Antibodies specifically binding cd38 and methods of making and using the same

Abstract

The present application relates to antibodies that specifically bind CD38 and methods of making and using the same. In particular, the present application provides an anti-CD38 nanobody or binding fragment thereof. The antibodies of the present application have good specificity, and the antibodies and use of the antibodies for preparing immune effector cells targeting CD38 show good therapeutic effects in treating or ameliorating diseases with positive expression of CD38.

Description

[0001] This application is a divisional application of Chinese patent application 202480033489.4, filed on April 28, 2024, entitled "Antibody that specifically binds to CD38 and its preparation and application". Technical Field

[0002] This invention relates to the field of engineered immunotherapy, and more specifically to antibodies that specifically bind to CD38, their preparation methods, and applications. Background Technology

[0003] Acute myeloid leukemia (AML) is a cancer of the blood cells in the bone marrow, characterized by the rapid growth of immature blood cells (“bronchi”) that accumulate in the bone marrow and blood and interfere with normal blood cells. AML can spread to other organs, such as the liver, spleen, and brain. Without treatment, AML progresses rapidly and is often fatal within weeks or months. AML has several subtypes, and the treatment and outcomes for these subtypes can differ. Typically, AML is initially treated with chemotherapy, sometimes in conjunction with targeted therapy drugs. Subsequently, patients may continue with stem cell transplantation, other chemotherapy, surgery, or radiation therapy. AML is most common in older adults, some of whom are less healthy and cannot tolerate intensive chemotherapy, thus leading to poor clinical outcomes. Although current therapies for AML often achieve disease remission, almost all patients eventually relapse. Effective immunotherapies are needed to treat AML.

[0004] Chimeric antigen receptors (CARs) are artificial receptors that mimic the function of T-cell receptors, combining the specificity of antigen-antibody or ligand-receptor recognition and binding with the killing ability of effector T cells against recognized tumor cells. A CAR consists of a signal peptide, an antigen recognition region (ligand, single-chain antibody, or Fab fragment), a transmembrane region, and a series of signal transduction domains of the T cell (CD28, CD3, CD137 intracellular signal transduction domains). After modification, the CAR expressed on the surface of T cells first binds to tumor cell surface antigens through the antigen recognition region, and then transmits the activation signal into the cell through its signal transduction domain, targeting and activating the T cell's tumor-killing activity. The DNA sequence expressing the CAR is cloned into a lentiviral expression vector and used to infect T cells isolated from the patient's blood, causing the T cells to express the corresponding CAR. These modified T cells are then reinfused into the patient, allowing them to target and kill tumor cells expressing the relevant antigen, thus achieving tumor cell elimination.

[0005] Camel-derived antibodies (or single-domain antibodies) have advantages such as stability, good water solubility, low immunogenicity, stronger affinity, small molecular weight, and stronger tissue penetration than traditional antibodies. Single-domain antibodies can be used as diagnostic tools and also for the delivery of CAR-T and targeted drugs.

[0006] Therefore, there is an urgent need in this field to develop a new type of CAR immune cell with higher specificity and / or lower immunogenicity and optimal affinity for the treatment of acute myeloid leukemia. Summary of the Invention

[0007] The purpose of this invention is to provide an antibody with high affinity and high bioactivity that can specifically recognize the CD38 antigen and its application.

[0008] In a first aspect of the invention, a nanobody that specifically binds to CD38 is provided, wherein the complementarity-determining region (CDR) of the VHH chain in the nanobody has the following CDR1, CDR2, and CDR3: (a) CDR1: Its sequence is shown in SEQ ID No: 6 or 11; (b) CDR2: Its sequence is shown in SEQ ID No: 7, 9, 12 or 14; (c) CDR3: Its sequence is shown in SEQ ID No:8, 10, 13 or 15.

[0009] In another preferred embodiment, the complementarity-determining region (CDR) of the VHH chain in the nanobody is selected from the group consisting of: (Y1) CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:14, and CDR3 shown in SEQ ID NO:15; (Y2) CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:9, and CDR3 shown in SEQ ID NO:10; (Y3) CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: 7, and CDR3 shown in SEQ ID NO: 8; (Y4) CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: 9, and CDR3 shown in SEQ ID NO: 10; (Y5) CDR1 shown in SEQ ID NO: 11, CDR2 shown in SEQ ID NO: 12, and CDR3 shown in SEQ ID NO: 13.

[0010] In another preferred embodiment, CDR1, CDR2 and CDR3 are separated by the frame regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0011] In another preferred embodiment, the nanobody that specifically binds to CD38 includes humanized antibodies, camel-derived antibodies, and chimeric antibodies.

[0012] In another preferred embodiment, the amino acid sequence of the CD38-specifically binding nanobody is as shown in any one of SEQ ID NO:1 to 5.

[0013] In another preferred embodiment, the amino acid sequence of the VHH chain of the nanobody is selected from the group consisting of SEQ ID NO: 1 to 5, or a combination thereof.

[0014] In another preferred embodiment, the CDR region of the nanobody VHH chain contains an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 99% sequence similarity to any one of SEQ ID NO: 1-5.

[0015] In another preferred embodiment, the amino acid sequence of the CDR region of the nanobody VHH chain contains one or more amino acid substitutions compared to any of SEQ ID NO: 1 to 5, preferably conserved amino acid substitutions.

[0016] In another preferred embodiment, any of the amino acid sequences described above further includes a derived sequence which has optionally been added, deleted, modified and / or substituted at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and retains the ability to specifically bind to CD38.

[0017] In another preferred embodiment, the CD38 is a CD38 of a human or non-human mammal.

[0018] In another preferred embodiment, the CD38 is a CD38 of a human, mouse, rat, or non-human primate (such as a monkey).

[0019] In a second aspect of the invention, a multivalent antibody or an antibody targeting multiple epitopes that specifically binds to CD38 is provided, said multivalent antibody or antibody targeting multiple epitopes comprising at least one antibody element targeting the CD38 epitope, said antibody element being the anti-CD38 nanobody described in the first aspect of the invention.

[0020] In another preferred embodiment, the anti-CD38 multivalent antibody or antibody targeting multiple epitopes comprises one or more anti-CD38 nanobodies.

[0021] In another preferred embodiment, the anti-CD38 antibody comprises a monomer, a bivalent (bivalent antibody), a tetravalent (tetravalent antibody), and / or a multivalent (multivalent antibody).

[0022] In another preferred embodiment, the anti-CD38 antibody comprises one or more VHH chains having an amino acid sequence as shown in any of SEQ ID NO: 1 to 5.

[0023] In another preferred embodiment, the anti-CD38 antibody comprises two VHH chains having amino acid sequences as shown in SEQ ID NO: 1-5.

[0024] In another preferred embodiment, the antibody is selected from: animal-derived antibodies, chimeric antibodies, humanized antibodies, or combinations thereof.

[0025] In a third aspect of the invention, a recombinant protein is provided, said recombinant protein having: (i) The CD38-specific binding nanobody described in the first aspect of the present invention, or the CD38-specific multivalent antibody or antibody targeting multiple epitopes described in the second aspect of the present invention; and (ii) Optional tag sequences to assist in expression and / or purification.

[0026] In another preferred embodiment, the antibody is a multivalent antibody.

[0027] In another preferred embodiment, the tag sequence includes an Fc tag, an HA tag, a Flag tag, and a 6His tag.

[0028] In another preferred embodiment, the Fc tag comprises mIgG2aFc.

[0029] In another preferred embodiment, the recombinant protein specifically binds to the CD38 protein.

