, 308, 321, 333, 345 and 357; wherein the antibody specifically binds GPRC5D, e.g., human GPRC5D.
In certain embodiments, the antibody comprises: (i) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 124; (ii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 125; (iii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 126; (iv) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 127; (v) a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:128; and (vi) a light chain variable region CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 129.
In certain embodiments, the antibody comprises: (i) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 130; (ii) a heavy chain variable region CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 131; (iii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 132; (iv) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 133; (v) a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:134; and (vi) a light chain variable region CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 135.
In certain ments, the antibody comprises: (i) a heavy chain le region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 136; (ii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 137; (iii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 138; (iv) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 139; (V) a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:l40; and (vi) a light chain variable region CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 141.
In certain embodiments, the antibody comprises: (i) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 142; (ii) a heavy chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID NO: 143; (iii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 144; (iv) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 145; (v) a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:l46; and (vi) a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 147.
In certain ments, the antibody comprises: (i) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 148; (ii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 149; (iii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 150; (iv) a light chain variable region CDRl sing amino acids having the sequence set forth in SEQ ID NO: 151; (v) a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:l52; and (vi) a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 153.
In certain embodiments, the antibody comprises: (i) a heavy chain variable region CDRl sing amino acids having the sequence set forth in SEQ ID NO: 154; (ii) a heavy chain variable region CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 155; (iii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 156; (iv) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 157; (v) a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:158; and (vi) a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 159.
In certain embodiments, the antibody comprises: (i) a heavy chain variable region CDRl sing amino acids having the sequence set forth in SEQ ID NO: 160; (ii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 161; (iii) a heavy chain variable region CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 162; (iv) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 163; (v) a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:164; and (vi) a light chain le region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 165.
In certain embodiments, the antibody comprises: (i) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 166; (ii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 167; (iii) a heavy chain variable region CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 168; (iv) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 169; (V) a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:170; and (vi) a light chain le region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 171.
In n ments, the antibody ses: (i) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 172; (ii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 173; (iii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 174; (iv) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 175; (v) a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:176; and (vi) a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 177.
In certain embodiments, the antibody comprises: (i) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 178; (ii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 179; (iii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 180; (iv) a light chain variable region CDRl sing amino acids having the sequence set forth in SEQ ID NO: 181; (v) a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:182; and (vi) a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 183.
In certain embodiments, the antibody comprises: (i) a heavy chain le region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 184; (ii) a heavy chain le region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 185; (iii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 186; (iv) a light chain le region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 187; (v) a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:188; and (vi) a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 189.
In certain embodiments, the antibody comprises: (i) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 190; (ii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 191; (iii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 192; (iv) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 193; (v) a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:194; and (vi) a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 195.
In certain embodiments, the antibody comprises: (i) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 196; (ii) a heavy chain le region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 197; (iii) a heavy chain le region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 198; (iv) a light chain le region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 199; (XXii) a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:200; and (vi) a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 201.
In certain ments, the antibody ses: (i) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 202; (ii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 203; (iii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 204; (iv) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 205; (XXii) a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:206; and (vi) a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 207.
In certain embodiments, the antibody comprises: (i) a heavy chain le region CDRl sing amino acids having the sequence set forth in SEQ ID NO: 208; (ii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 209; (iii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 210; (iv) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 211; (XXii) a light chain variable region CDR2 comprising amino acids having the ce set forth in SEQ ID NO:2l2; and (vi) a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 213.
In n embodiments, the antibody comprises: (i) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 214; (ii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 215; (iii) a heavy chain variable region CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 216; (iv) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 217; (XXii) a light chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID NO:218; and (vi) a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 219.
In certain embodiments, the antibody comprises: (i) a heavy chain le region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 220; (ii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 221; (iii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 222; (iv) a light chain variable region CDRl comprising amino acids having the ce set forth in SEQ ID NO: 223; (XXii) a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:224; and (vi) a light chain variable region CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 225.
In certain embodiments, the antibody comprises: (i) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 226; (ii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 227; (iii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 228; (iv) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 229; (XXii) a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:230; and (vi) a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 231.
In certain embodiments, the antibody comprises: (i) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 232; (ii) a heavy chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID NO: 233; (iii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 234; (iv) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 235; (XXii) a light chain le region CDR2 comprising amino acids having the sequence set forth in SEQ ID ; and (vi) a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 237.
In n embodiments, the antibody comprises: (i) a heavy chain le region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 238; (ii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 239; (iii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 240; (iv) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 241; (XXii) a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:242; and (vi) a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 243.
In certain embodiments, the antibody comprises: (i) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 244; (ii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 245; (iii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 246; (iv) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 247; (XXii) a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:248; and (vi) a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 249.
In certain embodiments, the antibody comprises: (i) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 250; (ii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 251; (iii) a heavy chain variable region CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 252; (iv) a light chain le region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 253; (XXii) a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID ; and (vi) a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 255.
In certain embodiments, the antibody comprises: (i) a heavy chain variable region CDRl comprising amino acids having the ce set forth in SEQ ID NO: 256; (ii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 257; (iii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 258; (iv) a light chain le region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 259; (XXii) a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID ; and (vi) a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 261.
In certain embodiments, the antibody comprises: (i) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 262; (ii) a heavy chain le region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 263; (iii) a heavy chain variable region CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 264; (iv) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 265; (XXii) a light chain le region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:266; and (vi) a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 267.
In certain embodiments, the dy comprises: (i) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 268; (ii) a heavy chain variable region CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 269; (iii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 270; (iv) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 271; (XXii) a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:272; and (vi) a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 273.
In certain embodiments, the antibody comprises: (i) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 280; (ii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 281; (iii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 282; (iv) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 283; (XXii) a light chain le region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:284; and (vi) a light chain variable region CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 285.
In certain embodiments, the antibody comprises: (i) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 292; (ii) a heavy chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID NO: 293; (iii) a heavy chain le region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 294; (iv) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 295; (XXii) a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:296; and (vi) a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 297.
In certain embodiments, the antibody comprises: (i) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 303; (ii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 304; (iii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 305; (iv) a light chain le region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 306; (XXii) a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 307; and (vi) a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 308.
In certain embodiments, the antibody comprises: (i) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 316; (ii) a heavy chain variable region CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 317; (iii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 318; (iv) a light chain le region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 319; (XXii) a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 320; and (vi) a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 321.
In certain embodiments, the antibody comprises: (i) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 328; (ii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 329; (iii) a heavy chain le region CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 330; (iv) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 331; (XXii) a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 332; and (vi) a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 333.
In certain embodiments, the antibody ses: (i) a heavy chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 340; (ii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 341; (iii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 342; (iv) a light chain variable region CDRl comprising amino acids having the ce set forth in SEQ ID NO: 343; (XXii) a light chain variable region CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 344; and (vi) a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 345.
In certain embodiments, the antibody comprises: (i) a heavy chain variable region CDRl sing amino acids having the sequence set forth in SEQ ID NO: 352; (ii) a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 353; (iii) a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 354; (iv) a light chain variable region CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 355; (XXii) a light chain variable region CDR2 sing amino acids having the sequence set forth in SEQ ID NO: 356; and (vi) a light chain le region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 357.
The constant region/framework region of the anti—GPRC5D antibodies disclosed herein can be altered, for example, by amino acid substitution, to modify the properties of the antibody (e.g., to increase or decrease one or more of: antigen binding affinity, Fc receptor binding, antibody carbohydrate, for example, glycosylation, fucosylation etc the number of cysteine residues, effector cell function, effector cell function, complement function or uction of a conjugation site).
In certain embodiments, a presently disclosed PRC5D antibody is a fully—human antibody, e.g., any one of ETl50—l53, ETl50—l66, ETl50—l70, ETl50— l7l, l75, ETl50-l54, ETl50-l56, ETl50-l57, ETl50—l59, l60, ET150-l6l, ETl50-l62, ETl50-l63, ETl50-l5l, ETl50-l52, ETl50-l55, ET150- 158, ET150-168, ET150-165, ET150-167, ET150-169, ET150-172, ET150-173, ET150-024, ET150-026, ET150-028, ET150-029, ET150—030, ET150—031, ET150— 032 and ET150—033. Fully—human mAbs are preferred for therapeutic use in humans because murine antibodies cause an immunogenicity reaction, known as the HAMA (human anti—mouse antibodies) response (Azinovic I, et al. Survival t associated with human anti—mouse antibody (HAMA) in patients with B—cell malignancies.
