Antigen-binding protein targeting CD38 and use thereof

Abstract

The present application relates to an antigen-binding protein targeting CD38 and the use thereof, and specifically relates to an isolated antigen-binding protein, which is capable of binding to CD38. The antigen-binding protein contains at least one CDR in an antibody heavy chain variable region. Further provided in the present application are a chimeric antigen receptor containing the antigen-binding protein, a fusion protein, a modified immune cell, an immunoconjugate, a pharmaceutical composition, a nucleic acid encoding the antigen-binding protein, a vector containing the nucleic acid molecule, and a cell containing the vector. Further provided in the present application is the use of the antigen-binding protein in the prevention and / or treatment of a disease.

Description

Antigen-binding proteins targeting CD38 and their applications Technical Field

[0001] This application relates to the field of biomedicine, specifically to an antigen-binding protein that targets CD38 and its applications. Background Technology

[0002] CD38, also known as cyclic ADP-ribose hydrolase, is a type II transmembrane glycoprotein with a long C-terminal extracellular domain and a short N-terminal cytoplasmic domain. It has the ability to convert NAD+ into cyclic ADP-ribose (cADPR). CD38 protein is one of the antigens expressed on malignant plasma cells and is expressed in various hematological malignancies, such as multiple myeloma, lymphoma, and leukemia cells. Functionally, CD38 protein participates in receptor-mediated activities such as adhesion, signal transduction, and ecto-enzyme activity. CD38 is also associated with autoimmune diseases. CD38 has diverse functions and participates in various physiological and pathological processes.

[0003] Therefore, CD38 is a target with great development potential in tumor treatment, and there is an urgent need to develop more therapeutic drugs that can effectively bind to it to meet the treatment needs of a wide range of diseases. Summary of the Invention

[0004] This application provides an antigen-binding protein targeting CD38. In this application, the antigen-binding protein has one or more of the following properties: (1) it can bind CD38 and has good binding activity; (2) it can bind CD38 expressed on the surface of target cells; (3) it can exert a tumor-killing effect. This application also provides chimeric antigen receptors, fusion proteins, modified immune cells, immune conjugates, and pharmaceutical compositions comprising the antigen-binding protein.

[0005] On one hand, this application provides an isolated antigen-binding protein capable of binding CD38, wherein the antigen-binding protein comprises an antibody heavy chain variable region VH, wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:26.

[0006] In some embodiments, the amino acid sequence of HCDR3 is as shown in SEQ ID NO:3 or SEQ ID NO:16.

[0007] In some embodiments, the amino acid sequence of HCDR2 is shown in SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:15.

[0008] In some embodiments, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:20 or SEQ ID NO:23.

[0009] In some embodiments, the amino acid sequence of HCDR1 is as shown in SEQ ID NO:29 or SEQ ID NO:14.

[0010] In some embodiments, the amino acid sequence of HCDR1 is as shown in SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:14 or SEQ ID NO:19.

[0011] In some embodiments, the amino acid sequences of HCDR1, HCDR2, and HCDR3 are selected from any combination of the following:

[0012] a) The amino acid sequence of HCDR1 is shown in SEQ ID NO:29, the amino acid sequence of HCDR2 is shown in SEQ ID NO:27, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:26.

[0013] b) The amino acid sequence of HCDR1 is shown in SEQ ID NO:29, the amino acid sequence of HCDR2 is shown in SEQ ID NO:28, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:26; and

[0014] c) The amino acid sequence of HCDR1 is shown in SEQ ID NO:14, the amino acid sequence of HCDR2 is shown in SEQ ID NO:15, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:26.

[0015] In some embodiments, the amino acid sequences of HCDR1, HCDR2, and HCDR3 are selected from any combination of the following:

[0016] a) The amino acid sequence of HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:3.

[0017] b) The amino acid sequence of HCDR1 is shown in SEQ ID NO:9, the amino acid sequence of HCDR2 is shown in SEQ ID NO:10, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:3.

[0018] c) The amino acid sequence of HCDR1 is shown in SEQ ID NO:14, the amino acid sequence of HCDR2 is shown in SEQ ID NO:15, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:16.

[0019] d) The amino acid sequence of HCDR1 is shown in SEQ ID NO:19, the amino acid sequence of HCDR2 is shown in SEQ ID NO:20, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:3; and

[0020] e) The amino acid sequence of HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:23, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:3.

[0021] In some embodiments, the amino acid sequence of the VH is as shown in SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:22 or SEQ ID NO:25.

[0022] In some embodiments, the antigen-binding protein further includes an immunoglobulin constant region.

[0023] In some embodiments, the immunoglobulin constant region is the IgG Fc region.

[0024] In some embodiments, the immunoglobulin constant region is the human IgG Fc region.

[0025] In some embodiments, the antigen-binding protein is an antibody or its antigen-binding fragment.

[0026] In some embodiments, the antigen-binding fragment is a Fab, (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH, or dAb fragment of the antibody.

[0027] In some embodiments, the antigen-binding protein is VHH.

[0028] In some embodiments, the antigen-binding protein is a chimeric antibody, a humanized antibody, or a fully human antibody.

[0029] In some embodiments, the antigen-binding protein is a monovalent antibody, a bivalent antibody, or a multivalent antibody.

[0030] In some embodiments, the antigen-binding protein is a monospecific antibody, a bispecific antibody, or a multispecific antibody.

[0031] On the other hand, this application provides a chimeric antigen receptor comprising the antigen-binding protein.

[0032] In some embodiments, the chimeric antigen receptor includes an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain.

[0033] In some embodiments, the antigen-binding domain contains the antigen-binding protein.

[0034] In some embodiments, the chimeric antigen receptor further includes a co-stimulatory domain.

[0035] In some embodiments, the chimeric antigen receptor further includes a hinge region.

[0036] On the other hand, this application provides a fusion protein comprising the antigen-binding protein.

[0037] On the other hand, this application provides modified immune cells, wherein the immune cells comprise the chimeric antigen receptor and / or the fusion protein.

[0038] In some embodiments, the immune cells are T cells, NK cells, iNKT cells, dendritic cells, and / or macrophages.

[0039] In some embodiments, the immune cells also contain and / or express low-density lipoprotein receptor-associated protein or fragments thereof.

[0040] In some embodiments, the low-density lipoprotein receptor-associated protein or a fragment thereof comprises one or more selected from the group consisting of low-density lipoprotein receptor-associated proteins 1-12 and their functional fragments.

[0041] In some embodiments, the low-density lipoprotein receptor-associated protein or a fragment thereof is low-density lipoprotein receptor-associated protein 5 and / or 6 or a fragment thereof.

[0042] In some embodiments, the low-density lipoprotein receptor-associated protein or a fragment thereof comprises the amino acid sequence shown in SEQ ID NO:36.

[0043] In some embodiments, the low-density lipoprotein receptor-associated protein or a fragment thereof is an exogenous low-density lipoprotein receptor-associated protein or a fragment thereof.

[0044] On the other hand, this application provides an immune conjugate comprising the antigen-binding protein.

[0045] On the other hand, this application provides isolated nucleic acid molecules that encode the antigen-binding protein, the chimeric antigen receptor, and / or the fusion protein.

[0046] On the other hand, this application provides a vector containing the nucleic acid molecule.

[0047] On the other hand, this application provides cells that contain the nucleic acid molecules and / or the vector.

[0048] On the other hand, this application provides a pharmaceutical composition comprising the antigen-binding protein, the chimeric antigen receptor, the fusion protein, the modified immune cell, the immune conjugate, the nucleic acid molecule, the carrier and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0049] On the other hand, this application provides a kit comprising the antigen-binding protein, the chimeric antigen receptor, the fusion protein, the modified immune cells, the immune conjugate, the nucleic acid molecule, the carrier, the cells, and / or the pharmaceutical composition.

[0050] On the other hand, this application provides the use of the antigen-binding protein, the chimeric antigen receptor, the fusion protein, the modified immune cell, the immune conjugate, the nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of diseases and / or conditions.

[0051] In some implementations, the disease and / or condition is a tumor and / or an autoimmune disease.

[0052] In some implementations, the disease and / or condition is multiple myeloma, lymphoma, leukemia, and / or autoimmune disease.

[0053] On the other hand, this application provides a method for preventing and / or treating diseases and / or conditions, comprising administering to a subject in need the antigen-binding protein, the chimeric antigen receptor, the fusion protein, the modified immune cell, the immune conjugate, the nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition.

[0054] In some implementations, the disease and / or condition is a tumor and / or an autoimmune disease.

[0055] In some implementations, the disease and / or condition is multiple myeloma, lymphoma, leukemia, and / or autoimmune disease.

[0056] On the other hand, this application provides the antigen-binding protein, the chimeric antigen receptor, the fusion protein, the modified immune cell, the immune conjugate, the nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition for the prevention and / or treatment of diseases and / or conditions.

[0057] In some implementations, the disease and / or condition is a tumor and / or an autoimmune disease.

[0058] In some implementations, the disease and / or condition is multiple myeloma, lymphoma, leukemia, and / or autoimmune disease.

[0059] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0060] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0061] Figure 1 shows the CD38 protein selection strategy described in this application.

[0062] Figure 2 shows the results of the binding activity of the CD38 antibody to the CD38 protein described in this application.

[0063] Figure 3 shows the results of the specific binding activity of the CD38 antibody described in this application.

[0064] Figure 4 shows the binding activity results of the CD38 antibody described in this application with target cells expressing different CD38 target antigen abundances.

[0065] Figure 5 shows a schematic diagram of the CD38 chimeric antigen receptor structure described in this application.

[0066] Figure 6 shows a schematic diagram of the CD38 chimeric antigen receptor lentiviral vector system described in this application.

[0067] Figure 7 shows the results of CD38 CAR-T killing MM.1S target cells as described in this application.

[0068] Figure 8 shows the results of CD38 CAR-T killing NCIH929 target cells as described in this application.

[0069] Figure 9 shows the IL-2 secretion level results when CD38 CAR-T cells described in this application are co-incubated with target cell lines.

[0070] Figure 10 shows the IFN-γ secretion level results when CD38 CAR-T cells described in this application are co-incubated with target cell lines.

[0071] Figure 11 shows the TNF-α secretion level results when CD38 CAR-T cells described in this application are co-incubated with target cell lines.

[0072] Figure 12 shows the results of the non-specific binding activity of the CD38 antibody described in this application. Detailed Implementation

[0073] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0074] Terminology Definition

[0075] In this application, the terms "CD38" and "ADP-ribosylcyclase 1" are generally used interchangeably and typically refer to a type II transmembrane glycoprotein. In this application, CD38 may consist of a long C-terminal extracellular domain and a short N-terminal cytoplasmic domain. In this application, CD38 may be a cyclic ADP-ribohydrolase. In this application, CD38 may be the complete CD38 and its functionally active fragments, homologs, analogs, variants, or derivatives. For example, CD38 may be full-length CD38 or a truncated CD38 that retains its functional activity. In this application, CD38 may be of any species origin. For example, CD38 may be human CD38. In this application, CD38 may be wild-type or artificially modified. For example, CD38 may be a modified CD38.