[0030] In a fourth aspect of the invention, a chimeric antigen receptor (CAR) fusion protein is provided, characterized in that the chimeric antigen receptor (CAR) fusion protein comprises, from the N-terminus to the C-terminus: (i) Specifically binds to the antigen-binding domain of CD38, wherein the antigen-binding domain contains the nanobody described in the first aspect of the present invention; (ii) Transmembrane domains; (iii) At least one co-stimulatory domain; and (iv) Activate the structural domain.

[0031] In another preferred embodiment, the antigen-binding domain is monovalent or polyvalent.

[0032] In another preferred embodiment, the antigen-binding domain is derived from the nanobody described in the first aspect of the invention or the recombinant protein described in the third aspect of the invention.

[0033] In another preferred embodiment, the CAR has the structure shown in Formula Ia: L-VHH-FH-TM-C-CD3ζ (Ia) In the formula, Each "-" independently represents a linking peptide or peptide bond; L represents the signal peptide sequence; VHH is the antigen-binding domain that specifically binds to CD38; F stands for Flag tag; H represents the hinge area; TM represents a transmembrane domain; C is the co-stimulation signal structure domain; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type or its mutants / modifiers).

[0034] In another preferred embodiment, L is a signal peptide derived from CD8.

[0035] In another preferred embodiment, the L comprises an amino acid sequence as shown in SEQ ID NO: 20.

[0036] In another preferred embodiment, the amino acid sequence of the VHH is as shown in any of SEQ ID NO:1 to 5.

[0037] In another preferred embodiment, H includes the hinge region of CD28.

[0038] In another preferred embodiment, the TM includes a transmembrane region derived from CD28.

[0039] In another preferred embodiment, the amino acid sequences of H and TM comprise the sequence shown in SEQ ID No: 22.

[0040] In another preferred embodiment, C is a transmembrane region of a protein selected from the group consisting of CD28, 4-1BB, CD8a, or a combination thereof.

[0041] In another preferred embodiment, the C comprises a co-stimulatory signaling molecule derived from 4-1BB.

[0042] In another preferred embodiment, C comprises an amino acid sequence as shown in SEQ ID No: 23.

[0043] In another preferred embodiment, the CD3ζ comprises an amino acid sequence as shown in SEQ ID No: 24. In another preferred embodiment, the CAR fusion protein has any of the amino acid sequences shown in SEQ ID NO: 25-29.

[0044] In a fifth aspect of the invention, an antibody-drug conjugate is provided, the antibody-drug conjugate comprising: (a) The nanobody described in the first aspect of the present invention, the multivalent antibody or antibody targeting multiple epitopes described in the second aspect of the present invention, and the recombinant protein described in the third aspect of the present invention; and (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

[0045] In another preferred embodiment, the antibody portion is coupled to the coupling portion via a chemical bond or a linker.

[0046] In a sixth aspect of the invention, a polynucleotide is provided, the polynucleotide encoding a protein selected from the group consisting of: nanobodies described in the first aspect of the invention, or multivalent antibodies or antibodies targeting multiple epitopes described in the second aspect of the invention, recombinant proteins described in the third aspect of the invention, or CAR fusion proteins described in the fourth aspect of the invention.

[0047] In a seventh aspect of the invention, an expression vector is provided, the expression vector containing the polynucleotide described in the sixth aspect of the invention.

[0048] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

[0049] In another preferred embodiment, the expression vector is a lentiviral vector.

[0050] In an eighth aspect of the invention, a host cell is provided, the host cell containing the expression vector of the seventh aspect of the invention, or having integrated the polynucleotide of the sixth aspect of the invention into its genome, or expressing the nanobody of the first aspect of the invention, the multivalent antibody or antibody against multiple epitopes of the second aspect of the invention, the recombinant protein of the third aspect of the invention, or the CAR fusion protein of the fourth aspect of the invention.

[0051] In another preferred embodiment, the cells are isolated cells, and / or the cells are genetically engineered cells.

[0052] In another preferred embodiment, the cell is a mammalian cell.

[0053] In another preferred embodiment, the cell is a T cell.

[0054] In another preferred embodiment, the host cell is an engineered immune cell.

[0055] In another preferred embodiment, the engineered immune cells are selected from the group consisting of: (i) Chimeric antigen receptor αβ T cells (CAR-T cells); (ii) Chimeric antigen receptor γδ T cells (CAR-T cells); (iii) Chimeric antigen receptor NKT cells (CAR-NKT cells); (iv) Chimeric antigen receptor NK cells (CAR-NK cells); (v) Chimeric antigen receptor macrophages.

[0056] In a ninth aspect of the present invention, a method for preparing engineered immune cells is provided, comprising the following steps: transducing a nucleic acid molecule as described in the sixth aspect of the present invention or a vector as described in the seventh aspect of the present invention into T cells or NK cells, thereby obtaining the engineered immune cells, wherein the engineered immune cells express the CAR fusion protein as described in the fourth aspect of the present invention.

[0057] In another preferred embodiment, the method further includes the step of: testing the function and effectiveness of the obtained engineered immune cells.

[0058] In a tenth aspect of the invention, a formulation is provided comprising the nanobody described in the first aspect of the invention, the multivalent antibody or antibody targeting multiple epitopes described in the second aspect of the invention, the recombinant protein described in the third aspect of the invention, the CAR fusion protein described in the fourth aspect of the invention, or the carrier described in the seventh aspect of the invention, or the host cell described in the eighth aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0059] In another preferred embodiment, the host cell is an engineered immune cell.

[0060] In an eleventh aspect of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising: (i) the nanobody described in the first aspect of the present invention, or the multivalent antibody or antibody targeting multiple epitopes described in the second aspect of the present invention, or the recombinant protein described in the third aspect of the present invention, or immune cells expressing the CAR fusion protein described in the fourth aspect of the present invention; and (ii) Pharmaceutically acceptable carriers.

[0061] In another preferred embodiment, the conjugation portion of the immunoconjugate is a drug, a toxin, and / or a therapeutic isotope.

[0062] In another preferred embodiment, the pharmaceutical composition further contains other drugs for treating immune system diseases or tumor diseases.

[0063] In another preferred embodiment, the pharmaceutical composition is used to prepare a medicament for the prevention and / or treatment of diseases or conditions related to CD38.

[0064] In another preferred embodiment, the CD38-related disease or condition is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), or a combination thereof.

[0065] In a twelfth aspect of the invention, a kit is provided containing the nucleic acid molecule described in the sixth aspect of the invention, the recombinant protein described in the third aspect of the invention, or the vector described in the seventh aspect of the invention.

[0066] In another preferred embodiment, the kit is used to prepare immune cells expressing the receptor CAR fusion protein described in the fourth aspect of the present invention.

[0067] In a thirteenth aspect of the invention, the use of the nanobody described in the first aspect of the invention, or the multivalent antibody or antibody targeting multiple epitopes described in the second aspect of the invention, or the recombinant protein described in the third aspect of the invention, or immune cells expressing the CAR fusion protein described in the fourth aspect of the invention, is provided for the preparation of a medicament for the prevention and / or treatment of CD38-related tumors.

[0068] In a fourteenth aspect of the invention, a method of treating a disease is provided, comprising administering to a subject requiring treatment: a nanobody as described in the first aspect of the invention, or a multivalent antibody or antibody targeting multiple epitopes as described in the second aspect of the invention, or a recombinant protein as described in the third aspect of the invention, or immune cells expressing a CAR fusion protein as described in the fourth aspect of the invention.

[0069] In another preferred embodiment, the disease is a CD38-positive tumor.