Cancer Immunol Immunother 2006; 55(12):1451—8; Tjandra JJ, et al. Development of human anti—murine antibody (HAMA) response in patients. l Cell Biol 1990; 68(6):367—76), when administered to humans, causing serious side effects, including anaphylaXis and hypersensitivity reactions. This immunogenicity on is triggered by the human immune system recognizing the murine antibodies as n because of slightly different amino acid sequences from natural human antibodies. Humanization methods known in the art (Riechmann L, et al. Reshaping human antibodies for therapy. Nature 1988; 332 (6162): 3; Queen C, et al. A humanized antibody that binds to the interleukin 2 or. Proc Natl Acad Sci USA 1989; 86 (24): 10029—33) can be employed to reduce the immunogenicity of murine—derived antibodies (Gerd R, et al. gical Analysis of Human uman dy ses in Colon Cancer Patients Treated with Repeated Doses of Humanized Monoclonal Antibody A33. Cancer Res 2001; 61, 6851—6859).
The use of phage display libraries has made it possible to select large numbers of Ab repertoires for unique and rare Abs against very defined epitopes (for more s on phage display see McCafferty et al., Phage antibodies: filamentous phage displaying antibody variable domains. Nature, 348: 552—554.) The rapid identification of human Fab or single chain Fv (ScFV) fragments highly specific for tumor antigen—derived peptide—MHC complex molecules has thus become possible (19—22). Recently, immuno—toxins, generated by fusing TCR—like Fab specific for ma Ag MART—1 26—35/A2 or gp100 280—288/A2 to a ted form of Pseudomonas endotoxin, have been shown to t human melanoma growth both in vitro and in vivo (Klechevsky E, et al. Antitumor activity of immunotoxins with T— cell receptor—like specificity against human melanoma xenografts. Cancer Res 2008; 68 (15): 6360— 6367). In addition, by engineering full—length mAb using the Fab fragments, it is possible to directly te a therapeutic human mAb, bypassing months of time—consuming work, normally needed for developing therapeutic mAbs.
The presently disclosed subject matter involves the pment of a fully human mAb that recognizes, for example, a human GPRC5D polypeptide (e. g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:97) for cancer y. 3. Homologous Antibodies In certain embodiments, an antibody of the presently disclosed subject matter comprises heavy and light chain variable regions comprising amino acid sequences that are homologous to the amino acid sequences of the antibodies described herein (e.g., ET150-153, ET150-166, ET150-170, ET150-171, ET150-175, ET150-154, ET150-156, ET150-157, 159, ET150-160, ET150-161, ET150- 162, ET150-163, ET150-151, 152, ET150-155, ET150—158, ET150—168, ET150-165, ET150-167, ET150-169, ET150-172, ET150-173, ET150-024, ET150- 026, ET150—028, ET150—029, ET150—030, ET150—031, ET150—032 and ET150—033 antibodies), and wherein the dies retain the desired functional properties of the anti—PGPRC5D antibodies of the presently sed t matter.
For example, the tly disclosed subject matter provides an ed antibody, or antigen—binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein: (a) the heavy chain variable region comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to an amino acid ce selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 274, 286, 298, 310, 322, 334, 346 and 358; and (b) the light chain variable region comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 275, 287, 299, 311, 323, 335, 347 and 359; and the antibody binds to human GPRC5D with a Kd of 1 X 10'7 M or less.
In certain embodiments, the VH and/or VL amino acid sequences can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequences set forth above. An dy having VH and VL regions having high (i.e., 80% or greater) homology to the VH and VL regions of the sequences set forth above, can be ed by mutagenesis (e. g., site—directed or PCR—mediated mutagenesis), followed by testing of the d altered dy for retained function (i.e., the binding affinity) using the binding assays described herein.
As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical ons shared by the ces (i.e., % homology 2 # of identical positions/total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of t ty between two ces can be lished using a mathematical algorithm, as described in the non—limiting examples below.
The percent homology between two amino acid sequences can be determined using the thm of E. Meyers and W. Miller t. Appl. Biosci., 4:11—17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length y of 12 and a gap penalty of 4. In addition, the percent homology between two amino acid sequences can be ined using the Needleman and Wunsch (J. Mol. Biol. 48:444—453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 , and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, , or 6.
Additionally or alternatively, the protein sequences of the presently disclosed subject matter can further be used as a "query sequence" to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215 :403—10. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the antibody molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389—3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. (See www.ncbi.nlm.nih.gov). 4. Antibodies with Conservative Modifications In certain embodiments, an antibody of the presently disclosed t matter comprises a heavy chain variable region comprising CDRl, CDR2 and CDR3 sequences and a light chain variable region comprising CDRl, CDR2 and CDR3 sequences, wherein one or more of these CDR sequences comprise specified amino acid sequences based on the preferred antibodies bed herein (e.g., ET150—153, ET150-166, ET150-170, ET150-171, ET150-175, ET150-154, ET150-156, ET150- 157, ET150-159, ET150-160, ET150-161, ET150-162, ET150-163, ET150-151, ET150-152, ET150-155, ET150-158, ET150-168, ET150-165, ET150-167, ET150— 169, 172, ET150-173, ET150-024, 026, ET150—028, 029, ET150—030, ETl50—031, ET150—032 and 033 antibodies), or vative modifications thereof, and wherein the antibodies retain the d functional properties of the anti—GPRC5D antibodies of the presently disclosed subject matter.
The presently disclosed subject matter provides an isolated antibody, or antigen—binding portion thereof, sing a heavy chain variable region comprising CDRl, CDR2, and CDR3 sequences and a light chain variable region comprising CDRl, CDR2, and CDR3 sequences, wherein: (a) the heavy chain le region CDR3 sequence comprises an amino acid sequence selected from the group ting of amino acid sequences of SEQ ID NOs: 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, 240, 246, 252, 258, 264, 270, 282, 294, 305, 318, 330, 342 and 354, and conservative modifications thereof; (b) the light chain variable region CDR3 sequence comprises an amino acid sequence selected from the group consisting of amino acid sequence of SEQ ID NOs: 129, 135, 141, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, 213, 219, 225, 231, 237, 243, 249, 255, 261, 267, 273, 285, 297, 308, 321, 333, 345 and 357, and 431, and conservative modifications thereof; and the antibody exhibits binds to human GPRCSD with a Kd of 1 x 10'7 M or less.
In certain embodiments, the heavy chain variable region CDR2 sequence comprises an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOs: 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197, 203, 209, 215, 221, 227, 233, 239, 245, 251, 257, 263, 269, 281, 293, 304, 317, 329, 341 and 353, and conservative modifications thereof; and the light chain variable region CDR2 sequence comprises an amino acid sequence selected from the group ting of amino acid sequences of SEQ ID NOs: 128, 134, 140, 146, 152, 158, 164, 170, 176, 182, 188, 194, 200, 206, 212, 218, 224, 230, 236, 242, 248, 254, 260, 266, 272, 284, 296, 307, 320, 332, 344 and 356, and conservative cations thereof.
In certain embodiments, the heavy chain variable region CDR1 sequence comprises an amino acid sequence selected from the group consisting of amino acid ces of SEQ ID NOs: 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196, 202, 208, 214, 220, 226, 232, 238, 244, 250, 256, 262, 268, 280, 292, 303, 316, 328, 340 and 352, and conservative cations thereof; and the light chain variable region CDR1 sequence comprises an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOs: 127, 133, 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, 211, 217, 223, 229, 235, 241, 247, 253, 259, 265, 271, 283, 295, 306, 319, 331, 343 and 355, and conservative modifications thereof.
As used , the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not icantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions.
Modifications can be introduced into an antibody of the invention by standard techniques known in the art, such as site—directed nesis and PCR—mediated nesis.
Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having r side chains have been defined in the art.
Exemplary conservative amino acid substitutions are shown in Table 33. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e. g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC. In certain embodiments, a sequence disclosed herein, e.g., a CDR sequence, a VH sequence or a VL ce, can have up to about one, up to about two, up to about three, up to about four, up to about five, up to about siX, up to about seven, up to about eight, up to about nine or up to about ten amino acid residues that are modified and/or substituted.
Table 33 Original Residue Exemplary vative amino acid Substitutions Ala (A) Val; Leu; 116 Arg (R) Lys; Gln; Asn Asn (N) Gln; His; Asp, Lys; Arg Cys (C) Ser; Ala Gln (Q) Asn; Glu Glu (E) Asp; Gln Gly (G) Ala His (H) Asn; Gln; Lys; Arg 116 (1) Leu; Val; Met; Ala; Phe AAA AAA Pro (P) Ala Ser (S) Thr AAAAAA Amino acids may be grouped according to common side—chain properties: 0 hydrophobic: Norleucine, Met, Ala, Val, Leu, He; 0 neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; 0 acidic: Asp, Glu; 0 basic: His, Lys, Arg; 0 residues that in?uence chain orientation: Gly, Pro; 0 ic: Trp, Tyr, Phe.
Non—conservative substitutions will entail exchanging a member of one of these classes for another class.