[0076] In this application, the term "antigen-binding protein" generally refers to a protein capable of binding antigens. In this application, the antigen-binding protein may comprise an antigen-binding portion and optionally, allow the antigen-binding portion to employ a scaffold or framework portion that facilitates the antigen-binding portion's conformation for binding antigens. In this application, the antigen-binding protein may be wild-type or artificially engineered. In this application, the antigen-binding protein may comprise, for example, an antibody-derived protein scaffold or an alternative protein scaffold or artificial scaffold with a transplanted CDR or CDR derivative. In this application, the CDR can be determined using various coding systems. For example, the CDR can be determined using CCG, Kabat, Chothia, IMGT, AbM, or a combination of Kabat / Chothia, etc. For example, the CDR can be determined using the IMGT coding system. In this application, the CDR encompasses CDR sequences partitioned according to any CDR partitioning method. In this application, the CDR may encompass its variants. For example, the amino acid sequence of the CDR can be substituted, deleted, and / or added with one or more amino acids, such as 1-30, 1-20, or 1-10, or for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids. For example, the CDR encompasses homologs. For example, the homolog can be an amino acid sequence having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher) sequence homology with the amino acid sequence of the CDR. In this application, the antigen-binding protein can be artificially synthesized. For example, the antigen-binding protein can be derived from plasma, hybridoma, or recombinant cell lines. In this application, the antigen-binding protein can be of animal or non-animal origin. In this application, the antigen-binding protein may be an antibody or its antigen-binding fragment, as well as its variants, homologs, derivatives, or analogs. In this application, the antigen-binding protein may include, but is not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv, VHH, and / or dAb), immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, as long as they exhibit the desired antigen-binding activity. In this application, the antibody may be a chimeric antibody, a humanized antibody, or a fully human antibody. In this application, the antibody may be a recombinant, hybrid, mutated, or transplanted antibody. In this application, the antigen-binding protein may be VHH. In this application, the antigen-binding protein may specifically bind to CD38.

[0077] In this application, the terms "VHH," "nanobody," and "single-domain antibody" are generally used interchangeably and typically refer to an antibody structure composed of a heavy chain variable region. In this application, the VHH may consist of VHs. In this application, the VHH may contain a complementarity-determining region (CDR) and a framework region (FR). In this application, the VHH contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In this application, the VHH may be artificially synthesized. In this application, the VHH may be of animal or non-animal origin. In this application, the VHH may be a chimeric antibody, a humanized antibody, or a fully human antibody.

[0078] In this application, the terms "immunoglobulin constant region" and "constant region" are generally used interchangeably, referring to a region of the antibody's non-variable region. In this application, the constant region does not directly participate in antigen binding but exhibits various effector functions. In this application, the constant region can be the entire antibody non-variable region or a portion of it. For example, the constant region can be a heavy chain constant region. For example, the constant region can be an Fc region. In this application, the constant region can be derived from animals. For example, the constant region can be derived from humans, goats, rabbits, rats, or guinea pigs. For example, the Fc region can be a human Fc region.

[0079] The protein and / or amino acid sequences involved in this application should also be understood to include at least the following range: variants or homologs having the same or similar functions as the protein. In this application, the variant can be a protein or polypeptide that has undergone substitution, deletion, or addition of one or more amino acids in the amino acid sequence of the protein (e.g., the antigen-binding protein described in this application). For example, the functional variant may comprise a protein or polypeptide that has undergone amino acid alterations through substitution, deletion, and / or insertion of at least one, such as 1-30, 1-20, or 1-10, or even 1, 2, 3, 4, or 5 amino acids. The functional variant can substantially retain the biological properties of the protein or polypeptide before the alteration (e.g., substitution, deletion, or addition). For example, the functional variant can retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen-binding capacity) of the protein or polypeptide before the alteration. For example, the substitution can be a conserved substitution. In this application, a portion of the amino acid sequence of the antigen-binding protein may be homologous to the corresponding amino acid sequence in an antibody from a specific species, or belong to a specific category. For example, both the variable region and the constant region of the antibody may be derived from the variable region and the constant region of an antibody from an animal species (such as a human). In this application, the homolog may be a protein or polypeptide having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology to the amino acid sequence of the protein and / or the polypeptide (e.g., the antigen-binding protein described in this application).

[0080] In this application, the terms "chimeric antigen receptor" and "CAR" are generally used interchangeably and typically refer to a recombinant polypeptide. In this application, the chimeric antigen receptor may include an extracellular domain, a transmembrane domain, and an intracellular domain that specifically binds to an antigen or target. In this application, the extracellular domain may include an antigen-binding domain. In this application, the extracellular domain may also include a hinge region. In this application, the extracellular domain may also include a signal peptide. In this application, the intracellular domain may include an intracellular signal transduction domain. In this application, the intracellular domain may also include a co-stimulatory domain. In this application, the CAR may be secreted by cells or artificially synthesized. In this application, the chimeric antigen receptor may also include a low-density lipoprotein receptor-associated protein or a fragment thereof.

[0081] In this application, the term "fusion protein" generally refers to a recombinant polypeptide. In this application, the fusion protein may include an extracellular domain and a transmembrane domain that specifically bind to an antigen or target. In this application, the extracellular domain may include an antigen-binding domain. In this application, the extracellular domain may also include a hinge region. In this application, the extracellular domain may also include a signal peptide. In this application, the fusion protein may also include an intracellular domain. In this application, the intracellular domain may include a co-stimulatory domain. In this application, the intracellular domain may also include an intracellular signal transduction domain. In this application, the fusion protein may be secreted by cells or artificially synthesized. In this application, the fusion protein may also include a low-density lipoprotein receptor-associated protein or a fragment thereof.

[0082] In this application, the term "signal peptide" generally refers to a leader sequence located at the amino terminus (N-terminus) of a nascent CAR that can guide the nascent protein to the endoplasmic reticulum for expression during or after translation.

[0083] In this application, the term "hinge region" generally refers to a peptide, polypeptide, or protein molecule. In this application, the hinge region may be a polypeptide molecule. In this application, the hinge region may be flexible, allowing independent movement of the antigen-binding domain. In this application, the hinge region may be subdivided into upper, middle, and lower hinge domains. In this application, the hinge region may include hinge regions derived from the following proteins: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

[0084] In this application, the term "transmembrane domain" generally refers to a domain of a peptide, polypeptide, or protein that can cross the cell membrane, and these domains can be used to anchor extracellular domains to the cell membrane. For example, the transmembrane region may comprise a transmembrane domain of one or more proteins selected from the group consisting of: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

[0085] In this application, the term "co-stimulatory domain" generally refers to a portion of the intracellular signaling region capable of transducing effector signals by a CAR. For example, the co-stimulatory domain may include a co-stimulatory domain derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.

[0086] In this application, the term "intracellular signaling domain" generally refers to an intracellular region capable of generating signals that promote the function of immune effectors in CAR-containing cells (e.g., CAR-T cells or CAR-iNKT cells). For example, the intracellular signaling region may include intracellular signaling regions of one or more proteins selected from the group consisting of: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (KSHV), DAP10, DAP-12, and a domain containing at least one ITAM.

[0087] In this application, the term "immune cell" generally refers to a cell involved in an immune response. For example, the immune cell may be an immune cell that performs effector functions. For example, performing effector functions may include clearing foreign antigens or promoting immune effector responses. In this application, the immune effector cells may include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, iNKT cells, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphoprogenitor cells, and / or pluripotent stem cells. For example, the immune effector cell may be a T cell.

[0088] In this application, the term "low-density lipoprotein receptor-related protein" refers to a cell surface protein belonging to the endocytic receptor family. It is widely distributed in organisms and exhibits significant interstitial variability. Its main function is to take up cholesterol into cells for cell proliferation and the synthesis of steroid hormones and bile salts. For example, the low-density lipoprotein receptor-related protein can be derived from any vertebrate. For example, the low-density lipoprotein receptor-related protein or a fragment thereof can be located at the C-terminus of the intracellular signaling region. For example, the low-density lipoprotein receptor-related protein or a fragment thereof can comprise one or more selected from the group consisting of low-density lipoprotein receptor-related proteins 1-12 and their functional fragments. For example, the low-density lipoprotein receptor-related protein or a fragment thereof can be low-density lipoprotein receptor-related protein 6 or a fragment thereof.

[0089] In this application, the term "immunoconjugate" generally refers to a conjugate formed by the conjugation of other active ingredients with the antigen-binding protein. In this application, the active ingredient may be covalently linked to the antigen-binding protein via a linker molecule. In this application, the immunoconjugate may be an antigen-binding protein conjugated to a payload. For example, the payload may be a toxin, polymer, protein, drug, radioisotope, nucleic acid compound, or glucocorticoid. In this application, the immunoconjugate may be an antibody-drug conjugate (ADC), a PROTAC-antibody conjugate (DAC), or a targeted radiopharmaceutical (RDC). In this application, the conjugate can deliver the payload to the target cell by specifically binding the antigen to the antigen on the target cell via the antigen-binding protein. For example, the target cell may be a tumor cell.

[0090] In this application, the term "pharmaceuticalally acceptable carrier" generally refers to a non-toxic material that does not interfere with the effectiveness of the bioactivity of the active ingredient. For example, a pharmaceutically acceptable carrier includes pharmaceutically acceptable excipients or stabilizers that are non-toxic to cells or mammals exposed to them at the doses and concentrations used. For example, a physiologically acceptable carrier may be water, salt, protein, polysaccharide, lipid, or inactive viral particles.

[0091] In this application, the term "prevention and / or treatment" generally refers to the prevention and / or treatment of a disease. For example, the prevention and / or treatment could be preventing the onset of the disease, slowing or reversing the disease progression, preventing or slowing the onset of one or more symptoms associated with the disease, reducing or alleviating one or more symptoms associated with the disease, reducing the severity and duration of the disease and any symptoms associated with it, or preventing further increases in the severity of the disease and any symptoms associated with it. In this application, the disease could be a tumor. For example, preventing or alleviating the onset of one or more symptoms associated with a tumor, or reducing the severity and duration of the tumor and any related symptoms.

[0092] In this application, the term "tumor" generally refers to any new pathological tissue proliferation containing tumor antigens that can be recognized by the immune system. In this application, the tumor may include benign or malignant tumors (cancer). In this application, the cancer may be metastatic or non-metastatic. In this application, the tumor may include solid tumors and / or hematologic malignancies. In this application, a solid tumor generally refers to a tangible tumor that can be detected by clinical examination. For example, a solid tumor may include growths or solid lesions formed by abnormal cell growth. In this application, a hematologic malignancy generally refers to a class of hematopoietic system diseases. In this application, a hematologic malignancy may include various types of leukemia, multiple myeloma, or malignant lymphoma. In this application, the tumor may be a CD38-positive tumor. For example, the tumor may be multiple myeloma, lymphoma, and / or leukemia.

[0093] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.

[0094] In this application, the term "comprising" generally means including the explicitly specified features, but does not exclude other elements.