[0070] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0071] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0072] Figure 1 To visualize the expression of target cell surface antigens.

[0073] Figure 2 To show the CAR positivity rate of CD38-CAR T cells.

[0074] Figure 3To demonstrate the expression of CD38 on the surface of WT CD38-CAR T cells.

[0075] Figure 4 To show the changes in the CAR positivity rate of CD38-CAR T cells during the expansion culture process.

[0076] Figure 5 To demonstrate the viability and fold expansion of CD38-CAR T cells during the expansion culture process.

[0077] Figure 6 To demonstrate the binding ability of CD38 CAR to CD38 antigen.

[0078] Figure 7 To demonstrate the killing effect of CD38-CAT T on Hela-WT and CD38-overexpressing Hela cells (RTCA method).

[0079] Figure 8 To demonstrate the killing effect of CD38-CAR T on CD38-positive target cells (luciferase method).

[0080] Figure 9 To demonstrate the inhibitory effect of CD38-CAR T on Molm13 cells inoculated into immunodeficient mice and the change in mouse body weight after reinfusion.

[0081] Figure 10 To show the CAR positivity rate after CAR-NK92 sorting.

[0082] Figure 11 To demonstrate the killing effect of CAR-NK92 on tumor cells (luciferase method).

[0083] Figure 12 To demonstrate the multi-round kill results of CAR-N92K. Detailed Implementation

[0084] Through extensive and in-depth research, the inventors have unexpectedly obtained, for the first time, a class of anti-CD38 antibodies with excellent affinity and high anti-tumor activity. Specifically, through extensive screening, the inventors have obtained, for the first time, multiple nanobodies that specifically bind to CD38. Based on nanobodies, this invention further prepared recombinant antibodies and humanized antibodies, as well as chimeric antigen receptors targeting CD38. Based on alpaca heavy chain antibodies that specifically bind to CD38, this invention provides CAR-T cells with superior affinity, stronger specificity to the target antigen, and lower immunogenicity compared to traditional antibodies. The CAR-T cells of this invention exhibit excellent target cell killing activity and in vivo tumor suppression effects. This invention was completed based on these findings.

[0085] Specifically, this invention utilizes human CD38 antigen protein to immunize alpacas, obtaining a high-quality immune nanobody gene library. Then, CD38 protein molecules are coupled to an ELISA plate to display the correct spatial structure of the CD38 protein. Next, using this antigen, an immune nanobody gene library (alpaca heavy chain antibody phage display gene library) is screened using phage display technology, thereby obtaining nanobody genes specifically targeting the CD38 protein. This gene is then transformed into *E. coli*, resulting in highly specific nanobody strains that can be efficiently expressed in *E. coli*.

[0086] the term To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0087] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0088] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0089] Sequence identity is determined by comparing two aligned sequences along a predetermined comparison window (which may be 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the length of a reference nucleotide sequence or protein) and determining the number of positions where identical residues occur. This is typically expressed as a percentage. The measurement of nucleotide sequence identity is a method well-known to those skilled in the art.

[0090] As used in this article, the terms "heavy chain variable region" and "V" are used interchangeably. H "They can be used interchangeably."

[0091] As used in this article, the terms “variable region” and “complementarity determining region (CDR)” are used interchangeably.

[0092] In this invention, the terms "antibody of the invention," "single-domain antibody of the invention," "protein of the invention," or "peptide of the invention" are used interchangeably and all refer to antibodies that specifically bind to CD38, such as proteins or peptides having a heavy chain variable region (such as the amino acid sequence shown in SEQ ID NO: 1-5). They may or may not contain an initiating methionine.

[0093] CD38 Human CD38 antigen (also called cyclic ADP ribose hydrolase) is a 45 kDa single-chain type II transmembrane glycoprotein. Its overall structure can be divided into an N-terminal intracellular region, a single transmembrane structure, and a C-terminal extracellular region. Its extracellular region catalyzes the synthesis and degradation of cyclic ADP-ribose (cADPR). CD38 is a marker of cell activation and is associated with HIV infection, leukemia, myeloma, and solid tumors.

[0094] CD38 can be detected on the surface of many immune cells, including B cells and NK cells. CD38 is also highly expressed on the surface of various tumors, including multiple myeloma, diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), plasma cell leukemia (PCL), acute myeloid leukemia (AML), follicular lymphoma (FL), mantle cell lymphoma (MCL), and lung cancer.

[0095] Antibody As used in this article, the term "antibody" refers to an immunoglobulin, which is a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds.

[0096] As used herein, the terms "single-domain antibody (VHH)" and "nanobody" have the same meaning: to clone the variable region of an antibody heavy chain to construct a single-domain antibody (VHH) consisting of only one heavy chain variable region. It is the smallest antigen-binding fragment with complete function. Typically, antibodies that are naturally missing the light chain and the heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting of only one heavy chain variable region.

[0097] Existing antibody numbering schemes include: 1. The Kabat scheme (Kabat et al., 1991) is based on the location of highly variable regions between sequences of the same domain type, with different numbering for antibody heavy (VH) and light (Vλ and Vκ) variable domains.

[0098] 2. Chothia's scheme (Al-Lazikani, 1997) is similar to Kabat's scheme, but corrects the placement of the annotations around the first VH complementarity determination region (CDR) to correspond to the structural loop. Similarly, the enhanced Chothia scheme (Abhinandan and Martin, 2008) further structurally modifies the insertion position.

[0099] 3. In contrast to these Kabat-like schemes, IMGT (Lefranc, 2003) and AHo (Honegger and Plückthun, 2001) have defined unique schemes for variable domains of antibodies and T-cell receptors (TCRs) (Vα and Vβ). Therefore, equivalent residue positions can be readily compared between domain types. IMGT and AHo differ in the number of positions they annotated (128 and 149, respectively) and in the locations where they believe indels occur.

[0100] Immunoglobulins differ in the amino acid composition and sequence of their heavy chain constant regions, thus exhibiting different antigenicities. Based on this, immunoglobulins can be classified into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of the heavy chain region and the number and position of disulfide bonds in the heavy chain can further lead to different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as κ or λ chains based on their constant regions. Each of the five classes of Ig can possess either a κ or λ chain. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well-known to those skilled in the art.

[0101] The antibody light chain of the present invention may further include a light chain constant region, wherein the light chain constant region comprises a human or mouse κ, λ chain or a variant thereof.

[0102] In this invention, the antibody heavy chain may further include a heavy chain constant region, which contains human or mouse IgG1, IgG2, IgG3, IgG4, or variants thereof. The sequence of approximately 110 amino acids near the N-terminus of the antibody heavy and light chains varies considerably and is called the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is called the constant region. The variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FR). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDR). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the terminal terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0103] In this invention, the term "mouse antibody" refers to a monoclonal antibody against CD38 prepared according to the knowledge and skills in the art. Preparation involves injecting the test subject with CD38 antigen, followed by isolating hybridomas expressing antibodies with the desired sequence or functional characteristics. In a preferred embodiment of the invention, the mouse CD38 antibody or its antigen-binding fragment may further comprise a light chain constant region of a mouse κ, λ chain, or a variant thereof, or further comprise a heavy chain constant region of mouse IgG1, IgG2, IgG3, or a variant thereof.

[0104] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by murine antibodies.

[0105] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody generated by grafting a mouse CDR sequence into the variable region framework of a human antibody, i.e., a different type of human germline antibody framework sequence. Humanized antibodies can overcome the heterologous response induced by chimeric antibodies, which carry a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences or from publicly available references. To avoid a decrease in immunogenicity along with a decrease in activity, minimal reverse or reversion mutations can be performed on the human antibody variable region framework sequence to maintain activity.