. Anti-GPRCSD Antibodies that Cross-compete for Binding to GPRCSD with Anti-GPRCSD Antibodies of the Invention The presently disclosed subject matter provides antibodies that cross— compete with any of the disclosed anti—GPRCSD antibodies for binding to GPRCSD (e.g., human GPRCSD). For example, and not by way of limitation, the cross— competing dies can bind to the same epitope region, e.g., same epitope, adjacent epitope, or overlapping as any of the anti—GPRCSD antibodies of the presently sed subject . In certain embodiments, the reference antibody for cross— competition studies can be any one of the anti—GPRCSD antibodies disclosed herein, e.g., ET150-153, ET150-166, ET150-170, ET150-171, ET150-175, ET150-154, ET150-156, ET150-157, ET150-159, ET150-160, ET150-161, ET150-162, ET150- 163, ET150-151, ET150-152, 155, ET150-158, ET150—168, ET150—165, ET150-167, ET150-169, ET150-172, ET150-173, ET150-024, ET150-026, ET150- 028, ET150—029, ET150—030, ET150-031, 032 and ET150-033 antibodies.
In certain embodiments, the cross—competing dy binds to an e region comprising amino acids 14—22 of SEQ ID NO: 97. In n embodiments, the cross—competing dy binds to one, two, three, four, five, six, or seven epitope regions selected from the group consisting of amino acids 5—17, 10—17, 1—27, 15—23, 16—23, 16—25, 85—93, 85—95, 145—167, 157—164, 157-167, 226-239, 230-237, 229-237, 3 and 227-237 of SEQ ID NO: 97.
Such cross—competing antibodies can be identified based on their ability to cross—compete with any one of the presently disclosed anti—GPRCSD antibodies in standard GPRCSD binding assays. For example, Biacore analysis, ELISA assays or ?ow cytometry can be used to demonstrate cross—competition with the antibodies of the presently disclosed subject matter. The ability of a test antibody to t the binding of, for example, any one of the presently sed GPRCSD antibodies (e.g., ET150-153, ET150-166, ET150-170, ET150-171, ET150-175, ET150-154, ET150- 156, 157, ET150-159, ET150-160, ET150-161, ET150-162, ET150-163, 151, ET150-152, ET150-155, ET150-158, ET150-168, ET150—165, ET150— 167, ET150-169, ET150-172, ET150-173, ET150-024, ET150-026, ET150—028, 029, ET150—030, ET150-031, ET150-032 and ET150-033 antibodies) to human GPRCSD demonstrates that the test antibody can compete with any one of the presently disclosed PRCSD antibodies for binding to human GPRCSD and thus binds to the same epitope region on human GPRCSD as any one of the presently disclosed anti—GPRCSD antibodies. In certain embodiments, the cross—competing antibody binds to the same e on human GPRCSD as any one of the presently disclosed anti—GPRCSD antibodies. 6. Characterization of Antibody Binding to Antigen Antibodies of the presently disclosed subject can be tested for binding to GPRCSD by, for example, standard ELISA. To determine if the selected anti— GPRCSD antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Competition studies using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be performed using GPRCSD coated—ELISA plates as described above. Biotinylated mAb binding can be detected with a strep—avidin—alkaline phosphatase probe.
To determine the e of purified antibodies, isotype ELISAs can be performed using ts specific for antibodies of a particular isotype. Anti— GPRCSD human IgGs can be further tested for reactivity with GPRCSD antigen by Western blotting.
In certain embodiments, Kd is measured by a radiolabeled antigen g assay (RIA). In certain ments, an RIA is performed with the Fab version of an dy of interest and its antigen. For example, solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of (1251)— labeled antigen in the presence of a titration series of unlabeled n, then capturing bound n with an anti—Fab antibody—coated plate (see, e.g., Chen et al., J. Mol.
Biol. 293:865—881(l999)).
In certain embodiments, Kd is measured using a BIACORE® surface plasmon nce assay. For example, an assay using a E®—2000 or a E ®—3000 (BIAcore, Inc., Piscataway, NJ). e Mapping In certain embodiments, the antibody or an antigen—binding fragment thereof binds to a human GPRCSD polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 97. In certain embodiments, the antibody or an n— binding fragment thereof binds to one, two, three or four of N—terminal region (amino acids l—27 of SEQ ID NO:97), ECLl region (amino acids 85—93 of SEQ ID NO:97), ECL2 region (amino acids 145—167 of SEQ ID NO:97), and ECL3 region (amino acids 226—239 of SEQ ID NO:97). In certain embodiments, an antibody or an antigen— binding fragment thereof of the presently disclosed subject matter binds to an epitope region in the N—terminal region, including, but not d to, an epitope region comprising amino acids 16—23 of SEQ ID NO:97, and an epitope region comprising amino acids 10—17 of SEQ ID NO:97. In certain embodiments, the epitope region in the N—terminal region comprises amino acids 15—23 of SEQ ID NO:97. In n embodiments, the epitope region in the N—terminal region comprises amino acids 16— of SEQ ID NO:97. In n embodiments, the epitope region in the N—terminal region comprises amino acids 10—17 of SEQ ID NO:97. In certain embodiments, the e region in the N—terminal region comprises amino acids 5—17 of SEQ ID NO:97.
In certain embodiments, an antibody or an antigen—binding fragment thereof of the presently disclosed subject matter binds to an epitope region in the ECLl region, including, but not limited to, an epitope region comprising amino acids 85—95 of SEQ ID NO:97.
In certain embodiments, an antibody or an antigen—binding fragment thereof of the presently sed subject matter binds to an epitope region in the ECL2 region, including, but not limited to, an epitope region comprising amino acids 157—164 of SEQ ID NO:97. In certain ments, the epitope region in the ECL2 region comprises amino acids 157—164 of SEQ ID NO:97. In certain embodiments, the epitope region in the ECL2 region comprises amino acids 157—167 of SEQ ID NO:97.
In certain embodiments, an antibody or an antigen—binding fragment thereof of the presently disclosed subject matter binds to an e region in the ECL3 region, including, but not limited to, an epitope region comprising amino acids 230—237 of SEQ ID NO:97. In certain embodiments, the epitope region in the ECL3 region comprises amino acids 7 of SEQ ID NO:97. In certain embodiments, the e region in the ECL3 region comprises amino acids 230—243 of SEQ ID NO:97. In certain embodiments, the epitope region in the ECL3 region comprises amino acids 227—237 of SEQ ID NO:97.
In certain embodiments, the antibody or an antigen—binding nt thereof binds to an epitope region comprising amino acids 16—25 of SEQ ID NO:97, an epitope region comprising amino acids 157—164 of SEQ ID NO:97, and an epitope region comprising amino acids 229—237 of SEQ ID NO:97. For example, the antibody or an antigen—binding nt thereof comprises a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:57 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:58, ally with (iii) a linker sequence, for example a linker peptide, n the heavy chain variable region and the light chain variable region. In certain embodiments, the linker comprises amino acids having the ce set forth in SEQ ID NO:98. In certain embodiments, the antibody or an antigen—binding fragment f is a scFv. In n embodiments, the antibody or an antigen—binding nt thereof is a scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 15. In certain embodiments, the antibody or an antigen—binding nt thereof comprises a VH comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence set forth in SEQ ID NO:57. In certain embodiments, the antibody or an antigen—binding fragment thereof comprises a VH comprising amino acids having the ce set forth in SEQ ID NO:57. In n embodiments, the antibody or an antigen—binding fragment thereof comprises a VL comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence set forth in SEQ ID NO:58. In certain embodiments, the antibody or an antigen—binding fragment thereof comprises a VL sing amino acids having the sequence set forth in SEQ ID NO:58. In certain embodiments, the antibody or an antigen—binding fragment thereof comprises a VH comprising amino acids having the ce set forth in SEQ ID NO:57 and a VL comprising amino acids having the sequence set forth in SEQ ID NO:58. In certain embodiments, the antibody or an antigen—binding fragment thereof comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:208 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 209 or conservative modifications thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 210 or conservative modifications thereof. In certain embodiments, the antibody or an antigen—binding fragment thereof comprises a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 211 or conservative modifications thereof, a VL CDR2 sing amino acids having the sequence set forth in SEQ ID NO: 212 or conservativemodifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 213 or conservative modifications thereof. In certain embodiments, the antibody or an antigen—binding nt thereof comprises a VH CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 208 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 209 or conservative modifications f, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 210 or conservative modifications thereof, a VL CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 211 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 212 or conservativemodifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 213 or conservative modifications thereof. In certain embodiments, the antibody or an antigen—binding fragment thereof comprises a VH CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 208, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 209, a VH CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 210, a VL CDR1 comprising amino acids having the ce set forth in SEQ ID NO: 211, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 212, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 213. In certain embodiments, the antibody or an n—binding fragment thereof is ET150—2 scFv (or ET150—152 scFv).
