CN117229407A

CN117229407A - Single-domain antibody targeting GPRC5D, chimeric antigen receptor and application thereof

Info

Publication number: CN117229407A
Application number: CN202311506682.9A
Authority: CN
Inventors: 周丹; 何雨辰; 周霞; 黄智威; 李红梅; 孙海
Original assignee: Chengdu Yousainuo Biotechnology Co ltd
Current assignee: Chengdu Yousainuo Biotechnology Co ltd
Priority date: 2023-11-14
Filing date: 2023-11-14
Publication date: 2023-12-15
Anticipated expiration: 2043-11-14
Also published as: CN117229407B

Abstract

The invention relates to the technical field of immunotherapy, and provides a single-domain antibody targeting GPRC5D, a chimeric antigen receptor and application thereof. Specifically, the invention provides a single domain antibody targeting GPRC5D, a Chimeric Antigen Receptor (CAR) targeting GPRC5D constructed using the single domain antibody, and an engineered immune effector cell comprising the CAR. The invention also provides the use of a single domain antibody, chimeric antigen receptor, which targets GPRC5D, and engineered immune effector cells comprising the CAR in the preparation of a medicament for diagnosing, preventing and/or treating a disease or disorder associated with GPRC5D expression.

Description

Single-domain antibody targeting GPRC5D, chimeric antigen receptor and application thereof

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to a single domain antibody specifically targeting GPRC5D, a chimeric antigen receptor targeting GPRC5D constructed by the single domain antibody, an engineered immune effector cell containing the chimeric antigen receptor, and application of the chimeric antigen receptor in diagnosis, prevention and/or treatment of diseases related to GPRC5D expression.

Background

Multiple Myeloma (MM) is a malignant disease caused by abnormal proliferation of cloned plasma cells, classified as B-cell related malignant tumor, and is a common hematological malignancy ranked 2 in many countries, accounting for about 2% of all cancer death cases. Symptoms common to multiple myeloma clinics include manifestations of myeloma-related organ function impairment, i.e., "CRAB" symptoms (increased blood calcium (Calcium elevation), impaired renal function (Renal insufficiency), anemia (Anemia), bone disease (Bone disease)), and related manifestations of secondary amyloidosis. The current preferred treatment for multiple myeloma is autologous hematopoietic stem cell transplantation (ASCT) (see China's society of physicians and blood science, china's society of medical science, guidelines for diagnosis and treatment of multiple myeloma in China (revised 2022) [ J ]. J.Zhonghua J.2022, 61 (5): 480-487). Traditional treatments for multiple myeloma, such as those including chemotherapy, radiotherapy, stem cell transplantation, bone marrow transplantation, etc., although current therapies generally result in remission of the disease, almost all patients eventually relapse. For primary patients with multiple myeloma, common first-line therapeutic drugs include proteasome inhibitors, immunomodulatory drugs, and alkylating agents. For most patients, the first-line treatment commonly used can stabilize the disease condition of the patients for 3-5 years, but a small part of patients show primary drug resistance during primary treatment, and the disease condition cannot be effectively controlled. Most primary patients who are effective in treatment inevitably enter the relapse/refractory stage after the period of disease stabilization. Thus, there is still an unmet medical need for a significant number of multiple myeloma patients, particularly relapsed/refractory multiple myeloma patients.

In selecting therapeutic targets, unlike other B cell malignancies, CD19 expression is only observed in 2% of myeloma patients, and the common extracellular immunophenotype markers (CD 138, CD38 and CD 56) in myeloma are all co-expressed on other basal cell types. At present, BCMA is mainly used as a target in immunotherapy of multiple myeloma, but there are a considerable number of patients with multiple myeloma with BCMA negative or BCMA low expression, and the problem of BCMA negative relapse caused by antigen immune escape after BCMA targeted therapy is adopted. Thus, there remains a great unmet need for immunotherapeutic approaches developed based on new therapeutic targets that can be effectively used to treat multiple myeloma.

GPRC5D (G protein-coupled receptor, class C, group 5, membrane D), namely G protein-coupled receptor family group C5 member D, is a member of the G protein-coupled receptor family, and the G protein-coupled receptor is a membrane protein receptor which has important roles in human body, and GPRC5D is taken as a novel G protein-coupled receptor drug target point, and an endogenous ligand thereof is not discovered temporarily, so that the GPRC belongs to an orphan receptor. The existing studies show that GPRC5D is specifically highly expressed in multiple myeloma cells and hardly expressed on normal tissues. The unique expression of GPRC5D on the plasma cell line also makes it potentially an ideal target for anti-myeloma treatment. Studies have also shown that GPRC 5D-targeted CAR-T cells have anti-tumor activity in tumor recurrence models caused by BCMA antigen loss, and GPRC5D would be a very potential specific therapeutic target in alleviating the problem of tumor recurrence caused by BCMA escape.

Single-domain antibodies (sdabs) differ from traditional 4-chain antibodies by having a single variable domain of a monomeric antibody. For example, camelids and sharks produce antibodies that naturally lack light chains, which are referred to as heavy chain-only antibodies, or simply heavy chain antibodies (hcabs). The antigen binding fragment in each arm of a camelid heavy chain antibody has a single heavy chain variable domain, known as a heavy chain single domain antibody (VHH), which can have high affinity for antigens without the aid of light chains. The camelid-derived VHH antibodies are referred to as the smallest functional antigen binding fragments and have a molecular weight of only about 15kD and are thus also referred to as Nanobodies (Nbs). VHH antibodies have the natural advantages of good solubility, high stability, strong penetration and wide binding epitopes, and since they were discovered, they have been increasingly focused by researchers in the field of immunotherapy, and various studies on them have become mature. In application, the preparation method gradually enters a clinical research stage aiming at malignant tumors, autoimmune diseases, virus infection and the like of a blood system, and has great advantages in the aspects of treating the malignant tumors, autoimmune diseases, anti-infection and the like.

Chimeric antigen receptor (Chimeric Antigen Receptor, CAR) modified T cells are receiving wide attention and application as an immunotherapeutic strategy in tumor therapy, especially in hematological malignancies. The chimeric antigen receptor is a recombinant polypeptide construct, and the principle is that a receptor structure (for example, a single-chain antibody) capable of specifically recognizing a tumor cell surface antigen is expressed by a T cell through genetic engineering modification, and after the receptor is specifically combined with the tumor cell surface antigen, an immune co-stimulatory factor and the T cell at the downstream of the receptor are activated, so that the T cell is activated to secrete related cytokines, the tumor cell is specifically killed, and the restriction of a main histocompatibility complex (Major Histocompatibility Complex, MHC) with specificity for a target tumor antigen is avoided. Since this process is targeting T cells to the target in a non-MHC-restricted manner, the primary mechanism of tumor escape is bypassed. The representative structure of a CAR consists of four parts, an extracellular antigen binding domain (typically a single chain antibody with antigen recognition function), a hinge region, a transmembrane domain, and an intracellular signaling domain. Classical CAR structures are currently classified into the first generation (without costimulatory molecules), the second generation (comprising one costimulatory molecule) and the third generation (comprising two costimulatory molecules) depending on whether or not costimulatory molecules are added to the intracellular signaling domain and the number, the second generation CAR structures being the most used by date in the market products and clinical research stage. Because CAR-T cell manufacturing techniques require the use of single chain antibodies that have good binding activity and high binding epitope efficiency, one of the key technical parts of CAR-T cell therapies is the screening of high affinity antibodies that have good specificity, strong binding capacity, and efficient binding epitope.

Thus, there remains a broad need to screen and develop improved GPRC 5D-targeting single domain antibodies, GPRC 5D-targeting chimeric antigen receptors constructed using the same, and engineered immune effector cells. In particular, specific GPRC 5D-targeting single domain antibodies suitable for more effective and more efficient CAR-T cell therapies were developed.

Disclosure of Invention

The invention aims to provide a GPRC 5D-targeted single-domain antibody and application thereof. The targeted GPRC5D single-domain antibody has the advantages of natural single-chain structure, small molecular weight, high solubility, high stability, low immunogenicity, high tissue permeability and no need of additional folding and assembling steps or linker optimization transformation, so that the targeted GPRC5D single-domain antibody becomes a promising alternative scheme of scFv single-chain antibodies with larger molecular weight, and the single-domain antibody has remarkable specific tumor cell killing capacity after being constructed into CAR-T cells.

According to a first aspect of the present disclosure, there is provided a single domain antibody targeting GPRC 5D. The invention provides a single domain antibody targeting GPRC5D, comprising CDR1, CDR2 and CDR3 regions, wherein CDR1 comprises any one of the amino acid sequences as shown in SEQ ID NOS: 1-18, wherein CDR2 comprises any one of the amino acid sequences as shown in SEQ ID NOS: 19-33, and wherein CDR3 comprises any one of the amino acid sequences as shown in SEQ ID NOS: 34-50.

In some embodiments, the invention provides a single domain antibody targeting GPRC5D comprising CDR1, CDR2 and CDR3 regions, wherein CDR1 is an amino acid sequence as shown in any one of SEQ ID NOS: 1-18, CDR2 is an amino acid sequence as shown in any one of SEQ ID NOS: 19-33, and CDR3 is an amino acid sequence as shown in any one of SEQ ID NOS: 34-50.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising CDR1, CDR2, and CDR3 regions, wherein CDR1, CDR2, and CDR3 comprise the amino acid sequences as shown in table 1.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising CDR1, CDR2, and CDR3 regions, wherein CDR1, CDR2, and CDR3 are amino acid sequences as shown in table 1.

In some embodiments, the present invention provides a GPRC 5D-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 1, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 19, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 34.

In some embodiments, the present invention provides a GPRC 5D-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 1, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 20, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 35.

In some embodiments, the present invention provides a GPRC 5D-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 2, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 21, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 36.

In some embodiments, the present invention provides a GPRC 5D-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 3, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 21, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 37.

In some embodiments, the present invention provides a GPRC 5D-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 4, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 21, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 37.

In some embodiments, the present invention provides a GPRC 5D-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 5, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 21, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 37.

In some embodiments, the present invention provides a GPRC 5D-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 2, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 22, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 37.

In some embodiments, the present invention provides a GPRC 5D-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 2, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 22, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 38.

In some embodiments, the present invention provides a GPRC 5D-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 6, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 22, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 38.

In some embodiments, the present invention provides a GPRC 5D-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 7, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 22, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 38.

In some embodiments, the present invention provides a GPRC 5D-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 8, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 23, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 39.

In some embodiments, the present invention provides a GPRC 5D-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 9, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 24, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 40.

In some embodiments, the present invention provides a GPRC 5D-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 10, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 25, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 41.

In some embodiments, the present invention provides a GPRC 5D-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 11, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 26, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 42.

In some embodiments, the present invention provides a GPRC 5D-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 12, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 26, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 43.

In some embodiments, the present invention provides a GPRC 5D-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 13, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 27, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 44.

In some embodiments, the present invention provides a GPRC 5D-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 14, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 28, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 45.

In some embodiments, the present invention provides a GPRC 5D-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 15, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 29, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 46.

In some embodiments, the present invention provides a GPRC 5D-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 15, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 30, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 47.

In some embodiments, the present invention provides a GPRC 5D-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 16, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 31, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 48.

In some embodiments, the present invention provides a GPRC 5D-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 17, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 32, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 49.

In some embodiments, the present invention provides a GPRC 5D-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 18, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 33, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 50.

The technical scheme disclosed by the invention, wherein the specific amino acid sequence information shown in SEQ ID NO 1-50 is shown in Table 1.

TABLE 1

The invention provides a single domain antibody targeting GPRC5D, which comprises a CDR1, a CDR2 and a CDR3 region; wherein the determination of CDR1, CDR2 and CDR3 is according to the IMGT numbering scheme, the Kabat numbering scheme, the AbM numbering scheme, the Chothia numbering scheme or the Contact numbering scheme.

The invention provides a single domain antibody targeting GPRC5D, which comprises a CDR1, a CDR2 and a CDR3 region; wherein the determination of CDR1, CDR2 and CDR3 is according to the IMGT numbering scheme.

The single domain antibodies or CDR regions thereof provided herein encompass CDRs defined by the numbering scheme described above, other known numbering schemes, and CDRs determined by other methods. For example, it will be understood by those skilled in the art that CDR regions determined by any numbering scheme or any method fall within the scope of the present invention as long as they are identical to, or comprise, the CDR regions of the present invention.

The present invention provides a single domain antibody targeting GPRC5D comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of the amino acid sequences shown in Table 2 or SEQ ID NO. 51-72.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 51.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 52.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 53.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 54.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 55.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 56.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 57.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 58.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO 59.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 60.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 61.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 62.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 63.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 64.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 65.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 66.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 67.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 68.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 69.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 70.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 71.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 72.