[0106] The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that maintain its ability to specifically bind to an antigen (e.g., CD38). It has been shown that fragments of full-length antibodies can be used for antigen-binding function. Examples of binding fragments included in the term "antigen-binding fragment of an antibody" include... (i) Fab fragment, a monovalent fragment composed of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragment, which is a divalent fragment containing two Fab fragments connected by a disulfide bridge on the chain region; (iii) Fd fragments composed of VH and CH1 domains; (iv) The Fv fragment consisting of the VH and VL domains of the single arm of the antibody.

[0107] Fv antibodies contain variable regions on the heavy and light chains, but no constant regions, and are the smallest antibody fragments with all antigen-binding sites. Typically, Fv antibodies also contain a polypeptide linker between the VH and VL domains and can form the structure required for antigen binding.

[0108] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. One of the most commonly used definitions of these six CDRs is provided by Kabat EA et al., (1991) Sequences of proteins of immune interest. NIH Publication, No. 91-3242.

[0109] The term "epitope" or "antigenic determinant" refers to a site on an antigen where an immunoglobulin or antibody specifically binds (e.g., a specific site on the CD38 molecule). Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique spatial conformation.

[0110] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to a pre-defined epitope on an antigen. Typically, antibodies bind at a concentration of approximately less than 10... -7 M, for example, approximately less than 10 -8 M, 10 -9 M or 10 10 M or lower affinity (KD) binding.

[0111] The term "competitive binding" refers to an antibody that recognizes the same epitope (also called an antigenic determinant) or a portion of the same epitope on the extracellular region of CD38 as the monoclonal antibody of the present invention and binds to said antigen. An antibody that binds to the same epitope as the monoclonal antibody of the present invention refers to an antibody that recognizes and binds to the amino acid sequence of CD38 recognized by the monoclonal antibody of the present invention.

[0112] The term "KD" or "Kd" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. Typically, the antibodies of this invention have a dissociation equilibrium constant of less than approximately 10. -7 M, for example, less than approximately 10 -8 M, 10 -9 M or 10 -10 M or a smaller dissociation equilibrium constant (KD) is combined with CD38.

[0113] As used herein, the term "antigen determinant" refers to a discontinuous three-dimensional spatial site on an antigen that is recognized by the antibody or antigen-binding fragment of the present invention.

[0114] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0115] In this invention, antibodies include mouse, chimeric, humanized, or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including both human and non-human portions, can be prepared using DNA recombination techniques well known in the art.

[0116] As used herein, the term "monoclonal antibody" refers to an antibody secreted by a clone derived from a single cell. Monoclonal antibodies are highly specific, targeting a single antigenic epitope. The cell may be a eukaryotic, prokaryotic, or phage clone.

[0117] In this invention, the antibody can be monospecific, bispecific, trispecific, or more multiple specific.

[0118] In this invention, the antibody also includes its conserved variants, which are polypeptides formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of this invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A below.

[0119] Table A .

[0120] Anti-CD38 humanized antibody This invention provides a humanized antibody against CD38 (hereinafter referred to as CLDN 18.2 antibody). Specifically, this invention provides a humanized antibody with high specificity and high affinity against CD38, comprising a heavy chain and a light chain, wherein the heavy chain contains a heavy chain variable region (VH) amino acid sequence and the light chain contains a light chain variable region (VL) amino acid sequence.

[0121] In antibody humanization, there are generally two choices for the human backbone region: known mature antibodies and human Germline sequences. Known mature antibody backbone regions often contain somatic mutation sites, potentially introducing immunogenicity. Compared to mature antibodies, human Germline sequence backbone regions theoretically have lower immunogenicity, are more structurally flexible and malleable, and readily accept different CDR regions. The frequency of use of human antibody Germline genes in the human body exhibits a certain bias; antibodies humanized from frequently used Germline backbone regions have advantages such as low immunogenicity, high expression levels, and structural stability.

[0122] In a preferred embodiment of the present invention, during humanization, multiple factors (including similarity and frequency of human use) were considered. After extensive experimental screening, a preferred backbone region was selected for humanization. The human antibody Germline backbone region was used for CDR transplantation, resulting in a more stable humanized antibody structure with higher expression levels, lower immunogenicity, and higher drugability.

[0123] In another preferred embodiment, the heavy chain constant region and / or light chain constant region may be a humanized heavy chain constant region or a light chain constant region. More preferably, the humanized heavy chain constant region or light chain constant region is a heavy chain constant region of human IgG1, IgG2, etc., or a light chain constant region of human kappa, Lambda.

[0124] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or substituting at least one amino acid sequence preferably has a homology of at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the amino acid sequence.

[0125] The antibodies of the present invention can be double-chain or single-chain antibodies, and are preferably fully humanized antibodies.

[0126] The antibody derivatives described in this invention may be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2, or other known antibody derivatives in the field, as well as any one or more of IgA, IgD, IgE, IgG, and IgM antibodies or other subtypes of antibodies.

[0127] The antibodies of this invention may be humanized antibodies targeting CD38, CDR-grafted and / or modified antibodies.

[0128] In the above-described content of the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably no more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, and more preferably 15-20%.

[0129] Antibody preparation Any method suitable for producing monoclonal antibodies can be used to produce the CD38 antibody of this invention. For example, animals can be immunized with linked or naturally occurring CD38 protein or fragments thereof. Suitable immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, or one or more routes.

[0130] Any suitable form of CD38 can serve as an immunogen (antigen) for generating non-human antibodies specific to CD38 and screening for the biological activity of said antibodies. The immunogen can be used alone or in combination with one or more immunogenic enhancers known in the art. The immunogen can be purified from a natural source or produced in genetically modified cells. The DNA encoding the immunogen can be genomic or non-genomic (e.g., cDNA). The DNA encoding the immunogen can be expressed using suitable genetic vectors, including but not limited to adenovirus vectors, baculovirus vectors, plasmids, and nonviral vectors.

[0131] Humanized antibodies can be selected from any type of immunoglobulin, including IgM, IgD, IgG, IgA, and IgE. Similarly, any class of light chains can be used in the compounds and methods described herein. Specifically, κ, λ chains, or variants thereof, can be used in the compounds and methods of this invention.

[0132] The DNA sequences of the antibodies or fragments thereof of this invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Furthermore, the coding sequences of the light and heavy chains can be fused together to form single-chain antibodies.

[0133] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0134] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences are obtained by first synthesizing multiple small fragments and then ligating them. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0135] The term "nucleic acid molecule" refers to both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0136] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one implementation, the vector is a "plasmid," which refers to a circular double-stranded DNA loop to which an additional DNA segment can be linked.

[0137] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0138] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant or animal cells (such as mammalian cells).

[0139] The steps of transforming host cells with recombinant DNA as described in this invention can be performed using techniques well known in the art. The obtained transformants can be cultured using conventional methods, and the transformants express the polypeptide encoded by the gene of this invention. Depending on the host cell used, the cells are cultured in a conventional culture medium under suitable conditions.

[0140] Typically, host cells transformed with the antibody are cultured under conditions suitable for antibody expression according to the present invention. The antibody of the present invention is then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, which are well known to those skilled in the art.

[0141] The obtained monoclonal antibodies can be identified using conventional methods. For example, the binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)).