In certain embodiments, the antibody or an antigen—binding nt thereof binds to an epitope region comprising amino acids 5—17 of SEQ ID NO:97, an epitope region comprising amino acids 85—95 of SEQ ID NO:97, and an e region comprising amino acids 157—164 of SEQ ID NO:97. For example, the antibody or an antigen—binding fragment thereof comprises a heavy chain variable region sing amino acids having the sequence set forth in SEQ ID NO:61 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:62, optionally with (iii) a linker sequence, for example a linker e, between the heavy chain variable region and the light chain variable region. In certain ments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the antibody or an antigen—binding fragment thereof is a scFv. In certain embodiments, the antibody or an antigen—binding fragment thereof is a scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 16. In certain embodiments, the antibody or an antigen—binding fragment thereof comprises a VH comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid ce set forth in SEQ ID NO:6l. In certain embodiments, the antibody or an antigen—binding fragment thereof comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:6l. In certain embodiments, the antibody or an antigen—binding fragment thereof comprises a VL comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid ce set forth in SEQ ID NO:62. In certain embodiments, the antibody or an antigen—binding fragment f comprises a VL sing amino acids having the sequence set forth in SEQ ID NO:62. In certain embodiments, the dy or an antigen—binding nt thereof comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:6l and a VL comprising amino acids having the sequence set forth in SEQ ID NO:62. ain embodiments certain embodiments, the antibody or an antigen—binding fragment thereof comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:2l4 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 215 or conservative modifications thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 216 or conservative modifications thereof. In certain embodiments, the antibody or an antigen—binding nt thereof comprises a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 217 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 218 or conservativemodifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 219 or conservative modifications thereof. In certain embodiments, the antibody or an antigen—binding fragment thereof comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 214 or conservative cations f, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 215 or vative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 216 or conservative modifications thereof, a VL CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 217 or vative modifications f, a VL CDR2 comprising amino acids having the ce set forth in SEQ ID NO: 218 or conservativemodifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 219 or conservative modifications thereof. In certain embodiments, the antibody or an n—binding fragment thereof comprises a VH CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 214, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 215, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 216, a VL CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 217, a VL CDR2 sing amino acids having the sequence set forth in SEQ ID NO: 218 and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 219. In certain embodiments, the antibody or an antigen—binding fragment thereof is ET150—155 scFv (or ET150—5 scFv).
In certain embodiments, the antibody or an n—binding fragment f binds to an epitope region comprising amino acids 15—23 of SEQ ID NO:97, and an epitope region comprising amino acids 230—243 of SEQ ID NO:97, For example, the antibody or an antigen—binding fragment thereof ses a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:65 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:66, optionally with (iii) a linker sequence, for example a linker peptide, between the heavy chain variable region and the light chain variable region. In certain embodiments, the linker ses amino acids having the sequence set forth in SEQ ID NO:98. In certain embodiments, the antibody or an antigen—binding fragment thereof is a scFv. In certain embodiments, the antibody or an n—binding fragment thereof is a scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 17. In certain embodiments, the antibody or an antigen—binding fragment thereof comprises a VH comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence set forth in SEQ ID NO:65. In certain embodiments, the antibody or an n—binding fragment thereof comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:65. In certain embodiments, the antibody or an antigen—binding fragment thereof comprises a VL comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence set forth in SEQ ID NO:66, as shown in Table 17. In n embodiments, the antibody or an antigen—binding fragment thereof comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:66. In certain embodiments, the antibody or an antigen—binding fragment thereof comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:65 and a VL comprising amino acids having the sequence set forth in SEQ ID NO:66. In n embodiments, the antibody or an n—binding nt thereof comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO:220 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 221 or conservative modifications thereof, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 222 or conservative modifications thereof. In certain embodiments, the antibody or an n—binding fragment thereof comprises a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 223 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 224 or conservativemodifications f, and a VL CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 225 or conservative modifications thereof. In n embodiments, the antibody or an antigen—binding fragment thereof comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 220 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 221 or conservative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 222 or conservative modifications thereof, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 223 or conservative cations thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 224 or conservativemodifications thereof, and a VL CDR3 sing amino acids having the sequence set forth in SEQ ID NO: 225 or conservative modifications thereof. In certain embodiments, the antibody or an antigen—binding fragment thereof comprises a VH CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 220, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 221, a VH CDR3 comprising amino acids having the ce set forth in SEQ ID NO: 222, a VL CDRl comprising amino acids having the sequence set forth in SEQ ID NO: 223, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 224, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 225. In certain embodiments, the antibody or an antigen—binding fragment thereof is ET150—8 scFv (or ET150—158 scFv).
In n embodiments, the antibody or an antigen—binding fragment thereof binds to an epitope region comprising amino acids 10—17 of SEQ ID NO:97, an epitope region comprising amino acids 157—167 of SEQ ID NO:97, and an epitope region comprising amino acids 227—237 of SEQ ID NO:97, For example, the antibody or an antigen—binding fragment thereof comprises a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:69 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:70, optionally with (iii) a linker sequence, for e a linker peptide, between the heavy chain variable region and the light chain variable region. In certain embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO:98. In certain ments, the antibody or an antigen—binding fragment thereof is a scFv. In n ments, the antibody or an antigen—binding fragment thereof is a scFv—Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 18. In certain embodiments, the antibody or an antigen—binding fragment thereof comprises a VH comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence set forth in SEQ ID NO:69. In certain embodiments, the dy or an n—binding fragment thereof comprises a VH sing amino acids having the sequence set forth in SEQ ID NO:69. In certain ments, the antibody or an antigen—binding fragment thereof comprises a VL comprising an amino acid ce that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence set forth in SEQ ID NO:70. In certain embodiments, the antibody or an antigen—binding fragment thereof comprises a VL comprising amino acids having the sequence set forth in SEQ ID NO:70. In certain embodiments, the antibody or an antigen—binding fragment thereof comprises a VH comprising amino acids having the sequence set forth in SEQ ID NO:69 and a VL comprising amino acids having the sequence set forth in SEQ ID NO:70. In certain embodiments, the antibody or an n—binding nt thereof ses a VH CDRI comprising amino acids having the sequence set forth in SEQ ID NO:226 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 227 or conservative modifications f, and a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 228 or conservative modifications thereof. In n embodiments, the antibody or an antigen—binding fragment thereof comprises a VL CDRI comprising amino acids having the sequence set forth in SEQ ID NO: 229 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 230 or vativemodifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 231 or conservative modifications thereof. In certain embodiments, the antibody or an antigen—binding fragment f comprises a VH CDRI comprising amino acids having the sequence set forth in SEQ ID NO: 226 or conservative modifications thereof, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 227 or vative modifications thereof, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 228 or vative modifications thereof, a VL CDRI comprising amino acids having the sequence set forth in SEQ ID NO: 229 or conservative modifications thereof, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 230 or conservativemodifications thereof, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 231 or conservative modifications thereof. In certain ments, the antibody or an antigen—binding fragment thereof comprises a VH CDRI comprising amino acids having the sequence set forth in SEQ ID NO: 226, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 227, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 228, a VL CDRI comprising amino acids having the sequence set forth in SEQ ID NO: 229, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 230, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 231. In certain embodiments, the antibody or an antigen—binding fragment thereof is ETlSO—lS scFv (or ETlSO—l68 scFv). 7. Immunoconjugates The presently disclosed subject provides an anti—GPRCSD antibody, or a fragment thereof, conjugated to a therapeutic moiety, such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin. Such conjugates are referred to herein as "immunoconjugates". Immunoconjugates that include one or more cytotoxins are referred to as "immunotoxins." A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. es include taxol (such as ricin, diphtheria, gelonin), alasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, stine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, ntrone, mithramycin, actinomycin D, l— dehydrotestosterone, glucocorticoids, ne, tetracaine, ine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents also include, for example, calecheamicin, aureastatin, antimetabolites (e.g., methotrexate, 6— mercaptopurine, 6—thioguanine, cytarabine, S—?uorouracil decarbazine), alkylating agents (e. g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), hosphamide, busulfan, omannitol, streptozotocin, mitomycin C, and cis—dichlorodiamine platinum (ll) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e. g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin , and itotic agents (e. g., vincristine and vinblastine).
Other examples of therapeutic xins that can be conjugated to an anti—GPRCSD antibody disclosed herein e duocarmycins, calicheamicins, sines and auristatins, and derivatives thereof. An example of a calicheamicin antibody conjugate is commercially available (MylotargTM; Wyeth—Ayerst).
Cytoxins can be conjugated to anti—GPRCSD dy disclosed herein using linker technology available in the art. Examples of linker types that have been used to conjugate a cytotoxin to an antibody include, but are not d to, hydrazones, thioethers, esters, disulfides and peptide—containing linkers. A linker can be chosen that is, for example, susceptible to cleavage by low pH within the lysosomal compartment or susceptible to cleavage by proteases, such as proteases preferentially expressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D). For further discussion of types of cytotoxins, linkers and methods for conjugating therapeutic agents to antibodies, see also Saito, G. et al. (2003) Adv. Drug Deliv. Rev. 55:199- 215; Trail, RA. et al. (2003) Cancer Immunol. Irnmunother. 52:328—337; Payne, G. (2003) Cancer Cell 3:207—212; Allen, TM. (2002) Nat. Rev. Cancer 2:750—763; Pastan, I. and Kreitman, R. J. (2002) Curr. Opin. Investig. Drugs 3:1089—1091; , PD. and Springer, C]. (2001) Adv. Drug Deliv. Rev. —264.