The single domain antibody for targeting GPRC5D provided by the invention comprises any one of amino acid sequences shown in a table 2 or SEQ ID NO. 51-72.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising the amino acid sequence shown as SEQ ID NO. 51.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising the amino acid sequence shown as SEQ ID NO. 52.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising the amino acid sequence shown as SEQ ID NO. 53.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising the amino acid sequence shown as SEQ ID NO. 54.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising the amino acid sequence shown as SEQ ID NO. 55.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising the amino acid sequence shown as SEQ ID NO. 56.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising the amino acid sequence shown as SEQ ID NO. 57.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising the amino acid sequence shown as SEQ ID NO. 58.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising the amino acid sequence shown as SEQ ID NO. 59.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising the amino acid sequence shown as SEQ ID NO. 60.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising the amino acid sequence shown as SEQ ID NO. 61.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising the amino acid sequence shown as SEQ ID NO. 62.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising the amino acid sequence shown as SEQ ID NO. 63.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising the amino acid sequence shown as SEQ ID NO. 64.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising the amino acid sequence shown as SEQ ID NO. 65.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising the amino acid sequence shown as SEQ ID NO. 66.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising the amino acid sequence shown as SEQ ID NO. 67.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising the amino acid sequence shown as SEQ ID NO. 68.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising the amino acid sequence shown as SEQ ID NO. 69.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising the amino acid sequence shown as SEQ ID NO. 70.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising the amino acid sequence shown as SEQ ID NO. 71.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D comprising the amino acid sequence shown as SEQ ID NO. 72.

The single domain antibody targeting GPRC5D provided by the invention is any one of amino acid sequences shown in table 2 or SEQ ID NO. 51-72.

In some embodiments, the single domain antibody targeting GPRC5D provided by the invention is an amino acid sequence shown as SEQ ID NO. 51.

In some embodiments, the single domain antibody targeting GPRC5D provided by the invention is an amino acid sequence shown as SEQ ID NO. 52.

In some embodiments, the single domain antibody targeting GPRC5D provided by the invention is an amino acid sequence shown as SEQ ID NO. 53.

In some embodiments, the single domain antibody targeting GPRC5D provided by the invention is an amino acid sequence shown as SEQ ID NO. 54.

In some embodiments, the single domain antibody targeting GPRC5D provided by the invention is an amino acid sequence shown as SEQ ID NO. 55.

In some embodiments, the single domain antibody targeting GPRC5D provided by the invention is an amino acid sequence shown as SEQ ID NO. 56.

In some embodiments, the present invention provides a single domain antibody targeting GPRC5D, which is an amino acid sequence as shown in SEQ ID NO. 57.

In some embodiments, the single domain antibody targeting GPRC5D provided by the invention is an amino acid sequence as shown in SEQ ID NO. 58.

In some embodiments, the single domain antibody targeting GPRC5D provided by the invention is an amino acid sequence as shown in SEQ ID NO. 59.

In some embodiments, the single domain antibody targeting GPRC5D provided by the invention is an amino acid sequence shown as SEQ ID NO. 60.

In some embodiments, the single domain antibody targeting GPRC5D provided by the invention is an amino acid sequence shown as SEQ ID NO. 61.

In some embodiments, the single domain antibody targeting GPRC5D provided by the invention is an amino acid sequence shown as SEQ ID NO. 62.

In some embodiments, the single domain antibody targeting GPRC5D provided by the invention is an amino acid sequence shown as SEQ ID NO. 63.

In some embodiments, the single domain antibody targeting GPRC5D provided by the invention is an amino acid sequence shown as SEQ ID NO. 64.

In some embodiments, the single domain antibody targeting GPRC5D provided by the invention is an amino acid sequence shown as SEQ ID NO. 65.

In some embodiments, the single domain antibody targeting GPRC5D provided by the invention is an amino acid sequence shown as SEQ ID NO. 66.

In some embodiments, the single domain antibody targeting GPRC5D provided by the invention is an amino acid sequence shown as SEQ ID NO. 67.

In some embodiments, the single domain antibody targeting GPRC5D provided by the invention is an amino acid sequence as shown in SEQ ID NO. 68.

In some embodiments, the single domain antibody targeting GPRC5D provided by the invention is an amino acid sequence shown as SEQ ID NO. 69.

In some embodiments, the single domain antibody targeting GPRC5D provided by the invention is an amino acid sequence shown as SEQ ID NO. 70.

In some embodiments, the single domain antibody targeting GPRC5D provided by the invention is an amino acid sequence shown as SEQ ID NO. 71.

In some embodiments, the single domain antibody targeting GPRC5D provided by the invention is an amino acid sequence shown as SEQ ID NO. 72.

The technical scheme disclosed by the invention, wherein the specific amino acid sequence information shown in SEQ ID NO 51-72 is shown in Table 2.

TABLE 2

According to a second aspect of the present disclosure there is provided a chimeric antigen receptor comprising a single domain antibody of the invention that targets GPRC 5D.

In some embodiments, the chimeric antigen receptor provided herein comprises an extracellular antigen binding domain comprising a single domain antibody that targets GPRC5D as described herein.

In some embodiments, the chimeric antigen receptor provided herein can further comprise one or more of the following structures: a linker (e.g., a peptide linker), a signal peptide, a hinge region, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain.

In some embodiments, the present invention provides chimeric antigen receptors comprising:

(a) An extracellular antigen-binding domain,

(b) A transmembrane domain, and

(c) An intracellular signaling domain;

wherein the extracellular antigen-binding domain comprises a single domain antibody that targets GPRC5D according to the first aspect of the disclosure.

(a) An extracellular antigen-binding domain,

(b) A transmembrane domain, and

(c) An intracellular signaling domain;

wherein the extracellular antigen-binding domain comprises a single domain antibody targeting GPRC5D, the single domain antibody comprising CDR1, CDR2, and CDR3 regions, wherein CDR1, CDR2, and CDR3 regions comprise CDR1, CDR2, and CDR3 regions of a single domain antibody targeting GPRC5D according to the first aspect of the present disclosure.

(a) An extracellular antigen-binding domain,

(b) A transmembrane domain, and

(c) An intracellular signaling domain;

wherein the extracellular antigen-binding domain comprises a single domain antibody targeting GPRC5D, the single domain antibody comprising CDR1, CDR2, and CDR3, wherein CDR1, CDR2, and CDR3 are CDR1, CDR2, and CDR3 regions of a single domain antibody targeting GPRC5D according to the first aspect of the present disclosure.

(a) An extracellular antigen-binding domain,

(b) A transmembrane domain, and

(c) An intracellular signaling domain;

wherein the extracellular antigen-binding domain comprises a single domain antibody that targets GPRC 5D; wherein the single domain antibody comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any of the amino acid sequences shown as SEQ ID NO:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 72.

(a) An extracellular antigen-binding domain,

(b) A transmembrane domain, and

(c) An intracellular signaling domain;

wherein the extracellular antigen-binding domain comprises a single domain antibody that targets GPRC 5D; wherein the single domain antibody comprises any one of the amino acid sequences shown as SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 or SEQ ID NO: 72.

(a) An extracellular antigen-binding domain,

(b) A transmembrane domain, and

(c) An intracellular signaling domain;

wherein the extracellular antigen-binding domain comprises a single domain antibody that targets GPRC 5D; wherein the single domain antibody is any one of amino acid sequences shown as SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO. 56, SEQ ID NO. 57, SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71 or SEQ ID NO. 72.

In some embodiments, the present invention provides chimeric antigen receptors wherein the transmembrane domain is derived from CD8 a, CD28, cd3ζ, cd3γ, cd3δ, cd3ε, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, tcra, tcrβ, tcrγ, tcrδ, tcrζ, OX40, ICOS, LAG-3, 2B4, BTLA, CTLA-4, PD-1, or any combination thereof.

In some embodiments, the present invention provides chimeric antigen receptors in which the transmembrane domain is derived from CD8 a.

In some embodiments, the present invention provides chimeric antigen receptors in which the transmembrane domain comprises the amino acid sequence shown as SEQ ID NO. 75.

In some embodiments, the present invention provides chimeric antigen receptors in which the transmembrane domain is the amino acid sequence shown as SEQ ID NO. 75.

In some embodiments, the present invention provides chimeric antigen receptors in which the intracellular signaling domain comprises the primary intracellular signaling domain of an immune effector cell (e.g., T cell). In some embodiments, the intracellular signaling domain is derived from fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, cd3ζ, CD22, CD79a, CD79b, and CD66d, or any combination thereof.

In some embodiments, the present invention provides chimeric antigen receptors in which the intracellular signaling domain is derived from cd3ζ.

In some embodiments, the present invention provides chimeric antigen receptors in which the intracellular signaling domain comprises the amino acid sequence shown as SEQ ID NO. 77.

In some embodiments, the present invention provides chimeric antigen receptors in which the intracellular signaling domain is the amino acid sequence shown as SEQ ID NO. 77.

In some embodiments, the invention provides chimeric antigen receptors wherein the intracellular signaling domain further comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from an MHC class I molecule, BTLA, and Toll ligand receptor. In some embodiments, the costimulatory signaling domain is derived from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD134 (OX 40), CD2, CD7, CD27, CD28, CD30, CD40, CD83, ICAM, 4-1BB (CD 137), CD276 (B7-H3), CD278 (ICOS), GITR, LIGHT, HVEM (light), BTLA, CD8 a, LFA-1, NKG2C, LAT, SLP-76, DAP10, PD-1, TRIM, ZAP70 ligand, or any combination thereof.

In some embodiments, the present invention provides chimeric antigen receptors wherein the costimulatory signaling domain is derived from 4-1BB (CD 137).

In some embodiments, the present invention provides chimeric antigen receptors in which the costimulatory signaling domain comprises the amino acid sequence depicted as SEQ ID NO. 76.

In some embodiments, the present invention provides chimeric antigen receptors in which the costimulatory signaling domain is the amino acid sequence depicted as SEQ ID NO. 76.

In some embodiments, the chimeric antigen receptor provided herein further comprises a hinge region located between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain.

In some embodiments, the present invention provides chimeric antigen receptors wherein the hinge region is derived from CD8 a, CD28, CD137, igG4, igG1, or any combination thereof.

In some embodiments, the invention provides chimeric antigen receptors in which the hinge region is derived from CD8 a.

In some embodiments, the present invention provides chimeric antigen receptors in which the hinge region comprises the amino acid sequence set forth in SEQ ID NO. 74.

In some embodiments, the present invention provides chimeric antigen receptors in which the hinge region is the amino acid sequence shown as SEQ ID NO. 74.

In some embodiments, the chimeric antigen receptor provided herein further comprises a signal peptide located at the N-terminus of the chimeric antigen receptor polypeptide.

In some embodiments, the present invention provides chimeric antigen receptors wherein the signal peptide is derived from HLA-A, CD8 a, CD4, CD33, CD137, GM-CSFR a, igG1, ig kappa, IL-2, or any combination thereof.

In some embodiments, the present invention provides chimeric antigen receptors wherein the signal peptide is derived from HLA-A.

In some embodiments, the present invention provides chimeric antigen receptors wherein the signal peptide is derived from CD8 a.

In some embodiments, the present invention provides chimeric antigen receptors in which the signal peptide comprises the amino acid sequence shown as SEQ ID NO. 73.

In some embodiments, the present invention provides chimeric antigen receptors wherein the signal peptide is the amino acid sequence shown as SEQ ID NO. 73.

The technical scheme disclosed by the invention, wherein the specific amino acid sequence information shown in SEQ ID NO 73-77 is shown in Table 3.

TABLE 3 Table 3

In some embodiments, the chimeric antigen receptor polypeptides provided herein comprise, in order from N-terminus to C-terminus: HLA-A signal peptide or CD8 a signal peptide, extracellular antigen binding domain, CD8 a hinge region, CD8 a transmembrane domain, costimulatory signal domain derived from 4-1BB (CD 137) and intracellular signaling domain derived from cd3ζ.

The chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to an amino acid sequence shown in Table 4 or comprising an amino acid sequence shown in SEQ ID NOS: 78-99.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 78.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 79.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 80.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 81.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 82.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 83.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 84.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 85.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 86.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 87.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 88.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 89.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 90.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 91.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 92.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 93.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 94.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 95.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 96.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 97.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 98.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO 99.

The chimeric antigen receptor provided by the invention comprises an amino acid sequence shown in table 4 or SEQ ID NO. 78-99.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 78.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 79.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as shown in SEQ ID NO. 80.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as shown in SEQ ID NO. 81.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 82.

In some embodiments, the chimeric antigen receptor provided by the invention comprises the amino acid sequence shown as SEQ ID NO. 83.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 84.

In some embodiments, the chimeric antigen receptor provided by the invention comprises the amino acid sequence as set forth in SEQ ID NO. 85.

In some embodiments, the chimeric antigen receptor provided by the invention comprises the amino acid sequence as shown in SEQ ID NO. 86.

In some embodiments, the chimeric antigen receptor provided by the invention comprises the amino acid sequence shown as SEQ ID NO. 87.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as shown in SEQ ID NO. 88.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 89.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as shown in SEQ ID NO. 90.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 91.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 92.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 93.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 94.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 95.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 96.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 97.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 98.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 99.

The chimeric antigen receptor provided by the invention is shown in the table 4 or the amino acid sequence shown in SEQ ID NO. 78-99.

In some embodiments, the chimeric antigen receptor provided by the invention is an amino acid sequence as shown in SEQ ID NO. 78.