[0142] antibody preparations Antibodies exhibit varying degrees of stability in different formulation buffers, manifesting as changes in charge heterogeneity, antibody molecule degradation, and polymerization. These changes in quality properties are related to the antibody's inherent physicochemical properties. Therefore, in antibody drug development, it is necessary to screen suitable formulation buffers based on the physicochemical properties of different antibodies. Currently, commonly used antibody formulation buffer systems include phosphate buffer, citrate buffer, and histidine buffer. Depending on the antibody's properties, different concentrations of salt ions or excipients such as sorbitol, trehalose, and sucrose, as well as appropriate amounts of surfactants such as Tween, are added to maintain antibody stability.

[0143] Pharmaceutical Composition The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the aforementioned antibody or its active fragment or fusion protein or its ADC or corresponding CAR-T cell, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0144] The antibodies of the present invention can also be expressed in cells by nucleotide sequences for cell therapy, such as chimeric antigen receptor T-cell immunotherapy (CAR-T).

[0145] The pharmaceutical compositions of the present invention can be directly used to bind to CD38 protein molecules, and are therefore used for the prevention and treatment of CD38-related diseases. Furthermore, other therapeutic agents can be used simultaneously.

[0146] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described monoclonal antibody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0147] When using a pharmaceutical composition, a safe and effective amount of the composition is administered to a mammal, wherein the safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight, preferably about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0148] Detection uses and kits The antibodies of this invention can be used in detection applications, such as for testing samples, to provide diagnostic information.

[0149] In this invention, the samples used include cells, tissue samples, and biopsy specimens. The term "biopsy" as used in this invention should include all types of biopsies known to those skilled in the art. Therefore, biopsies used in this invention can include tissue samples prepared, for example, by endoscopic methods or by puncture or needle biopsy of organs.

[0150] The samples used in this invention include fixed or preserved cell or tissue samples.

[0151] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be immobilized on a detection plate.

[0152] Other implementation methods Implementation Method 1. A nanobody that specifically binds to CD38, characterized in that the complementarity-determining region (CDR) of the VHH chain in the nanobody has the following CDR1, CDR2, and CDR3: (a) CDR1: Its sequence is shown in SEQ ID No: 6 or 11; (b) CDR2: Its sequence is shown in SEQ ID No: 7, 9, 12 or 14; (c) CDR3: Its sequence is shown in SEQ ID No:8, 10, 13 or 15.

[0153] Implementation Method 2. The nanobody as described in Implementation Method 1, characterized in that the complementarity-determining region (CDR) of the VHH chain in the nanobody is selected from the group consisting of: (Y1) CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:14, and CDR3 shown in SEQ ID NO:15; (Y2) CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:9, and CDR3 shown in SEQ ID NO:10; (Y3) CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: 7, and CDR3 shown in SEQ ID NO: 8; (Y4) CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: 9, and CDR3 shown in SEQ ID NO: 10; (Y5) CDR1 shown in SEQ ID NO: 11, CDR2 shown in SEQ ID NO: 12, and CDR3 shown in SEQ ID NO: 13.

[0154] Implementation Method 3. The nanobody as described in Implementation Method 1, characterized in that the amino acid sequence of the nanobody that specifically binds to CD38 is as shown in any one of SEQ ID NO: 1 to 5.

[0155] Embodiment 4. A multivalent antibody or antibody targeting multiple epitopes that specifically binds to CD38, characterized in that the multivalent antibody or antibody targeting multiple epitopes includes at least one antibody element targeting the CD38 epitope, wherein the antibody element is the anti-CD38 nanobody described in Embodiment 1.

[0156] Embodiment 5. A recombinant protein, characterized in that the recombinant protein has: (i) The CD38-specific nanobody described in Embodiment 1, or the CD38-specific multivalent antibody or antibody targeting multiple epitopes described in Embodiment 4; and (ii) Optional tag sequences to assist in expression and / or purification.

[0157] Embodiment 6. A chimeric antigen receptor (CAR) fusion protein, characterized in that the chimeric antigen receptor (CAR) fusion protein comprises, from the N-terminus to the C-terminus: (i) Specifically binds to the antigen-binding domain of CD38, wherein the antigen-binding domain contains the nanobody described in Embodiment 1; (ii) Transmembrane domains; (iii) At least one co-stimulatory domain; and (iv) Activate the structural domain.

[0158] Embodiment 7. The CAR fusion protein as described in Embodiment 6, characterized in that the CAR has the structure shown in Formula Ia: L-VHH-FH-TM-C-CD3ζ (Ia) In the formula, Each "-" independently represents a linking peptide or peptide bond; L represents the signal peptide sequence; VHH is an antigen-binding domain that specifically binds to CD38; the amino acid sequence of the VHH is shown in any of SEQ ID NO: 1 to 5; F stands for Flag tag; H represents the hinge area; TM represents a transmembrane domain; C is the co-stimulation signal structure domain; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type or its mutants / modifiers).

[0159] Embodiment 8. The CAR fusion protein as described in Embodiment 6, characterized in that the CAR fusion protein has any of the amino acid sequences shown in SEQ ID NO: 25-29.

[0160] Implementation Method 9. An antibody-drug conjugate, characterized in that the antibody-drug conjugate comprises: (a) Nanobodies as described in Embodiment 1, multivalent antibodies or antibodies targeting multiple epitopes as described in Embodiment 4, and recombinant proteins as described in Embodiment 5; and (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

[0161] Embodiment 10. A polynucleotide, characterized in that the polynucleotide encodes a protein selected from the group consisting of: the nanobody described in Embodiment 1, or the multivalent antibody or antibody targeting multiple epitopes described in Embodiment 4, the recombinant protein described in Embodiment 5, or the CAR fusion protein described in Embodiment 6. Embodiment 11. An expression vector, characterized in that the expression vector contains the polynucleotide described in Embodiment 10.

[0162] Embodiment 12. A host cell, characterized in that the host cell contains the expression vector of Embodiment 11, or its genome integrates the polynucleotide of Embodiment 10, or expresses the nanobody of Embodiment 1, the multivalent antibody of Embodiment 4 or the antibody against multiple epitopes, the recombinant protein of Embodiment 5, or the CAR fusion protein of Embodiment 6.

[0163] Embodiment 13. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises: (i) The nanobody described in Embodiment 1, or the multivalent antibody or antibody targeting multiple epitopes described in Embodiment 4, or the recombinant protein described in Embodiment 5, or immune cells expressing the CAR fusion protein of Embodiment 6; and (ii) Pharmaceutically acceptable carriers.

[0164] Embodiment 14. Use of the nanobody of Embodiment 1, or the multivalent antibody or antibody targeting multiple epitopes of Embodiment 4, or the recombinant protein of Embodiment 5, or the immune cells expressing the CAR fusion protein of Embodiment 6, characterized in that it is used to prepare a drug for the prevention and / or treatment of diseases related to CD38 positive expression.

[0165] Implementation Method 15. A method for treating a disease, characterized in that it includes administering to a subject requiring treatment: the nanobody described in Implementation Method 1, or the multivalent antibody or antibody targeting multiple epitopes described in Implementation Method 4, or the recombinant protein described in Implementation Method 5, or immune cells expressing the CAR fusion protein described in Implementation Method 6.

[0166] The main advantages of this invention include: (a) The single-domain antibody (VHH domain) of CD38 of the present invention has high specificity and optimal affinity.

[0167] (b) The CD38 CAR T cells of the present invention exhibit highly specific in vitro cytotoxicity against CD38-positive target cells.

[0168] (c) The CD38 CAR T cells of the present invention can effectively inhibit the growth of CD38 positive tumors in vivo and show a long-lasting anti-tumor effect.