Anti—GPRCSD antibodies of the presently disclosed subject matter also can be ated to a ctive e to te cytotoxic radiopharmaceuticals, also referred to as radioimmunoconjugates. Examples of radioactive isotopes that can be conjugated to antibodies for use diagnostically or therapeutically include, but are not limited to, 90Y, 1311, 225Ac, 213Bi, 223Ra and 227Th. Methods for preparing radioimmunconjugates are established in the art. Examples of radioimmunoconjugates are commercially available, including nTM (IDEC Pharmaceuticals) and BexxarTM (Corixa Pharmaceuticals), and r methods can be used to prepare radioimmunoconjugates using the antibodies of the invention.
The dy conjugates of the presently disclosed subject matter can be used to modify a given ical response, and the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, an enzymatically active toxin, or active fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor (TNF) or interferon—y; or, biological response modifiers such as, for example, lymphokines, interleukin—1 ("IL—1"), interleukin—2 ("IL—2"), interleukin—6 ("IL—6"), granulocyte macrophage colony stimulating factor ("GM—CSF"), granulocyte colony stimulating factor ("G—CSF"), or other growth factors.
Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Amon et al., "Monoclonal Antibodies For targeting Of Drugs In Cancer Therapy", in Monoclonal dies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243—56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623—53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody rs Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475—506 ; "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer y", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303—16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic ties Of Antibody—Toxin Conjugates", Irnmunol. Rev., 62:119—58 (1982). 8. i?c Molecules The presently disclosed subject matter provides bispecific molecules comprising an anti—GPRCSD antibody, or a fragment thereof, disclosed herein. An antibody of the presently disclosed subject matter, or antigen—binding portions thereof, can be derivatized or linked to r onal molecule, e.g., another peptide or protein (e. g., another dy or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibody of the presently disclosed subject matter can in fact be derivatized or linked to more than one other functional molecule to generate multispecific les that bind to more than two different binding sites and/or target molecules; such multispecific molecules are also ed to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule, a presently disclosed anti— GPRCSD antibody can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, such that a bispecific molecule results.
The presently disclosed subject matter provides ific molecules comprising at least a first binding icity for GRPCSD and a second binding specificity for a second target epitope. The second target epitope can be a GPRCSD epitope, or a non—GPRCSD epitope, e. g., a different antigen. In certain embodiments, the bispecific molecule is multispecific, the molecule can further e a third binding specificity. Where a first portion of a ific antibody binds to an antigen on a tumor cell for example and a second portion of a bispecific antibody recognizes an antigen on the surface of a human immune effector cell, the antibody is capable of ting the activity of that effector cell by specifically binding to the effector antigen on the human immune effector cell. In certain embodiments, bispecific antibodies, ore, are able to form a link n or cells, for example, T cells and tumor cells, thereby enhancing or function. In certain embodiments, a bispecific antibody of the present disclosure comprises at least a first binding to GPRC5D and at least a second binding to an immune cell. For e, and not by way of limitation, a bispecific antibody of the present sure ses at least a first binding to GPRC5D and at least a second binding to a receptor present on the surface of an immune cell, e.g., CD3.
The bispecific molecules of the presently disclosed subject matter can be prepared by conjugating the constituent binding specificities using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to one r. When the binding specificities are proteins or peptides, a variety of coupling or cross—linking agents can be used for covalent conjugation. Examples of cross—linking agents include protein A, carbodiimide, N—succinimidyl—S—acetyl—thioacetate (SATA), 5, 5'—dithiobis(2— nitrobenzoic acid) (DTNB), ylenedimaleimide (oPDM), N—succinimidyl—3—(2— pyridyldithio)propionate (SPDP), and uccinimidyl 4—(N—maleimidomethyl) cyclohaxane—l—carboxylate (sulfo—SMCC) (see e.g., Karpovsky et al. (1984) J. Exp.
Med. 160:1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118—132; Brennan et al. (1985) Science 229:81—83), and Glennie et al. (1987) J. Immunol. 139: 2367—2375). red ating agents are SATA and sulfo—SMCC, both available from Pierce Chemical Co. (Rockford, IL).
When the binding specificities are antibodies, they can be conjugated via sulfhydryl bonding of the C—terminus hinge regions of the two heavy chains. In certain embodiments, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.
Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful where the bispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab')2 or ligand x Fab fusion protein.
Binding of the ific molecules to their specific targets can be med by, for example, enzyme—linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e. g., growth tion), or n Blot assay. Each of these assays generally detects the presence of protein— antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest. Alternatively, the complexes can be detected using any of a variety of other immunoassays. For example, the antibody can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by such means as the use of a y counter or a scintillation counter or by autoradiography.
Selectin ahi ha ini ScFva ainstaGPRCSD 01 e tide The next step is to the selection of phage that bind to the target antigen of interest with high affinity, from phage in a human phage display y that either does not bind or that binds with lower ty. This is accomplished by iterative binding of phage to the antigen, which is bound to a solid support, for example, beads or mammalian cells followed by removal of non—bound phage and by elution of specifically bound phage. In certain embodiments, antigens are first biotinylated for lization to, for example, streptavidin—conjugated Dynabeads M—280. The phage library is incubated with the cells, beads or other solid support and non binding phage is removed by washing. Clones that bind are selected and tested.
Once selected, positive scFv clones are tested for their binding to GPRCSD (human GPRCSD) on live 3T3 cell surfaces by ?ow cytometry. Brie?y, phage clones are incubated with 3T3 cells over—expressing GPRCSD. The cells are washed and then with a mouse anti—M13 coat protein mAb. Cells are washed again and labeled with a oat anti—mouse Ig prior to ?ow cytometry.
In other embodiments, the anti—GPRCSD antibodies can comprise one or more framework region amino acid tutions designed to improve n ity, antibody g, expression levels or to introduce a site for conjugation of therapeutic agents. These scFv are then used to produce recombinant human monoclonal Igs in ance with methods known to those of skill in the art.
I 0. Engineering full length mAb using the selected ScFv fragments Phage display technology allows for the rapid selection and production of antigen—specific scFv and Fab fragments, which are useful in and of themselves, or which can be further ped to provide complete antibodies, antigen binding proteins or n binding fragments thereof. Complete mAbs with PC domains have a number of ages over the scFv and Fab antibodies. First, only full length Abs exert immunological function such as CDC and ADCC mediated via Fc domain.
Second, bivalent mAbs offer stronger n—binding affinity than monomeric Fab Abs. Third, plasma half— life and renal clearance will be different with the Fab and bivalent mAb. The particular features and advantages of each can be matched to the planned effector strategy. Fourth, bivalent mAb may be internalized at different rates than scFv and Fab, altering immune function or carrier on. Alpha rs, for example, do not need to be internalized to kill the targets, but many drugs and toxins will benefit from internalization of the immune complex. In certain embodiments, therefore, once scFv clones specific for GRPCSD were obtained from phage display ies, a full length IgG mAb using the scFv fragments was produced.
To produce inant human monoclonal IgG in Chinese hamster ovary (CHO) cells, a full length IgG mAb can be ered based on a method known to those of skill in the art atsu et al., Production of human monoclonal antibodies against FceRIa by a method combining in vitro immunization with phage y. Biosci Biotechnol Biochem 73(7): 1465—1469 2009). Brie?y, antibody variable regions can be subcloned into mammalian expression vectors, with matching Lambda or Kappa light chain constant sequences and IgG1 ss PC (for example) (Lidija P, et al. An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries. Gene 1997; 187(1 ): 9—18; Lisa JH, et al. Crystallographic structure of an intact lgGl monoclonal antibody. l of Molecular Biology 1998; 275 (5): 861—872). Kinetic binding analysis (Yasmina NA, et al. Probing the binding mechanism and affinity of tanezumab, a recombinant humanized anti—NGF monoclonal antibody, using a repertoire of biosensors. Protein Science 2008; 17(8): 335) can be used to confirm specific binding of full length IgG to GRPCSD, with a KD in nanomolar range.
Pharmaceutical Comgositions and Methods of Treatment Anti—GPRCSD antibodies of the presently disclosed subject matter can be administered for therapeutic treatments to a patient suffering from a tumor (e.g., multiple myeloma) in an amount ient to prevent, inhibit, or reduce the progression of the turmor. Progression includes, e.g, the growth, invasiveness, metastases and/or recurrence of the tumor. Amounts effective for this use will depend upon the ty of the e and the general state of the patient's own immune . Dosing schedules will also vary with the disease state and status of the patient, and will typically range from a single bolus dosage or continuous infusion to multiple administrations per day (e.g., every 4—6 hours), or as indicated by the treating ian and the patient's condition.