In some embodiments, the chimeric antigen receptor provided by the invention is an amino acid sequence as shown in SEQ ID NO. 79.

In some embodiments, the chimeric antigen receptor provided by the invention is an amino acid sequence as shown in SEQ ID NO. 80.

In some embodiments, the chimeric antigen receptor provided by the invention is an amino acid sequence as shown in SEQ ID NO. 81.

In some embodiments, the chimeric antigen receptor provided by the invention is an amino acid sequence as shown in SEQ ID NO. 82.

In some embodiments, the chimeric antigen receptor provided by the invention is an amino acid sequence as shown in SEQ ID NO. 83.

In some embodiments, the chimeric antigen receptor provided by the invention is an amino acid sequence as shown in SEQ ID NO. 84.

In some embodiments, the chimeric antigen receptor provided by the invention is an amino acid sequence as shown in SEQ ID NO. 85.

In some embodiments, the chimeric antigen receptor provided by the invention is an amino acid sequence as shown in SEQ ID NO. 86.

In some embodiments, the chimeric antigen receptor provided by the invention is an amino acid sequence as shown in SEQ ID NO. 87.

In some embodiments, the chimeric antigen receptor provided by the invention is an amino acid sequence as shown in SEQ ID NO. 88.

In some embodiments, the chimeric antigen receptor provided by the invention is an amino acid sequence as shown in SEQ ID NO. 89.

In some embodiments, the chimeric antigen receptor provided by the invention is an amino acid sequence as shown in SEQ ID NO. 90.

In some embodiments, the chimeric antigen receptor provided by the invention is an amino acid sequence as shown in SEQ ID NO. 91.

In some embodiments, the chimeric antigen receptor provided by the invention is an amino acid sequence as shown in SEQ ID NO. 92.

In some embodiments, the chimeric antigen receptor provided by the invention is an amino acid sequence as shown in SEQ ID NO. 93.

In some embodiments, the chimeric antigen receptor provided by the invention is an amino acid sequence as shown in SEQ ID NO. 94.

In some embodiments, the chimeric antigen receptor provided by the invention is an amino acid sequence as shown in SEQ ID NO. 95.

In some embodiments, the chimeric antigen receptor provided by the invention is an amino acid sequence as shown in SEQ ID NO. 96.

In some embodiments, the chimeric antigen receptor provided by the invention is an amino acid sequence as shown in SEQ ID NO. 97.

In some embodiments, the chimeric antigen receptor provided by the invention is an amino acid sequence as shown in SEQ ID NO. 98.

In some embodiments, the chimeric antigen receptor provided by the invention is an amino acid sequence as shown in SEQ ID NO. 99.

The technical scheme disclosed by the invention, wherein the specific amino acid sequence information shown in SEQ ID NO 78-99 is shown in Table 4.

TABLE 4 Table 4

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According to a third aspect of the present disclosure, there is provided an isolated nucleic acid encoding a chimeric antigen receptor according to the present invention.

In some embodiments, the isolated nucleic acids provided herein are in the form of DNA or RNA. In some embodiments, the DNA provided by the present invention includes cDNA, genomic DNA, or synthetic DNA. In some embodiments, the DNA provided by the invention is single-stranded DNA or double-stranded DNA. In some embodiments, the DNA provided by the invention is coding strand DNA or non-coding strand DNA.

The isolated nucleic acids provided by the invention comprise the nucleic acid sequences shown in Table 5 or as shown in SEQ ID NOS.100-121.

In some embodiments, the isolated nucleic acids provided herein comprise a nucleic acid sequence as set forth in SEQ ID NO. 100.

In some embodiments, the isolated nucleic acids provided herein comprise a nucleic acid sequence as set forth in SEQ ID NO. 101.

In some embodiments, the isolated nucleic acids provided herein comprise a nucleic acid sequence as set forth in SEQ ID NO. 102.

In some embodiments, the isolated nucleic acids provided herein comprise a nucleic acid sequence as set forth in SEQ ID NO. 103.

In some embodiments, the isolated nucleic acids provided herein comprise a nucleic acid sequence as set forth in SEQ ID NO. 104.

In some embodiments, the isolated nucleic acids provided herein comprise a nucleic acid sequence as set forth in SEQ ID NO. 105.

In some embodiments, the isolated nucleic acids provided herein comprise a nucleic acid sequence as set forth in SEQ ID NO. 106.

In some embodiments, the invention provides an isolated nucleic acid comprising a nucleic acid sequence as set forth in SEQ ID NO. 107.

In some embodiments, the isolated nucleic acids provided herein comprise a nucleic acid sequence as set forth in SEQ ID NO. 108.

In some embodiments, the isolated nucleic acids provided herein comprise a nucleic acid sequence as set forth in SEQ ID NO. 109.

In some embodiments, the invention provides an isolated nucleic acid comprising a nucleic acid sequence as set forth in SEQ ID NO. 110.

In some embodiments, the isolated nucleic acids provided herein comprise a nucleic acid sequence as set forth in SEQ ID NO. 111.

In some embodiments, the invention provides an isolated nucleic acid comprising a nucleic acid sequence as set forth in SEQ ID NO. 112.

In some embodiments, the invention provides an isolated nucleic acid comprising a nucleic acid sequence as set forth in SEQ ID NO. 113.

In some embodiments, the invention provides an isolated nucleic acid comprising a nucleic acid sequence as set forth in SEQ ID NO. 114.

In some embodiments, the isolated nucleic acids provided herein comprise a nucleic acid sequence as set forth in SEQ ID NO. 115.

In some embodiments, the isolated nucleic acids provided herein comprise a nucleic acid sequence as set forth in SEQ ID NO. 116.

In some embodiments, the isolated nucleic acids provided herein comprise a nucleic acid sequence as set forth in SEQ ID NO. 117.

In some embodiments, the isolated nucleic acids provided herein comprise a nucleic acid sequence as set forth in SEQ ID NO. 118.

In some embodiments, the isolated nucleic acids provided herein comprise a nucleic acid sequence as set forth in SEQ ID NO. 119.

In some embodiments, the isolated nucleic acids provided herein comprise a nucleic acid sequence as set forth in SEQ ID NO. 120.

In some embodiments, the isolated nucleic acids provided herein comprise a nucleic acid sequence as set forth in SEQ ID NO. 121.

The technical scheme disclosed by the invention, wherein the specific nucleic acid sequence information shown in SEQ ID NO. 100-121 is shown in Table 5.

TABLE 5

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According to a fourth aspect of the present disclosure there is provided a vector comprising an isolated nucleic acid according to the present invention.

In some embodiments, the vector is suitable for replication and integration in eukaryotic cells, such as mammalian cells. In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentiviral vectors, vaccinia vectors, herpes simplex virus vectors, and derivatives thereof.

In some embodiments, the vector is comprised in a host cell.

In some embodiments, the host cell is a killer cell.

In some embodiments, the killer cell is a T cell or an NK cell.

In some embodiments, the NK cells are primary NK cells.

In some embodiments, the T cells are peripheral blood T lymphocytes, umbilical cord blood T lymphocytes.

According to a fifth aspect of the present disclosure, there is provided an engineered immune effector cell comprising a chimeric antigen receptor or an isolated nucleic acid of the present invention.

In some embodiments, the engineered immune effector cells provided herein may be selected from T cells, B cells, NK cells, macrophages, dendritic cells, immune effector cells differentiated from induced pluripotent stem cells (ipscs), or any combination thereof.

In some embodiments, the engineered immune effector cells provided herein are T cells. For example, the T cells may be selected from the group consisting of CD4+/CD8+ T cells, CD4+/CD 8-T cells, CD4-/CD8+ T cells, CD4-/CD 8-T cells, CD4+ helper T cells (e.g., th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, γδ -T cells, αβ -T cells, NKT cells, DNT cells (double negative T cells).

In some embodiments, the T cell is a CD4+/CD 8-T cell, a CD4-/CD8+ T cell, a CD4+/CD8+ T cell, a CD4-/CD 8-T cell, or a combination thereof.

In some embodiments, the T cells produce cytokines such as IL-2, IFN-gamma, and/or TNF-alpha after expressing the chimeric antigen receptor and binding to target cells (e.g., GPR5D+ tumor cells). In some embodiments, cd8+ T cells lyse antigen-specific target cells after expression of the chimeric antigen receptor and binding to the target cells.

In some embodiments, the immune effector cell is an NK cell. In some embodiments, the immune effector cell may be an established cell line, such as NK-92 cells.

In some embodiments, the immune effector cells may be differentiated from stem cells, such as ipscs.

According to a sixth aspect of the present disclosure there is provided a pharmaceutical composition comprising a GPRC5D single domain antibody, chimeric antigen receptor, engineered immune effector cells and one or more pharmaceutically acceptable excipients and/or carriers according to the present invention.

Excipients and/or carriers are also commonly referred to as adjuvants. Pharmaceutically acceptable excipients and/or carriers include, but are not limited to, fillers, binders, disintegrants, coatings, adsorbents, anti-adherent agents, glidants, antioxidants, flavoring agents, colorants, sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, wetting agents, preservatives, emulsifiers, coating agents, isotonicity agents, absorption delaying agents, stabilizers and tonicity adjusting agents, diluents, solubilizers, emulsifiers, preservatives and/or adjuvants. Those skilled in the art will appreciate that pharmaceutically acceptable excipients and/or carriers are non-toxic or substantially non-toxic to recipients at the dosages and concentrations employed, are pharmacologically and/or physiologically compatible with the active ingredients of the present invention, e.g., do not affect their viability or efficacy. In some embodiments, the pharmaceutical composition may contain substances for improving, maintaining or retaining, for example, pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or permeation of the composition. The optimal pharmaceutical composition can be determined depending on the intended route of administration, the mode of delivery and the dosage required. Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, remington's Pharmaceutical Sciences (18 th edition, 1990).

In some embodiments, the pharmaceutical compositions of the present invention are provided in the form of a sterile formulation. Sterilization is achieved by filtration through sterile filtration membranes. In lyophilizing a composition, the method may be used to sterilize prior to or after lyophilization, reconstitution, dilution. Sterile formulations such as isotonic aqueous solutions, suspensions, emulsions, dispersions and the like. In some embodiments, the pharmaceutical compositions of the present invention, wherein the pharmaceutically acceptable excipients and/or carriers include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, polyol. Compositions for parenteral administration may be stored in lyophilized form or in solution. For example, by using physiological saline or an aqueous solution containing glucose and other auxiliary agents by conventional methods. Parenteral compositions are typically placed in a container having a sterile access port, such as an intravenous solution bag or vial having a needle-penetratable stopper. Alternatively, the composition may be selected for inhalation or delivery through the digestive tract (such as orally). The preparation of the pharmaceutically acceptable compositions is within the skill of the art. Other pharmaceutical compositions will also be apparent to those skilled in the art, including formulations comprising antibodies or engineered immune effector cells in sustained or controlled release delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery means, such as liposome carriers, bioerodible particles or porous beads, and depot injections, are also well known to those skilled in the art. Once formulated, the pharmaceutical compositions are stored in sterile vials as solutions, suspensions, gels, emulsions, viscous compositions, solids, crystals, or as lyophilized powders. The formulation may be stored in a ready-to-use form or reconstituted for use in a treated form prior to administration.

The pharmaceutical composition may be used in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The pharmaceutical compositions of the invention may be injected, for example, orally, nasally, intravenously, intraperitoneally, intracerebrally (intraparenchymally), intracerebroventricular, intramuscularly, intraocularly, intraarterially, portal vein or intralesionally, and may also be administered by a sustained release system or by an implanted device. In some embodiments, administration is accomplished parenterally. Parenteral delivery methods include topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual, or intranasal administration.

The therapeutically effective amount of the pharmaceutical composition comprising the GPRC 5D-targeting single domain antibodies or engineered immune effector cells of the invention to be employed will depend, for example, on the extent of treatment and the goal. Those skilled in the art will appreciate that the appropriate dosage level for treatment will vary depending in part on the molecule delivered, the indication, the route of administration, and the patient's condition (body weight, body surface or organ size) and/or condition (age and general health). In certain embodiments, the clinician may titrate the dose and alter the route of administration to obtain the optimal therapeutic effect.

The frequency of administration will depend on the pharmacokinetic parameters of the GPRC 5D-targeted single domain antibody or engineered immune effector cells in the formulation used. The clinician typically administers the pharmaceutical composition until a dose is reached that achieves the desired effect. The pharmaceutical composition may thus be administered as a single dose, or over time as two or more doses (which may or may not contain the same amount of the desired molecule), or as a continuous infusion through an implanted device or catheter.

According to a seventh aspect of the present disclosure, the present invention provides the use of a single domain antibody, chimeric antigen receptor, engineered immune effector cell, or pharmaceutical composition as described previously that targets GPRC5D in the manufacture of a medicament for the diagnosis, prevention and/or treatment of a disease or disorder.

The invention also provides a method of treating a subject suffering from a disease associated with GPRC5D expression comprising administering to the subject an effective amount of an engineered immune effector cell or pharmaceutical composition according to the invention. Accordingly, the present invention provides the use of a single domain antibody, chimeric antigen receptor, engineered immune effector cell, or pharmaceutical composition of the invention that targets GPRC5D in the manufacture of a medicament for the diagnosis, prevention and/or treatment of a disease or disorder.