[0095] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0096] Invention Details

[0097] antigen-binding proteins

[0098] The CDR (Complementarity Determinant Region) of an antibody, also known as the complementarity-determining region, is part of the variable region. Amino acid residues in this region can contact antigens or antigenic epitopes. Antibody CDRs can be determined using various coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, and a combination of Kabat / Chothia. These coding systems are known in the art and can be found, for example, at http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can determine the CDR region using different coding systems based on the antibody's sequence and structure. The CDR region may differ when using different coding systems. In this application, the term CDR encompasses CDR sequences partitioned according to any CDR partitioning method; it also encompasses variants of the CDR, including amino acid sequences with substitutions, deletions, and / or additions of one or more amino acids. For example, substitutions, deletions, and / or insertions of 1-30, 1-20, or 1-10 amino acids, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids; this also includes homologs, which can be amino acid sequences having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher) sequence homology with the amino acid sequence of the CDR. For example, the CDR of the isolated antigen-binding protein described in the sequence listing of this application can be determined using IMGT.

[0099] On one hand, this application provides an isolated antigen-binding protein capable of binding CD38, wherein the antigen-binding protein comprises at least one CDR in the variable region of the antibody heavy chain.

[0100] In this application, the antigen-binding protein may include an antibody heavy chain variable region VH, and the VH may include heavy chain complementarity-determining regions HCDR1, HCDR2 and / or HCDR3.

[0101] In this application, the antigen-binding protein has one or more of the following properties: (1) it can bind CD38 and has good binding activity; (2) it can bind CD38 expressed on the surface of target cells; (3) it can kill tumors.

[0102] In this application, the antigen-binding protein is capable of binding to CD38 with an affinity / binding activity similar to or higher than that of the control antibody Daratumumab. For example, the antigen-binding protein is capable of binding to CD38 with a KD value similar to or higher than that of the control antibody Daratumumab. For example, the antigen-binding protein is capable of binding to CD38 with an EC50 value similar to or lower than that of the control antibody Daratumumab. In this application, the EC50 value or KD value can be determined using conventional techniques in the art. In this application, the KD value can be determined using Octet, SPR, ELISA, competitive ELISA, BIACORE, or KINEXA. In this application, the specific binding can be determined using FACS.

[0103] In this application, the VH may contain HCDR3, the amino acid sequence of which is shown in SEQ ID NO:26.

[0104] In this application, the VH may contain HCDR3, the amino acid sequence of which is shown in SEQ ID NO:3 or SEQ ID NO:16.

[0105] In this application, the VH may contain HCDR2, the amino acid sequence of which is shown in SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:15.

[0106] In this application, the VH may contain HCDR2, and the amino acid sequence of the HCDR2 is shown in SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:20 or SEQ ID NO:23.

[0107] In this application, the VH may contain HCDR1, the amino acid sequence of which is shown in SEQ ID NO:29 or SEQ ID NO:14.

[0108] In this application, the VH may contain HCDR1, and the amino acid sequence of the HCDR1 is shown in SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:14 or SEQ ID NO:19.

[0109] In this application, AAGTKVYGYSEY X1 X2 (SEQ ID NO:26), where X1 can be D or T, and X2 can be F or Y.

[0110] In this application, I X1S X2DG X3T (SEQ ID NO:27), wherein X1 can be N or S, X2 can be R or S, and X3 can be G or I.

[0111] In this application, INWGG X1 X2T (SEQ ID NO:28), where X1 can be G or S, and X2 can be G or S.

[0112] In this application, G X1ILSRN X2 (SEQ ID NO:29), wherein X1 can be D, R or S, and X2 can be I, T or V.

[0113] In this application, the antigen-binding protein may include an antibody heavy chain variable region VH, and the VH may include heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO:29 or SEQ ID NO:14, the amino acid sequence of HCDR2 is shown in SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:15, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:26.

[0114] In this application, the antigen-binding protein may include an antibody heavy chain variable region VH, and the VH may include heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:14, or SEQ ID NO:19. The amino acid sequence of HCDR2 is shown in SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:20, or SEQ ID NO:23. The amino acid sequence of HCDR3 is shown in SEQ ID NO:3 or SEQ ID NO:16.

[0115] In this application, the antigen-binding protein may include an antibody heavy chain variable region VH, and the VH may include heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO:29, the amino acid sequence of HCDR2 is shown in SEQ ID NO:27, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:26; or the amino acid sequence of HCDR1 is shown in SEQ ID NO:29, the amino acid sequence of HCDR2 is shown in SEQ ID NO:28, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:26; or the amino acid sequence of HCDR1 is shown in SEQ ID NO:14, the amino acid sequence of HCDR2 is shown in SEQ ID NO:15, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:26.

[0116] In this application, the antigen-binding protein may include an antibody heavy chain variable region VH, and the VH may include heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:3; or the amino acid sequence of HCDR1 is shown in SEQ ID NO:9, the amino acid sequence of HCDR2 is shown in SEQ ID NO:10, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:3; or the amino acid sequence of HCDR1 is shown in SEQ ID NO:14, the amino acid sequence of HCDR2 is shown in SEQ ID NO:15, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:16; or the amino acid sequence of HCDR1 is shown in SEQ ID NO:19, the amino acid sequence of HCDR2 is shown in SEQ ID NO:20, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:3; or the amino acid sequence of HCDR1 is shown in SEQ ID NO:19. As shown in NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:23, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:3.

[0117] In this application, the VH may include H-FR1, H-FR2, H-FR3 and / or H-FR4.

[0118] In this application, the VH may contain H-FR1, the amino acid sequence of which is shown in SEQ ID NO:4.

[0119] In this application, the VH may contain H-FR2, the amino acid sequence of which is shown in SEQ ID NO:5, SEQ ID NO:11 or SEQ ID NO:24.

[0120] In this application, the VH may contain H-FR3, the amino acid sequence of which is shown in SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:17 or SEQ ID NO:21.

[0121] In this application, the VH may contain H-FR4, the amino acid sequence of which is shown in SEQ ID NO:7.

[0122] In this application, the VH comprises H-FR1, H-FR2, H-FR3 and H-FR4, the amino acid sequence of H-FR1 is shown in SEQ ID NO:4, the amino acid sequence of H-FR2 is shown in SEQ ID NO:5, SEQ ID NO:11 or SEQ ID NO:24, the amino acid sequence of H-FR3 is shown in SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:17 or SEQ ID NO:21, and the amino acid sequence of H-FR4 is shown in SEQ ID NO:7.

[0123] In this application, the VH may comprise H-FR1, H-FR2, H-FR3, and H-FR4, wherein the amino acid sequence of H-FR1 is shown in SEQ ID NO:4, the amino acid sequence of H-FR2 is shown in SEQ ID NO:5, the amino acid sequence of H-FR3 is shown in SEQ ID NO:6, and the amino acid sequence of H-FR4 is shown in SEQ ID NO:7; or the amino acid sequence of H-FR1 is shown in SEQ ID NO:4, the amino acid sequence of H-FR2 is shown in SEQ ID NO:11, the amino acid sequence of H-FR3 is shown in SEQ ID NO:12, and the amino acid sequence of H-FR4 is shown in SEQ ID NO:7; or the amino acid sequence of H-FR1 is shown in SEQ ID NO:4, the amino acid sequence of H-FR2 is shown in SEQ ID NO:5, the amino acid sequence of H-FR3 is shown in SEQ ID NO:17, and the amino acid sequence of H-FR4 is shown in SEQ ID NO:7; or the amino acid sequence of H-FR1 is shown in SEQ ID NO:4. As shown in NO:4, the amino acid sequence of H-FR2 is shown in SEQ ID NO:5, the amino acid sequence of H-FR3 is shown in SEQ ID NO:21, and the amino acid sequence of H-FR4 is shown in SEQ ID NO:7; or the amino acid sequence of H-FR1 is shown in SEQ ID NO:4, the amino acid sequence of H-FR2 is shown in SEQ ID NO:24, the amino acid sequence of H-FR3 is shown in SEQ ID NO:12, and the amino acid sequence of H-FR4 is shown in SEQ ID NO:7.

[0124] In this application, the C-terminus of H-FR1 can be directly or indirectly connected to the N-terminus of HCDR1. In this application, H-FR2 can be located between HCDR1 and HCDR2. In this application, H-FR3 can be located between HCDR2 and HCDR3. In this application, the N-terminus of H-FR4 can be directly or indirectly connected to the C-terminus of HCDR3.

[0125] In this application, the antigen-binding protein may include HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3, and H-FR4. The C-terminus of H-FR1 is directly or indirectly connected to the N-terminus of HCDR1. H-FR2 is located between HCDR1 and HCDR2. H-FR3 is located between HCDR2 and HCDR3. The N-terminus of H-FR4 is directly or indirectly connected to the C-terminus of HCDR3.

[0126] In this application, the amino acid sequence of the FR can be a FR from any species. For example, the FR can be a FR from mouse, rabbit, goat, alpaca, or human. For example, the FR can be a human FR.

[0127] In this application, the amino acid sequence of FR can be adjusted as needed. For example, FR can be a wild-type sequence. For example, during antibody humanization, the amino acid sequence of FR can be altered without reducing the binding activity / affinity of the antigen-binding protein to CD38. For example, during antibody humanization, one or more amino acid sequences in FR can be mutated or optimized without reducing the binding activity / affinity of the antigen-binding protein to CD38. For example, FR can be a variant thereof, which includes the substitution, deletion, and / or addition of one or more amino acids in the amino acid sequence of FR. For example, 1-30, 1-20, or 1-10 amino acids, or for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions. For example, the FR can be a homolog, which can be an amino acid sequence having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology with the amino acid sequence of the FR.

[0128] In this application, the direct or indirect connection can be achieved through intermolecular forces or through connectors.

[0129] In this application, the amino acid sequence of the VH may be as shown in SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:22, or SEQ ID NO:25. For example, the VH may be a wild-type sequence. For example, one or more amino acid sequences in the VH may be mutated or optimized without reducing the binding activity / affinity of the antigen-binding protein to CD38. For example, the VH may be a variant thereof, the variant comprising substitution, deletion, and / or addition of one or more amino acids to the amino acid sequence of the VH. For example, the VH may also contain a sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher homology to the amino acid sequences shown in SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:22, or SEQ ID NO:25.

[0130] In this application, the antigen-binding protein may further comprise an immunoglobulin constant region. In this application, the immunoglobulin constant region may be an immunoglobulin Fc region. In this application, the Fc region may be a wild-type sequence or may be mutated or optimized. In this application, the Fc region may be an Fc region of any species origin. For example, the immunoglobulin constant region may be an IgG Fc region. For example, the immunoglobulin constant region may be an IgG1 Fc region. For example, the Fc region may be derived from the Fc region of mouse, rabbit, goat, alpaca, or human. For example, the immunoglobulin constant region may be a human IgG Fc region. For example, the immunoglobulin constant region may be a human IgG1 Fc region.

[0131] In this application, the antigen-binding protein may be an antibody or its antigen-binding fragment. For example, the isolated antigen-binding protein described in this application may include, but is not limited to, antibodies, antigen-binding fragments, immunoconjugates, multispecific antibodies, antibody fragments, antibody derivatives, antibody analogs, or fusion proteins.