[0169] (d) The CD38 CAR T cell knockout / knockdown of endogenous CD38 expression of the present invention can effectively reduce cell fratricide and improve the in vivo and in vitro expansion of CAR-T cells.

[0170] (e) The antibodies of the present invention can be used for the specific detection of CD38 protein.

[0171] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0172] Example 1. Alpaca Antibody Screening Antibodies were screened from a phage antibody library. The process of establishing the alpaca antibody library is briefly described as follows: The antigen CD38 was mixed with an adjuvant and emulsified. Alpacas were immunized by subcutaneous injection five times. After the five immunizations, blood samples were collected to isolate PBMCs. Total RNA was extracted from the PBMCs and then reverse transcribed into cDNA. VHH was amplified by nested PCR. The VHH fragment was then inserted into the phage particles. The phages were amplified and the antibody library was obtained.

[0173] The CD38 antibody screening process using protein panning is briefly described as follows: CD38 antigen is coated overnight, and the antibody library is added and incubated for 1 hour after blocking the next day. After incubation, the cells are washed 8-10 times, and the bound phages are eluted and amplified. The amplified phages are isolated, purified, and then screened for the next round.

[0174] After enrichment was observed, the enriched phage was used to infect the host bacteria. Antibiotic plates were then plated, and 96 single clones / libraries were randomly selected for culturing. The overnight culture supernatant was collected, and positive single clones were identified using a Phage ELISA assay. Based on the OD value of the positive antigen wells (Pro) and the difference between the Pro and control wells (PVA) (Pro / PVA), multiple single clones were selected for sequencing. Clones with abnormal sequencing sequences and those with identical sequences were further removed.

[0175] result Based on the affinity sequencing of positive monoclonal antibodies and the frequency of antibody enrichment, five single-domain antibodies binding to CD38 were finally obtained, and their amino acid sequences and corresponding SEQ ID Nos of CDRs are shown in Tables 1, 2 and 3 below.

[0176] Table 1. Amino acid sequence of CD38 single-domain antibody .

[0177] Table 2. CDR amino acid sequences of CD38 single-domain antibodies .

[0178] Table 3. Sequence numbers (SEQ ID No) of CD38 single-domain antibodies .

[0179] Example 2. Cell Culture and Construction Molm13-Luc cells, KG1-LucG cells, HL60-LucG cells, U937-LucG cells, and K562-LucG cells were all cultured in RPMI 1640 medium. 293T and wild-type HeLa cells expressing CD38, CLL1, and both CLL1 and CD38 (HeLa-WT, HeLa-CD38, HeLa-CLL1, HeLa-CLL1-CD38) were cultured in DMEM medium. All media were supplemented with 10% (v / v) fetal bovine serum, 100 U / ml penicillin and streptomycin, 2 ml M glutamine, and 1 mM sodium pyruvate. Cells were cultured at 37°C, 5% CO2, and saturated humidity.

[0180] Among them, HeLa cells expressing CD38 are stable cell lines obtained by transfecting the CD38 antigen into a lentiviral vector, and can specifically express CD38 protein molecules; HeLa cells expressing CLL1 are stable cell lines obtained by transfecting the CLL1 antigen into a lentiviral vector, and can specifically express CLL1 protein molecules; HeLa cells expressing both CLL1 and CD38 are stable cell lines obtained by transfecting the CLL1-CD38 (T2A-linked) antigen into a lentiviral vector, and can specifically express CLL1 and CD38 protein molecules. Molm13-Luc cells are stable cell lines obtained by infection with a lentivirus expressing firefly luciferase and selected from stable cell lines. KG1-LucG cells, HL60-LucG cells, U937-LucG cells, and K562-LucG cells are stable cell lines obtained by infection with a lentivirus expressing firefly luciferase-GFP (T2A-linked) and selected from stable cell lines.

[0181] The expression of the target cell surface antigens used is as follows: Figure 1 As shown. Hela-CD38 is a cell overexpressing CD38, Hela-CLL1 is a cell overexpressing CLL1, Hela-CLL1-CD38 is a cell overexpressing both CLL1 and CD38, Hela-WT and K562 are CLL1 and CD38 double-negative cells, Molm13 is a CD38 single-positive cell, and HL60, KG1, and U937 are CLL1 and CD38 double-positive cells.

[0182] Example 3. Construction of CD38 CAR vector and preparation of virus The CD38 CAR gene consists of a CD8 signal peptide, a VHH or scFv that recognizes CD38, a CD8 hinge and transmembrane region, a 4-1BB signal region, and a CD3z signal region. The CD38 CAR gene is placed under the EF1α (EF-1α) promoter to form a CD38 CAR expression vector. CD38 CAR expression vectors are numbered B1, B2, B3, B5, and B8 according to their VHH sequences; the CD38 CAR expression vector constructed using the scFv of the CD38-targeting antibody daratumumab is named dara.

[0183] The amino acid sequence of the CD38 C-CAR in this embodiment is shown in SEQ ID No: 25-29, while the amino acid sequence of the scFv-based daratumumab CAR is shown in SEQ ID No: 30.

[0184] 2.5×10 6 293T cells were seeded at a 150cm depth. 2 Cells were cultured overnight at 37°C, 5% CO2 and saturated humidity in DMEM medium containing 10% FBS in culture dishes before transfection.

[0185] On the second day, the helper plasmid and CD38 CAR expression vector were added to a centrifuge tube containing 13.8 mL of Opti MEM medium, followed by the addition of 80 μg of PEI to obtain a mixture. The mixture was allowed to stand at room temperature for 20 minutes, and then 12 mL of Opti MEM medium was added to obtain the transfection medium. For transfection, after removing the medium, 293T cells were incubated with the transfection medium for 4–6 hours, and then the transfection medium was replaced with 20 mL of DMEM medium containing 2% FBS. After 72 hours, the medium was collected and centrifuged at 3000 g, 4 °C for 15 min. The supernatant was further centrifuged at 27000 g, 4 °C for 2 hours. The precipitate was collected and resuspended in 400 μL of pre-chilled X-VIVO medium to obtain a CD38 CAR lentiviral suspension, which was kept overnight at 4 °C. On the second day, the viral suspension was aliquoted for further use.

[0186] Example 4. Preparation of CD38-CAR T cells T cells were cultured in X-VIVO medium supplemented with 1% human serum albumin, 4% serum substitute, and 300 IU / mL human interleukin-2 (hIL-2). Pan T cells were incubated with CD3 / CD28 Dynabeads at a 1:1 ratio (CD3 / CD28 Dynabeads:T cells) and 300 IU / mL IL-2 to activate the cells. After 2 days, the activated cells were divided into two groups: one group was directly transfected with a virus to prepare wild-type CD38-CAR T cells (WT CD38-CAR T cells), and the other group underwent electroporation to knock out the CD38 gene before viral transfection to prepare CD38KO CD38-CAR T cells.

[0187] The preparation process of WT CD38-CAR T is as follows: After activating CD3 / CD28 Dynabeads for 2 days, the Dynabeads are removed using a magnetic column, and then directly heated at 37°C at a rate of 1×10⁻⁶. 6 T cells were transfected with CD38 CAR virus at a cell density of 100 cells / mL. The transfected cells were then cultured and expanded. During culture and before cryopreservation, the CAR positivity rate of T cells was detected using CD38 antigen, and the expression of CD38 on the cell surface was detected using CD38 antibody. Half of the culture medium was replaced every 2-3 days. WT CD38-CAR T cells were harvested on day 8 after removing CD3 / CD28 Dynabeads.