In some embodiments, the disease or disorder comprises a disease or disorder associated with GPRC5D expression.

In some embodiments, the disease or disorder is a cancer, malignancy, autoimmune and/or inflammatory disease associated with GPRC5D expression.

In some embodiments, the disease or disorder associated with GPRC5D expression is a malignancy associated with GPRC5D expression, an autoimmune disease associated with GPRC5D expression, and/or an inflammatory disease.

In some embodiments, the disease or disorder is systemic lupus erythematosus, rheumatoid arthritis, waldenstrom's Macroglobulinemia), marginal zone lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinum large B-cell lymphoma, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, follicular lymphoma, burkitt's lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, hairy cell leukemia, precursor B-cell leukemia, non-hodgkin's lymphoma, high grade B-cell lymphoma, and/or multiple myeloma.

In some embodiments, the disease or disorder is multiple myeloma.

The single domain antibodies, nanobodies or heavy chain antibodies of the invention can be prepared using methods conventional in the art, such as phage display techniques well known in the art. Alternatively, the various antibodies of the invention may be expressed in other cell lines. Suitable mammalian host cells may be transformed with sequences encoding the various antibodies of the invention. Transformation may be performed using any known method, including, for example, packaging the polynucleotide in a virus (or viral vector) and transducing the host cell with the virus (or vector). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide in liposomes, and direct microinjection of DNA into the nucleus, etc. Host mammalian cell lines useful for expression are well known in the art, for example, a variety of immortalized cell lines available from the American Type Culture Collection (ATCC) including, but not limited to, chinese Hamster Ovary (CHO) cells, heLa cells, baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatocellular carcinoma (HepG 2) cells, and the like. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with basic GPRC5D binding properties.

Chimeric antigen receptors of the invention can be prepared using methods conventional in the art, see, for example, park et al, trends Bio technol., 29:550-557,2011; grupp et al, N Engl J med, 368:1509-1518, 2013; han et al, j.Hematol.Oncol., 6:47, 2013.

As is well known to those skilled in the art, due to the degeneracy of the genetic code, a very large number of nucleic acids can be made, all of which encode the chimeric antigen receptor of the present invention. Thus, where a particular amino acid sequence has been identified, one of skill in the art can prepare any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not alter the amino acid sequence encoding the protein. Thus, the present invention also relates to polynucleotides which hybridize to the above polynucleotide sequences and which have at least 70%, preferably at least 80%, more preferably at least 90% identity between the two sequences. The invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention.

The full-length nucleic acid sequences of the various chimeric antigen receptors of the invention or fragments thereof can be obtained generally by PCR amplification, recombinant methods or synthetic methods. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the heavy chain coding sequence and the expression tag (e.g., 6 His) may be fused together to form a fusion protein.

The invention also relates to nucleic acid constructs, such as expression vectors and recombinant vectors, comprising the above-described nucleic acid sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein. Vectors typically contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. The sequences (collectively referred to as "flanking sequences" in certain embodiments) typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a leader sequence encoding for secretion of the polypeptide, a ribosome binding site, a polyadenylation sequence, a multiple linker region for inserting a nucleic acid encoding an antibody to be expressed, and selectable marker elements.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, polypeptides, etc.) to which the present invention relates include biomolecules that exist in isolated form. At present, it is entirely possible to obtain DNA sequences encoding the polypeptides of the invention (or fragments or derivatives thereof) by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can be introduced into the polypeptide sequences of the invention by chemical synthesis.

In some embodiments, the nucleic acid encoding the chimeric antigen receptor is operably linked to a constitutive promoter. Constitutive promoters allow for constitutive expression of a heterologous gene (also referred to as a transgene) in a host cell. Exemplary constitutive promoters of the invention include, but are not limited to, the Cytomegalovirus (CMV) promoter, the human elongation factor-1 alpha (hEF 1 alpha) promoter, the ubiquitin C (Ubic) promoter, the phosphoglycerate kinase (PGK) promoter, the simian virus 40 (SV 40) early promoter, and the chicken beta-actin coupled to CMV early enhancer (CAGG) promoter. The efficiency of such constitutive promoters in driving transgene expression has been widely compared in a number of studies. For example Michael C.Milone et al (Molecular Therapy, 17 (8): 1453-1464 (2009)) compared the efficiencies of CMV, hEF 1. Alpha., ubic and PGK in driving chimeric antigen receptor expression in human primary T cells and concluded that the hEF 1. Alpha. Promoter not only induced the highest levels of transgene expression, but was also optimally maintained in CD4 and CD8 human T cells. In some embodiments, the nucleic acid encoding the chimeric antigen receptor is operably linked to the hef1α promoter.

In some embodiments, the nucleic acid encoding the chimeric antigen receptor is operably linked to an inducible promoter. Inducible promoters are among the regulatory promoters. Inducible promoters may be induced by one or more conditions, e.g., physical conditions, inducers, etc.

In some embodiments, the induction conditions induce expression of an endogenous gene in the engineered mammalian cell and/or in a subject receiving the pharmaceutical composition. In some embodiments, the induction conditions are selected from: inducer, radiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox status, tumor environment, and activation status of the engineered mammalian cells.

Viral vector technology is well known in the art and is described in Sambrook et al Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, cold Spring harbor (2001) ([ molecular cloning: laboratory Manual ], cold Spring harbor Laboratory, new York Cold Spring harbor, 2001), and other virology and molecular biology manuals.

Many virus-based systems have been developed in the art for transferring genes into mammalian cells. The heterologous nucleic acid can be inserted into the vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated in vitro or ex vivo and delivered to engineered mammalian cells. Many retroviral systems are known in the art. In some embodiments, an adenovirus vector is used. Many adenoviral vectors are known in the art. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying an immunomodulatory agent (e.g., immune checkpoint inhibitor) coding sequence and/or self-inactivating lentiviral vectors carrying a chimeric antigen receptor may be packaged using protocols known in the art. In some embodiments, lentiviral vectors are used. The resulting lentiviral vector may be used to transduce mammalian cells (e.g., primary human T cells) using methods known in the art.

The nucleic acid may be cloned into a vector using any molecular cloning method known in the art, including, for example, using restriction endonuclease sites and one or more selection markers. In some embodiments, the nucleic acid is operably linked to a promoter. The prior art has disclosed a variety of promoters for gene expression in mammalian cells, and any promoter known in the art may be used in the present invention. Promoters can be further classified as constitutive or regulated, e.g., inducible.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which can take up DNA, can be obtained after the exponential growth phase and then treated with CaCl ₂ The process is carried out using procedures well known in the art. Another approach is to use MgCl ₂ . Alternatively, transformation may be performed by electroporation. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.

The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the nucleic acid molecule of the present invention. The medium used in the culture may be selected from various conventional media, such as a serum-containing medium or a serum-free medium, depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.

The polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations of these methods.

The engineered immune effector cells of the invention are prepared by introducing a chimeric antigen receptor into an immune effector cell (e.g., T cell).

Nucleic acid sequences encoding chimeric antigen receptors can be introduced into immune effector cells using conventional methods known in the art (e.g., by transfection, transduction, transformation, etc.).

Methods for introducing vectors or isolated nucleic acids into immune effector cells are known in the art. The vectors described can be transferred into immune effector cells by physical, chemical or biological means.

Physical methods for introducing the vector into immune effector cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for preparing cells comprising vectors and/or exogenous nucleic acids are well known in the art and are described in, for example, sambrook, j., fritsch, e.f. and Maniatis, t. (2001) Molecular Cloning: a Laboratory Manual Cold Spring Harbor Laboratory Press, cold Spring harbor, and other virology and molecular biology manuals. In some embodiments, the vector is introduced into the immune effector cell by electroporation.

Biological methods for introducing vectors into immune effector cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian cells (e.g., human cells).

Chemical methods for introducing the carrier into immune effector cells include colloidal dispersion systems, including, for example, macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including, for example, oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro delivery vehicle is a liposome.

In some embodiments, the vector further comprises a selectable marker gene or reporter gene to select cells expressing the chimeric antigen receptor from a population of host cells transfected with the lentiviral vector. The selectable marker and the reporter gene may be flanked by appropriate regulatory sequences for expression in the host cell. For example, the vector may contain transcription and translation terminators, initiation sequences, and promoters for regulating expression of the nucleic acid sequences.

Reporter genes can be used to identify potentially transfected cells and to evaluate the function of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by a recipient organism or tissue and which encodes a polypeptide that expresses some readily detectable property, such as enzymatic activity. The expression of the reporter gene is determined at a suitable time after introduction of the DNA into the recipient cell. Suitable reporter genes may include genes encoding luciferases, beta-galactosidases, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or green fluorescent protein (see Kumiko Ui-tei, FEBS Letters, 479:79-82 (2000)). Suitable expression systems are known in the art and may be prepared or commercially available using known techniques. Other methods of confirming the presence of a nucleic acid encoding a CAR in an engineered immune effector cell include: molecular biological testing methods well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; biochemical assay methods, such as detecting the presence or absence of a particular peptide; immunological methods such as ELISA.

In some embodiments, a nucleic acid molecule encoding any CAR described herein can be prepared by conventional methods (e.g., in vitro transcription), and then introduced into immune effector cells by known methods such as mRNA electroporation (see Peter M rabinovich, human Gene Therapy, 17:1027-1035 (2006)).

In some embodiments, the transduced or transfected immune effector cells are propagated in vitro following introduction of the vector or isolated nucleic acid. In some embodiments, the transduced or transfected immune effector cells are cultured to proliferate for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 14 days. In some embodiments, the transduced or transfected immune effector cells can be further evaluated or screened to select for engineered immune effector cells.

Term interpretation:

unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

The term "sequence identity" as used herein refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at identical positions in an alignment, and is typically expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies with identical sequences have 100% identity. One skilled in the art knows that several algorithms can be used to determine sequence identity, such as Blast (Altschul et al (1997) Nucleic acids Res.25:3389-3402), blast2 (Altschul et al (1990) J.mol.biol.215:403-410), smith Waterman (Smith et al (1981) J.mol.biol.147:195-197), and Clustal W. In addition, sequence analysis software may be used to perform the determination, such as computer programs BLAST, and in particular BLASTP or TBLASTN, using default parameters.

The term "derived from" as used herein refers to a relationship between the two, generally referring to structural similarity between the two. For example, in the case of an intracellular signaling domain derived from cd3ζ, the intracellular signaling domain retains sufficient cd3ζ structure such that it has the desired function, i.e., the ability to generate a signal under appropriate conditions. It does not imply or include limitations on the particular process by which the intracellular signaling domain is generated, e.g., it does not mean that in order to provide the intracellular signaling domain, unwanted sequences must be started from the cd3ζ sequence and deleted, or mutations imposed, to reach the intracellular signaling domain.

The term "antibody" as used herein refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Including, for example, monoclonal antibodies (including intact antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), diabodies, and single chain molecules, and antibody fragments or synthetic polypeptides, particularly antigen-binding fragments, such as Fab, F (ab') 2, and Fv, bearing one or more CDR sequences, which are capable of exhibiting the desired biological activity. In some embodiments of the invention, the terms "immunoglobulin (Ig)" and "antibody" are used interchangeably.

"variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively. These domains are typically the most variable parts of an antibody (relative to other antibodies of the same type) and contain antigen binding sites.

Heavy chain antibodies are antibodies derived from camelidae or cartilaginous fish organisms. In contrast to 4-chain antibodies, heavy-chain antibodies lack the light and heavy chain constant region 1 (CH 1), comprising only 2 heavy chains consisting of variable regions (VHH) linked to the constant region by a hinge-like structure and other constant regions. Each heavy chain of a camelidae heavy chain antibody comprises 1 variable region (VHH) and 2 constant regions (CH 2 and CH 3), and each heavy chain of a cartilaginous fish heavy chain antibody comprises 1 variable region and 5 constant regions (CH 1-CH 5). Antigen binding fragments of heavy chain antibodies include VHH and single chain heavy chain antibodies. Heavy chain antibodies can have CH2 and CH3 of human IgG Fc by fusion to the constant region of human IgG Fc.

The term "antigen" or "Ag" as used herein refers to a molecule that causes an immune response. The immune response may involve antibody production or activation of specific immunocompetent cells or both. The skilled artisan will appreciate that virtually any macromolecule, including all proteins or peptides, can act as an antigen. Furthermore, the antigen may be derived from recombinant or genomic DNA. The skilled artisan will appreciate that any DNA comprising a nucleotide sequence or portion of a nucleotide sequence encoding a protein that elicits an immune response, thus encodes an "antigen". The antigen may be synthetically produced or may be derived from a biological sample or may be a macromolecule other than a polypeptide. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or fluids having other biological components.

The term "antigen-binding fragment" or "antibody fragment" as used herein refers to at least a portion of an intact antibody or recombinant variant thereof, typically comprising the antigen-binding site of the intact antibody and thus retaining the ability to bind antigen. Examples of antibody fragments include, but are not limited to: fab, scFv, heavy chain variable region (VH) of an antibody, linear antibodies, single domain antibodies, nanobodies, natural ligands of an antigen or functional fragments thereof, and the like.