[0132] In this application, the antigen-binding fragment may be a Fab, (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH, or dAb fragment of the antibody. For example, the antigen-binding fragment may be VHH, and the amino acid sequence of the VHH is shown in SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:22, or SEQ ID NO:25.

[0133] In this application, the antigen-binding protein may be a chimeric antibody, a humanized antibody, or a fully human antibody. For example, the antigen-binding protein may have its FR and / or constant regions mutated / optimized without reducing its binding activity / affinity with CD38. For example, the antigen-binding protein may have low immunogenicity without reducing its binding activity / affinity with CD38.

[0134] In this application, the antigen-binding protein may be a monovalent antibody, a bivalent antibody, or a multivalent antibody. For example, the antigen-binding protein may have one, two, or more antigen-binding sites.

[0135] In this application, the antigen-binding protein may be a monospecific antibody, a bispecific antibody, or a multispecific antibody. For example, the antigen-binding protein may contain one, two, or more targeting moieties.

[0136] In this application, the antigen-binding protein can be expressed as a fusion protein. For example, the antigen-binding protein can be fused with peptides, polypeptides, amino acids, or proteins. In this application, the fusion protein can be a Fab fusion protein, an Fc fusion protein, or a single-chain antibody fusion protein. For example, the antigen-binding protein can be fused with one or more functional molecules. For example, the functional molecule can be one or more antigen-binding proteins. For example, the functional molecule can be one or more Fc regions.

[0137] In this application, the antigen-binding protein may be wild-type. For example, the antigen-binding protein may contain a wild-type sequence. In this application, the antigen-binding protein may be recombinant. For example, the antigen-binding protein may be produced using recombinant DNA technology. In this application, the antigen-binding protein may be modified. For example, the antigen-binding protein may be mutated or optimized. For example, the antigen-binding protein may be substituted, deleted, and / or added with one or more amino acids. For example, 1-30, 1-20, or 1-10 amino acids, or for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids substituted, deleted, and / or inserted.

[0138] Antigen recognition receptors and fusion proteins

[0139] On the other hand, this application provides an antigen recognition receptor comprising the antigen-binding protein.

[0140] On the other hand, this application provides a fusion protein comprising the antigen-binding protein.

[0141] In this application, the antigen recognition receptor may be a chimeric antigen receptor (CAR). In this application, the antigen recognition receptor may be a T-cell receptor (TCR).

[0142] In this application, the TCR may include the antigen-binding protein.

[0143] In this application, the TCR can specifically bind to CD38.

[0144] In this application, the TCR may include an α chain and a β chain, wherein the α chain includes an α chain variable region (TRAV) and an α chain constant region (TRAC), and the β chain includes a β chain variable region (TRBV) and a β chain constant region (TRBC).

[0145] For example, the TRAV and / or TRBV may contain three high-variable regions CDR1, CDR2 and CDR3.

[0146] In this application, the chimeric antigen receptor may include an extracellular domain and a transmembrane domain that specifically bind to an antigen or target. In this application, the extracellular domain may include an antigen-binding domain. In this application, the extracellular domain may also include a hinge region. In this application, the extracellular domain may also include a signal peptide. In this application, the fusion protein may also include an intracellular domain. In this application, the intracellular domain may include a co-stimulatory domain. In this application, the intracellular domain may also include an intracellular signal transduction domain.

[0147] In this application, the chimeric antigen receptor may include an antigen-binding domain. In this application, the antigen-binding domain may include the antigen-binding protein. For example, the antigen-binding domain may be an antibody or an antigen-binding fragment thereof. For example, the antigen-binding fragment may be a Fab, (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH, or dAb fragment. For example, the antigen-binding fragment may be a VHH. For example, the VHH may include HCDR1, HCDR2, and HCDR3. In this application, the VHH may include H-FR1, HCDR1, H-FR2, HCDR2, H-FR3, HCDR3, and H-FR4.

[0148] In this application, the fusion protein may include an extracellular domain and a transmembrane domain that specifically bind to an antigen or target. In this application, the extracellular domain may include an antigen-binding domain. In this application, the extracellular domain may also include a hinge region. In this application, the extracellular domain may also include a signal peptide. In this application, the fusion protein may also include an intracellular domain. In this application, the intracellular domain may include a co-stimulatory domain. In this application, the intracellular domain may also include an intracellular signal transduction domain.

[0149] In this application, the fusion protein may include an antigen-binding domain. In this application, the antigen-binding domain may contain the antigen-binding protein. For example, the antigen-binding domain may be an antibody or an antigen-binding fragment thereof. For example, the antigen-binding fragment may be Fab, (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH, or dAb fragments. For example, the antigen-binding fragment may be VHH. For example, the VHH may contain HCDR1, HCDR2, and HCDR3. In this application, the VHH may contain H-FR1, HCDR1, H-FR2, HCDR2, H-FR3, HCDR3, and H-FR4.

[0150] In this application, the antigen-binding domain may include an antigen-binding protein, the antigen-binding protein may include an antibody heavy chain variable region VH, the VH may include heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, the amino acid sequence of HCDR1 is shown in SEQ ID NO:29 or SEQ ID NO:14, the amino acid sequence of HCDR2 is shown in SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:15, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:26.

[0151] In this application, the antigen-binding domain may comprise an antigen-binding protein, the antigen-binding protein may comprise an antibody heavy chain variable region VH, the VH may comprise heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, the amino acid sequence of HCDR1 is shown in SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:14, or SEQ ID NO:19, the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:20, or SEQ ID NO:23, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:3 or SEQ ID NO:16.

[0152] In this application, the antigen-binding domain may comprise an antigen-binding protein, the antigen-binding protein may comprise an antibody heavy chain variable region VH, the VH may comprise heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, the amino acid sequence of HCDR1 is shown in SEQ ID NO:29, the amino acid sequence of HCDR2 is shown in SEQ ID NO:27, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:26; or the amino acid sequence of HCDR1 is shown in SEQ ID NO:29, the amino acid sequence of HCDR2 is shown in SEQ ID NO:28, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:26; or the amino acid sequence of HCDR1 is shown in SEQ ID NO:14, the amino acid sequence of HCDR2 is shown in SEQ ID NO:15, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:26.

[0153] In this application, the antigen-binding domain may comprise an antigen-binding protein, which may comprise an antibody heavy chain variable region (VH). The VH may comprise heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3). The amino acid sequence of HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:3; or the amino acid sequence of HCDR1 is shown in SEQ ID NO:9, the amino acid sequence of HCDR2 is shown in SEQ ID NO:10, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:3; or the amino acid sequence of HCDR1 is shown in SEQ ID NO:14, the amino acid sequence of HCDR2 is shown in SEQ ID NO:15, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:16; or the amino acid sequence of HCDR1 is shown in SEQ ID NO:19, the amino acid sequence of HCDR2 is shown in SEQ ID NO:20, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:19. The amino acid sequence of HCDR1 is shown in SEQ ID NO:3; or the amino acid sequence of HCDR2 is shown in SEQ ID NO:23; and the amino acid sequence of HCDR3 is shown in SEQ ID NO:3.

[0154] In this application, the antigen-binding domain may include an antigen-binding protein, and the antigen-binding protein may include an antibody heavy chain variable region VH. The amino acid sequence of the VH may be as shown in SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:22 or SEQ ID NO:25.

[0155] In this application, the signal peptide may be a CD8 signal peptide. In this application, the amino acid sequence of the signal peptide may be as shown in SEQ ID NO:30.

[0156] In this application, the hinge region may include hinge regions derived from the following proteins: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

[0157] In this application, the hinge region may be the hinge region of CD8. In this application, the amino acid sequence of the hinge region may be as shown in SEQ ID NO:31.

[0158] In this application, the transmembrane domain comprises a transmembrane domain derived from any of the following proteins: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

[0159] In this application, the transmembrane domain may be a CD8 transmembrane domain. In this application, the amino acid sequence of the transmembrane domain may be as shown in SEQ ID NO:32.

[0160] In this application, the co-stimulatory domain may comprise a co-stimulatory domain derived from one or more of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.

[0161] In this application, the co-stimulatory domain may be a 4-1BB co-stimulatory domain. In this application, the amino acid sequence of the co-stimulatory domain may be as shown in SEQ ID NO:33.

[0162] In this application, the intracellular signal transduction domain may include an intracellular signal transduction domain derived from the following proteins: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (KSHV), DAP10, DAP-12, or a domain containing at least one ITAM.

[0163] In this application, the intracellular signal transduction domain may be the intracellular signal transduction domain of CD3ζ. In this application, the amino acid sequence of the intracellular signal transduction domain may be as shown in SEQ ID NO:34.

[0164] In this application, the chimeric antigen receptor and / or fusion protein may further comprise low-density lipoprotein receptor-associated protein (LDL-AAP) or fragments thereof. For example, the LDL-AAP or fragments thereof may be located at the C-terminus of the chimeric antigen receptor and / or fusion protein. For example, the LDL-AAP or fragments thereof may comprise LDL-AAP 1-12 and their functional fragments. For example, the LDL-AAP or fragments thereof may be LDL-AAP 5 and / or 6 or fragments thereof. For example, the LDL-AAP or fragments thereof may be LDL-AAP 6 or fragments thereof. In this application, the LDL-AAP or fragments thereof may comprise the amino acid sequence shown in SEQ ID NO:36. In this application, the LDL-AAP or fragments thereof may be exogenous LDL-AAP or fragments thereof.

[0165] In this application, the sequence of the low-density lipoprotein receptor-associated protein or a fragment thereof in the chimeric antigen receptor and / or fusion protein can be linked to the C-terminal sequence of the chimeric antigen receptor and / or fusion protein via a self-cleaving peptide (e.g., 2A peptides such as T2A, P2A, and E2A). For example, the low-density lipoprotein receptor-associated protein or a fragment thereof can be linked to the C-terminus of an intracellular signaling region via T2A. In this application, the amino acid sequence of the self-cleaving peptide may be as shown in SEQ ID NO:35.

[0166] In this application, the chimeric antigen receptor may include an antigen-binding domain targeting CD38, a transmembrane domain, and an intracellular signaling domain. Alternatively, the chimeric antigen receptor may include a CD38-targeting antigen-binding domain, a hinge region, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain. For example, the chimeric antigen receptor may include a CD38-targeting VHH, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ intracellular signaling domain. Another example is a signal peptide, a CD38-targeting VHH, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ intracellular signaling domain.

[0167] For example, the chimeric antigen receptor may comprise a VHH targeting CD38, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ intracellular signal transduction domain, the amino acid sequence of which is shown in SEQ ID NO:18. Alternatively, the chimeric antigen receptor may comprise a signal peptide, a VHH targeting CD38, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ intracellular signal transduction domain, the amino acid sequence of which is shown in SEQ ID NO:18.

[0168] For example, the chimeric antigen receptor may comprise a VHH targeting CD38, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ intracellular signal transduction domain, the amino acid sequence of which is shown in SEQ ID NO:22. Alternatively, the chimeric antigen receptor may comprise a signal peptide, a VHH targeting CD38, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ intracellular signal transduction domain, the amino acid sequence of which is shown in SEQ ID NO:22.