The identification of medical conditions treatable by anti—GPRCSD antibodies of the presently sed subject matter is well within the ability and knowledge of one skilled in the art. For example, human duals who are either suffering from multiple myeloma or who are at risk of developing multiple myeloma are suitable for administration of the tly disclosed anti—GPRCSD antibodies. A clinician d in the art can y determine, for example, by the use of clinical tests, physical examination and medical/family history, if an individual is a candidate for such ent.
In certain embodiments, the presently sed subject matter es a method of treating a tumor by stering a presently disclosed anti—GPRCSD antibody in combination with one or more other agents. For example, the presently disclosed subject matter provides a method of treating a tumor by administering a presently disclosed anti—GPRCSD antibody with an antineoplastic agent. The anti— GPRCSD antibody can be chemically or biosynthetically linked to one or more of the antineoplastic agents. miting examples of suitable tumors include multiple myeloma and Waldenstrom’s Macroglobulinemia. In certain embodiments, the tumor is multiple myeloma.
Any suitable method or route can be used to administer a presently disclosed anti—GPRCSD antibody, and optionally, to coadminister antineoplastic agents. Routes of administration include, for example, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration. It should be emphasized, however, that the presently sed subject matter is not limited to any particular method or route of administration.
It is noted that presently disclosed anti—GPRCSD antibody can be administered as a conjugate, which binds specifically to the receptor and delivers a toxic, lethal payload following ligand—toxin internalization.
It is understood that anti—GPRCSD antibodies of the presently disclosed subject matter can be administered in the form of a composition additionally comprising a pharmaceutically acceptable carrier. le pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Pharmaceutically acceptable carriers may further se minor amounts of ary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the binding proteins. The compositions of the ion can, as is well known in the art, be formulated so as to provide quick, sustained or delayed release of the active ient after stration to the .
The presently disclosed subject matter also provides use of antibodies and nucleic acids that encode them for treatment of a tumor (e. g., multiple a), for diagnostic and stic applications as well as use as research tools for the detection of GPRCSD in cells and tissues. Pharmaceutical compositions comprising the disclosed antibodies and c acids are encompassed by the tly disclosed subject matter. Vectors comprising the nucleic acids of the presently disclosed t matter for antibody—based treatment by vectored immunotherapy are also plated by the presently disclosed t matter. Vectors include expression vectors which enable the expression and secretion of antibodies, as well as vectors which are directed to cell surface expression of the antigen binding proteins, such as chimeric antigen receptors.
Cells comprising the nucleic acids, for example cells that have been transfected with the vectors of the invention are also encompassed by the presently disclosed subject matter.
The presently disclosed subject matter provides kits for the treatment or prevention of a tumor (e.g., le myeloma). In certain embodiments, the kit comprises a therapeutic composition containing an effective amount of an anti— GPRCSD antibody in unit dosage form. In certain embodiments, the kit comprises a sterile container which contains a therapeutic or prophylactic vaccine; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister—packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
If desired, the anti—GPRCSD antibody is provided together with instructions for stering the cell to a subject having or at risk of developing a tumor (e. g., multiple myeloma). The instructions will generally include information about the use of the composition for the treatment or prevention of a tumor (e.g., multiple myeloma). In other embodiments, the instructions include at least one of the ing: description of the therapeutic agent; dosage le and administration for treatment or prevention of a neoplasia (e.g., multiple myeloma) or symptoms thereof; precautions; warnings; indications; counter—indications; sage information; adverse reactions; animal pharmacology; al s; and/or references. The instructions may be printed directly on the ner (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
Methods Flow cytometry analysis. For cell e staining, cells can be incubated with appropriate mAbs for 30 minutes on ice, , and incubated with secondary antibody reagents when necessary. Flow cytometry data can be collected on a FACS Calibur (Becton Dickinson) and analyzed with FlowJo V8.7.l and 9.4.8 software.
Selection and characterization of scFv specific for GPRCSD. A human scFv antibody phage display library is used for the selection of mAb clones. In brief, biotinylated ns can be first mixed with the human scFv phage library, then the antigen—scFv antibody complexes can be pulled down by streptavidin—conjugated Dynabeads M—280 through a magnetic rack. Bound clones can be then eluted and used to infect E. Coli XLl —Blue. The scFv phage clones expressed in the bacteria can be purified (Yasmina NA, et al. Probing the g mechanism and affinity of tanezumab, a recombinant humanized anti—NGF onal antibody, using a repertoire of biosensors. Protein Science 2008; 17(8): 1326—1335; Roberts WK, et al.
Vaccination with CD20 peptides induces a biologically active, specific immune response in mice. Blood 2002: 99 (10): 3748—3755). Panning can be med for 3— 4 cycles to enrich scFv phage clones binding to GPRCSD specifically. Positive clones can be determined by ?ow cytometry method against biotinylated single chain GPRCSD. Positive clones can be further tested for their binding to GPRCSD on live cell surfaces by ?ow cytometry, using a GPRC5D+ cell line, 3T3. The cells can be washed, and the staining can be performed in following steps.
The cells can be first stained with purified scFv phage clones, and ed by staining with a mouse anti—M13 mAb, and finally the goat anti—mouse lg's ate to FlTC. Each step of the staining can be done between 30—60 minutes on ice and the cells were washed twice between each step of the staining.
Engineering full length mAb using the selected ScFv fragments. Full— length human IgG of the ed phage clones can be produced in HEK293 and Chinese hamster ovary (CHO) cell lines, as described (Caron PC, Class K, Laird W, Co MS, Queen C, Scheinberg DA. Engineered humanized dimeric forms of IgG are more effective antibodies. J Exp Med 176:1 191 —1 195 (1992). In brief, antibody le regions can be subcloned into mammalian expression vectors, with matching human lambda or kappa light chain nt region and human lgG constant region sequences. Molecular weight of the purified full length IgG antibodies can be measured under both reducing and non—reducing conditions by electrophoresis.
Characterization of the full-length human IgG for . Initially, specificities of the fully human IgG mAbs for the GPRCSD can be determined by staining 3T3 cells transduced to overexpress GPRCSD, followed by secondary goat anti—human lgG mAb conjugate to PE or FlTC. The ?uorescence intensity can be measured by ?ow cytometry. The same method can be used to determine the binding of the mAbs to fresh tumor cells and cell lines.
Antibody-dependent cellular cytotoxicity (ADCC). Target cells used for ADCC can be 3T3 cells over—expressing GPRCSD. Anti—GPRCSD antibody or its control human IgG at various concentrations can be incubated with target cells and fresh PBMCs at different effector:target (E:T) ratio for 16 hrs. The supernatant can be harvested and the cytotoxicity can be ed by LDH e assay using Cytotox 96 nonradioactive kit from Promega following their ction. Cytotoxicity can also be measured by standard 4 hours 51 Cr—release assay.
EXAMPLES The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the antibodies, bispecific antibodies, compositions comprising thereof, screening, and therapeutic methods of the presently disclosed subject matter, and are not ed to limit the scope of what the inventors regard as their presently disclosed t matter.
It is understood that various other embodiments may be practiced, given the general description provided above.
Example 1 - GPRCSD Expression in various tissues The sion of human GPRCSD was evaluated in various malignant and normal tissues by igating gene expression profiles in databases such as the cancer cell line encyclopedia and BioGPS. As shown in Figure 2, human GPRCSD was highly expressed in multiple a, but not in other malignant s.
Normal expression appeared limited to plasma cells. Potential GPRCSD targeted CAR T cell eradication of this normal cell type may not have significant adverse effects based on inventors’ patient experience with CD19 targeted CAR T cells. Any lack of physiologic antibody production can be addressed with intravenous immunoglobulin treatment.
Example 2 — Selection of ScFv specific for GPRCSD using a fully human phage display library.
Phage display against GPRCSD was performed for 4 panning rounds to enrich the scFv phage clones binding to GPRCSD specifically. Four independent pannings with 12 different phage libraries were carried out against GPRCSD overexpressing 3T3 cells identifying 80 positive clones. Individual scFv phage clones positive for the GPRCSD were determined by ELISA and the clones that possessed unique DNA coding sequences were ted to further characterization. To test if the ScFv bound to GPRCSD on live cells, the positive phage clones were tested for binding to a GPRCSD—positive cell line, 3T3. 72 positive clones were identified out of 80 clones screened FACS; the positive clone rate was 90%. After sequencing, 32 unique and GPRCSD—3T3 positive binding clones were found out of 72 sequenced positive ; the unique clone rate was 45%.
Example 3 - Epitope Mapping ofAnti-GPRCSD Antibodies Four anti—GPRCSD antibodies: ET150—2, ET150—5, ET150—8, and ET150— 18 mIgGl. "mlgGl" used in all es represents that the variable region is fully human and the Fc part is mouse lgGl. See Table 34.