[0188] The preparation of CD38KO CD38-CAR T cells requires electroporation to knock out the CD38 gene in T cells activated by CD3 / CD28 Dynabeads for 2 days. The specific procedure is as follows: 0.25 nmol of gRNA targeting the CD38 gene and 16.5 μg of Cas9 protein are mixed thoroughly and incubated at 37°C for 15 min to prepare RNPs; during incubation, 18 μL of supplement buffer is added to 82 μL of Nucleofector Solution to prepare 100 μL of Lonza P3 electroporation buffer; simultaneously, CD3 / CD28 Dynabeads are removed using a magnetic column, and 1×10⁻⁶ cells are collected. 7 Activated T cells were centrifuged at 400 g for 5 min, and the cell pellet was collected. After incubation, 1 × 10⁶ cells were resuspended in 100 μL Lonza P3 electroporation buffer. 7 The T cell pellet was then added to RNP and mixed thoroughly. The mixture was then transferred to an electroporation cuvette and placed in a Lonza 2B electroporator. Electroporation was performed using the FI-115 program. The electroporated cells were incubated overnight with 300 IU / mL IL2.

[0189] On the second day after electroporation, at 37℃, at a rate of 1× 10 6 T cells were transfected with CD38 CAR virus at a cell density of 100 cells / mL. The transfected cells were then cultured and expanded. During culture and before cryopreservation, the CAR positivity rate of T cells was detected using CD38 antigen, and the expression of CD38 on the cell surface was detected using CD38 antibody. Half of the culture medium was replaced every 2-3 days. CD38KO CD38-CAR T cells were harvested on day 8 after removing CD3 / CD28 Dynabeads.

[0190] Figure 2 This indicates that CD38 CAR expression can be detected on the surface of T cells after viral transfection using the CD38 antigen.

[0191] Because of the presence of the CD38 protein on the surface of T cells, the expression of CD38 CAR can lead to cannibalism, resulting in the elimination of T cells expressing CD38. Figure 3 A statistical analysis of the changes in the percentage of CD38+ cells during cell expansion culture showed that in WT CD38-CAR T cells, the percentage of CD38+ cells in B3 and dara cells rapidly decreased to CD38-negative and did not recover during culture; the percentage of CD38+ cells in B1, B2, B5, and B8 cells gradually decreased in the early stage of culture, and the percentage of CD38+ cells in B1, B5, and B8 cells recovered in the later stage of culture. Figure 3 B shows the expression of CD38 on the surface of WT CD38-CAR T cells in each group on the day of cryopreservation. B3 and dara were CD38 negative. B1, B2, B5, and B8 were similar to NT, showing partial CD38 positivity, but the average fluorescence intensity of the CD38+ portion of B1, B2, B5, and B8 cells was lower than that of NT cells.

[0192] Similarly, the cannibalistic effects caused by CD38 CAR expression lead to changes in the percentage of CAR+ cells. The dynamic changes in CD38 CAR expression on the cell surface are shown below. Figure 4 As shown, the CAR positivity rate was continuously upregulated in unedited WT CD38-CAR T cells, while the CAR positivity rate did not change drastically in CD38KO CD38-CAR T cells.

[0193] The presence of cannibalism can also affect cell viability and proliferation. Figure 5 A statistical analysis of changes in cell viability during cell expansion revealed that CD38KO CD38-CAR T cells exhibited significantly higher viability than WT CD38-CAR T cells. Figure 5B analyzed the cell expansion fold, and the expansion fold of CD38KO CD38-CAR T cells was significantly higher than that of WT CD38-CAR T cells; Figure 5 C analyzed the expansion of WT (KO) CD38-CAR T cells relative to WT (KO) NT cells and found that the expansion of WT CD38-CAR T cells was significantly weaker than that of WT NT cells.

[0194] Example 5. Analysis of CD38 CAR binding antigen ability The CD38 antigen was serially diluted and used to determine the CAR positivity rate of different CD38-CAR T cells, which can be used to analyze the binding ability of different CD38 CARs to the CD38 antigen.

[0195] Analysis results as follows Figure 6 As shown, B1, B2, B3, B5, and B8 can bind less CD38 antigen compared to dara, and they also have higher average fluorescence intensity than dara under the same antigen dosage conditions.

[0196] Example 6. In vitro killing of CD38-CAR T cells The CD38-CAR T cells obtained above were subjected to in vitro killing experiments. The RTCA method was used to test the killing effect of CAR-T cells on HeLa cell lines overexpressing CD38.

[0197] The results are as follows Figure 7 As shown in the RTCA assay. Under both effector-to-target ratio conditions, the NT control group did not kill Hela-CD38 cells, while CD38-CAR T cells did kill Hela-CD38 cells. In Hela-WT cells, WT-dara showed some non-specific killing, but other groups did not show non-specific killing against Hela-WT cells.

[0198] The cytotoxic ability of tumor target cells labeled with luciferase was detected. The luciferase gene was transferred into target cells, and stable cell lines KG1-LucG and K562-LucG were obtained after clone selection. During the experiment, a luciferin substrate was added, and the luciferase reacted with luciferin to produce fluorescence. The activity of luciferase was determined by detecting the intensity of the fluorescence, and the cytotoxic effect of each CAR-T cell was obtained by detecting the cell survival rate.

[0199] Figure 8 At all E:T ratios, NT cells showed no cytotoxic function, CD38-CAR T cells exhibited dose-dependent cytotoxicity against KG1-LucG cells, and CD38-CAR T cells did not have cytotoxicity against K562-LucG cells.

[0200] In summary, after co-culturing CD38-CAR T cells with target cells (CD38-overexpressing HeLa cells, CD38-positive tumor cells, and KG1 cells), the target cells can be lysed by CD38-targeting CAR T cells; and except for WT-dara, the other groups of CD38-CAR T cells do not exhibit non-specific killing of non-target cells (wild-type HeLa cells and CD38-negative K562 cells).

[0201] Example 7. In vivo pharmacodynamic study of CD38-CAR T cells Select 4-6 week old NOG-dKO mice and inject them via tail vein with 2×10 6 Molm13-Luc cells. Tumor burden was assessed by small animal in vivo imaging six days later. On the same day, mice were grouped and injected with KO CD38-CAR T cells and NT cells. After T cell treatment, tumor burden in mice was assessed twice a week by small animal in vivo imaging.

[0202] The results are as follows Figure 9 As shown, compared to the NT control group, tumor growth was inhibited in mice injected with KO CD38-CAR T cells; KO-B5 and KO-B8 showed more durable tumor suppression than KO-dara. The reinfusion of CD38-CAR T cells in each group did not significantly affect the body weight of the mice.

[0203] Example 8. NK92 cells and culture method NK92 cells were cultured in Alpha MEM supplemented with 12.5% ​​FBS, 12.5% ​​Horse Serum, 1% Pen / strep, 1% sodium pyruvate, 1% L-glutamine, and 100U IL-2. HL60, Molm13, KG-1, THP-1, and K562 cells were cultured in RPMI 1640 supplemented with 10% FBS, 1% Pen / strep, 1% sodium pyruvate, and 1% L-glutamine. Cells were cultured at 37°C, 5% CO2, and saturated humidity.