[0169] Modified immune cells

[0170] On the other hand, this application provides a modified immune cell, wherein the immune cell may contain the chimeric antigen receptor, T cell receptor and / or fusion protein.

[0171] In this application, the immune cells can be T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, iNKT cells, TIL cells, CIK cells, γδT cells, DNT cells, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphoprogenitor cells, and / or pluripotent stem cells. For example, the immune cells can be immune effector cells. For example, the immune cells can be T cells. For example, the immune cells can be a mixture, and the mixture can contain different types of immune cells; for example, the mixture can contain one or more types of immune cells.

[0172] In this application, the immune cells may contain and / or express one or more antigen recognition receptors and / or fusion proteins.

[0173] In this application, the immune cells may contain and / or express one or more chimeric antigen receptors and / or fusion proteins.

[0174] In this application, the immune cells may contain and / or express the chimeric antigen receptor and / or fusion protein and the low-density lipoprotein receptor-associated protein or fragments thereof.

[0175] Immunoconjugates

[0176] On the other hand, this application also provides an immunoconjugate, wherein the immunoconjugate may contain the antigen-binding protein. In this application, the immunoconjugate can specifically bind to CD38.

[0177] In this application, the immunoconjugate may comprise an antigen-binding protein, a linker, and a payload. In this application, the portions of the immunoconjugate are directly or indirectly connected. For example, the portions of the immunoconjugate are indirectly connected. For example, the portions of the immunoconjugate are connected via linkers.

[0178] In this application, the payload may be a toxin, polymer, protein, drug, radioactive isotope, nucleic acid compound, or glucocorticoid.

[0179] In this application, the immune conjugate may be an antibody-drug conjugate (ADC), a PROTAC-antibody conjugate (DAC), or a targeted radiopharmaceutical (RDC).

[0180] In this application, the antibody-drug conjugate may comprise the antigen-binding protein, a linker, and a cytotoxic drug. In this application, the cytotoxic drug may be a chemotherapeutic agent, a tubulin inhibitor, a DNA damaging agent, or a topoisomerase I inhibitor. In this application, the chemotherapeutic agent may be vinblastine or doxorubicin. In this application, the tubulin inhibitor may be oliquistatin, eribulin, or maytansine. In this application, the DNA damaging agent may be chachiomycin, buprofen, or an atrazomycin derivative (PBD). In this application, the topoisomerase I inhibitor may be camptothecin, hydroxycamptothecin, irinotecan, or topotecan.

[0181] In this application, the PROTAC-antibody conjugate may comprise the antigen-binding protein, a linker, and a PROTAC. In this application, the PROTAC may comprise a target protein ligand, a linker, and an E3 ligase ligand. In this application, the E3 ligase ligand may be CRBN, VHL, IAP, MDM2, DCF15, RNF114, DCAF16, KEAP1, or FEM1B.

[0182] In this application, the targeted radiopharmaceutical may comprise the antigen-binding protein, a linker, and a radioisotope. In this application, the radioisotope may be iodine-131, radium-223, thallium-201, or arsenic-211.

[0183] Nucleic acid molecules, vectors and cells

[0184] On the other hand, this application also provides isolated nucleic acid molecules that can encode the antigen-binding protein, the chimeric antigen receptor, and / or the fusion protein.

[0185] In this application, the nucleic acid molecule may also contain a sequence encoding a signal peptide.

[0186] In this application, the nucleic acid molecule may further comprise a sequence encoding the low-density lipoprotein receptor-associated protein or a fragment thereof. For example, the nucleic acid molecule may comprise a sequence capable of expressing the chimeric antigen receptor and / or fusion protein in cells, and the low-density lipoprotein receptor-associated protein or a fragment thereof. In this application, the nucleic acid sequence encoding the chimeric antigen receptor and / or fusion protein may be linked to the nucleic acid sequence encoding the low-density lipoprotein receptor-associated protein or a fragment thereof via a cleavage peptide.

[0187] In this application, the nucleic acid molecule may be generated or synthesized by: (i) in vitro amplification, for example by polymerase chain reaction (PCR) amplification; (ii) clonal recombination; (iii) purification, for example by enzyme digestion and gel electrophoresis fractionation; or (iv) synthesis, for example by chemical synthesis.

[0188] In this application, the nucleic acid molecule may be DNA and / or RNA. In this application, the nucleic acid molecule may be a synthetically produced nucleic acid analogue.

[0189] In this application, the nucleic acid molecule may be a modified nucleic acid molecule.

[0190] On the other hand, this application provides a vector containing the nucleic acid molecule.

[0191] In this application, the vector may contain one or more of the aforementioned nucleic acid molecules.

[0192] In this application, the vector can be an expression vector or a cloning vector. In this application, the vector can be a viral vector or a non-viral vector. In this application, the vector can be a viral vector, a plasmid vector, a bacteriophage vector, or other vectors commonly used in, for example, genetic engineering. For example, the viral vector can be adenovirus, adeno-associated virus, or retrovirus (including lentivirus). In this application, the vector can be a fusion vector or a non-fusion vector.

[0193] In this application, the vector may also contain other genes. For example, the other genes may be marker genes.

[0194] In this application, the vector may contain various elements that control expression. For example, the vector may include a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and / or a reporter gene. For example, the vector may also contain a replication initiation site. For example, the vector may include components that facilitate entry into the cell. To enable the nucleic acid molecule to replicate in the vector, the 5' and 3' ends of the nucleic acid molecule may also contain long terminal repeat sequences.

[0195] For example, the promoter could be the EF1α promoter.

[0196] On the other hand, this application provides a cell that contains the nucleic acid molecule and / or the vector.

[0197] In this application, the cell may include the offspring of a single cell. Due to natural, accidental, or intentional mutations, the offspring may not necessarily be completely identical to the original parent cell (in terms of the morphology of the total DNA complement or in the genome).

[0198] In this application, the cell may be a prokaryotic cell (e.g., a bacterial cell), a CHO cell, an NS / O cell, a HEK293T cell or a HEK293A cell, or other eukaryotic cells, such as fungal or yeast cells.

[0199] In this application, the cell may contain one or more of the aforementioned nucleic acid molecules and / or one or more vectors. In this application, the vector may contain one or more of the aforementioned nucleic acid molecules and / or one or more vectors.

[0200] In this application, the vector can be introduced into the cells using methods known in the art. For example, the method may be electroporation, Lipofectine transfection, or Lipofectamine transfection.

[0201] On the other hand, this application provides a method for preparing the antigen-binding protein, chimeric antigen receptor and / or fusion protein, the method comprising culturing the cells under conditions that cause the antigen-binding protein, chimeric antigen receptor and / or fusion protein to be expressed.

[0202] Pharmaceutical Composition

[0203] On the other hand, this application also provides pharmaceutical compositions that may comprise the antigen-binding protein, the chimeric antigen receptor, the fusion protein, the modified immune cell, the immune conjugate, the nucleic acid molecule, the carrier and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0204] In this application, the pharmaceutical composition may comprise suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical compositions of this application may include liquid, freeze-dried, and lyophilized compositions.

[0205] In this application, the pharmaceutically acceptable carrier may comprise any and all solvents, dispersion media, coatings, isotonic agents and absorption delay agents compatible with drug administration, generally safe and non-toxic, and neither biologically nor otherwise undesirable.

[0206] In this application, the pharmaceutical composition may be administered parenterally, percutaneously, intracavitarily, intra-arterially, intrathecally, and / or intranasally, or directly injected into tissues. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In some embodiments, the pharmaceutical composition may be administered in various ways, such as intravenously, intraperitoneally, subcutaneously, intramuscularly, locally, or intradermally.

[0207] Drug combination

[0208] On the other hand, this application provides a pharmaceutical composition comprising the antigen-binding protein, and the pharmaceutical composition may further comprise one or more active ingredients other than the antigen-binding protein.

[0209] For example, the drug combination may also include substances related to the immune response. For example, the drug combination may also include drugs related to the immune response.

[0210] On the other hand, this application provides a drug combination comprising the antigen-binding protein, the nucleic acid molecule and / or the cell, and a therapeutic agent. In this application, the therapeutic agent may be selected from one or more of the following groups: antitumor drugs, chemotherapeutic agents, radioisotopes, or immune checkpoint inhibitors.

[0211] On the other hand, this application also provides a method for using the antigen-binding protein in combination with one or more other active ingredients. For example, the antigen-binding protein may be used in combination with one or more other therapeutic agents. In this application, the drug combination may be administered separately, simultaneously, or sequentially. In this application, the drug combination may be administered at the same or different doses or methods of administration. For example, the active ingredients in the drug combination may be administered to the patient as separate entities at the same / different doses or methods of administration. In this application, the components in the drug combination may be administered to the patient simultaneously as a single entity or dose. For example, the components in the drug combination may be administered to the patient simultaneously, jointly, or sequentially as separate entities. In this application, the specific method of administration may be determined according to the category of the active ingredient, and the specific dosage may be adjusted according to the severity of the subject's condition, the subject's physical condition, etc.

[0212] In this application, the different active ingredients in the drug combination can be placed together or separately. For example, the active ingredients can be placed in the same container. Or, the active ingredients can be placed in different containers.

[0213] Preparation method

[0214] On the other hand, this application provides methods for preparing the antigen-binding protein, the chimeric antigen receptor, the fusion protein, the modified immune cells, the immune conjugate, the nucleic acid molecule, the carrier, the cells, and / or the pharmaceutical composition. For example, the method may include culturing the cells under conditions that cause the antigen-binding protein, the chimeric antigen receptor, and / or the fusion protein to be expressed. For example, the method may include introducing the carrier into the immune cells.

[0215] use

[0216] On the other hand, this application also provides the use of the antigen-binding protein, the chimeric antigen receptor, the fusion protein, the modified immune cell, the immune conjugate, the nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition in the preparation of a medicament that can be used for the prevention, diagnosis, and / or treatment of diseases and / or conditions.

[0217] On the other hand, this application also provides a method for preventing, diagnosing, and / or treating diseases and / or conditions, which may include administering the antigen-binding protein, the chimeric antigen receptor, the fusion protein, the modified immune cells, the immune conjugate, the nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition to a subject in need.

[0218] On the other hand, this application also provides the antigen-binding protein, the chimeric antigen receptor, the fusion protein, the modified immune cell, the immune conjugate, the nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition, which can be used for the prevention, diagnosis, and / or treatment of diseases and / or conditions.

[0219] In this application, the disease and / or condition may be a CD38-related disease and / or condition.

[0220] In this application, the disease and / or condition is a tumor and / or an autoimmune disease.

[0221] In this application, the disease and / or condition can be a tumor. In this application, the tumor can be a solid tumor and / or a hematologic malignancy. In this application, the tumor can be a CD38-positive tumor. In this application, the hematologic malignancy can include various types of leukemia, multiple myeloma, and / or malignant lymphoma. In this application, the tumor can be multiple myeloma, lymphoma, and / or leukemia.

[0222] In this application, the disease and / or condition may be multiple myeloma, lymphoma, leukemia and / or autoimmune disease.