Table 34 \\\\\\\\\\\\§\\\\\\\\\\\\\\§®"\\§&\\§\§\§\\\\\\\\\\X\\\\\\\ $1119:15 migm mm; IF ETla?-SE mig?l mouseFt: ‘\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\‘ The target protein is human GPRCSD having the amino acid sequence set forth in SEQ ID NO: 97. The N—terminal region of human GPRCSD has amino acids 1—27 of SEQ ID NO:97. The extracellular loop I (ECLl) region of human GPRCSD has amino acids 85—93 of SEQ ID NO:97. The extracellular loop 2 (ECL2) region of human GPRCSD has amino acids 145—167 of SEQ ID NO:97. The extracellular loop 3 (ECL3) region of human GPRCSD has amino acids 226—239 of SEQ ID NO:97.
Methods The principles of clips technology. CLIPS technology structurally fixes peptides into defined three—dimensional structures. This results in functional mimics of even the most complex binding sites. CLIPS technology is now routinely used to shape peptide libraries into single, double or triple looped structures as well as sheet— and helix—like folds (Figure 2).
Combinatorial clips y screening in detail. CLIPS library screening starts with the conversion of the target protein into a library of up to 10,000 overlapping e constructs, using a combinatorial matrix design. On a solid r, a matrix of linear peptides is synthesized, which are subsequently shaped into spatially defined CLIPS constructs (Figure 3). ucts representing both parts of the discontinuous epitope in the correct conformation bind the antibody with high ty, which is detected and quantified. Constructs presenting the incomplete epitope bind the dy with lower affinity, whereas constructs not containing the epitope do not bind at all. Affinity information is used in iterative screens to define the sequence and conformation of es in detail.
Heat map analysis. A heat map is a graphical representation of data where the values taken by a variable in a two—dimensional map are represented as colors. For double—looped CLIPS peptides, such a two—dimensional map can be derived from the independent sequences of the first and second loops. For example, the sequences of the 16 CLIPS peptides depicted in Figure 5 are effectively permutations of 4 unique sub—sequences in loop 1 (colored in blue in Figure 4) and 4 unique sub—sequences in loop 2 (colored in green in Figure 4). Thus, the observed ELISA data (colored in red in Figure 5A) can be d in a 4x4 matrix, where each X coordinate corresponds to the sequence of the first loop, and each Y coordinate corresponds to the ce of the second loop. For instance, the ELISA value observed for CLIPS peptide CLSSERERVEDLFEYECELLTSEPlFHCRQEDC (indicated with an arrow in Figure 4A) can be found at the third row, third column of Figure 5B (indicated with an arrow and a red square). To further facilitate the visualization, ELISA values can be replaced with colors from a continuous gradient.
In this case, extremely low values are colored in green, extremely high values are colored in red, and average values are d in black (see Figure 5C). For the aforementioned example, the average value is 0.71. When this color map is applied to the data matrix depicted in Figure 5B, a color heat map is obtained (see Figure 5D, the original data is still indicated for extra clarity).
Synthesis of es. To reconstruct epitopes of the target molecule a library of peptides was synthesized. An amino functionalized polypropylene support was obtained by grafting with a proprietary hydrophilic polymer ation, followed by reaction with t—butyloxycarbonyl—hexamethylenediamine (BocHMDA) using dicyclohexylcarbodiimide (DCC) with Nhydroxybenzotriazole (HOBt) and subsequent cleavage of the Boc—groups using roacetic acid (TFA). rd Fmoc—peptide synthesis was used to synthesize peptides on the amino—functionalized solid support by custom ed JANUS liquid handling ns n Elmer).
Synthesis of structural mimics was done using Pepscan’s proprietary Chemically Linked Peptides on Scaffolds (CLIPS) technology. CLIPS technology allows to structure peptides into single loops, doubleloops, triple loops, sheet—like folds, helix— like folds and combinations thereof. CLIPS templates are coupled to cysteine residues.
The side—chains of multiple cysteines in the peptides were d to one or two CLlPS templates. For example, a 0.5 mM solution of the P2 CLIPS (2,6— bis(bromomethyl)pyridine) was dissolved in ammonium bicarbonate (20 mM, pH 7.8)/acetonitrile (l:3(v/v)). This solution was added onto the peptide arrays. The CLlPS te bound to side—chains of two cysteines as present in the solid—phase bound peptides of the peptide—arrays (455 wells plate with 3 ul wells). The peptide arrays were gently shaken in the solution for 30 to 60 s while completely covered in solution. Finally, the peptide arrays were washed extensively with excess of H20 and sonicated in disrupt—buffer containing 1 % SDS/0.l % beta— mercaptoethanol in PBS (pH 7.2) at 70°C for 30 s, followed by sonication in H20 for another 45 minutes. The T3 CLIPS carrying peptides were made in a similar way but now with three cysteines.
ELISA ing. The binding of antibody to each of the synthesized es was tested in a PEPSCAN—based ELISA. The peptide arrays were incubated with primary antibody solution (overnight at 4°C). After washing, the peptide arrays were incubated with a 1/1000 dilution of an appropriate antibody peroxidase ate (SBA) for one hour at 25°C. After washing, the peroxidase substrate 2,2’— azino—di—3—ethylbenzthiazoline sulfonate (ABTS) and 2 ul/ml of 3 percent H202 were added. After one hour, the color pment was measured. The color development was quantified with a charge coupled device (CCD) — camera and an image processing system.
Data processing. The values obtained from the CCD camera ranged from 0 to 3000 mAU, similar to a standard 96—well plate ELISA—reader. The results were quantified and stored into the Peplab database. Occasionally a well contained an air— bubble resulting in a false—positive value, the cards were ly inspected and any values caused by an air—bubble were scored as 0. sis quality control —— To verify the quality of the synthesized peptides, a separate set of positive and negative control peptides was synthesized in parallel. These were screened with antibody 57.9 (ref. Posthumus et al., J. Virology, 1990, 64:3304—3309).
Results ing. Antibody binding depends on a combination of factors, including concentration of the dy and the amounts and nature of ing proteins in the ELISA buffer. Also, the pre—coat conditions (the specific treatment of the e arrays prior to incubation with the experimental sample) affected binding.
These details are summed up in Table 35. For the Pepscan Buffer and Preconditioning (SQ), the numbers indicate the relative amount of competing protein (a combination of horse serum and ovalbumin).
Table 35. Screening conditions E‘HSB- ' 33$ 3% so .,_ :I- \ gag) . \ \ \ \\\\\\\\\\\\\\\\\\\\\\\\\\\\\ \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ \\\\\\\\\\\\\\\\\\\\\\\\\\\ \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ Antibody ETlSO—Z. When tested under moderate stringency conditions antibody ET150—2 avidly bound peptides from all sets (Figure 6). tive data analysis shows that the antibody ize a tinuous epitope composed of peptides stretches 16CDAEGPWGIIZ5 (N—term), 157MFVNMTPC164 (ECL2) and QRQPQW237 (ECL3), where peptide stretches 16CDAEGPWGII25 and 229PQFQRQPQW237 alone suffice for binding.
Antibody ETlSO—S. When tested under high stringency conditions antibody ETlSO—S avidly bound peptides from all sets (Figure 7). Cumulative data analysis shows that the antibody recognizes a discontinuous epitope composed of peptide hes GDYFLLCD17 (N—term), 35NQQTAPVRYFL95 (ECLl) and NMTPC164 (ECL2), where peptide stretch 5CIESTGDYFLCD17 alone suffices for binding.
Antibody ETlSO—lS. When tested under high stringency conditions antibody ETlSO—lS bound es from set 4 and set 7, ning structurally constrained peptides. No significant binding was recorded on sets containing linear peptides (Figure 8). Cumulative data analysis shows that the antibody recognizes a discontinuous epitope composed of stretches 10GDYFLLCD17 (N—term), 157MFVNMTPCQLN167 (ECL2) and 227GNPQFQRQPQW237 (ECL3). Peptide stretches 10GDYFLLCD17 and 227GNPQFQRQPQW237 represent the epitope’s core, as both peptide stretches separately suffice for binding.
Antibody ETlSO—S. When tested under high ency conditions antibody ETlSO—S bound peptides from all sets, except for set 2 (Figure 9).
Cumulative data analysis shows that the antibody recognizes a discontinuous epitope composed of peptides stretches 15LCDAEGPWG23 (N—term) and 230QFQRQPQWDDPVVC243 (ECL3) where peptide stretch 15LCDAEGPWG23 is the dominant part of the epitope, as it alone es for binding. Moreover, comparison of the results obtained on set 1 (linear) and set 4 (loop) shows that introduction of structural constrains to epitope mimics enhances binding of peptides, especially in case of peptides containing sequence 230QFQRQPQWDDPVVC243.
Conclusions All dies investigated recognized discontinuous epitopes, which were mapped using Pepscan arrays. Core tentative epitopes are listed in Table 36. All antibodies commonly recognized overlapping regions at the N—terminus of the protein in ation with regions from one or two ECLs. Two antibodies ETlSO—lS and S showed a requirement for ural constraints to t antibody binding, suggesting that these two antibodies recognize not only discontinuous, but also conformational epitopes. dies ET150—2 and ETlSO—S did not show notable discrepancies in peptide binding between linear and looped peptides.