[0204] Example 9. Packaging and transfection of CAR-NK92 lentivirus Resuscitation 293T was administered to one 150cm site. 2 The culture dishes were placed in a CO2 incubator and incubated for 72 hours. After two subcultures, the cultures were inoculated into 150 cm³ plates. 2Culture dishes were used for transfection. A four-plasmid system consisting of the lentiviral expression vector, helper plasmids gag / pol, Rev, and VSV-G was mixed with PEI transfection reagent and added to a certain volume of serum-free DMEM. The mixture was incubated for 15 min. The solution was then added to a 150 cm² culture dish containing 293T cells. 2 In a culture dish, gently mix and incubate at 37°C in a 5% CO2 cell culture incubator for 6 hours. After 6 hours, replace with fresh culture medium and continue culturing. Collect lentivirus culture supernatant after 48 and 72 hours for infection. Transfer the harvested supernatant to centrifuge tubes and centrifuge at 4000 rpm for 10 minutes at an acceleration of 9 and a deceleration of 9 to remove cell debris. Transfer all centrifuged LVV supernatant to a 0.45 μm filter for filtration and clarification, then transfer the filtrate to a new centrifuge tube. Add the clarified lentivirus supernatant to an ultracentrifuge tube and balance on a balance. Place the balanced ultracentrifuge tube in a hanging cup, position the hanging cup in the corresponding position on the rotor, and then centrifuge together at 4°C, 100,000 g, for 90 minutes, with the acceleration and deceleration set to maximum. After ultracentrifugation, discard the supernatant, add 0.5 mL of culture medium to each tube, and resuspend at 2-8°C for 2 hours. The harvested virus was added to 1e6 NK92 and mixed in a 24-well plate, and then incubated in a CO2 incubator for 72 hours.

[0205] Example 10. CAR-NK92 Flow Cytometry Detection and Sorting Remove the cell suspension, wash three times with DPBS, centrifuge at 300g for 5 min, discard the supernatant, add antibody mixture (anti-FLAG-APC, 1:100 in DPBS), mix well, incubate at 4℃, stain for 30 min, wash three times with DPBS, centrifuge at 300g for 5 min. Resuspend in DPBS for flow cytometry detection and sorting. During sorting, circle PE-positive cells and collect them in 5 ml flow cytometry tubes. After sorting, centrifuge at 300g for 5 min, discard the supernatant, resuspend the cells in preheated culture medium, and incubate at 37℃ in a 5% CO2 cell culture incubator.

[0206] After sorting, CAR-NK92 cells were stained with anti-FLAG-APC antibody, and the expression of CAR was detected by flow cytometry.

[0207] The results of flow cytometry detection are as follows Figure 10 As shown, the CAR-NK92 cells after sorting achieved a CAR positivity rate of over 97%.

[0208] Example 11. CAR-NK92 lethality experiment The cytotoxic ability of tumor target cells labeled with luciferase was detected. Stable cell lines expressing the luciferase gene (Molm13, HL60, THP-1, and KG1) were obtained by transfecting the luciferase gene into target cells. During the experiment, a luciferin substrate was added, and the luciferase reacted with luciferin to produce fluorescence. The activity of luciferase was determined by detecting the intensity of the fluorescence, and the cytotoxic effect of each CAR NK92 cell line was obtained by detecting the cell survival rate.

[0209] The results are as follows Figure 11 As shown, NK92 B8 exhibited more significant killing effects on all target cells than NK92, suggesting CAR-mediated killing effects.

[0210] Example 12. CAR-NK92 Multi-round Kill Experiment The day before the experiment, THP1 cells containing fluorescent target cells were resuspended and counted, and the cell density was adjusted to 8 × 10⁻⁶. 4 / ml, seeded at 100ul per well in a 96-well plate. On the day of the experiment, add the appropriate number of NK92 and CAR-NK92 cells to the wells. Place in Cellcyte to begin the experiment, taking pictures every 4 hours through bright-field and fluorescence channels. Each round lasts two days. After one round of killing, remove part of the supernatant, transfer to a newly seeded plate of target cells, and place in Cellcyte to begin a new round of killing. The KillingIndex is calculated by the decrease in the number of fluorescent cells after adjusting for zero-crossing time and normal cell growth error.

[0211] Results of multiple rounds of lethality experiments as follows Figure 12 As shown, NK92 B8 cells exhibited stronger killing ability than NK92 cells, suggesting that CAR-mediated killing plays a sustained role.

[0212] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A nanobody that specifically binds to CD38, characterized in that, The complementarity-determining region (CDR) of the VHH chain in the nanobody has the following CDR1, CDR2, and CDR3: (a) CDR1: Its sequence is shown in SEQ ID No: 6 or 11; (b) CDR2: Its sequence is shown in SEQ ID No: 7, 9, 12 or 14; (c) CDR3: Its sequence is shown in SEQ ID No:8, 10, 13 or 15.

2. The nanobody as described in claim 1, characterized in that, The complementarity-determining region (CDR) of the VHH chain in the nanobody is selected from the following group: (Y1) CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:14, and CDR3 shown in SEQ ID NO:15; (Y2) CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:9, and CDR3 shown in SEQ ID NO:10; (Y3) CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: 7, and CDR3 shown in SEQ ID NO: 8; (Y4) CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: 9, and CDR3 shown in SEQ ID NO: 10; (Y5) CDR1 shown in SEQ ID NO: 11, CDR2 shown in SEQ ID NO: 12, and CDR3 shown in SEQ ID NO:

13.

3. The nanobody as described in claim 1, characterized in that, The amino acid sequence of the CD38-specifically binding nanobody is shown in any one of SEQ ID NO: 1 to 5.

4. A multivalent antibody that specifically binds to CD38 or an antibody targeting multiple epitopes, characterized in that, The multivalent antibody or antibody targeting multiple epitopes includes at least one antibody element targeting the CD38 epitope, said antibody element being the anti-CD38 nanobody of claim 1.

5. A recombinant protein, characterized in that, The recombinant protein has the following characteristics: (i) the CD38-specific binding nanobody of claim 1, or the CD38-specific multivalent antibody or antibody targeting multiple epitopes of claim 4; and (ii) Optional tag sequences to assist in expression and / or purification.

6. A chimeric antigen receptor (CAR) fusion protein, characterized in that, The chimeric antigen receptor (CAR) fusion protein comprises, from the N-terminus to the C-terminus: (i) Specifically binds to the antigen-binding domain of CD38, wherein the antigen-binding domain contains the nanobody of claim 1; (ii) Transmembrane domains; (iii) At least one co-stimulatory domain; and (iv) Activate the structural domain.

7. The CAR fusion protein as described in claim 6, characterized in that, The CAR described above has the structure shown in Formula Ia: L-VHH-FH-TM-C-CD3ζ (Ia) In the formula, Each "-" independently represents a linking peptide or peptide bond; L represents the signal peptide sequence; VHH is an antigen-binding domain that specifically binds to CD38; the amino acid sequence of the VHH is shown in any of SEQ ID NO: 1 to 5; F stands for Flag tag; H represents the hinge area; TM represents a transmembrane domain; C is the co-stimulation signal structure domain; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type or its mutants / modifiers).

8. The CAR fusion protein as described in claim 6, characterized in that, The CAR fusion protein has any of the amino acid sequences shown in SEQ ID NO: 25-29.

9. An antibody-drug conjugate, characterized in that, The antibody-drug conjugate contains: (a) The nanobody of claim 1, the multivalent antibody or antibody targeting multiple epitopes of claim 4, and the recombinant protein of claim 5; as well as (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

10. A polynucleotide, characterized in that, The polynucleotide encodes a protein selected from the group consisting of: the nanobody of claim 1, or the multivalent antibody or antibody targeting multiple epitopes of claim 4, the recombinant protein of claim 5, or the CAR fusion protein of claim 6.

Images