[0223] In this application, the prevention, diagnosis and / or treatment may be to prevent the onset of the disease, slow down or reverse the disease progression, prevent or slow down the onset of one or more symptoms associated with the disease, reduce or alleviate one or more symptoms associated with the disease, reduce the severity and duration of the disease and any symptoms associated therewith, or prevent further increase in the severity of the disease and any symptoms associated therewith.

[0224] On the other hand, this application provides the use of the antigen-binding protein in the preparation of diagnostic agents, wherein the diagnostic agents are used to diagnose diseases and / or conditions associated with the expression of CD38 protein.

[0225] On the other hand, this application provides the antigen-binding protein for the diagnosis of diseases and / or conditions associated with the expression of CD38 protein.

[0226] In this application, the diagnostic agent can be used alone or in combination with instruments, appliances, devices, or systems. In the prevention, diagnosis, treatment monitoring, prognosis observation, health status evaluation, and prediction of hereditary diseases, the diagnostic agent can be used to perform in vitro testing on human samples (e.g., various body fluids, cells, tissue samples, etc.).

[0227] On the other hand, this application also provides a kit that may include the antigen-binding protein, the kit being used to detect the presence and / or content of CD38 in a sample or subject. For example, the kit may be used for the prevention, diagnosis, and / or treatment of diseases and / or conditions.

[0228] For example, this application relates to immunoassay kits used in immunoassay methods such as antigen-binding protein binding ELISA, immunohistochemistry, Western blotting, and flow cytometry.

[0229] On the other hand, the use of the antigen-binding protein, the chimeric antigen receptor, the fusion protein, the modified immune cell, the immune conjugate, the nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition described in this application in the preparation of a kit for diagnostic purposes.

[0230] In this application, the container components of the kit will generally include at least one vial, test tube, flask, bottle, syringe, or other container components in which antibodies or preferably appropriately aliquots of antibodies can be placed. The kit of this application will also typically include components for containing antibodies, antigens, and any other reagents in a sealed form for commercial sale. Such containers may include injection-molded or blow-molded plastic containers in which the aforementioned vials are held.

[0231] On the other hand, this application also provides a method for detecting the presence and / or content of CD38, which may include using the antigen-binding protein.

[0232] On the other hand, this application provides a method for diagnosing diseases and / or conditions related to the expression of CD38 protein in a subject, the method comprising: contacting a sample derived from the subject with the antigen-binding protein, and determining the presence and / or content of a substance in the sample capable of specifically binding the antigen-binding protein.

[0233] On the other hand, this application provides a method for detecting CD38 in a sample or subject, the method comprising administering the antigen-binding protein. In this application, the administration can be performed in various ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, local, or intradermal administration.

[0234] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the antigen-binding protein, preparation method and use of this application, and are not intended to limit the scope of the invention.

[0235] Example

[0236] Example 1: Screening of CD38-targeting antibodies using the artificially synthesized nanobody library NanoOri_1.0

[0237] By comparing 296 nanobodies with non-repetitive sequences and their structures in the Protein Data Bank (PDB) database, the CDR mutation strategy was determined, and the lengths of the CDR3 amino acid were identified as three possibilities: 14, 17, and 21. Using the commercially available nanobody Caplacizumab as the backbone, the nucleotide sequence of Caplacizumab was synthesized from its entire genome and then cloned into the HP153 phage vector. Single-stranded DNA was then extracted from HP153, and the CDRs of the nanobodies were mutated using the Kunkel Mutagenesis method to obtain double-stranded DNA. The double-stranded DNA was electroporated into competent E. coli SS320 cells pre-infected with M13KO7 helper phage. After overnight culture, the phage supernatant was collected, ultimately constructing a strain with a diversity of 1.44 × 10⁻⁶. 10 The artificially synthesized nanobody library NanoOri_1.0 (Yuanqi Biotechnology) was used for subsequent antibody sequence screening.

[0238] The CD38 protein selection strategy is shown in Figure 1, which selects recombinant human CD38. ECD -His protein (constructed using the original Qi Biomolecular Cloning Platform, expressed and purified using the protein platform; the amino acid sequence of human CD38 protein is shown in Uniprot identifier: P28907-1; the amino acid sequence of human His is HHHHHH) was alternately sorted using an artificially synthesized nanobody library, for a total of four rounds of sorting.

[0239] After four rounds of panning, polyclonal ELISA showed significant enrichment of specific antibody sequences. The panned phage antibody clones were identified using monoclonal ELISA: human CD38ECD-His protein was coated onto 96-well ELISA plates at a concentration of 1 μg / mL and incubated overnight at 4°C. Non-specific binding sites were blocked with 5% skim milk powder. After thorough washing, the supernatant of the monoclonal phage was added to the 96-well plates and incubated at 37°C for 2 h. After thorough washing, Anti-M13-HRP (GE healtcare, 27-9421-01) was added, and the plates were incubated at 37°C for 45 min. After thorough washing, TMB was added for color development, and the reaction was incubated at room temperature for 5-10 min. Finally, the reaction was terminated with sulfuric acid, and the OD value of each well was measured at 450 nm.

[0240] Phage antibody clones that specifically bind to human CD38 were identified by monoclonal ELISA. After sequencing, the single-stranded VHH gene sequence was obtained. The antibody clones were named 381F3, 383A8, 383F12, 385C10 and 386C2, respectively. Their sequences are shown in Table 1. CDRs were divided according to IMGT.

[0241] Table 1. Schematic diagram of antibody sequences targeting CD38

[0242] Example 2: Construction, eukaryotic expression, purification, and identification of CD38 VHH-hFc

[0243] Phage antibody clones 381F3, 383A8, 383F12, 385C10, and 386C2, along with the positive control antibody Daratumumab (CN106456731B), were redesigned and constructed into a eukaryotic expression vector containing human IgG1 Fc. Primers were designed to amplify the phage antibody clone VHH by PCR. The PCR product was then recombinantly cloned into the pcDNA3.4-hFc vector, which had been digested with SfiI and NotI restriction endonucleases. After confirming correct Sanger sequencing, the plasmid was transfected into Expi293 cells for transient expression. The protein was purified using a Protein A affinity column. The resulting CD38 VHH-hFc proteins of the antibody and the positive control antibody were subjected to HPLC-SEC monomer purity identification. The results showed that the overall monomer purity of the purified proteins was high, and they can be used for subsequent protein-level functional identification.

[0244] Example 3: Detection of the binding activity of CD38-targeting antibody and CD38 protein by ELISA

[0245] The binding affinity of CD38 VHH-hFc prepared in Example 2 was determined using an ELISA assay. Human CD38 was then subjected to PBS. ECD The His protein was diluted to a final concentration of 1 μg / mL, and 100 μL / well was coated onto a 96-well ELISA plate and incubated overnight at 4°C. The plate was washed three times with PBST (containing 0.05% Tween 20), and then blocked with 5% skim milk powder at room temperature for 1 h. After blocking, the plate was washed three times with PBST, and then 30 μg / mL serially diluted CD38 VHH-hFc was added and incubated at room temperature for 2 h. After washing the plate three times with PBST, goat anti-human IgG Fc-HRP (1:5000) was added and incubated at room temperature for 1 h. After washing the plate seven times with PBST, TMB was added for color development, and the plate was incubated at room temperature in the dark for 5-10 min. The reaction was terminated with ELISA stop solution, and the absorbance of each well was measured at 450 nm using a microplate reader.

[0246] The binding activity results are shown in Figure 2 and Table 2. The highest OD value of the CD38 antibody prepared in this application binding to the CD38 protein is higher than that of the positive control antibody, indicating that the CD38 antibody prepared in this application has high binding activity to the CD38 protein.

[0247] Table 2. Results of the binding activity between anti-CD38 antibody and CD38 protein.

[0248] The specific binding activity is shown in Figure 3. Further testing of the specific binding activity of the CD38 VHH-hFc prepared in this application revealed that the antibody prepared in this application did not exhibit binding activity with the CD7 protein, indicating that the CD38 antibody prepared in this application has the ability to specifically bind. Furthermore, the isotype control Isotype-huIgG1 Fc showed no binding activity with the CD38 protein, while the positive control TH69-hFc (anti-CD7 TH69-huIgG1 Fc, WO2003051926A3) showed significant binding activity with the CD7 protein, further demonstrating the specificity of the antibody in this application.

[0249] Experimental results show that the CD38 antibody prepared in this application has high binding activity to CD38 protein and has specific binding ability.

[0250] Example 4: Detection of the binding activity of CD38-targeting antibody to target cells with different CD38 antigen expression abundances.

[0251] The binding activity of CD38 VHH-hFc to target cell lines MM.1S and NCIH929 with different CD38 target antigen expression levels was determined using flow cytometry fluorescence sorting (FACS) with an iQue Screener flow cytometer (IntelliCyt). In short, PBS containing 0.1% BSA was used as the buffer to prepare a final concentration of 1×10⁻⁶. 6Target cells (MM.1S and NCIH929) were added to 96-well conical plates (corning 3894), 30 μL per well. Antibody concentrations of 30 μg / mL were prepared and serially diluted 2.5 or 3-fold to create 11 concentration gradients. 30 μL of each prepared antibody concentration was added to the plated target cells and mixed thoroughly. The cells were incubated at 4°C for 1 h. 150 μL of buffer was added to each well, centrifuged at 300g for 5 min, the supernatant was discarded, and the cells were gently shaken to loosen the cells. The washing was repeated once. Fluorescent secondary antibody (ab98593) was prepared at a 1:200 ratio using buffer, 30 μL was added to each well, and the cells were mixed thoroughly. The cells were incubated at 4°C for 30 min. 150 μL of buffer was added to each well, centrifuged at 300g for 5 min, the supernatant was discarded, and the cells were gently shaken to loosen the cells. The washing was repeated once. 35 μL of buffer was added to each well, and the cells were analyzed by flow cytometry.

[0252] As shown in Figure 4 and Table 3, the CD38 antibody prepared in this application exhibits binding activity with MM.1S and NCIH929 target cell lines with different CD38 target antigen expression abundances, demonstrating binding affinity.

[0253] Table 3. Binding of anti-CD38 antibody to CD38 protein on target cell surface (EC) 50 Results Table

[0254] Example 5: Detection of surface plasmon resonance (SPR) affinity of CD38-targeted antibody

[0255] The binding kinetics between antigen and antibody were analyzed using biomembrane interferometry (BLI) with a Fortebiooctet RED384 molecular interaction analyzer. The binding dynamics of CD38 VHH-hFc to CD38 were also measured. ECD - Affinity to His protein. In short, using PBS (FB buffer) containing 0.1% BSA and 0.02% Tween-20 as buffer, adjust the initial concentration of CD38 VHH-hFc to 200 nM, serially dilute in 3-fold increments, and add 200 μL / well to a 96-well microplate. Add CD38 to a final concentration of 50 nM. ECD -His protein, 200 μL / well. Different concentrations of CD38 VHH-hFc samples were immobilized using an AHC sensor for 3 min. Then, CD38 was added... ECD-His protein solution was bound for 3 min and dissociated for 5 min. The obtained data were fitted and analyzed using Octet Data Analysis 10.0 software (Fortebio) (Langmuir, local fitting, 1:1 binding model) to calculate the binding strength between the antibody and the antigen, the affinity KD value, the binding constant Ka (1 / Ms) value, and the dissociation constant Kd (1 / s) value.