Table 36. List of"epitopes r«auxin/"#111111", Figure 10 is an illustration of the results of the study with respect to overall organization of GPCRs. As the N—terminus is highly ?exible and unstructured, it likely transiently cts with ECLs forming discontinuous immunodominant regions.
Differences and commonalities in e binding can be illustrated with a scatter plot analysis in Figure ll. Data points in the top left and bottom right comers point to the differences in the binding. Despite significant epitope overlap, the fine specificities of epitopes of the individual antibodies differ to a large extent. e 4 - Screening Datafor Anti-GPRCSD Antibodies FACS Screening. Figure 12 shows FACS analysis of the GPRCSD— specific phage antibody clones (ETlSO—l, ET150—2, ETlSO—S, ETlSO—S, ETlSO—lS).
Phage clones were ted with 3T3—GPRC5D cell line, then with anti—M13 mouse antibody. Finally APC—labeled anti—mouse IgG 2nd dy was added to the reaction after washing again. The g was measured by FACS and expressed as mean ?uorescence intensity (MFI). Cells incubated with M13 K07 helper phage and cells only were used as negative controls.
Example 5 - Binding Af?nity ofAnti-GPRCSD dies Figure 13 shows FACS analysis of GPRCSD—specific phage dy clones -2, ETlSO-S, ETlSO-S, ETlSO-lS). Each antibody (ETlSO-l, 2, ETlSO—S, ETlSO—S, lS) was incubated with 3T3 or 3T3—GPRC5D cells at 10 or 1 ug/mL, then with anti—M13 mouse antibody. Finally PE—labeled anti—mouse IgG 2nd antibody was added to the reaction. The binding was measured by FACS and expressed as mean ?uorescence intensity (MFI) (Figure 13). Cells incubated with 2nd dy alone, ET90l mIgGl isotype control and cells only were used as negative controls.
Example 6 - Bispecific Antibodies Specificfor GPRCSD and CD3.
Figure 14 shows FACS analysis of the anti—GPRCSD/anti—CD3 ific antibodies generated using the ETlSO—2, S, ETlSO—S, ETlSO—lS clones disclosed herein. Each antibody was incubated with 3T3 or 3T3—GPRC5D cells at 10 ug/ml, followed by the incubation with a FlTC—conjugated anti—His tag antibody. The binding was measured by FACS and sed as mean ?uorescence ity (MFI).
Cells incubated with 2nd antibody alone, ET90l bispecific antibody control and cells only were used as negative controls. As shown in Figure 14, the anti—GPRCSD/CD3 bispecific antibodies generated using the disclosed scFvs specifically bound to 3T3 cells expressing GPRCSD.
Although the foregoing presently disclosed subject matter has been described in some detail by way of ration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the presently disclosed subject matter. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.

Claims (42)

What we claim is:
1. An anti-G-protein coupled receptor family C group 5 member D (GPRC5D) antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID ; a CDR2 comprising the amino acid ce set forth in SEQ ID NO:221; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:222; and a light chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:223; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:224; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:225.
2. An anti-G-protein coupled receptor family C group 5 member D (GPRC5D) antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising a CDR1, a CDR2, and a CDR3 of the heavy chain variable region ce set forth in SEQ ID NO:65, and a light chain variable region comprising a CDR1, a CDR2, and a CDR3 of the light chain variable region sequence set forth in SEQ ID NO:66.
3. The dy or an antigen-binding fragment thereof of claim 1 or claim 2, wherein the heavy chain variable region comprises an amino acid sequence that has at least about 90% identity to the ce set forth in SEQ ID NO:65, and the light chain le region comprises an amino acid sequence that has at least about 90% identity to the sequence set forth in SEQ ID NO:66.
4. The antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:65, and the light chain le region comprises the amino acid sequence set forth in SEQ ID NO:66.
5. The antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein the antigen-binding fragment of the antibody is a Fab, Fab', F(ab')2, Fv or single chain Fv (scFv).
6. The antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the antigen-binding fragment of the antibody is a single chain variable fragment (scFv).
7. The antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the dy comprises a human variable region framework region.
8. The antibody or antigen-binding fragment thereof of any one of claims 1-7, which is a fully human antibody or an antigen-binding nt thereof.
9. The antibody or antigen-binding fragment thereof of any one of claims 1-7, which is a chimeric antibody or an n-binding fragment thereof.
10. The antibody or antigen-binding fragment thereof of any one of claims 1-7 and 9, which is a humanized antibody or an antigen-binding fragment thereof.
11. The antibody or antigen g fragment thereof of any one of claims 1-10, sing the amino acid sequence set forth in SEQ ID NO: 116.
12. The antibody or antigen g fragment f of any one of claims 1-11, wherein the antibody, or antigen-binding fragment thereof binds to human GPRC5D with a binding affinity (Kd) of from about 1 x 10-9 M to about 1 x 10-8 M.
13. The antibody or antigen binding fragment thereof of any one of claims 1-12, wherein the GPRC5D ses the amino acid sequence set forth in SEQ ID NO:97.
14. The antibody or antigen binding fragment thereof of any one of claims 1-13, wherein the antibody or antigen-binding fragment thereof binds to an epitope region comprising amino acids 15-23 of SEQ ID NO:97.
15. The antibody or antigen binding fragment thereof of any one of claims 1-13, wherein the antibody or antigen-binding fragment thereof binds to an epitope region comprising amino acids 230-243 of SEQ ID NO:97.
16. A composition comprising the dy or antigen-binding nt thereof of any one of claims 1-15, and a pharmaceutically acceptable carrier.
17. An immunoconjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-15, linked to a therapeutic agent.
18. The immunoconjugate of claim 17, wherein the therapeutic agent is a drug, cytotoxin, or a radioactive isotope.
19. A composition comprising the immunoconjugate of claim 17 or claim 18 and a pharmaceutically acceptable carrier.
20. A bispecific molecule comprising the antibody or antigen-binding nt thereof of any one of claims 1-15, linked to a second functional moiety.
21. The bispecific le of claim 20, wherein the second functional moiety has a different binding specificity than the antibody or n binding fragment thereof.
22. The bispecific molecule of claim 20 or claim 21, wherein the second functional moiety has a binding specificity for an immune cell.
23. The bispecific molecule of any one of claims 20-22, wherein the second functional moiety has a binding specificity for CD3.
24. A composition comprising the bispecific molecule of any one of claims 20-23 and a pharmaceutically acceptable carrier.
25. A nucleic acid molecule that encodes an antibody or antigen-binding nt thereof of any one of claims 1-15.
26. An expression vector comprising the nucleic acid molecule of claim 25.
27. The expression vector of claim 26, wherein the expression vector is a viral vector.
28. An isolated host cell comprising the expression vector of claim 26 or claim 27.
29. An ex vivo method for detecting GPRC5D in a whole cell or tissue, comprising: ting a cell or tissue with the antibody or antigen-binding fragment thereof of any one of claims 1-15, wherein the antibody or n-binding fragment thereof comprises a detectable label; and determining the amount of the labeled antibody or antigen-binding fragment thereof bound to the cell or tissue by measuring the amount of detectable label ated with the cell or tissue, wherein the amount of bound antibody or antigen-binding fragment thereof indicates the amount of GPRC5D in the cell or tissue.
30. Use of a composition for the manufacture of a ment for the ent of a tumor in a subject, wherein the composition comprises an effective amount of the antibody, or antigenbinding fragment thereof, of any one of claims 1-15.
31. The use of claim 30, wherein the medicament reduces the number of tumor cells in the subject.
32. The use of claim 30 or claim 31, wherein the medicament reduces the size of the tumor in the subject.
33. The use of any one of claims 30-32, wherein the medicament eradicates the tumor in the subject.
34. The use of any one of claims 30-33, wherein the tumor is multiple myeloma or Waldenstrom’s Macroglobulinemia.
35. The use of any one of claims 30-34, wherein the tumor is multiple myeloma.
36. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-15 for use in treating a tumor in a subject.
37. The composition of claim 36, wherein the tumor is multiple myeloma or strom’s Macroglobulinemia.
38. The composition of claim 36 or claim 37, wherein the tumor is multiple myeloma.
39. A kit for treating a tumor, comprising the antibody, or antigen-binding fragment thereof, of any one of claims 1-15.
40. The kit of claim 39, wherein the kit further comprises written instructions for using the antibody or antigen-binding fragment f for ng the tumor.
41. The kit of claim 39 or claim 40, wherein the tumor is multiple myeloma or Waldenstrom’s lobulinemia.
42. The kit of any one of claims 39-41, wherein the tumor is multiple myeloma. Memorial Sloan-Kettering Cancer Center Eureka Therapeutics, Inc Patent Attorneys for the Applicant/Nominated Person SPRUSON & FERGUSON
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