The term "functional variant" or "functional fragment" as used herein refers to a variant that substantially comprises the amino acid sequence of a parent but that contains at least one amino acid modification (e.g., substitution, deletion, or insertion) as compared to the parent amino acid sequence, provided that the variant retains the biological activity of the parent amino acid sequence. In some embodiments, the amino acid modification is a conservative modification.

Single domain antibodies (sdabs) may have the same or different sources, and have the same or different sizes. Exemplary sdabs include, but are not limited to, heavy chain variable domains (e.g., VHH) from heavy chain-only antibodies, binding molecules that are naturally devoid of light chains, single domains (e.g., VH or VL) derived from conventional 4-chain antibodies, human single domain antibodies produced by transgenic mice or rats expressing human heavy chain fragments, and engineering domains and single domain scaffolds that are not derived from antibodies. Any sdAb known in the art or disclosed by the invention, including the single domain antibodies disclosed herein, can be used to construct a CAR described herein. The sdAb may be derived from any species, including but not limited to mouse, rat, human, camel, llama, lamprey, shark, goat, rabbit, and cow. The single domain antibodies of the invention also include naturally occurring single domain antibody molecules from species other than camelidae and shark.

In the present invention, "single domain antibody", "single domain antibody targeting GPRC 5D", "heavy chain single domain antibody", "VHH", "nanobody" are used interchangeably and refer to a single domain antibody specifically recognizing and binding to GPRC 5D. Typically, single domain antibodies contain three CDR regions and four FR regions. Single domain antibodies are the smallest functional antigen binding fragments. Typically, after an antibody is obtained which naturally lacks the light and heavy chain constant regions 1 (CH 1), the variable regions of the heavy chain of the antibody are cloned, and a single domain antibody consisting of only one heavy chain variable region is constructed.

The term "complementarity determining regions" or "CDRs" as used herein refers to sequences of amino acids within the variable regions of an antibody that confer antigen specificity and binding affinity. Generally, there are three CDRs (e.g., HCDR1, HCDR2, and HCDR 3) in each heavy chain variable region, and three CDRs (LCDR 1, LCDR2, and LCDR 3) in each light chain variable region.

The portion of the chimeric antigen receptor comprising the antibody or antibody fragment thereof may exist In a variety of forms, for example, wherein the antigen binding domain is expressed as part of a polypeptide chain (including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv), or, for example, a human or humanized antibody), see Harlow et al, 1999, in: using Antibodies A Laboratory Manual, cold Spring Harbor Laboratory Press, NY, harlow et al, 1989, in: antibodies A Laboratory Manual, cold Spring Harbor, new York, houston et al, 1988, proc, natl. Acad. Sci. USA 85:5879-5883, bird et al, 1988, science 242:423-426.

An "antigen binding domain" refers to the portion of a chimeric antigen receptor that specifically binds to a target antigen, which can be used to direct T cells and/or other immune cells to a selected target using its antigen binding properties. The antigen binding domain may typically comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), but it need not comprise both variable regions. For example, fab fragments, fab' fragments, single Fv fragments such as scFv, single domain antibodies, and the like, which have antigen binding activity, can be included.

A "transmembrane domain" is an extracellular domain and an intracellular domain that are used to connect a chimeric antigen receptor. The transmembrane domain may be natural or synthetic, and may be derived from any membrane-bound or transmembrane protein. Non-limiting examples of transmembrane domains include tcrα, tcrβ, tcrγ, tcrδ, tcrζ, CD28, cd3ζ, cd3ε, cd3γ, cd3δ, CD45, CD4, CD5, cd8α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD152, CD154, OX40, ICOS, LAG-3, 2B4, BTLA, CTLA-4, PD-1.

An "intracellular signaling domain" refers to a functional portion of a protein, i.e., a functional portion of an intracellular signaling domain sufficient to transduce an effector function signal. Intracellular signaling domains are responsible for intracellular primary signaling after antigen binding by the antigen binding region, leading to activation of immune cells and immune responses. In other words, the intracellular signaling domain is responsible for activating at least one of the normal effector functions of the immune cells in which the CAR is expressed. For example, the effector function of a T cell may be cytolytic activity or helper activity, including secretion of cytokines. In one embodiment, the intracellular signaling domains comprised by the chimeric antigen receptor of the invention may be cytoplasmic sequences of T cell receptors and co-receptors that function together to elicit signaling upon antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences having the same or similar function. The intracellular signaling domain may contain a number of immunoreceptor tyrosine-activating motifs (Immunoreceptor Tyrosine-based Activation Motifs, ITAM). Non-limiting examples of intracellular signaling domains include, but are not limited to, fcrγ, fcrβ, TCR ζ, cd3γ, cd3δ, cd3ε, cd3ζ, CD5, CD22, CD79a, CD79b, CD278 (ICOS), fcεri, DAP10, DAP12, or CD66d.

"hinge region" refers to any oligopeptide or polypeptide used to connect a transmembrane domain and an antigen binding domain. In particular, the hinge region serves to provide greater flexibility and accessibility to the antigen binding domain. The hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. The hinge region may be derived in whole or in part from a natural molecule, for example, in whole or in part from the extracellular region of CD8 or CD28, or in whole or in part from the antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a fully synthetic hinge sequence.

The different domains of the CAR may also be fused to each other via a peptide linker. Depending on the structural and/or functional characteristics of the single domain antibody and/or the various domains, each peptide linker in the CAR may have the same or different length and/or sequence. Each peptide linker can be independently selected and optimized by one skilled in the art. In some embodiments, the peptide linker consists of amino acids linked together by peptide bonds, wherein the amino acids are selected from the group consisting of 20 naturally occurring amino acids: glycine, alanine, valine, leucine, isoleucine, serine, cysteine, threonine, methionine, proline, phenylalanine, tyrosine, tryptophan, histidine, lysine, arginine, aspartic acid, glutamic acid, asparagine and glutamine. As will be appreciated by those skilled in the art, one or more of these amino acids may be glycosylated. In some embodiments, the peptide linker comprises flexible residues (e.g., glycine and serine) such that adjacent domains can move freely relative to each other. For example, glycine-serine duplex may be a suitable peptide linker.

The peptide linker may have any suitable length. In some embodiments, the peptide linker is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or more amino acids in length. In some embodiments, the peptide linker is no more than about 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or less amino acids in length. In some embodiments, the peptide linker is about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids in length.

The peptide linker may have a naturally occurring sequence or a non-naturally occurring sequence. For example, sequences derived from the hinge region of heavy chain-only antibodies may be used as linkers. See, e.g., WO1996/34103. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include, but are not limited to, glycine polymer (G) n, glycine-serine polymer (e.g., (GS) n, (GSG) n, (GGGS) n, and (GGGGS) n, where n is an integer of at least 1), glycine-alanine polymer, alanine-serine polymer, and other flexible linkers known in the art.

The "costimulatory signaling domain" may be an intracellular functional signaling domain from a costimulatory molecule, comprising the entire intracellular portion of the costimulatory molecule, or a functional fragment thereof. "costimulatory molecule" refers to a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (e.g., proliferation) of the T cell. Non-limiting examples of co-stimulatory domains include, but are not limited to, the intracellular regions of the following proteins: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), activating NK cell receptors, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD134 (OX 40), CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CDS, ICAM, CD137 (4-1 BB), CD276 (B7-H3), CD278 (ICOS), GITR, BAFFR, LIGHT, HVEM (light), BTLA, kirs 2, SLAMF7, NKp80 (KLRF 1), NKp44, NKp30, NKp46, CD19, CD4, CD8 a, CD8 β, IL2rβ, IL2rγ, IL7rα, ITGA4, VLA1, IL2rβ; CD49a, ITGA4, IA4, CD49D, ITGA, VLA-6, CD49f, ITGAD, CD11D, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11B, ITGAX, CD c, ITGB1, CD29, ITGB2, ITGB7, NKG2D, NKG2C, TRANCE/RANKL, DNAM1 (CD 226), SLAMF4 (CD 244, 2B 4), CD84, CD96 (Tactive), CEACAM1, CRTAM, ly9 (CD 229), CD160 (BY 55), PSGL1, CD100 (SEMA 4D), CD69, SLAMF6 (NTB-A, ly 108), SLAM (SLAMF 1, CD150, IPO-3), BLAME (SLAMF 8), PLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, CD28-OX40, CD 28-4-BB, TRIAP 10, PD-1, ZAP70, or any combination thereof.

The "signal peptide" may be such that when the chimeric antigen receptor is expressed in a cell (e.g., a T cell), the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface. In general, the core of the signal peptide may contain long hydrophobic amino acid segments that have a tendency to form a single α -helix. The signal peptide directs the transport and/or secretion of the translated protein across the membrane. At the end of the signal peptide, there is typically an amino acid segment that is recognized and cleaved by the signal peptidase. The signal peptidase may cleave during or after translocation to produce the free signal peptide and the mature protein. The free signal peptide is then digested by a specific protease. The signal peptide may also be referred to as a targeting signal, transit peptide, localization signal or signal sequence. For example, the signal sequence may be a co-translated or post-translated signal peptide.

By "immune effector cell" is meant an immune cell that can perform immune effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC). For example, the immune effector cells may be T cells, macrophages, dendritic cells, monocytes, NK cells and/or NKT cells, or immune cells derived from stem cells, such as adult stem cells, embryonic stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, hematopoietic stem cells, or the like. When the immune effector cell is a T cell, the T cell may be any T cell, such as an in vitro cultured T cell, e.g., a primary T cell, or a T cell from an in vitro cultured T cell line, e.g., jurkat, supT1, etc., or a T cell obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats and transgenic species thereof. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. T cells may also be concentrated or purified. T cells may be at any stage of development, including, but not limited to, cd4+/cd8+ T cells, cd4+ helper T cells (e.g., th1 and Th2 cells), cd8+ T cells (e.g., cytotoxic T cells), CD4-/CD 8-T cells, tumor infiltrating cells, memory T cells, naive T cells, γδ -T cells, αβ -T cells, and the like.

The terms "patient", "subject", "individual", "subject" as used herein are used interchangeably and include any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, etc.), and most preferably a human.

"treatment" refers to the use of the methods of treatment of the invention to achieve at least one positive therapeutic effect (e.g., reduced number of cancer cells, reduced tumor volume, reduced rate of infiltration of cancer cells into peripheral organs, or reduced rate of tumor metastasis or tumor growth) in a subject. The therapeutic regimen effective to treat a patient can be adjusted according to a variety of factors, such as the disease state, age, weight, and ability of the patient to elicit an anti-cancer response in the subject.

Drawings

Figure 1 shows the expression rate of GPRC5D CAR molecules on each set of cd4+ CAR-T cells prepared.

Figure 2 shows the expression rate of GPRC5D CAR molecules on each set of cd8+ CAR-T cells prepared.

Figure 3 shows the fold proliferation of each group of cd4+ CAR-T cells prepared after 3 days of culture.

Figure 4 shows the fold proliferation of each group of cd8+ CAR-T cells prepared after 3 days of culture.

FIG. 5 shows that each group of prepared CAR-T cells was cultured for 13 days, wherein T _SCM +T _CM The proportion of the components is as follows.

FIG. 6 shows the proportion of CAR-T cells expressing Tim3 in each group of CAR-T cells prepared after 13 days of culture.

FIG. 7 shows the separate determination of various target cells: expression of GPRC5D antigen on surfaces of Raji-1F7 cells, raji-BE6 cells and Raji-CF10 cells.

Fig. 8 shows the results of in vitro killing rate flow assay of Raji-1F7 cells by each group of effector cells on day 1 when the effective target ratio (E: T) =1:3.

Fig. 9 shows the results of in vitro killing rate flow assay of Raji-1F7 cells by each group of effector cells on day 3 when the effective target ratio (E: T) =1:3.

FIG. 10 shows the results of in vitro killing rate flow assay of Raji-BE6 cells by each set of effector cells on day 1 at an effective target ratio (E: T) =1:3.

FIG. 11 shows the results of in vitro killing rate flow assay of Raji-BE6 cells by each set of effector cells on day 3 at an effective target ratio (E: T) =1:3.

Fig. 12 shows the results of in vitro killing rate flow assay of Raji-CF10 cells by each group of effector cells on day 3 when the effective target ratio (E: T) =1:3.

FIG. 13 shows that target cells (Raji-1F 7) were mixed with each group of CAR-T cells for 6 days after in vitro culture at an effective target ratio (E: T) =1:3, wherein T _SCM +T _CM The proportion of the components is as follows.

Fig. 14 shows the proportion of CAR-T cells expressing Tim3 after 6 days in vitro mixed culture of target cells (Raji-1F 7) with CAR-T cells of each group at an effective target ratio (E: T) =1:3.

Fig. 15 shows the fold proliferation of cd4+ CAR-T cells after 9 days of mixed culture of target cells (Raji-1F 7) with each group of effector cells in vitro at an effective target ratio (E: T) =1:3.