[0256] The affinity results are shown in Table 4. The CD38 VHH-hFc prepared by 383F12, 385C10, and 386C2 showed higher binding rates and similar dissociation rates than the positive control antibody Daratumumab-huIgG1 Fc, and their KD values ​​were all lower than the positive control. The experimental results indicate that the CD38 antibody prepared in this application has high binding activity to the CD38 protein.

[0257] Table 4. CD38 affinity of CD38-targeting antibodies

[0258] Ka: Binding rate constant; Kd: Dissociation rate constant; KD: Equilibrium dissociation constant, equal to Kd / Ka.

[0259] Example 6: Construction of a lentiviral expression vector targeting the CD38-specific chimeric antigen receptor

[0260] The CAR lentiviral core empty vector plasmid (constructed using the Yuanqi Biomolecular Cloning Platform, referred to as the CAR lentiviral empty vector) was double-digested with restriction endonucleases SphI and NotI (purchased from NEB) to generate a linearized fragment. After gel recovery, the fragment was mixed with the CD38 VHH fragment at a molar ratio of 1:3 for homologous recombination. The resulting fragment was then transformed into DH5α competent cells, plated on kanamycin-resistant plates, and incubated overnight at 37°C inverted in a CO2 incubator. Single colonies were then picked and sequenced for identification. The components and linkage order of the CAR in the CAR lentiviral empty vector and CD38 core plasmid are shown in Figure 5. Considering that T cells themselves contain CD38, to avoid CAR-T cell cannibalism affecting the test results, we introduced PEBL linked by a self-cleaving peptide based on the traditional second-generation CAR structure to block the expression of endogenous CD38 protein on the T cell membrane, thus enabling subsequent related verification.

[0261] Amino acid sequences: CD8 signal peptide (SEQ ID NO:30), CD8 hinge region (SEQ ID NO:31), CD8 transmembrane domain (SEQ ID NO:32), 4-1BB co-stimulatory domain (SEQ ID NO:33), CD3ζ intracellular signal transduction domain (SEQ ID NO:34), low-density lipoprotein receptor-associated protein (SEQ ID NO:36), T2A (SEQ ID NO:35), and PEBL (sequence derived from patent CN 110268049 A, SEQ ID NO:37).

[0262] Example 7: Lentiviral Packaging and Viral Titer Determination Targeting CD38-Specific Chimeric Antigen Receptor

[0263] The lentiviral vector system used to construct this invention consists of four plasmids, the structure of which is shown in Figure 6: the PRH1 plasmid encoding the Gag-Pol protein, the PMH2 plasmid encoding the envelope protein VSV-G, the PVH3 plasmid encoding the Rev protein, and a CD38 VHH core plasmid. The expression of the CAR gene in each CAR lentiviral plasmid is regulated by the elongation factor-1α (EF-1α) promoter. The lentiviral packaging process is as follows: 1×10 6 293T cells were suspended in 2 mL of DMEM medium containing 10% FBS and cultured overnight in 6-well plates. 345 μL of the original medium was aspirated, and 345 μL of Opti-MEM medium (50 times the plasmid mass) containing 6.90 μg of packaging plasmid (PRH1:PMH2:PVH3:core plasmid = 2:1:1:2) and 20.7 μL of FuGENEHD transfection reagent was added evenly. The mixture was gently mixed and incubated at 37°C for 12 hours. The plasmid-containing medium was removed, and the cells were washed once with PBS and replaced with 2 mL of DMEM medium containing 5% FBS. The cells were cultured for 48 hours. 2 mL of viral supernatant was collected, centrifuged at 3000 rpm for 10 min, aliquoted, and stored at -80°C for later use. The viral titer was also determined.

[0264] When the positive rate of each test tube minus the negative control is 5%-20%, the results are usable, i.e.:

[0265] The titers of the aforementioned CAR viruses containing CD38 VHH ranged from 5 to 10 × 10⁻⁶. 6 TU / mL.

[0266] Example 8: Preparation of CAR-T cells and lentiviral infection

[0267] This embodiment uses the CD3 MicroBeads kit (Miltenyi Biotec) to sort CD3 in PBMCs.+ T cells, specifically, were revived using PBMCs (Ficoll density gradient centrifugation), centrifuged at 500g for 5 minutes to remove cryopreservation medium. They were then treated with 1×10⁻⁶ cells. 7 Resuspend the cells in 80 μL of pre-cooled sorting buffer. Repeat at a rate of 1 × 10⁶ cells / mL. 7 Add 20 μL of CD3 magnetic beads to the total cell count, mix thoroughly, and incubate at 4°C for 15 min. After incubation, repeat the process at 1×10⁻⁶ cells / mL. 7 Add 1.5 mL of pre-cooled sorting buffer to the total cell count, centrifuge at 500 g for 5 min, and discard the supernatant. Repeat at 1 × 10⁻⁶. 7 Resuspend the cells in 0.1 mL of room temperature separation buffer, mix thoroughly by pipetting, and transfer the cell suspension to a rinsed LS separation column (placed under a magnetic field). Collect CD3+ T cells, mix thoroughly, centrifuge at 500 g for 5 min, discard the supernatant, and adjust the cell density to 1.25 × 10⁻⁶ cells in X-VIVO complete medium. 6 T cells were activated by adding CD3 / CD28 DynaBeads magnetic beads at a cell number:bead number ratio of 1:3. The cells were cultured at 37°C and 5% CO2. PBMCs were then sorted using CD3-positive magnetic beads to obtain CD3-positive DynaBeads with a purity >95%. + T cells.

[0268] This will activate CD3 for 24 hours. + T lymphocytes were collected in 50 mL centrifuge tubes, centrifuged at 500 g for 5 min, the culture supernatant was discarded, and the cells were resuspended in X-VIVO complete medium for counting. The cells were counted at a rate of 7 × 10⁻⁶ cells / mL. 5 The total number of cells was determined, and the cells were seeded into 12-well plates with a multiplicity of infection (MOI) of 3. The required volume of lentivirus was added, and polybrene reagent (PEI) was added to a final concentration of 10 μg / mL. The mixture was thoroughly mixed, centrifuged at 1200 rpm for 60 min, and cultured at 37°C with 5% CO2. After 24 h, the cells were collected, centrifuged at 500 g for 5 min to remove the supernatant, and resuspended in X-VIVO complete medium. The cell density was adjusted to 5-7 × 10⁶ cells / well. 5 The cells were inoculated at a concentration of 10 cells / mL into 12-well cell culture plates and cultured at 37°C with 5% CO2.

[0269] Example 9: Detection of CAR gene expression efficiency and in vitro amplification in T cells

[0270] 9.1 CAR gene expression detection

[0271] T cells activated by CD3 / CD28 DynaBeads were cultured for 72 hours after lentiviral infection, and the CAR gene expression efficiency of CAR-T cells was detected. The details are as follows:

[0272] (1) Mix the cells, count the AO / PI ratio, and detect the CAR-T cell density. Take 4×10⁶ cells.5 The total number of cells was transferred to a flow cytometry tube, 2 mL of 1xPBS (containing 2% FBS) buffer was added, and the mixture was vortexed at 1600 rpm for 4 min.

[0273] (2) Remove the supernatant and resuspend the cells in 50 μL of 1xPBS (containing 2% FBS) buffer;

[0274] (3) Add 1 μL of APC-labeled CD38-His antibody, mix thoroughly, and incubate at 4°C for 1 h;

[0275] (4) Add 1 mL of 1xPBS (containing 2% FBS) buffer to wash, vortex to mix, 1600 rpm, 4 min;

[0276] (5) Wash once more;

[0277] (6) Resuspend in 300 μL of 1xPBS (containing 2% FBS) buffer, mix thoroughly, and then perform detection.

[0278] 9.2 In vitro expansion of CAR-T cells

[0279] CAR-T cells activated with CD3 / CD28 DynaBeads were infected with lentivirus and passaged according to culture times (Day 5, Day 7, Day 9, Day 12). After thorough mixing of the CAR-T cells, 20 μL of cell suspension was transferred to a 1.5 mL EP tube, and an equal volume of AO / PI reagent was added. 20 μL of the mixture was then added to a counting chamber, and cell counts were performed using an Auto2000 cell counter against a fluorescent background. Based on the obtained cell density, cell growth was recorded, including cell density, cell viability, and cell diameter, and a growth curve was plotted. Simultaneously, the cell passage density was adjusted to 5-7 × 10⁻⁷ cells / year. 5 Cells / mL were inoculated into 6-well plates and cultured for 10-12 days. Data were processed and analyzed using GraphPad 6.0.

[0280] The total expansion of CD38 CAR-T cells after 12 days of activation ranged from 80 to 200 times, and the CAR positivity rate after infection ranged from 65% to 85%, making them suitable for cell function experiments.

[0281] Example 10: Evaluation of the in vitro targeted killing activity of CD38 CAR-T cells

[0282] CAR-T cells activated by CD3 / CD28 DynaBeads were cultured for 9 days. The effector killing activity of these CAR-T cells against positive target cells was assessed using the Luciferase Assay System kit (Promega). The results were as follows: the total number of target cells carrying the Luciferase gene was 3.3 × 10⁻⁶. 4 CAR-T cells were seeded per well in 96-well plates. The prepared CAR-T cells were pre-tested for positivity. Effector cells:target cells (E:T) were mixed with corresponding target cells at different ratios of 3:1, 1:1, and 1:3, with three replicates per group. The cells were co-cultured at 37°C and 5% CO2 for 20-24 hours. After co-culture, the cells were thoroughly mixed, and 100 μL of the cell suspension was transferred to opaque 96-well plates. 100 μL of luciferase assay substrate was added, mixed thoroughly, and incubated at room temperature in the dark for 5 minutes. The luciferase luminescence intensity in the remaining target cells was measured using a multi-mode microplate reader. The luciferase activity of the remaining tumor cells after killing was quantitatively assessed to evaluate the killing efficiency.

[0283] Formula for calculating cell kill ratio:

[0284] The target cell killing results are shown in Figures 7 and 8. Compared with Mock T, the prepared CD38 CAR-T cells can significantly kill MM.1S and NCIH929 target cell lines with different CD38 target antigen expression levels. The experimental results show that the CD38 antibody prepared in this application has high binding activity to CD38 protein and high ability to kill target cells after being prepared as CAR-T.

[0285] Example 11 Assessment of CD38-targeted CAR-T cytokine secretion levels

[0286] Exemplary CD38-targeting CAR-T cells were cultured in vitro for 9 days, and CAR-T cytokine assays were performed using the BD Biosciences CBA Multi-Cytokine Assay Kit (catalog number 550749). The positivity rate and cell density of CAR-T cells were pre-tested. Lentivirally infected CAR-T cells were adjusted with blank T cells to ensure consistent cell counts across groups. CAR-T cells were co-incubated with MM.1S, NCIH929, and MOLP8 target cells at an effector-target ratio of 1:1, with a total CAR-positive cell count of 3 × 10⁻⁶ cells. 4 The total number of MM.1S cells was 3 × 10⁻⁶. 4 Each group was set up with 2 replicates. After incubation for 24 hours, the cells were centrifuged at 250g for 5 minutes, and the supernatant was collected. The amount of cytokine secretion was detected using a CBA kit.