Fig. 16 shows the fold proliferation of cd8+ CAR-T cells after 9 days of mixed culture of target cells (Raji-1F 7) with each group of effector cells in vitro at an effective target ratio (E: T) =1:3.

Figure 17 shows the fold proliferation of cd4+ CAR-T cells after 9 days of mixed culture of target cells (Raji-BE 6) with each group of effector cells in vitro at an effective target ratio (E: T) =1:3.

Fig. 18 shows the proliferation fold of cd8+ CAR-T cells after 9 days of mixed culture of target cells (Raji-BE 6) with each group of effector cells in vitro at an effective target ratio (E: T) =1:3.

Fig. 19 shows the results of LDH release assays for the in vitro killing rate of mm.1s cells by effector cells of each group on day 1 at an effective target ratio (E: T) =1:3.

FIG. 20 shows the results of IL-2 release experiments with target cells killed by each group of effector cells (Raji-1F 7) at an effective target ratio (E: T) =1:3.

Fig. 21 shows TNF- α release assay results when effector cells of each group kill target cells (Raji-1F 7) at an effective target ratio (E: T) =1:3.

Fig. 22 shows the results of IFN- γ release experiments when target cells (Raji-1F 7) were killed by each group of effector cells at an effective target ratio (E: T) =1:3.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the invention thereto. The experimental procedures, without specific conditions noted in the examples below, were selected according to methods and conditions conventional in the art, or according to the commercial specifications.

Example 1 preparation of a GPRC 5D-targeting Single-Domain antibody (VHH)

(1) Animal immunity and immune response test

Adult healthy alpaca was selected as the immunization subject, and 10mL of blood was taken as a negative serum control prior to immunization. The immune cycle was 14 days/time, 6 total times, and the immune process stimulated B cells to express antigen-specific nanobodies. The first immunization was performed by 1×10 CHOK1 human GPRC5D cells ⁷ Each was subcutaneously injected after isovolumetric emulsification with Complete Freund's Adjuvant (CFA). Immunization at 2-6 equal volumes of CHOK1 human GPRC5D cells with Incomplete Freund's Adjuvant (IFA) were emulsified and injected subcutaneously. Peripheral blood was taken after immunization and serum titers were determined by FACS. Cells in log phase were washed by centrifugation, resuspended in PBS and viable cells were counted by trypan blue staining. Cell density was adjusted to 4×10 using 1% horse serum/PBS ⁶ After that, 50. Mu.L of the cell suspension (containing 2X 10) ⁵ Individual cells) into EP tubes, serum was added and incubated for 1h at 4 ℃. After incubation was completed, the wash was added, centrifuged and the supernatant removed, and 50. Mu.L of diluted secondary anti-goat anti-alpaca IgG H was used&L conj-488 (NBbiolab, cat#S001A488, lot# 20211120) resuspended cells, after incubation for 1h at 4 ℃, the cells were washed twice and serum titers were detected after resuspension at 4 ℃. To the titer of 1X 10 ⁵ After the level, 50mL of peripheral blood was collected for banking.

(2) Construction of antibody phage libraries

1) Extraction and reverse transcription of RNA

After the animal is immunized, peripheral blood after immunization is taken and lymphocytes in the peripheral blood are isolated. Total RNA was extracted with RNAiso Plus reagent. The extracted RNA was reverse transcribed into cDNA using PrimeScript ™ II 1st Strand cDNA Synthesis Kit (Takara, cat# 6210A) reverse transcription kit, the procedure being as described.

2) PCR amplification

Specific antibody fragments were amplified from the reverse transcribed cDNA using the PCR method. The PCR product was digested, and a single VHH gene fragment having a size of about 750bp was recovered and purified, and the VHH gene fragment was purified using a DNA product purification kit Gel Extraction Kit (OMEGA, cat# D2500-01), and the fragment thus obtained was used as the target gene fragment.

Wherein, the PCR primer sequence is as follows:

first round upstream primer: 5 'CTTGGTGGTCCTGGCTGC 3'

First round downstream primer: 5 'GGTACGTGCTGTTGAACTGTTCC 3'

Second round upstream primer:

5’ CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC 3’

second round downstream primer:

5’ CATGCCATGACTCGCGGCCGGCCTGGCCGCTGGGGTCTTCGCTGTGGTGCG 3’ 。

3) Enzyme cutting

The vector and the target gene fragment were digested separately using restriction enzyme SfiI. The target gene fragment was ligated to pComb3XSS phage plasmid Vector at a ligation molar ratio VHH: vector=3:1 using T4 DNA ligase (NEB, cat# M0202L) to construct a recombinant plasmid.

4) Electric conversion

The recombinant plasmid was purified and electrotransformed 10 times into electrotransformed TG1 competent cells with the following electrotransformation parameters: electroporation apparatus BIO-RAD, gene Pulser Xcell Total System, voltage 2.5KV, electric cuvette 2mm. Immediately after electric shock, 1mL of 2YT medium (preheated at 37 ℃) was added to the cuvette, the electric shock product was sucked out and the cuvette was washed with the 2YT medium to obtain 100mL of liquid in total, and resuscitated at 180rpm at 37℃for 45 minutes as resuscitated product, 100. Mu.L was taken out therefrom, and diluted to 10 in a gradient ^-3 And 10 ^-4 Post-plating was used to determine the number of pool transformants. The remaining resuscitated product was centrifuged and resuspended in 8mL of 2YT medium before plating. The next day there were a total of 165 clones on the plates for the number of pool transformants. The pool capacity was determined and the pool built was verified for insertion rate and pool diversity by colony PCR.

(3) Packaging of phage libraries

Inoculating phage library to 2X 300mL 2YT+A (Amp) +G (Glu) cultureThe groups (Amp: 100. Mu.g/mL, glu: 1%) were varied until the initial OD600 was 0.1-0.2. Culturing at 37deg.C and 230rpm until OD600 is above 0.8. Helper phage M13KO7 (number of helper phages: number of bacteria=20:1) was added according to OD value, and mixed well, and after standing at 37℃for 30min, shaking at 180rpm at 37℃was continued for 30min. Next, the supernatant was discarded after centrifugation at 5000rpm for 10min, and the pellet was resuspended in an equal volume of 2YT+A (Amp) +K (Kan) medium (Amp: 100. Mu.g/mL, kan: 50. Mu.g/mL) and incubated at 30℃overnight at 220 rpm. After centrifuging overnight culture at 4℃and 10000rpm for 20min, collecting supernatant, transferring to a new centrifuge tube, centrifuging at 4℃and 10000rpm for 20min, and collecting supernatant. Adding PEG8000/NaCl solution with volume of 1/5 of the supernatant, mixing, ice-bath precipitating for at least 2 hr, centrifuging at 4deg.C and 10000rpm for 20min, and removing supernatant. The precipitate was suspended by adding 1mL of 1 XPBS, and then precipitated again by adding 1/5 of its volume of PEG8000/NaCl solution for 1h. After the secondary precipitation, the supernatant was removed by centrifugation at 12000rpm for 10min at 4℃and the precipitate was resuspended by adding 1 XPBS according to the amount of precipitate. Then adding 100% glycerol until the final concentration of the liquid is 50%, mixing, and preserving at-80 ℃. 10 mu L of phage library was diluted in gradient with 2YT medium from 10 ^-8 And 10 ^-9 10. Mu.L of the culture medium was added to 90. Mu.L of TG1 bacteria solution, and the mixture was gently mixed. The mixture was allowed to stand at 37℃for 15min, amp-resistant plates were applied, and incubated overnight at 37 ℃. The next day, at 10 ^-9 There were a total of 264 clones on titer plates and phage library titers were 2.64×10 ¹³ cfu/mL(264×10 ⁹ ×100)。

(4) Phage selection

1) Cell panning

GPRC5D (5X 10) ⁶ Cell) cells were centrifuged at 500g for 5min, resuspended in 5% serum-PBS and the Cell pellet washed, repeated twice to wash GPRC5D cells. 500 mu L of OVA blocking solution is added into the cell EP tube, and the cells are blocked for 1h by slight shaking at 4 ℃. Phage library was diluted with 5% serum-PBS and 3% BSA until BSA concentration was 2%, blocked at 37℃and used as phage library dilutions. Removing supernatant after sealing and centrifuging GPRC5D cells, adding 2×10 ¹¹ Dilutions of cfu phage library at 4 DEG CAfter 1h of gentle shaking, unbound phage were removed by centrifugation and the cells were washed 6 times with 5% serum-PBS. 100. Mu.L of Gly-HCl eluent was added and enriched at 37℃for 8min, the specifically bound phage was eluted, the eluent was transferred to a sterile centrifuge tube and immediately neutralized with 10. Mu.L of Tris-HCl neutralization buffer to obtain elutriation eluate. And (3) taking 10 mu L for gradient dilution, determining titer, and calculating the panning recovery rate.

2) Amplification of library after panning

The elutriation eluate was mixed with E.coli TG1 culture 20mL at the early stage of logarithmic growth, after standing at 37℃for 30min, 1mL of 20% glucose was added, and culturing at 220rpm for 30min, M13K07 phage and 4. Mu.L Amp were added in the ratio of cell: phase=1:20, and then standing at 37℃for 30min, 20mL of 2YT medium was added, and culturing at 220rpm for 30min. The culture was centrifuged at 5000rpm for 10min at 4℃and the resulting cell pellet was resuspended in 50 mL of 2 XYT+A+K liquid medium and cultured overnight with shaking at 250rpm at 30 ℃. The overnight culture was centrifuged at 10000rpm at 4℃for 20min, the supernatant was transferred to a new centrifuge tube and 1/5 volume of PEG/NaCl solution was added to the supernatant, and after mixing, it was allowed to stand at 4℃for at least 2h. After the completion of the standing, the mixture was centrifuged at 10000rpm at 4℃for 20 minutes, the supernatant was removed, the precipitate was resuspended in 1mL of PBS, and 1/5 of the PEG/NaCl solution was added thereto, followed by mixing and standing at 4℃for at least 1 hour. After the standing, the mixture was centrifuged again, and centrifuged at 12000rpm at 4℃for 2 minutes, the supernatant was removed, and the pellet was resuspended in 200. Mu.L PBS to obtain an amplified product. Titer was determined for the next round of panning or analysis.

Panning conditions were varied as in table 6 below, and a second and third round of cell panning was performed to gradually screen phage, each round of panning conditions as follows.

TABLE 6 affinity panning conditions

(5) Monoclonal ELISA detection

293T human GPRC5D cells and 293T cells were cultured until the whole plate was confluent. The cells were fixed by washing with PBS 2 times, adding 100. Mu.L of 4% paraformaldehyde, and allowing them to act at 25℃for 20-30 min. Washing 2 times with PBS, then adding 300. Mu.L of 5% skimmed milk per well, blocking at 37℃for 1h, washing 1 time with PBST, adding 50. Mu.L of phage culture bacteria supernatant and 50. Mu.L of 5% skimmed milk per well, and incubating at 37℃for 1h. After incubation, the wells were washed 5 times with PBST, and then 100. Mu.L of horseradish peroxidase-labeled anti-M13 antibody (diluted 1:10000 with PBS) was added to each well, and allowed to act at 37℃for 1h. After washing the plate 6 times with PBST, 100. Mu.L of TMB color developing solution was added to each well, the reaction was stopped by adding 50. Mu.L of stop solution per well after 7min reaction at 37℃and the optical density was measured at 450 nm. And (3) carrying out gene sequencing on the positive clone colony to obtain the gene sequence of the VHH single domain antibody.

Example 2 construction of a GPRC 5D-targeting chimeric antigen receptor and immune cell expression

(1) Construction of GPRC5D-CAR

Each set of GPRC 5D-targeting CAR nucleotide sequences (SEQ ID NO: 100-121) was designed and artificially synthesized, each set comprising the coding nucleotide sequences of the HLA-A signal peptide (SEQ ID NO: 73), the extracellular antigen binding domain of GPRC5D-VHH (SEQ ID NO: 51-72), the CD8 alpha hinge region (SEQ ID NO: 74), the CD8 alpha transmembrane domain (SEQ ID NO: 75), the 4-1BB (CD 137) costimulatory signal domain (SEQ ID NO: 76) and the CD3 zeta intracellular signal transduction domain (SEQ ID NO: 77) for expression of each experimental set of complete GPRC5D-CAR polypeptide molecules (SEQ ID NO: 78-99). The nucleotide sequence of the GPRC5D-CAR is inserted into the multiple cloning site of the lentiviral expression vector pK1 through homologous recombination to obtain the pK1-GPRC5D-CAR, and the successful construction of the lentiviral expression vector sequence is confirmed through electrophoresis and sequencing results.

In addition, a positive control CAR was constructed in the same manner, using the antibody sequence GC5B602 disclosed in patent document CN109715667a (see VL amino acid sequence SEQ ID No. 58 and VH amino acid sequence SEQ ID No. 88 in CN109715667 a) as the antigen binding domain of the positive control CAR. The complete amino acid sequence of the positive control CAR is shown as SEQ ID NO. 122 and the nucleic acid sequence of the positive control CAR is shown as SEQ ID NO. 123.