[0287] The cytokine secretion results are shown in Figures 9-11. The CD38 antibody prepared in this application, after being cultured as CAR-T cells alone and co-incubated with MM.1S, NCIH929, and MOLP8 target cell lines with different CD38 target antigen expression levels, showed significantly increased secretion levels of cytokines such as IL-2, IFN-γ, and TNF-α compared to Mock T cells. The experimental results indicate that the CD38 antibody prepared in this application, after being prepared as CAR-T cells and co-incubated with target cells, can significantly secrete cytokines and effectively kill target cells.

[0288] Example 12 Non-specific evaluation of CD38-targeting antibodies

[0289] Nonspecific binding assays of cell surface antigens to antibodies 383F12 and 385C10 were performed using flow cytometry fluorescence sorting (FACS) with an iQue Screener flow cytometer (IntelliCyt) and PBS containing 0.1% BSA as buffer. In short, a buffer concentration of 1×10⁻⁶ was used. 6 Target cell lines (in this example, normal or tumor cells that do not express CD38 antigen, such as human gastric epithelial cell line GES-1, human ovarian surface epithelial cell line IOSE-80, human prostate cancer cell line PC-3, human urinary tract epithelial cell line SV-HUC-1, etc.) were added to 96-well conical plates (corning 3894), 30 μL per well. Detection antibodies were prepared at a concentration of 30 μg / mL using buffer and serially diluted 3-fold to create 11 concentration gradients. 30 μL of each prepared antibody concentration was added to the plated target cells and mixed thoroughly. The cells were incubated at 4°C for 1 hour. 150 μL of buffer was added to each well, and the cells were centrifuged at 300g for 5 minutes. The supernatant was discarded, and the cells were loosened by shaking. The washing was repeated once. Fluorescent secondary antibody (ab98593) was prepared at a 1:200 ratio using buffer and 30 μL was added to each well and mixed thoroughly. The cells were incubated at 4°C for 30 minutes. Add 150 μL of buffer to each well, centrifuge at 300g for 5 min, discard the supernatant, loosen the cells by shaking, and repeat the washing once. Add 35 μL of buffer to each well, mix well, and then analyze using a flow cytometer.

[0290] The nonspecific binding activity is shown in Figure 12. The flow cytometry results indicate that the CD38 antibody prepared in this application exhibits virtually no nonspecific binding with nonspecific target cells. Combined with the results of Example 3, the experimental results further demonstrate that the CD38 antibody prepared in this application has high specificity and a low off-target probability.

Claims

1. An isolated antigen binding protein that is capable of binding CD38, wherein the antigen binding protein comprises an antibody heavy chain variable region VH comprising a heavy chain complementarity determining region HCDR1, HCDR2, and HCDR3, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO:

26.

2. The antigen binding protein of claim 1, wherein the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 3 or SEQ ID NO:

16.

3. The antigen binding protein of any one of claims 1-2, wherein the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO:

15.

4. The antigen binding protein of any one of claims 1-3, wherein the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 20, or SEQ ID NO:

23.

5. The antigen binding protein of any one of claims 1-4, wherein the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 29 or SEQ ID NO:

14.

6. The antigen binding protein of any one of claims 1-5, wherein the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 14, or SEQ ID NO:

19.

7. The antigen binding protein of any one of claims 1-6, wherein the amino acid sequences of the HCDR1, HCDR2, and HCDR3 are selected from any one of the following combinations: a) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 29, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 27, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 26; b) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 29, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 28, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 26; and c) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 14, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 15, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO:

26.

8. The antigen binding protein of any one of claims 1-7, wherein the amino acid sequences of the HCDR1, HCDR2, and HCDR3 are selected from any one of the following combinations: a) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 2, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 3; b) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 10, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 3; c) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 15, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 3; d) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 20, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 3; and e) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 23, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO:

3. b) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO:9, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 10, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO:3; c) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 14, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 15, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 16; d) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 19, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO:20, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO:3; and e) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO:23, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO:

3.

9. The antigen binding protein of any one of claims 1-8, wherein the amino acid sequence of the VH is set forth in SEQ ID NO:8, SEQ ID NO: 13, SEQ ID NO: 18, SEQ ID NO:22, or SEQ ID NO:

25.

10. The antigen binding protein of any one of claims 1-9, wherein the antigen binding protein further comprises an immunoglobulin constant region.

11. The antigen binding protein of claim 10, wherein the immunoglobulin constant region is an IgG Fc region.

12. The antigen binding protein of any one of claims 10-11, wherein the immunoglobulin constant region is a human IgG Fc region.

13. The antigen binding protein of any one of claims 1-12, wherein the antigen binding protein is an antibody or an antigen binding fragment thereof.

14. The antigen binding protein of claim 13, wherein the antigen binding fragment is a Fab, (Fab)2, F(ab’)2, scFv, di-scFv, Fv, VHH, or dAb fragment of the antibody.

15. The antigen binding protein of any one of claims 1-14, wherein the antigen binding protein is a VHH.

16. The antigen binding protein of any one of claims 1-15, wherein the antigen binding protein is a chimeric antibody, a humanized antibody, or a fully human antibody.

17. The antigen binding protein of any one of claims 1-16, wherein the antigen binding protein is a monovalent antibody, a bivalent antibody, or a multivalent antibody.

18. The antigen binding protein of any one of claims 1-17, wherein the antigen binding protein is a mono-specific antibody, a bi-specific antibody, or a multi-specific antibody.

19. A chimeric antigen receptor comprising the antigen binding protein of any one of claims 1-18.

20. The chimeric antigen receptor of claim 19, wherein the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain.

21. The chimeric antigen receptor of claim 20, wherein the antigen binding domain comprises the antigen binding protein of any one of claims 1-18.

22. The chimeric antigen receptor of any one of claims 20-21, wherein the chimeric antigen receptor further comprises a costimulatory domain.

23. The chimeric antigen receptor of any one of claims 20-22, wherein the chimeric antigen receptor further comprises a hinge region.

24. A fusion protein comprising the antigen binding protein of any one of claims 1-18.

25. A modified immune cell, wherein the immune cell comprises the chimeric antigen receptor of claims 19-23 and / or the fusion protein of claim 24.

26. The modified immune cell of claim 25, wherein the immune cell is a T cell, an NK cell, an iNKT cell, a dendritic cell, and / or a macrophage.

27. The modified immune cell of any one of claims 25-26, further comprising and / or expressing a low density lipoprotein receptor-related protein or a fragment thereof.

28. The modified immune cell of claim 27, wherein the low density lipoprotein receptor-related protein or a fragment thereof comprises one or more selected from the group consisting of low density lipoprotein receptor-related proteins 1-12 and functional fragments thereof.

29. The modified immune cell of any one of claims 27-28, wherein the low density lipoprotein receptor-related protein or a fragment thereof is low density lipoprotein receptor-related protein 5 and / or 6 or a fragment thereof.

30. The modified immune cell of any one of claims 27-29, wherein the low density lipoprotein receptor-related protein or a fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:

36.

31. The modified immune cell of any one of claims 27-30, wherein the low density lipoprotein receptor-related protein or a fragment thereof is an exogenous low density lipoprotein receptor-related protein or a fragment thereof.

32. An immunoconjugate comprising the antigen binding protein of any one of claims 1-18.

33. An isolated nucleic acid molecule encoding the antigen binding protein of any one of claims 1-18, the chimeric antigen receptor of any one of claims 19-23, and / or the fusion protein of claim 24.

34. A vector comprising the nucleic acid molecule of claim 33.

35. A cell comprising the nucleic acid molecule of claim 33 and / or the vector of claim 34.

36. A pharmaceutical composition comprising the antigen binding protein of any one of claims 1-18, the chimeric antigen receptor of any one of claims 19-23, the fusion protein of claim 24, the modified immune cell of any one of claims 25-31, the immunoconjugate of claim 32, the nucleic acid molecule of claim 33, the vector of claim 34, and / or the cell of claim 35, and optionally a pharmaceutically acceptable carrier.

37. A kit comprising the antigen binding protein of any one of claims 1-18, the chimeric antigen receptor of any one of claims 19-23, the fusion protein of claim 24, the modified immune cell of any one of claims 25-31, the immunoconjugate of claim 32, the nucleic acid molecule of claim 33, the vector of claim 34, the cell of claim 35, and / or the pharmaceutical composition of claim 36.

38. Use of the antigen binding protein of any one of claims 1-18, the chimeric antigen receptor of any one of claims 19-23, the fusion protein of claim 24, the modified immune cell of any one of claims 25-31, the immunoconjugate of claim 32, the nucleic acid molecule of claim 33, the vector of claim 34, the cell of claim 35, and / or the pharmaceutical composition of claim 36 for the manufacture of a medicament for the prevention and / or treatment of a disease and / or disorder.

39. The use according to claim 38, wherein the disease and / or disorder is a tumor and / or an autoimmune disease.

40. The use according to any one of claims 38-39, wherein the disease and / or disorder is multiple myeloma, lymphoma, leukemia and / or an autoimmune disease.

41. A method of preventing and / or treating a disease and / or disorder, comprising administering to a subject in need thereof the antigen binding protein of any one of claims 1-18, the chimeric antigen receptor of any one of claims 19-23, the fusion protein of claim 24, the modified immune cell of any one of claims 25-31, the immunoconjugate of claim 32, the nucleic acid molecule of claim 33, the vector of claim 34, the cell of claim 35, and / or the pharmaceutical composition of claim 36.

42. The method according to claim 41, wherein the disease and / or disorder is a tumor and / or an autoimmune disease.

43. The method according to any one of claims 41-42, wherein the disease and / or disorder is multiple myeloma, lymphoma, leukemia and / or an autoimmune disease.

44. The antigen binding protein of any one of claims 1-18, the chimeric antigen receptor of any one of claims 19-23, the fusion protein of claim 24, the modified immune cell of any one of claims 25-31, the immunoconjugate of claim 32, the nucleic acid molecule of claim 33, the vector of claim 34, the cell of claim 35, and / or the pharmaceutical composition of claim 36, for use in the prevention and / or treatment of a disease and / or disorder.

45. The antigen binding protein, chimeric antigen receptor, fusion protein, modified immune cell, immunoconjugate, nucleic acid molecule, vector, cell, and / or pharmaceutical composition of claim 44, wherein the disease and / or disorder is a tumor and / or an autoimmune disease.

46. The antigen binding protein, chimeric antigen receptor, fusion protein, modified immune cell, immunoconjugate, nucleic acid molecule, vector, cell, and / or pharmaceutical composition of any one of claims 44-45, wherein the disease and / or disorder is multiple myeloma, lymphoma, leukemia, and / or an autoimmune disease.

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