Amino acid sequence of positive control CAR:

AVMAPRTLLLLLSGALALTQTWADIQMTQSPSSLSASVGDRVTITCKASQNVATHVGWYQQKPGKAPKRLIYSASYRYSGVPSRFSGSGSGTEFTLTISNLQPEDFATYYCQQYNRYPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYNGDTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVALRVALDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR（SEQ ID NO:122）

nucleic acid sequence of positive control CAR:

GCTGTGATGGCCCCTAGAACCCTGCTGCTGCTGCTGAGCGGCGCCCTGGCCCTGACACAGACCTGGGCCGACATTCAGATGACACAGAGCCCTAGCAGCCTGAGCGCTAGCGTGGGCGACAGAGTGACCATCACCTGCAAAGCTAGCCAAAACGTGGCCACCCACGTGGGCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGAGACTGATCTACAGCGCTAGCTACAGATACAGCGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAACCTGCAGCCCGAGGACTTCGCCACCTACTACTGTCAGCAGTACAACAGATACCCCTACACCTTTGGCCAAGGCACCAAGCTGGAAATCAAGGGCGGGGGCGGCTCCGGCGGGGGCGGCTCCGGGGGCGGGGGCTCCCAAGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCTAGCGTGAAGGTGAGCTGCAAGGCTAGCGGCTACAGCTTCACCGGCTACACCATGAACTGGGTGAGACAAGCCCCCGGCCAAGGCCTGGAGTGGATGGGCCTGATCAACCCCTACAACGGCGACACCAACTACGCTCAGAAGCTGCAAGGCAGAGTGACAATGACAACCGACACAAGCACAAGCACCGCCTACATGGAGCTGAGAAGCCTGAGAAGCGACGACACCGCCGTGTACTACTGCGCCCGGGTGGCCCTGAGAGTGGCCCTGGACTACTGGGGGCAAGGCACACTCGTGACCGTGAGCAGCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC（SEQ ID NO:123）。

(2) Packaging of lentiviral vectors

293T cells were thawed and cultured in DMEM medium containing 10% FBS. After 2-3 generation cell expansion culture, the method is carried out according to 4 multiplied by 10 ⁴ Individual/cm ² Is seeded into a 2-layer cell factory. Plasmid transfection was performed 3 days after cell inoculation, 40mL of Opti-MEM was added to a sterile 50mL centrifuge tube for plasmid transfection, and then the viral packaging vector and viral envelope vector were added according to the ratio of pK1-GPRC5D-CAR: pLP1: pLP2: pLP-VSVG=5:4:3:1, and 800. Mu.L of PEI transfection reagent was added, mixed immediately, and incubated at room temperature for 15min. The plasmid/vector/transfection reagent complex was then added drop-wise to a culture flask of 293T cells, after 24h the virus supernatant was collected into a 50mL centrifuge tube, centrifuged at 250g for 5min, the supernatant was filtered with a 0.45 μm filter after centrifugation, and the filtered supernatant was ultracentrifuged (25000 g, 4 ℃ C., 3 h) to obtain concentrated GPRC5D-CAR lentivirus. Centrifuging, discarding supernatant, re-suspending lentivirus with PBS pre-cooled at 4deg.C, packaging the re-suspended GPRC5D-CAR lentivirus liquid, and storing at-80deg.C.

(3) Resuscitation and activation of T cells

The frozen cord blood was removed and thawed in a water bath at 38 ℃. Transferring cord blood into 50mL centrifuge tube, adding appropriate amount of RPMI1640 culture medium, mixing, sampling, counting, centrifuging under 300g for 5min, collecting lower cell layer, and re-suspending with complete culture medium (RPMI 1640 culture medium containing 10% FBS, and supplemented with glutamine, 2-mercaptoethanol, IL-7 (10 ng/mL), IL-15 (10 ng/mL), IL-2 (200U/mL)) to T lymphocyte density of 1×10 ⁶ Adding activated antibodies Anti-human CD3 Anti-ibody and Anti-human CD28 Anti-ibody according to the re-suspension volume, wherein the Anti-human CD3 Anti-ibody is used at a concentration of 0.15 mug/mL, the Anti-human CD28 Anti-ibody is used at a concentration of 0.625 mug/mL, and placing T cells into a chamber at 37 ℃ and 5% CO ₂ Culturing in an incubator.

(4) Sorting and purification of T cells

After T cells are activated for 2 days, 20 mu L of the mixture is evenly mixed and sampled, 10 mu L of the diluted fluorescent labeled antibody is added, the mixture is dyed for 10min, PBS is added for 10 times, the mixture is diluted and then is detected and counted by a flow cytometry, the densities of CD45+, CD3+, CD4+ and CD8+ T cells are recorded, and the expression condition of the activation markers CD69 and CD25 molecules is observed. Cell volumes were recorded and T cell population was confirmed. Transferring the cell suspension into a centrifuge tube for centrifugation (500 g, 5 min), and discarding the supernatant to collect the lower layer cells; after washing with MACS Buffer, the lower cells were collected after centrifugation (500 g, 5 min) and resuspended with the appropriate amount of MACS Buffer. The amounts of CD4+ beads (Miltenyi Biotec, 130-045-101) and CD8+ beads (Miltenyi Biotec, 130-045-201) used were calculated based on the amounts of cells and the bead specifications, and the sorted T cells were obtained by sorting using CD4+ and CD8+ beads according to the descriptions of the bead specifications.

(5) Preparation of GPRC 5D-targeted CAR-T cells

Adjusting cell density of T cells to 400 cells/μl, plating, adjusting cell volume to 500 μl, adding GPRC5D-CAR slow virus liquid according to actual cell number, mixing, and cooling to 37deg.C and 5% CO ₂ Culturing in an incubator for 3 days, and respectively detecting the expression rate of the GPRC5D-CAR molecules on the surfaces of CD4+ and CD8+ T cells and the proliferation condition of the cells. The expression rate of the positive control CAR molecule was detected using FITC anti-human GPRC5D antibody (FITC-Labeled Human GPRC D Protein, flag, his Tag (nanodisk), acro) using antibody GPD-HF2D7&Alexa Fluor 488 AffiniPure Goat Anti-Alpaca IgG, VHH domain, (Jackson ImmunoResearch, 128-545-230) examined the expression rate of CAR molecules from each experimental group. The expression rates of GPRC5D-CAR molecules of CD4+ and CD8+ T cells are shown in figures 1 and 2 respectively, which shows that the CAR molecules constructed by the invention are successfully expressed on the T cells and have higher expression rates. The proliferation conditions of CD4+ and CD8+ T cells are shown in figures 3 and 4 respectively, which show that the CAR-T cells obtained after the CAR molecules constructed by the invention are transduced into the T cells still have good proliferation capacity. The expansion of the CAR-T cells was observed every three days from plating and fresh complete medium was supplemented, and T in the CAR-T cells was detected after 13 days of culture _SCM +T _CM And the proportion of Tim 3-expressing CAR-T cells, T _SCM +T _CM As shown in FIG. 5, tim3 was expressedAs shown in fig. 6. Experimental results show that the CAR-T cells constructed by the invention can still maintain a high proportion of T _SCM +T _CM Phenotype, indicative of greater persistence in vivo. After 19 days of culture, CAR-T cells were harvested for subsequent in vitro killing experiments. Each CAR-T cell group constructed was G1 (SEQ ID NO: 89), G2 (SEQ ID NO: 78), G3 (SEQ ID NO: 94), G4 (SEQ ID NO: 86), G5 (SEQ ID NO: 99), G6 (SEQ ID NO: 88), G7 (SEQ ID NO: 79), G8 (SEQ ID NO: 83), G9 (SEQ ID NO: 97), G10 (SEQ ID NO: 92), G11 (SEQ ID NO: 91), G12 (SEQ ID NO: 95), G13 (SEQ ID NO: 81), G14 (SEQ ID NO: 98), G15 (SEQ ID NO: 96), G16 (SEQ ID NO: 82), G17 (SEQ ID NO: 80), G18 (SEQ ID NO: 85), G19 (SEQ ID NO: 93), G20 (SEQ ID NO: 87), G21 (SEQ ID NO: 84), G22 (SEQ ID NO: 90), and the negative control T cell group was UnT (non-transduced CAR-T cell group was GC5 (SEQ ID NO: 122).

Stem cell-like memory T cells (T _SCM ) Is a T cell subgroup with self-renewal and effector T cell generation capacity, has the characteristic of being capable of long-term survival in vivo, and plays a role in maintaining long-term anti-tumor capacity. Central memory T cell (T) _CM ) Is T _SCM The latter stage of differentiation is usually carried out by analysis of T _SCM +T _CM The proportion is used for judging the in vivo persistence and the therapeutic potential of effector T cells. The invention evaluates T by analyzing the percentage of CD45RO-CD62L+ T cells and CD45RO+CD62L+ T cells _SCM +T _CM (CD 45RO: APC/Cyanine7 anti-human CD45RO, biolegend, B350148; CD62L: PE/Cyanine7 anti-human CD62L, biolegend, B373155), the higher the ratio, the more durable the CAR-T cell in vivo is predicted. Thus, if a higher proportion of T can be maintained in CAR-T cells _SCM +T _CM It would also be advantageous for use in vivo therapy.

The present invention was performed by analyzing Tim3 expressing T cells (Brilliant Violet 421 ^TM anti-human CD366, biolegend, B355191) to determine the depletion of T cells, tim 3-expressing CAR-T cellsThe higher the example, the lower the effector function of the CAR-T cells is predicted.

Example 3 validation of tumor cell killing Effect of GPRC 5D-targeted CAR-T cells

(1) Determination of expression of GPRC5D antigen on target cell surface

Human multiple myeloma (MM.1S) cells (purchased from ATCC, CRL-2974), raji cells (purchased from ATCC, CCL-86) and Raji GPRC5D positive cells, each of which was cultured in RPMI 1640 medium, were used as target cells, respectively.

Preparation of Raji GPRC5D positive cells: lentiviruses expressing GPRC5D antigen were obtained by culturing 293T cells at 2X 10 ⁵ /cm ² In a T-vessel, transiently transfecting 293T cells with a plasmid, adding 40mL of Optim-MEM into a sterile 50mL centrifuge tube for plasmid transfection, adding a viral packaging vector and a viral envelope vector according to the ratio of pK1-GPRC5D: pLP1: pLP2: pLP-VSVG=5:4:3:1, adding 800 mu L of PEI transfection reagent, immediately mixing, incubating for 15min at room temperature, dropwise adding the plasmid/vector/transfection reagent complex into 293T cells, collecting the viral supernatant after 24h into a 50mL centrifuge tube, centrifuging for 5min at 250g, filtering the supernatant with a 0.45 mu m filter, ultracentrifugating the filtered supernatant (25000 g, 4 ℃ for 3 h) to obtain concentrated GPRC5D lentivirus, centrifuging, discarding the supernatant, resuspending the lentivirus with 4 ℃ pre-chilled PBS, sub-packaging the resuspended GPRC5D lentivirus solution, and storing at-80 ℃ for later use. Normal Raji cells were proliferated at 4X 10 ⁴ The density of individual cells/wells was plated in 96-well plates, GPRC5D slow virus solution was added at moi=25, and the volume was made up to a total volume of 200 μl from fresh growth medium; on day 3 after virus infection, cell GPRC5D antigen expression was detected, when cell GPRC5D expression was stable over 5 passages and the infection efficiency was higher than 90%, monoclonal plating was performed in 96-well plates using limiting dilution method, cell density was adjusted to 0.008/. Mu.L, the plating volume per well was 100. Mu.L, and the growth state of the monoclonal was observed at the phase points of 8h, D1, D7, and D14 after plating. After confirming that Raji cells were monoclonal, the cells were cultured for 2 months or more, and then the cells were subjected to the following description of GPRC5D fluorescent-labeled antibody And (3) carrying out light-proof staining on the Raji monoclonal cells, detecting GPRC5D antigen expression on the cell surfaces by using a flow cytometry, and obtaining the Raji GPRC5D positive cell strain after determining the GPRC5D antigen expression. According to GPRC5D fluorescent-labeled antibody (Human GPRC5D PE-conjugated Antibody, R&D SYSTEMS, FAB6300 RP), raji-1F7 cells, raji-BE6 cells and Raji cells (namely Raji-CF10 cells) were stained for 10min in the absence of light, and then the expression of the cell surface GPRC5D antigen was detected by a flow cytometer, and the results are shown in FIG. 7. The results showed that GPRC5D antigen was highly expressed on Raji-1F7 cells (Raji GPRC5D positive cells), was lowly expressed on Raji-BE6 cells (Raji GPRC5D positive cells), and was unexpressed on Raji-CF10 cells (Raji cells).

(2) In vitro killing effect flow cytometry assay of GPRC 5D-targeted CAR-T cells

Target cells (9X 10) were added separately to 24-well plates ⁵ Individual/well) Raji-1f7, raji-BE6, raji-CF10, and the CAR-T cells constructed in example 2 were added to 24-well plates in accordance with the respective effector cell amounts, based on the amount of effector cells that need to BE added, based on the cell amount of cd8+ CAR-T cells in the CAR-T cells, with an effective target ratio (E: T) of 1:3. The negative control UnT group of untransduced CARs also added the same amount of UnT cells and target cells according to the corresponding effective target ratio. All groups were supplemented with complete medium to 500. Mu.L/well and the well plates were placed in 37℃with 5% CO ₂ Culturing in an incubator, measuring the amount of Raji cells in each well by flow cytometry after culturing for 1 day and 3 days, and calculating the killing rate, and the results are shown in fig. 8, 9, 10, 11 and fig. 12. The killing experiment result shows that the CAR-T cell constructed by the invention has remarkable killing capacity on GPRC5D positive target cells; the CAR-T cells constructed by the invention hardly kill GPRC5D negative target cells, which shows that the CAR-T cells have specific killing capacity.

The calculation formula is as follows: killing% = target cell reduction/target cell plating cell amount x 100%.

(3) Post-killing proliferation of GPRC 5D-targeted CAR-T cells, memory T cell subpopulations, and CAR-T cell depletion assays

In practice6 days after the in vitro killing experiment of item (2) of example 3 was plated, 20. Mu.L of each group of CAR-T cell culture solutions with target cells of Raji-1F7 and an effective target ratio of 1:3 were collected, 10. Mu.L of a staining solution was added, after 7min of staining in a dark place, 170. Mu.L of DPBS was added, and each group of cell T was detected by using a flow cytometer _SCM +T _CM And the Tim3 expression, the results are shown in FIGS. 13 and 14, respectively. The results show that the CAR-T cells constructed by the invention can still maintain a higher proportion of T after killing _SCM +T _CM Indicating a stronger persistence in the body. After plating in vitro killing experiments, flow assays were performed every 3 days to determine proliferation of each group of CAR-T cells, and the cumulative fold proliferation of cd4+, cd8+ T cells after killing experiments was counted for different positive target cell groups (effective target ratio 1:3) on day 9, with the results shown in figures 15, 16, 17 and 18. Experimental results show that the CAR-T cells constructed by the invention not only have specific killing capacity, but also have good proliferation capacity after killing.

(4) In vitro killing effect Lactate Dehydrogenase (LDH) release assay of GPRC 5D-targeted CAR-T cells

6X 10 at an effective target ratio (E: T) of 1:3 ⁴ The target cells mm.1s were added to a 96-well plate, and CAR-T cells constructed in example 2 after 17 days of culture were added to the 96-well plate in accordance with the corresponding effector cell amounts, based on the cell amounts of cd8+ CAR-T cells in the CAR-T cells to be added. The final density was set at 300 target cells/. Mu.L, and 100 CD8+ CAR-T cells/. Mu.L, with a total system of 200. Mu.L for each well. The negative control UnT group of untransduced CARs also added the same amount of UnT cells and target cells according to the corresponding effective target ratio. At the same time, a target cell spontaneous release control well, a target cell maximum release control well, an effector cell spontaneous release well, a background well (200. Mu.L of medium) and a volume correction well (200. Mu.L of medium+20. Mu.L of lysate) were set, and 5 sets of multiplex wells were set for each set. The 96-well plate was placed at 37℃with 5% CO ₂ After 24h incubation in incubator, the mixture was taken out, centrifuged at 600g for 10min, 50. Mu.L of the supernatant was transferred to a new 96-well plate, and the mixture was measured according to the instructions of the LDH cytotoxicity detection kit (Cytotox 96-cube Non-Radioactive Cytotoxicity Assay G1780). The extent of damage to the target cells was judged by measuring the activity of LDH in the cell culture supernatants of each group.

The calculation formula is as follows:

cytotoxicity= (experimental group OD value-effector cell spontaneous release well OD value-target cell spontaneous release well OD value)/(target cell maximum release well OD value-target cell spontaneous release control well OD value) ×100%;

experimental group OD = experimental group measured OD-bottom hole OD;

effector spontaneous release well OD = effector spontaneous release Kong Shice OD-basal well OD;

target cell spontaneous release control well OD = target cell spontaneous release Kong Shice OD-basal well OD;

target cell maximum release well OD value = target cell maximum release control Kong Shice OD value-volume corrected well OD value.

The culture medium used in the above experimental procedure was: RPMI 1640+10% FBS+0.1% 2-mercaptoethanol+1% L-Glu.

As shown in fig. 19, unT cells have no obvious killing effect on mm.1s target cells, while all experimental groups of GPRC5D CAR-T cells constructed by the invention and positive control groups of CAR-T cells have obvious killing effect on mm.1s target cells, indicating that GPRC5D CAR-T cells can specifically kill mm.1s myeloma cell lines.

(5) Cytokine release assay of GPRC 5D-targeted CAR-T cells

The supernatant of each set of CAR-T cell cultures was collected 18h after the in vitro killing assay of example 3, item (2), and the cytokine release profile of each set of CAR-T cells (IL-2, TNF- α, IFN- γ) was measured by CBA assay kit LEGENDplex ™ HU Th Cytokine Panel (12-plex) w/FP V02 (biolegend, cat# 741027) and the results are shown in FIGS. 20, 21, 22. Experimental results show that the CAR-T cells constructed by the invention can secrete a large amount of inflammatory cytokines (IL-2, TNF-alpha and INF-gamma) in the killing process, which proves that the CAR-T cells have remarkable killing capacity.

The specific operation steps are as follows:

the CAR-T cell culture supernatants of each group were collected separately 18h after plating of the in vitro killing experiments. After the captured microspheres (Beads) were returned to room temperature, they were vortexed for 2 min to mix well and formulated to 15 μl per sample for use. The 20xWash Buffer was returned to room temperature to dissolve the salt therein sufficiently, and it was prepared into 1xWash Buffer with ultrapure water for use. The standard was dissolved and mixed with 250 μl Assay Buffer and subjected to gradient dilution, 4-fold dilution at a dilution multiple, 6 total dilutions to obtain 6 gradient concentration points, for establishing a cell supernatant standard curve. And adding 15uL of each of an Assay Buffer, a Beads and a standard substance into an EP tube, uniformly mixing, and preparing a standard substance hole. And adding 15 mu L of Assay Buffer, beads and each sample into the EP tube, and uniformly mixing to prepare sample holes. The standard wells and sample wells were shaken at 900rpm under light-protected conditions and incubated for 2h at room temperature. The sample is filtered in vacuum, 200 mu L of 1xWash buffer is added to each EP tube, and the mixture is stirred and mixed uniformly, and the washing is repeated once. Add 15. Mu.L of detection antibody and 15. Mu.L of Assay buffer to each EP tube, blow mix well, shake at 900rpm under dark conditions and incubate at room temperature for 1h under dark conditions. Then 15. Mu.L of streptavidin-phycoerythrin (SA-PE) was added to each EP tube, shaken at 900rpm under light-shielding conditions, and incubated at room temperature for 0.5h under light-shielding conditions to bind the capture microspheres (Beads), the target analyte, with biotinylated detection antibody, SA-PE. 200uL 1xWash buffer was added to each EP tube for a second wash, mixed well with shaking and the wash repeated once. 200. Mu.L of 1xWash buffer was added to each tube and tested in the dark. Each sample was mixed well by shaking 90 s before detection, and the flow cytometer parameters were selected to FSC, SSC, PE and APC channels to detect release of cytokines IL-2, TNF- α, IFN- γ, respectively.

Claims

1. A single domain antibody targeting GPRC5D, wherein the single domain antibody comprises CDR1, CDR2, and CDR3;

wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 1, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 19, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 34;

wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 1, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 20, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 35;

wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 2, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 21, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 36;

wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 3, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 21, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 37;

wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 4, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 21, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 37;

wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 5, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 21, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 37;

Wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 2, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 22, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 37;

wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 2, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 22, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 38;

wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 6, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 22, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 38;

wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 7, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 22, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 38;

wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 8, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 23, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 39;

wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 9, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 24, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 40;

wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 10, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 25, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 41;

Wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 11, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 26, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 42;

wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 12, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 26, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 43;

wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 13, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 27, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 44;

wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 14, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 28, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 45;

wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 15, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 29, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 46;

wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 15, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 30, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 47;

wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 16, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 31, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 48;

Wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 17, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 32, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 49; or (b)

Wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 18, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 33, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 50.

2. The single domain antibody of claim 1, wherein the single domain antibody comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any of the amino acid sequences shown in SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 or SEQ ID NO: 72.

3. The single domain antibody of claim 1, wherein the single domain antibody comprises any one of the amino acid sequences as set forth in SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 or SEQ ID NO: 72.

4. A chimeric antigen receptor comprising the single domain antibody of any one of claims 1-3 that targets GPRC 5D.

5. A chimeric antigen receptor comprising

(a) An extracellular antigen-binding domain,

(b) A transmembrane domain, and

(c) An intracellular signaling domain;

wherein the extracellular antigen-binding domain comprises the single domain antibody of any one of claims 1-3 that targets GPRGRC 5D.

6. The chimeric antigen receptor according to claim 5, wherein the transmembrane domain is derived from CD8 a, CD28, cd3ζ, cd3γ, cd3δ, cd3ε, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, tcra, tcrβ, tcrγ, tcrδ, tcrζ, OX40, ICOS, LAG-3, 2B4, BTLA, CTLA-4, PD-1, or any combination thereof.

7. The chimeric antigen receptor according to claim 6, wherein the transmembrane domain is derived from CD8 a.

8. The chimeric antigen receptor according to claim 5, wherein the intracellular signaling domain is derived from fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, cd3ζ, CD22, CD79a, CD79b, and CD66d, or any combination thereof.

9. The chimeric antigen receptor according to claim 8, wherein the intracellular signaling domain is derived from cd3ζ.

10. The chimeric antigen receptor according to claim 8, wherein the intracellular signaling domain further comprises a costimulatory signaling domain, wherein the costimulatory signaling domain is derived from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD134 (OX 40), CD2, CD7, CD27, CD28, CD30, CD40, CD83, ICAM, 4-1BB (CD 137), CD276 (B7-H3), CD278 (ICOS), GITR, LIGHT, HVEM (light), BTLA, CD8 a, LFA-1, NKG2C, LAT, SLP-76, DAP10, PD-1, TRIM, ZAP70 ligand, or any combination thereof.

11. The chimeric antigen receptor according to claim 10, wherein the costimulatory signaling domain is derived from 4-1BB (CD 137).

12. The chimeric antigen receptor according to claim 5, further comprising a hinge region between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain.

13. The chimeric antigen receptor according to claim 12, wherein the hinge region is derived from CD8 a, CD28, CD137, igG4, igG1, or any combination thereof.

14. The chimeric antigen receptor according to claim 5, further comprising a signal peptide at the N-terminus of the chimeric antigen receptor polypeptide.

15. The chimeric antigen receptor according to claim 14, wherein the signal peptide is derived from HLA-A, CD8 a, CD4, CD33, CD137, GM-csfra, igG1, igκ, IL-2, or any combination thereof.

16. A chimeric antigen receptor comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of the amino acid sequences shown as SEQ ID No. 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99.

17. A chimeric antigen receptor, characterized in that it comprises any one of the amino acid sequences shown as SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98 or SEQ ID NO: 99.

18. An isolated nucleic acid comprising a nucleic acid sequence encoding the chimeric antigen receptor of claims 4-17.

19. The isolated nucleic acid of claim 18, comprising any one of the nucleic acid sequences set forth in SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, or SEQ ID NO: 121.

20. A vector comprising the isolated nucleic acid of claim 18 or 19.

21. An engineered immune effector cell comprising the chimeric antigen receptor of any one of claims 4-17, the isolated nucleic acid of claim 18 or 19, or the vector of claim 20.

22. The engineered immune effector cell of claim 21, wherein the immune effector cell is selected from T cells, NKT cells, DNT cells, NK cells, B cells, macrophages, dendritic cells, immune effector cells differentiated from induced pluripotent stem cells, or any combination thereof.

23. A pharmaceutical composition comprising the single domain antibody of any one of claims 1-3, the chimeric antigen receptor of any one of claims 4-17, or the engineered immune effector cell of any one of claims 21-22, and one or more pharmaceutically acceptable excipients and/or carriers.

24. Use of the single domain antibody of any one of claims 1-3 that targets GPRC5D, the chimeric antigen receptor of any one of claims 4-17, the engineered immune effector cell of any one of claims 21-22, or the pharmaceutical composition of claim 23 in the manufacture of a medicament for diagnosing, preventing, and/or treating a disease or disorder associated with GPRC5D expression.

25. The use according to claim 24, wherein the disease or disorder associated with GPRC5D expression is a malignancy associated with GPRC5D expression, an autoimmune disease associated with GPRC5D expression and/or an inflammatory disease.

26. The use according to claim 25, wherein the malignancy associated with GPRC5D expression is selected from marginal zone lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinum large B-cell lymphoma, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, follicular lymphoma, burkitt's lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, hairy cell leukemia, precursor B-cell leukemia, non-hodgkin's lymphoma, high grade B-cell lymphoma, megaloblastic and/or multiple myeloma, wherein the autoimmune and/or inflammatory disease associated with GPRC5D expression is selected from systemic lupus erythematosus, and/or rheumatoid arthritis.